|UFDC Home||myUFDC Home | Help|
This item has the following downloads:
1 SELENIUM AS A POTENTIAL IMMUNOMODULATOR IN STRENUOUSLY EXERCISED HORSES By JILL BOBEL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Jill Bobel
3 To my parents
4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Lori Warren, for inspiring and believing in me and for hiring me with no prior lab experience. Her dedication to her students and her research continues to amaze and encourage me. I feel extremely lucky to be on the forefront of equine nutritional research. She has even stimulated my interest for a possible profession in academia; something I had never considered. I am also grateful to my committee members, Dr. Jeffrey Abbott and Dr. Lee McDowell, for taking the time to review my thesis and enhance my understanding in this scientific field. I would especially like to thank Dr. Abbott for his assistance with my immun ological assays as well as the use of his laboratory and supplies. There were several late nights and long discussions and I am grateful for all his support. Jan Kivipelto deserves a very special thank you. If it was not for her unwavering belief in me an d positive influence in my abilities, I do no t know if graduate school would have defined my future. Additionally, Jan was an instrumental part of the cell isolation process. These days would have been impossible without her help. I would like to thank C arlos Sulsona for the immense amount of time spent with contributed immensely to my success and I hope to be able to re pay the favor one day. Many thanks to the numerous in dividuals who made this trial and subsequent laboratory work a possibility; Sarah White, Heather Sorensen, Liliana Crosby, the crew of the Equine Science Center and all the undergraduate volunteers. And last but certainly not least, I would like to thank my parents for their unyielding without you
5 them and I do not know where I would be without their guidance. Even when the stress was overwhelming, I could always count on them to calm my nerves, tell me to breathe and then help me see a solution. I will forever carry the lessons they have taught me about priorities. I feel extremely lucky to hav e such amazing role models in my life whose expectations keep me striving for bigger and better things.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 REVIEW OF LITERATURE ................................ ................................ .................... 14 Overview of the Immune System ................................ ................................ ............ 14 Genera l Branches of the Immune System ................................ ........................ 14 Th1 and Th2 Responses ................................ ................................ .................. 15 Oxidative Stress ................................ ................................ ................................ ...... 19 Reactive Oxygen Species Production ................................ .............................. 19 ROS Production Good vs. Bad ................................ ................................ ......... 20 Exercise and the Immune System ................................ ................................ .......... 22 Exercise induced Muscle Damage and Repair ................................ ................. 22 Exercise Drives a Th2 Response ................................ ................................ ..... 23 Exercise induced Immunosuppression ................................ ............................. 26 Exercise induced Immunoenhancement ................................ .......................... 30 Selenium ................................ ................................ ................................ ................. 31 Role of Antioxidants ................................ ................................ ......................... 31 ................................ ................................ ... 32 Selenium in the Immune System ................................ ................................ ...... 34 Selenium Drives a Th1 Response ................................ ................................ .... 36 Selenium Requirement in Horses ................................ ................................ ..... 37 Effect of Selenium on Immune Function in Horses ................................ ........... 40 2 INTRODUCTION ................................ ................................ ................................ .... 44 3 METHODS AND MATERIALS ................................ ................................ ................ 4 6 Horses ................................ ................................ ................................ .............. 46 Dietary Treatments ................................ ................................ ........................... 46 Exercise Test ................................ ................................ ................................ .... 47 Blood Collection ................................ ................................ ............................... 48 PBMC Isolation ................................ ................................ ................................ 49 Complete Blood Count ................................ ................................ ..................... 50 Serum Se ................................ ................................ ................................ ......... 50
7 Lymphocyte Proliferation Assay ................................ ................................ ....... 50 Lymphocyte Viability Assay ................................ ................................ .............. 53 Statistical Analysis ................................ ................................ ............................ 55 4 RESULTS ................................ ................................ ................................ ............... 57 Serum Se ................................ ................................ ................................ ......... 57 Complete Blood Count ................................ ................................ ..................... 57 Lymphocyte Proliferation ................................ ................................ .................. 58 Lymphocyte Viability ................................ ................................ ......................... 59 5 DISCUSSION ................................ ................................ ................................ ......... 67 6 CONCLUSIONS ................................ ................................ ................................ ..... 78 APPENDIX A PRO TOCOL FOR PBMC ISOLATION ................................ ................................ .... 80 B PROTOCOL FOR CELL COUNTING ................................ ................................ ..... 82 C PROTOCOL FOR LYMPHOCYTE PROLIFERATION ................................ ............ 85 Thawing Cells from Liquid Nitrogen ................................ ................................ .. 85 Cell Counting Protocol for Lymphocyte Proliferation ................................ ........ 86 Calculations of Cells and Mitogens for Lymphocyte Proliferation Plate ............ 87 Addition of Tritiated Thymidine to Lymphocyte Proliferation Plate .................... 88 Lymphocyte Proliferation Cell Harvest ................................ ............................. 89 D PROTOCOL FOR H 2 O 2 LYMPHOCYTE VIABILITY ................................ ............... 91 E PROTOCOL TO MAXIMIZE CELLS DURING PBMC ISOLATION ......................... 92 LITERATURE CITED ................................ ................................ ................................ .... 93 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 102
8 LIST OF TABLES Table page 2 1 Th1 and Th2 cytokine profiles and their actions ................................ ................. 16 3 1 Nutrie nt composition of basal diet feeds and sodium selenite supplement ......... 56 4 1 Mean circulating leukocyte populations on d 0 (baseline), pre exercise (d 34), immediately after (0 h) and 6 h after ex ercise (d 35), and 24 h after exercise (d 36) ................................ ................................ ................................ .................. 65 4 2 Lymphoproliferative responses (CPM) to two concentrations of three different mitogens from samples obtained before exercise (Pre Ex) and 6 and 24 h post exercise in horses supplemented with 0.1 mg Se/kg DM (NRC S e) or 0.3 mg Se/kg DM (HIGH Se) ................................ ................................ .............. 66
9 LIST OF FIGURES Figure page 4 1 Serum Se concentrations in horses receiving 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se) for 34 d. Overall effects of time (P=0.3), treatment (P=0. 02), and time*treatment (P=0.14) ................................ ............... 60 4 2 Time effect on background lymphocyte proliferat ion without mitogen stimulation in samples obtained from horses before (Pre Ex) and 6 and 24 h after prolonged exercise ................................ ................................ ..................... 61 4 3 Lymphocyte stimulation index (SI) for concanavalin A (ConA), pokeweed (PWM) and phytohemmaglutinin (PHA) mitogens in samples obtained from horses before (Pre Ex) and 6 an d 24 h after prolonged exercise ....................... 62 4 4 Percen tage of dead perpheral blood mononuclear cells (PBMC) after exposure to 10 mM H 2 O 2 for 4 h in samples obtained before (Pre Ex) and 6 and 24 h after prolonged exercise from horses fed 0.1 mg Se/kg DM (NRC S e) or 0.3 mg Se/kg DM (HIGH Se) ................................ ................................ ... 63 4 5 Percentage of dead perpheral blood mononuclear cells (PBMC) not exposed to H 2 O 2 in samples obtained before (Pre Ex) and 6 and 24 h after prolonged exercise from horses fed 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se) ................................ ................................ ................................ ........... 64
10 LIST OF ABBREVIATION S 3 H Tritiated BAL Bronchoalveolar Lavage BW Bodyweight CAT Catalase CBC Complete Blood Count ConA Concanavalin A CPM Counts Per Minute DMSO Dimethyl Sulfoxide DTH Delayed Type Hypersensitivity EHV Equine Herpes Virus Ethd Ethidium H omodimer FAD Flavin Adenine Dehydrogenase FBS Fetal Bovine Serum GPx Glutathione Peroxidase IFAS Institute of Food and Agricultural Sciences IFN Interferon Ig Immunoglobulin IL Interleukin KLH Keyhole Limpet Hemocyanin LAK Lymphocyte Activated Killer LSM Lymphocyte Separation Medium MHC Major Histocompatibility Complex NF Nuclear Factor Kappa B NK Natural Killer Cells
11 PBMC Peripheral Blood Mononuclear Cells PBS Phosphate Buffered Saline PHA Phytohemmaglutinin PMA Phorbol Myristate Acetate PWM Pokeweed Mitogen ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute Se Selenium SI Stimulation Index SOD Superoxide Dismutase TB Trypan Blue Th T Helper TNF Tumor Necrosis Factor Trx Thiore doxin TrxR Thioredoxin Reductase WBC White Blood Cells
12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SELENIUM AS A PO TENTIAL IMMUNOMODULATOR IN STRENUOUSLY EXERCISED HORSES By Jill Bobel December 2011 Chair: Lori Warren Major: Animal Science s Exercise is widely known to cause physiological changes that can result in alterations in immune cell function and thus influ ence susceptibility to disease. The production of cytokines in response to exercise induced tissue trauma is believed to drive a T helper lymphocyte (Th) 2 immune response while suppressing the Th1 pathway, potentially making the host more susceptible to viral infections. Selenium (Se) is a potent antioxidant and has been shown to promote certain components of a Th1 type response. It was hypothesized that Se supplementation could modify the exercise induced shift towards a Th2 type response Such a shift c ould help decreas e risk of viral or intracellular pathogens infections that are common in performance horses. Twelve un conditioned Thoroughbred horse s were randomly assigned to receive either the current National Research Council recommendation of 0.1 mg S e/kg DM (n=6) or three times the recommend ation at 0.3 mg Se/kg DM (n=6) for 36 d. On d 35, the horses underwent a 2 h bout of sub maximal, aerobic exercise covering a distance of 25.8 km Blood samples were obtained before and up to 24 h post exercise for determination of serum Se and identification of leukocyte populations In addition, p eripheral blood mononuclear cells were isolated for immune assays. Serum Se concentrations were
13 higher in horses receiving 0.3 than 0.1 mg Se/kg DM. Prolonged exercise m ore than level of dietary Se, affected immune status and lymphocyte function. Exercise resulted in an increase (P<0.05) in circulating neutrophils and decreases (P<0.05) in lymphocytes and eosinophils which persisted through 24 h post exercise Lymphoprol iferative responses to three different mitogens were suppressed at 6 h (P<0.01) and 24 h (P<0.05) post ex ercise Overall lymphocyte cell viability was decreased (P<0.05) at 24 h post exercise. Lymphocyte cell viability following hydrogen peroxide exposure in vitro was decreased (P<0.05) at 24 h post exercise. These data indicate lymphocytes may be more vulnerable and not function properl y during recovery from exercise, which could leave horses at risk for infect ions following strenuous exercise Feeding a h igher level and or a higher available form of Se may help lymphocytes cope with exercise induced oxidative stress, but more study is needed before recommendations can be made.
14 CHAPTER 1 REVIEW OF LITERATURE Overview of the Immune System General Branches of the Immune System molecules and can launch entities To accomplish this, the immune system is divided into two functiona l divisions; innate and acquired (Calder, 2007). Innate immunity is considered first line of defense. The functional units of this division are physical barriers, circulating molecules and phagocytic cells (Calder, 2007). Physical barriers inclu de the skin, mucosal membranes, stomach acid and beneficial bacteria in the gut. The circulating proteins which help during an innate immune response are complement and macrophage derived cytokines. Phagocytic cells, such as monocyte s, macrophages and natural killer (NK) cells are responsible for the actual elimination of the pathogen. The events of an innate immune response trigger activation and involvement of the acquired immune response because this response relies on previous encounters with the same pathogens either through vaccination or natural exposure in the environment Acquired immunity involves B and T lymphocytes (also referred to as B and T cells) antibodies and lymphocyte d erived cytokines. Acquired immunity is further divided into humoral and cell mediated immunity (Calder, 2007). An immune response that is provoked by extracellular pathogens and requires B cells and antibodies is considered humoral immunity (Calder, 2007) Defense against p athogens which escape the humoral defense and cause infection by entering
15 cells require s cell mediated immunity. This method involves antigen specific T cells. T cells et al ., 2008). Alpha and beta T cells are distinguished by either a CD4 or a CD8 cell surface protein, respectively (Murphy et al ., 2008). CD8+ T cells are destined to become cytotoxic T cells and function in the killing of infected cells whereas CD4+ T cells can differentiate into two different types of T helper cells (Murphy et al ., 2008). Th1 and Th2 Responses It has been determined that T helper (Th) lymphocytes are a crucial aspect of immune function (Morel and Oriss, 1998). Two unique functional subsets of immune cells, Th1 and Th2 lymphocytes, are largely responsible for a working immune system. In general terms, Th1 cells are associated with a cell mediated immune response and the killing of intracellular pathogens, while Th2 cells are responsible for humo ral immunity and antibody production. At homeostasis, a balance between Th1 and Th2 cells and their responses exists. Certain immune events, like muscle damage or pathogen exposure, cause the activation of Th1 or Th2 precursor cells and tip the balance in favor of the now activated Th1 or Th2 cells. During an immune response, an antigen presenting cell presents to a nave T cell which causes the production of interleukin (IL) 2. This cytokine is responsible for up regulating the proliferation of more T cell s to help the immune response (Murphy et al ., 2008). Nave T cells differentiate into Th0 cells and depending on whether IL 12 or IL 4 is present, Th0 cells will differentiate into either Th1 or Th2 cell s respectively (Murphy et al 2008). Each Th subse t has a prevailing cytokine pattern, which allows for the (Table 2 1).
16 Table 2 1. Th1 and Th2 cytokine profiles and their actions Th1 Cytokines Main action Th2 Cytokines Main action IL 1 Th1 cell/macrophage activation IL 4 B cel l activation, Th2 differentiation IL 2 T cell proliferation IL 5 Eosinophil growth & differentiation IL 12 Activates NK cells, Th1 differentiation IL 9 Stimulates Th2 cells IFN Macrophage activation IL 10 Suppresses macrophages/Th1 cytokines TNF Pr omotes inflammation IL 13 Inhibits Th1, B cell growth & differentiation Th1 lymphocytes are activated by intracellular antigens and the presence of IL 12 produced by the antigen presenting cell (Murphy et al ., 2008). Once activated, Th1 cells produce a cytokine profile that makes this pathway distinguishable. Th1 cells produce additional IL is also produced by Th1 lymphocytes during this process and serves to activate monocytes, macrophages, cytotoxic T lymphocytes and NK cells, all of which can eliminate bacteria, viruses, fungi and tumor cells (Murphy et al ., 2008). Thus, INF potent stimulator of innate immunity. In macrophages, INF of nitric pathogens (Murphy et al., 2008). During a Th1 response, activated macrophages additionally produce IL et al ., 2010). INF
17 production of imm unoglobulin (Ig) G by B lymphocytes. IgG is the most common immunoglobulin in the blood and extracellular fluid and is responsible for opsonizing pathogens for engulfment by phagocytes, as well as activating the complement system (Murphy et al ., 2008). The expression of major histocompatibility complex (MHC) class I and II is also up regulated by INF infected cells to be more easily recognized and eliminated (Murphy et al ., 2008). In addition to IL 12 and IFN activated Th1 lymphocytes a lso produce IL 2, tumor 2 produced by these lymphocytes stimulates monocytes and NK cells, bu t its main job is to further up regulate the proliferation of T cells for immune defense (Murphy et al ., 2008). TNF uced by a variety of immune cells, including T lymphocytes, and acts as a potent pro inflammatory molecule. It is vitally important in the stimulation of endothelial cells at a site of injury to trigger coagulants which will prevent pathogens from entering the bloodstream and spreading throughout the body (Murphy et al ., 2008). It also promotes fibroblast proliferation and collagen production, which are often measured as markers of chronic inflammation. TNF ynthesis, and can prompt the killing of some tumor cells (Murphy et al ., 2008). TNF necrosis factor family and also can kill tumor cells and activate endothelial cells, B cells, macrophages, and neutrophils (Murphy et al ., 2008). T helper cells differentiate into Th2 lymphocytes in the presence of IL 4 (Calder 2007) These lymphocytes respond to extracellular pathogens and produce antibodies involved with humoral immunity. Cytokines produced by Th2 cells include IL 4, 5, 9, 1 0 and 13 (Murphy et al., 2008). Also in the presence of IL 4, B lymphocytes are
18 stimulated and produce IgE (Murphy et al ., 2008). IgE is involved with defense against parasitic infections and allergic reactions. IL 4 stimulates the development o f cytotoxic T cells and can even cause T helper cells to proliferate in the absence of IL 2 (Murphy et al ., 2008). In macrophages, IL 4 increases their antigen presenting ability, MHC class II expression and their cyotoxicity. IL 5 production by Th2 cells activates eosinophils which are vital for defense against extracellular pathogens (Murphy et al ., 2008) IL 9 is solely produced by Th2 cells and promotes the growth of both T helper cells and mast cells which also play a large role in defense against extr acellular pathogens (Murphy et al., 2008). This interleukin plays a role in the effect of IL 4 on IgE production and, therefore, also contributes to development of allergies. IL 10 produced by Th2 cells, acts as an immunosuppressant and an anti inflammator y (Murphy et al., 2008). This cytokine regulates T cell, NK cell and macrophage function and also has a large role in the cross regulation of the Th1 and Th2 pathways (Murphy et al ., 2008). IL 13 has structural similarities to IL 4 but cannot induce the di fferentiation of Th cells (Murphy et al., 2008). It does, however, have similar effects on B cells by stimulating their proliferation and IgE secretion. IL 10 and IL 13 also suppress some macrophage activity. As mentioned above, there is a cross regulati on of th e Th1 and Th2 responses. The up regulation of one of the T h pathways will induce the down regulation of the other. However, the opposing pathway is never completely shut off and does contribute somewhat to the immune defense. This cross regulation of the pathways is accomplished through the balance of specific cytokines produced by both T cell populations. IL 4 produced by Th2 cells, inhibits the differentiation and activity of Th1
19 cells, whereas INF tion and activity of Th2 cells (Morel a nd Oriss, 1998). IL 4 also down regulates IL 1, IL 6 and TNF secretion, while INF 10 inhibits the differentiation of Th1 cells and therefore also inhibits the secretion of IL 1, IL 6, and TNF 1998). Exercise, which can induce muscle damage, is one of the many stressors that tip the balance of the Th1 and Th2 responses. Oxidative Stress Reactive Oxygen Species Production Reactive oxygen species (ROS) are oxygen contai ning molecules that are highly reactive in tissues. The high reactivity of ROS results from the presence of one or more unpaired electrons in the ir valence shells (Bayir, 2005). At high levels, common ROS molecules such as superoxide, hydroxyl, nitric oxid e, and hydrogen peroxide are toxic and cause damage to cellular lipids, proteins and nucleic acids (Hoffmann, 2007). During normal cell respiration, antioxidant systems are able to efficiently reduce the amount of ROS produced, thereby minimizing the damag ing effects. ROS production is considered a normal biological process and plays an important role in maintaining cellular redox equilibrium (Finkel, 1998). ROS are essential for mitochondrial electron transport, cell signal transduction, enzymatic reaction s, activation of nuclear transcription factors, gene expression, and the functional ability of neutrophils and macrophages to destroy pathogens (Bayir, 2005). Exercise requires a dramatic increase in oxygen uptake by the entire body. The oxygen increase during exercise is mostly used for AT P production in the mitochondria, particularly in the skeletal muscle (Via et al ., 2000). A erobic metabolism used to generate ATP in the presence of oxygen results in an increased production of ROS that
20 ing a state of oxidative stress. The physiological state of oxidative stress occurs when the body is unable to maintain the crucial balance between beneficial ROS and harmful ROS. ROS Production Good vs. Bad Oxidative stress, which it categorized by excess ROS production, can lead to the depletion of antioxidants as well as damage to cellular lipids, proteins, and DNA. Cell membrane fluidity, an important functional requirement of a cell, is determined by the presence of polyunsaturated fatty acids within t he phospholipid membrane. These polyunsaturated fatty acids are very susceptible to attack by free radicals and without functional antioxidants available to neutralize free radicals, lipid peroxidation can quickly occur (Bayir, 2005). The propagation of li pid peroxidation can be prevented by lipid soluble antioxidants ( e.g. Vit E) or the reduction of lipid hydroperoxides by Se containing glutathione peroxidase (GPx). The by products of lipid oxidation can further lead to protein oxidation; most commonly th e formation of protein carbonyls and protein thiol oxidation. The amino acids c ysteine and methionine are thought to be the most prone to free radical attack (Bayir, 2005). Oxidative changes to these two important amino acids, such as the formation of thiy l radicals, disulfides, and methionine sulfoxides, can impair certain enzyme activities. Oxidative damage by ROS can also cause DNA base damage or deletion, single strand breaks, or damage to DNA protein cross links (Bayir, 2005). In contrast to the nega tive effects of excess ROS, the presence of low concentrations of ROS plays an important role in the functional ability of many immune cells. ROS are vital in phagocyte activation and functional ability. Macrophages and n eutrophils rely on toxic oxygen der ived products, such as nitric oxide, superoxide anion
21 and hydrogen peroxide because of their toxicity to bacteria (Murphy et al., 2008). Respiratory burst by macrophages and neutrophils requires the production of ROS and is a key defense strategy for the i nnate immune response. Without this process, microorganisms would easily bypass the innate immune system and establish an infection in the host. ROS production is also important for expression of antioxidant enzymes during exercise, as well as cellular si gnaling (Via et al ., 2006). Exhaustive exercise has been effectively used in numerous studies to induce oxidative stress (Cases et al ., 2006; Tauler et al ., 2006; Ferrer et al ., 2009; Fisher et al ., 2011). Thus, exercise also permits investigation of anti oxidant defenses within the body. It has recently been shown that ROS generated by exercise signals an increase in the production of antioxidant enzymes to cope with the increased ROS production (Gomez Cabrera et al ., 2006). Exercise and ROS were linked wi th the activation of nuclear factor kappa B (NF kB) in lymphocytes. NF kB is an important signaling pathway to the activation of antioxidant enzymes such as mitochondrial superoxide dis mutase, in ducible nitric oxide synthetase, and endothelial nitric oxide synthase (Gomez Cabrera et al ., 2006). When ROS p roduction during exercise was inhibited by allopurinol, the activation of these antioxidant enzymes through NF kB were abolished. It was also shown that oxidative stress did not occur with non exhaustive ex ercise because the increase in ROS production was counterbalanced by the ROS signal to increase antioxidant defenses (Gomez Cabrera et al ., 2006). A non exhaustive bout of exercise will cause an increase in the production of ROS; however, cells are able to adapt and effectively counteract the
22 excess ROS. In contrast, ROS produced during an exhaustive bout of exercise at high inte nsity and/or prolonged duration will cause damage to localized cells and tissues. Exercise and the Immune System Exercise induced Muscle Damage and Repair During exercise, mitochondria in muscles respire and produce ROS. Some of the ROS will be efficiently reduced to a non harmful state by antioxidant enzymes, such as GPx. However, ROS production by musc les is a necessary part of the tissue repair process following exercise Depending on the intensity and duration of the exercise, muscle fibers will incur a certain amount of damage. ROS serve as signaling molecules which recruit immune cells, such as monocytes, neutrophils and macroph ages, to the site of muscle damage. Monocytes and macrophages are phagocytes that are important for the removal of senescent and apoptotic cells (Carrick and Begg 2008). Activated macrophages are under the influence of certain cytokines. During exercise, cytokines IL 4, IL 10, IL 13 are commonly produced and cause macrophages to be alternatively activated into M2 macrophages, which are responsible for antigen presentation, tissue and wound healing, and repair (Carrick and Begg 2008). Neutrophils are the p rimary phagocyte and are quickly mobilized when signaled by inflammation or infection. Neutrophils are also responsible for the recruitment of more neutrophils and monocytes by the production of pro inflammatory molecules. In the case of exercise induced m uscle damage o nce phagocytosis of dead or dying tissue occurs by neutrophils the y undergo apoptosis. Monocytes and macrophages are responsible for clearing the apoptotic neutrophils which subsequently causes the inflammatory response to be directed toward repair or resolution. High intensity or long duration exercise will produce a substantial body wide inflammatory response. If the monocytes and macrophages are
23 unable to clear all the apoptotic neutrophils, the decomposing neutrophils release toxic metabo lites into the tissue and exacerbate the inflammation (Carrick and Begg 2008). Exercise Drives a Th2 Response Strenuous exercise is known to induce skeletal muscle damage and create oxidative stress (Smith, 2003). The tissue trauma induced by intense exer cise can cause certain cytokines to be produced, including IL 4, IL 6, IL 10 and TNF and Hoffma n Goetz, 2000). This cytokine profile drives a Th2 immune response and has been shown to increase both during and after exercise in humans (Pedersen and Hoffman Goetz, 2000). In marathon runners, a very strong Th2 cytokine pattern was shown by measuring the plasma level of several cytokines (Nieman et al ., 2001). Pre race cytokine levels were compared to those observed immediately post race and 90 min post race. There was a significant increase in the levels of IL 6 and IL 10 in both post race measurements. Interleukin 6 is produced by muscle cells and works with IL 4 to promote Th2 cell differentiation. Interleukin 10 is mainly produced during a Th2 im mune response. Interleukin 4 was also measured and found to remain stable (Nieman et al ., 200 1). However, even without an up regulation of IL 4, its presence is necessary for the development of Th2 cells. In sedentary women, a single bout of exercise at 50 % of maximal oxygen uptake, caused a significant decrease in the serum level of the Th1 cytokine IL 2, but a significant increase in the serum level of Th2 cytokines IL 4 and 6 (Giraldo et al ., 2009). This indicates that even with moderate exercise in unf it people, exercise can elicit a Th2 response. In the horse, there is conflicting data on the prevailing cytokine profile following exercise. Folsom et al (2001) measure d mRNA levels of cytokines from PBMC collected from unconditioned ponies after 5 d of an incremental, high intensity exercise.
24 Compared to the resting control group, there was a significant decrease in the mRNA level of INF PBMC in exercising ponies on d 1 to d 5. Since Th1 and NK cells are the only cells that produce this cytokine, these data indicated a suppression of the Th1 pathway caused by exercise (Folsom et al ., 2001). In cont rast, 11 physically fit horses did not show any significant differences in PBMC mRNA levels of IL 12, IFN IL 4 after exercise (Ainsworth et al ., 2003 ). In this particular study, horses underwent a 9 wk conditioning program followed by a 9 min maximal exercise test. Samples were obtained prior to the commencement of the conditioning program and compared to samples taken 24 h after the exercise test. These researchers hypothesized that a change in PBMC cytokine mRNA expression may have occurred before th e 24 h sample was collected and could explain why no change was seen Davis et al (2005) measured a significant increase in mRNA cytokine concentrations in bronchoalveolar lavage (BAL) fluid. A sub maximal exercise test was performed by physically fit hor ses and induced an increase in mRNA expression of IL 4, IL 5 and IL 10 5 h after the test suggesting an induction of the Th2 pathway mRNA expression of IL 2 and IL 6 also significantly increased, but to a lesser extent. No change was noted in the mRNA of IL 1, IFN or TNF in BAL fluid Also indicating Th2 pathway stimulation, Donovan and coworkers (2007) reported a 120 fold and a three fold increase in the leukocyte expression of mRNA for IL 6 and TNF exercise test. These horses also underwent a training period of several weeks before performing the exercise test. Another high intensity graded exercise test, per formed by unconditioned Standardbred horses, showed a significant increase in PBMC mRNA levels of TNF immediately post exercise, however IL 6 remained unaffected
25 (Streltsova et al ., 2006). These researchers concluded that acute high intensity exercise in horses resulted in the increase of TNF that the cellular source of IL 6 m ay not be peripheral blood cells which could explain why the mRN A level of IL 6 remained unaltered. Had they measured the plasma concentration of IL 6, a change may have been observed. In concurrence with Streltsova et al. (2006) a similar graded exercise test with un conditioned horses elicited the same increase in circulating TNF 6 mRNA level remained unchanged (Liburt et al ., 2010b). Unexpectedly, these researchers saw no increase in the circulating mRNA level of IFN of researchers performed another study on un conditioned Standardbred horses and showed significant increases in the mRNA of IFN 1 in the blood, as well as significant increases in mRNA of IFN 6 in the muscle (Liburt et al ., 2010a). By 24 h after the graded exercise test, all the mRNA levels had returned to pre exercise values. Davis and coworkers (2007) found a significant increase mRNA Th2 cytokines from BAL fluid in conditioned horses following exercise. Increases in mRNA o f IL 5 and IL 10 were seen 24 h after sub maximal exercise was performed. The prevailing cytokine pattern following exercise in horses is still controversial. Comparisons of studies such as these are impractical because of the use of different exercise tes ts, fitness levels of the horses, sampling times, and evaluation of tissue versus blood cytokine concentrations or mRNA expression. The maj ority of studies reported here utilized a high intensity, short duration exercise test in unconditioned horses to eli cit changes in mRNA cytokine levels with conflicting results. High intensity exercise in conditioned horses tends to produce negligible change, while the few
26 studies using sub maximal exercise in physically fit horses appear to lean toward a Th2 cytokine p rofile. The data seems to be lacking on the effect of moderate intensity, long duration exercise on mRNA cytokine levels in unconditioned horses. Exercise has also been shown to increase levels of cortisol and prostaglandin E 2 in horses (Snow and Rose, 19 81; Kurcz et al. 1988 ; Horohov et al., 1999; Nagata et al., 1999 ; Robson et al ., 2003 ). Cortisol has been shown to assist in the Th2 response by stimulating the production of IL 4 and suppressing the Th1 response by inhibiting the production of IL 12 ( Smi th 2003 ). Prostaglandin E 2 stimulates the synthesis of IL 10 via an IL 4 pathway; both cytokines being essential to Th2 response (Smith 2003). When Th2 precursor cells are activated, a suppression of the Th1 pathway occurs, potentially making the host more susceptible to intracellular pathogens. This could be a major problem for competitive horses that are subject ed to physically demanding exercise during training and competition, elevated stress from transport to competitions, and increased pathogen exposu re from contact with other horses at an event. Exercise induced Immunosuppression In general, the body undergoes several changes in response to strenuous exercise. Many of these changes cause important modulatory effects on immune function which have been well documented in humans and several animal species, that prolonged endurance exercise leads to altered immunity that can last between three and seventy two hours (Smith, 2003 ). During this time, it is possible for bacteria or viruses to gain a foothold thereby increasing the risk of infection. In humans, there is an increase in neutrophils, lymphocytes, NK cell activity and lymphocyte activated killer (LAK) cell activity du ring exercise (Pedersen and Hoffman
27 Goetz, 2000). The increase of lymphocytes observed during exercise is thought to be a redistribution of activated cells verses the generation of new cells (Pedersen and Hoffman Goetz, 2000). After moderate exercise, ther e tends to be an increase in neutrophil and monocyte numbers and a decrease in lymphocytes (Pedersen and Hoffman Goetz, 2000). In response to heavy exertion, the phagocytotic and oxidative burst capacity of neutrophils and monocytes has been shown to decre ase (Nieman, 1997). However, moderate exercise tends to have the opposite effect on these phagocytes by increasing their toxicity. Because of the damage endured by muscle cells during activity, this increase may be part of the inflammatory response wherea s the decre ase seen after intense activity may be due to overloading or stress on the cells. Compared to non exercised controls the capacity of phagocytes to ingest Escherichia coli was significantly reduced in athletes for three days following a 20 km ro ad race (Mns, 1994). Moreover, NK cell activity was shown to significantly decrease for at least six hours after endurance activities (Nieman, 1997). This occurrence closely parallels the drop in blood NK cell concentration. Both human and animal studies have shown decreased proliferative response to the T cell mitogens phytohemmaglutinin (PHA) and concanavalin A (ConA) up to several hours after intensive or prolonged exercise ( Nieman, 1997; Pedersen and Hoffman Goetz 2000 ). Like NK cell activity, this de creased response parallels a drop in blood T cell concentrations. In contrast, phorbol myristate acetate (PMA) + ionomycin, PHA, ConA, and pokeweed mitogen (PWM) elicited an unchanged proliferative response one hour after intense cycle exercise compared to pre exercise values (Field et al ., 1991).
28 Mice macrophages were shown to have suppressed MCH II expression after exhaustive exercise (Nieman, 1997 ; Woods et al ., 1997 ). The ability of macrophages to express MHC II is important for antigen presentation t o T cells (Nieman, 1997). Without proper expression and presentation, the ability of T cells to respond to an antigenic challenge, such as delayed typed hypersensitivity (DTH), is decreased. In vivo cell mediated immunity was tested two days after the com pletion of a triathlon and the DTH reaction was suppressed by 60% compared to controls (Bruunsgaard et al., 1997). Exercise research in horses has been a focus of our scientific community for several decades. Although the results of these studies have be en somewhat diverse, it generally can be agreed that exercise does influence the immune system similar to that observed in humans. Intensely exercised Quarter horses, showed a significant decrease in lymphocyte proliferation in response to T cell mitogens Con A and PHA (Kurcz et al. 1988). The proliferative response was suppressed at 30 min post exercise, but returned to baseline by 24 h post exercise. The researchers found that this suppression coincided with an elevation in cortisol levels, which has als o been reported in humans. Acutely exercised Thoroughbred horses showed a significant decrease in lymphocyte proliferation but a significant increase in LAK cell activity (Keadle et al ., 1993). Immediately after the exercise test, there was a significant r eduction in influenza virus stimulated proliferation and lymphocyte proliferation in response to PWM. On the contrary, LAK activity had a n early post exercise enhancement, but returned to baseline two hours post exercise. This enhancement was suggested as a possible evolutionary adaptation in horses due to their fitness requirement in the wild (Keadle et al ., 1993).
29 significantly decreased lymphoproliferative responses and increased WBC counts 12 16 h after the race (Nesse et al ., 2002). Lymphocytes isolated 12 16 h after the race from the competing horses, had a suppressed reaction to mitogens PHA, ConA and PWM compared to their pre race values and their unraced counterparts. A strenuous 5 d exercise program resulted in significant suppression of lymphoproliferative response and INF RNA production by PBMC and increased susc eptibility to influenza in unconditioned ponies (Folsom et al ., 2001). The ponies were vaccinated against and then challenged with the influenza virus. Three of the four exercised ponies exhibited clinical signs of influenza infection following the challenge. No significant differences were seen in the neutralizing antibody titers of the exercised ponies compared to the resting controls suggesting that there was no exercise induced change in antibodies contributing to disease susceptibility. The possible explanation instead is the decrease in INF role in immunity to viruses. There was also no noted change in IL 2 mRNA production by PBMC, ev en after adding recombinant equine IL 2 to the cultures. This suggested that the exercise stressed ponies were producing IL 2 but were less responsive to its effect. Conditioned horses showed no change in PBMC mRNA levels of IL 12, INF IL 4 at 24 h a fter a maximum exercise test (Ainsworth et al., 2003). The horses underwent a training program for 9 wk prior to the exercise test. These cytokines were chosen based on their association with a Th1 or Th2 response. Similarly, Colahan et al (2002) reported no change PBMC mRNA concentrations of IL 1, 2, 4, 6, 10 and TNF
30 in conditioned horses after a short bout of intense exercise. A 9 wk training protocol was used with blood samples taken weekly. At the end of 9 wk, the horses underwent an intense bout of exercise with additional blood samples obtained 1, 4, 8, and 24 h after exercise. Potentially, no quantifiable changes in PBMC mRNA levels were seen in the previous two studies because the duration of exerci se induced stress was too short and/or the horses were physically fit. A prolonged suppression of the inn ate immune system was seen in horses following an 80 km endurance race (Robson et al ., 2003). Immediately post race, monocyte and neutrophil counts were elevated and lymphocyte counts were depressed compared to pre race values. Oxidative burst activity of both neutrophils and monocytes decreased after the race and remained below pre race values after 3 d of rest. Also noted were an increase in serum cortisol and a decrease in plasma glucose following the race. Collectively, these findings indicate strenuou s exercise (either high intensity or prolonged duration) can result in impaired cell mediated immunity and therefore greater susceptibility to viruses. This is especially important to the sport horse industry because of the marked susceptibility of these a thletes to upper respiratory tract infections caused by equine herpesvirus 1 (EHV 1) or influenza. As seen by Robson et al (2003), a prolonged suppression of the innate immune system may account for the high incidence of respiratory infections in horses i n training. Exercise induced Immunoenhancement Several positive changes in immune function have been linked to moderate intensity exercise. In humans, NK cell activity was significantly higher in marathon runners relative to non athletic controls (Nieman, 1997). This phenomenon has been
31 reported in several other instances, comparing endurance athletes to sedentary individuals ( Pedersen et al ., 1989; Tvede et al ., 1991; Nieman, 2008). Both acute and chronic moderate intensity exercise training has been shown antibody response to an influenza vaccine ( Kohut et al., 2004 ; Edwards et al ., 2006 ; Lowder et al., 2006) The incidence of upper respiratory tract infections in humans has also been shown to decrease with moderate intensity physical activity (Nieman et al., 1990; Nieman et al., 1993; Nieman, 2008). Since respiratory diseases are so prevalent in the professional sport horse industry, it would be beneficial to know if progressive training could improve, verses suppress certain immune f unctions. In 4 to 5 yr old colts, progressive training induced an increase in chemotaxis and digestive capacity of neutrophils compared to untrained controls (Escribano et al ., 2002). The percentage of neutrophil s that were able to phagocytose was signifi cantly higher in trained colts. These results were confirmed in another study that showed trained horses to have increased oxidative metabolism in neutrophils at rest and immediately after exercise and greater phag ocytic response during recovery compared t o untrained horses (Escribano et al ., 2005). These data indicate the nonspecific immune response is actually improved with training and moderate intensity exercise. Selenium Role of Antioxidants In response to the harmful nature of free radicals the body has evolved certain antioxidant defense mechanisms to combat oxidative stress. Through preventative and repair mechanisms, physical defenses and antioxidant defenses, organisms are generally able to maintain the crucial balance between free radical produc tion and free
32 radical reduction. Without such systems to maintain this balance, the body would suffer irreparable damage and eventually death. The body has several enzymatic antioxidant defenses such as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT). Non enzymatic antioxidants include tocopherol (vitamin E), ascorbic acid (vitamin C), carotenoids, glutathione, and melatonin. Antioxidants prevent the oxidation of other molecules by reducing free radical intermediates which ot herwise would set off a chain reaction of oxidation to nearby molecules. Selenium (Se) exerts its biological e ffects in the body through its incorporation into selenoproteins as the amino acid selenocysteine. Selenocystein e has been termed the twenty first amino acid and is co translationally inserted into all selenoproteins. This integration requires translational recoding of one or more uracil guanine adenine (UGA) stop codons within the mRNA to a selenocysteine inserti on codon (Tinggi, 2008). Over 30 selenoproteins have been identified in rodents and/or humans, although the function of most has yet to be determined. Thioredoxin reductases and GPx are two groups of selenoproteins that are vital for antioxidant defenses ( Hoffmann, 2007). Glutathione peroxidase utilizes 33 40% of the total body Se (Kirem i djian Schumacher and Stotzky, 1987). Within the cell, the major role of this enzyme is to maintain low levels of hydrogen peroxide and therefore decreases the potential o f free radical damage (Tapiero et al tocopherol in the regulation of lipid peroxidation. In the pentose phosphate shunt, GPx and CAT degrade hydrogen peroxide to water via glutathione reductase and flavin adenine dehydrogenase ( FAD). Currently, there are six known members of the GPx family, with GPx1 being the most
33 abundant selenoprotein in mammals (Tapiero et al ., 2003). GPx1 is a cytosolic enzyme which is expressed in every cell type and thought to be a major antioxidant prote in in mammals. The activity of this enzyme is regulated by liver Se status (Tapiero et al ., 2003). GPx2 is found mostly in the gastrointestinal tract and is the closest homolog to GPx1. In the plasma, GPx3 is a secreted glycoprotein and the second most abu ndant selenoprotein. GPx4 is localized to the cytosol and the mitochondria and reduces fatty acid hydroperoxides esterified to phospholipids. Genetic deletion of GPx4 is lethal, indicating the essential role it plays in maintaining lipid membrane integrity (Hoffman n 2007). Thioredoxin (Trx) is a widely distributed redox protein and is responsible for the intracellular regulation of several redox dependent processes. Trx can also stimulate the proliferation of both normal and tumor cells. The Se containing thioredoxin reductase (TrxR) enzymes use Trx as a substrate to maintain this antioxidant system in a reduced state for removal of hydrogen peroxide (Tinggi, 2008). Three types of TrxR have been discovered, including cytosolic (TrxR1), mitochondrial (TrxR2 ) and spermatozoa specific (TrxR3) (Tinggi, 2008). In vitro Se supplementation of human endothelial cells was shown to increase activity of GPx and TrxR and decrease the amount of hydrogen peroxide within the extracellular medium (Tolando et al ., 2000). Se lenium deficient cells had a higher amount of hydrogen peroxide detectable in the extracellular medium. These data indicate that while some hydrogen peroxide will escape into the extracellular space, GPx and TrxR control much of the intracellular hydrogen peroxide pool.
34 Selenium in the Immune System Selenium deficiency or supplementation has long been shown to influence both innate and adaptive immune responses in humans and animals (Desowitz a nd Barnwell, 1980; Stable et al., 1991 ; Israel et al ., 1992; Sa ppey et al ., 1994; Beck, 2007; Hoffman n 2007; Kamada et al., 2007; Kumar et al., 2009 ) While a Se deficiency has often been shown to reduce immune responses to tumors and viruses (Baker and Cohen, 1983; Beck et al., 200 1 ; Safir et al ., 2003 ), supplementi ng adequate levels of Se has demonstrated an enhancement of immune responses ( Desowitz and Barnwell, 1980 ; Stable et al., 1991; Israel and Gougerot Pocidalo, 1997 ; Kamada et al., 2007 ; Kumar et al., 2009 ). However, Se supplementation above adequate levels of intake has produced mixed results. Upon immune challenge and phagocyte activation, macrophages and neutrophils exhibit a rapid increase in ROS, known as oxidative burst. This process allows phagocytes to play a crucial role in anti microbial activity, as well as intracellular and extracellular signaling. However, as discussed earlier, high levels of ROS can be detrimental. The positive and negative effects of ROS within phagocytes are crucially balanced by selenoproteins. In a Se deficient state, phagoc ytes may have insufficient oxidative burst function or may undergo oxidative induced death (Hoffman n 2007). Improper activation or function of phagocytes would be detrimental to the host. Decreased phagocyte activity, superoxide production and cytokine se cretion was demonstrated in stimulated Se deficient murine macrophages compared to Se adequate controls (Safir et al ., 2003). Baker and Cohen (1983) found that Se deficiency did not affect the number of neutrophils in rats, but it did greatly affect the ne function properly as oxidative burst was reduced. The loss of oxidative burst capacity
35 was thought to be due to inadequate metabolism of hydrogen peroxide. As mentioned before, proper levels of GPx1 are required for the regulation o f hydrogen peroxide, some of which is used for the activation of phagocytes (Hoffman n 2007). It has been well documented in humans that supplementing Se to Se deficient individuals will completely prevent the development of Keshan disease from the coxsac kievirus ( Beck, 2007; Hoffman n, 2007 ). In a Se deficient state, mutations of the coxsackievirus B3 genome, which leads to increased virulence and cardiac pathology, are more likely to occur. Mutations in the genomic RNA of the influenza virus, also causing an increase in virulence, have been shown in Se deficient mice (Nelson et al ., 2001). Selenium deficiency additionally influences components of antiviral immune response. One such study demonstrated higher numbers of total virus cells in Se deficient mice infected with influenza compared to Se adequate controls (Beck et al ., 2001). It has been suggested that for anti influenza viral responses, Se deficiency affects cell mediated immunity to a greater extent than humoral immunity (Beck et al., 2001). In sup port of this theory, the levels of CD8+ and CD4+ T cells of Se deficient influenza infected mice were lower compared to control mice (Beck et al., 2001). Selenium has also been suggested to mitigate the HIV 1 infection. Because HIV 1 infects major immune c ells which mount the attack against the virus, such as T cells and monocytes, the role that Se plays to enhance anti HIV 1 immune responses differs from other viruses. In HIV 1 infected cells, Se supplementation was shown to increase GPx 1 activity leadin g to lower hydrogen peroxide levels and decreased NF (Israel et al ., 1992; Sappey et al ., 1994). The reduction in ROS by increased GPx
36 activity mitigated the ROS dependent NF required for the activatio n of the HIV 1 infection (Israel and Gougerot Pocidalo, 1997). Selenium Drives a Th1 Response Based on some of the specific effects that Se has on immune cells, it is thought to promote a Th1 immune response. Selenium has been shown to increase lymphocyte IL 2 receptor expression and enhance proliferation and differentiation of lymphocytes in vitro and in vivo in mice (Roy et al., 1993). IL 2 is considered a Th1 profile cytokine and helps to activate macrophages and the cell mediated immune response by sign aling for T cell activation, proliferation and differentiation. In general, Se deficient animals have been shown to have a depressed cell mediated immune response, further suggesting that Se drives the Th1 pathway (Hoffmann, 2007). Additionally, with progr ession of the HIV 1 virus, there is a diminished production of Th1 interleukins, especially IL 2, and Se deficiency is common in these patients (Baum et al ., 2000). Holstein cows supplemented with two different sources of Se, showed an increase in serum co ncentration of IL 2 after a viral challenge (Covey et al ., 2010). IL activated during a Th1 response. In Se deficient mice, mRNA levels of IFN 2 from medias tinal lymph nodes were greatly decreased compared to Se adequate controls (Beck et al ., 2001). IL 4 and IL 5, which are usually considered Th2 cytokines, were decreased in the Se deficient mice 4 d after infection with influenza, but by d 14 had increase d beyond the levels shown in the Se adequate mice Moreover, after the mice were infected with influenza, the Se deficient mice had a significant increase in mRNA levels of IL 4, 5, 10, and 13 in mediastinal lymph nodes, which are all Th2 cytokines (Beck et al ., 2001). Conversely, supplementation of Se to Se adequate mice
37 resulted in no difference in phagocytosis or oxidative burst of granulocytes and decreased the production of IL 4 and IFN although it was not statistically significant (Albers et al ., 2003). However, these mice did not have an increase in serum Se level after Se supplementation. More recently, studies in humans have shown a correlation between Se supplementation and lymphocyte proliferation, whi ch is preceded by enhanced expression of interleukin 2 receptor ( IL 2R ) by lymphocytes (Roy et al 1994 ). Se lenium supplementation in humans has also resulted in a more robust Th1 immune response to a live attenuated poliovirus vaccine (Broome et al ., 200 4). In this study, Se supplemented groups had significant increases in the percentage of total T cells and CD4+ T cells. Additionally, the peak of whole blood IFN was not only greater in the Se group, but it also occurred 7 day s sooner than the placebo group. Based on these studies, as well as numerous others, there appears to be sufficient evidence that Se status affects the Th1/Th2 balance of immune responses towards the Th1 pathway following an immune challenge. Selenium Requ irement in Horses The current Se requirement for all ages and classes of horses is 0.1 mg/kg DM (NRC, 2007). The requirement was based principally on two studies which found plasma Se concentrations reached a plateau when mature, idle horses were fed 0.1 m g Se/kg DM, with no further increase in plasma Se at higher levels of supplementation (Stowe, 1967; Shellow et al ., 1985). Furthermore, this level of Se intake was sufficient to prevent problems associated with Se deficiency. Shellow et al (1985) further evaluated GPx activity in response to Se supplementation using t hree different diets supplemented with sodium selenite fed to 20 mature geldings over the course of 12 wk.
38 The basal diet consisted of 0.07 mg Se/kg DM and three of the diets were additionally supplemented with 0.11, 0.16, or 0.26 mg Se/kg DM. Whole blood and plasma concentrations of Se increased in the supplemented groups until wk 6, where they remained elevated until the end of the 12 wk study. GPx activity was found to be similar among all l evels of Se supplementation and the authors concluded that this enzyme was not a good indicator of Se status in mature horses. In contrast, two more studies in the GPx r esponse to parenteral Se was much higher compared to oral Se intake (Maylin et al ., 1980; Roneus and Lindholm, 1983). More recently, plasma Se and intake levels of numerous horses of various breeds were measured (Wichert et al ., 2002). Based on the intakes of the studied horses, only 25% were receiving the recommended daily amount of Se, as set by the NRC. Interestingly, no signs of Se deficiency were seen in horses with Se intake below 1.25 g/kg BW Based on a 500 kg horse, the majority of horses examined in this study were only receiving 0.6 mg Se/day, which is far below the current requirement of 1 mg Se/500 kg BW. This study suggests that for non reproducing, non performing adult horses, the NRC is possibly overestimated. However, the influence of low S e intake on the immune system was not assessed. The best source of Se and an accurate measure of Se status are still being investigated in the horse. Richardson et al (2006), supplemented sodium selenite or zinc L selenomethionine at approximately 4 X t he NRC requirements and found no change in plasma GPx3 activity or gluteal muscle Se concentration and a decrease in muscle GPx1 activity after 56 d of supplementation. Plasma Se concentration increased quickly and reached a plateau at d 28 in horses fed zinc L selenomethionine ;
39 however by d 56 the plasma Se concentration was similar across treatments. It was concluded that neither Se source exhibited an advantage over the other and plasma GPx activity may not be a sens itive marker of Se status in Se adeq uate horses. In a similar study that investigated the effects of dietary sodium selenite and Se yeast, a correlation was found between plasma GPx3 activity and plasma Se concentration. Horses were supplemented with organic Se yeast at 0.2 mg, 0.3 mg or 0.4 mg of total Se/kg DM or sodium selenite at 0.3 mg of total Se/kg DM for 112 d. Although both Se sources caused a linear dose effect, there was no statistically significant change in plasma GPx3 activity (Calamari et al., 2009). Total Se and GPx1 activity in whole blood and plasma were greater in all supplemented groups compared to a non supplemented control. The findings of these authors agree with Richardson et al. (2006) in that neither Se source represented a clear advantage over the others, and GPx ac tivity may not be an accurate marker of Se status in horses. Karren et al (2010) reported an increase in mare and foal plasma, muscle and colostrum Se concentration but no change in mare or foal plasma GPx activity when Se yeast was fed. Mare supplementat ion was initiated approximately 110 d before estimated foaling date. In contrast to Richarson et al (2006), these researchers did find an increase in muscle Se concentration following supplementation. However, they also reported no change in plasma GPx ac tivity. Taken collectively, these studies suggest that use of GPx activity to assess response to Se supplementation may not be a sensitive indicator of Se status particularly in Se sufficient horses GPx activity may be altered at a lower Se status where as a different response variable (e.g. immune response) may only be altered at a higher Se status
40 Se lenium requirements have not been studied in horses in great detail, although the recommendation to prevent clinical deficiency is likely 0.1 mg/kg DM. It is important to note that all the studies which contributed to the current requirement were performed using only mature, idle horses. Effect of Selenium on Immune Function in Horses With recent advances in our understanding of how Se affects the immune sy stem, it may be logical to expect an enhancement of immune features with an increase in Se intake. One of the earliest studies in horses evaluating the potential for Se to enhance immune function compared responses when horses were supplemented with Se, vi tamin E or a combination of both (Baalsrud and Overnes, 1986). Horses receiving vitamin E or both vitamin E and Se, had an increased humoral immune response to tetanus toxoid and equine influenza. However, because this study used Se in combination with vit amin E, the immune affects observed may not be attributed to Se alone. A 7 wk study using Shetland ponies ages six months to three years old, investigated the effect of Se supplementation on humoral antibody production (Knight and Tyznik, 1990). Unlike m ost studies, these ponies were depleted of Se for up to one year before the study began. The ponies were randomly assigned to either a low Se (0.02 mg/kg DM) or high Se (0.22 mg/kg DM) diet. (Note: the NRC requirement for horses was 0.1 mg/kg DM). The high er level of Se intake resulted in higher serum Se concentration and GPx activity. These values remained unchanged or decreased in the low Se diet group. At wk 0 and 2, the ponies were antigenically challenged using 2 mL of sheep red blood cells and serum I gG and IgM concentrations were assessed. The serum IgG concentrations and hemagglutination titers to sheep red blood cells were
41 elevated in the high Se diet. There was also an effect of age; the older ponies had higher IgG concentrations compared to the ye arlings. IgM concentrations were not affected by treatment. The authors concluded that the high er Se diet enhanced immune requirement, this study actually compared immune r esponses of a Se adequate diet to a Se deficient diet. More recently, it was found that foals from mares receiving 3 mg of Se per day (approximately 0.3 mg/kg DM) had greater antibodies to three strains of influenza virus than foals from mares who receive d only 1 mg of Se per day (approximately 0.1 mg/kg DM) (Janicki et al ., 2001). The mares were supplemented beginning 55 d prior to foaling through 56 d post foaling. Milk Se was found to be 29 38% higher in mares receiving 3 mg of Se per day and whole bloo d GPx activity was 17 30% higher in foals from mares receiving 3 mg per day. Greater influenza antibodies in the foals suggest either the foals had an increased antibody response possibly because additional Se increased B cell proliferation and differentia tion or that the amount of antibodies in the colostrum was greater in mares receiving the high do se of Se. Unfortunately, antibodies in colostrum were not measured. A Se deficient diet was shown to reduce vaccination response in horses (Brummer et al ., 2 011 ) Horses received either an adequate Se diet of 1.4 mg/day or a low Se diet of 0.7 mg/day for 28 wk. To ensure a primary immune response to the vaccine challenge, the novel antigen keyhole limpet hemocyanin (KLH) was used. After supplementation, whole blood Se and GPx activity was lower in the low Se group. The Se adequate horses responded quicker to vaccination by producing more KLH specific
42 IgG antibodies by 3 wk compared to the LS diet. However, after the second vaccination, KLH antibody levels of we re similar for the low and adequate Se groups. No significant differences were seen in lymphocyte proliferation or PBMC mRNA expression of INF and TNF PBMC mRNA expression of the transcription factor Tbet, which controls INF expression was found to be greater at 5 wk in Se adequate than low Se horses. Altho ugh no increased expression of INF adequate horses, it can be postulated that there was priming of a cellular immune response after the initial vaccine due to the increased Tbet expression. Given the decreased producti on of KLH antibodie s with no up regulation of the expression of Tbet, horses receiving a low Se diet appeared to produce an insufficient immune response to the vaccination challenge. A large proportion of horses in the United States are not at maintenance but athletes of th e highest caliber. Like all athletes, these horses undergo a great deal of stress associated with rigorous training schedules, traveling, stabling, exposure to unfamiliar horses, and the competitions themselves. Stressors such as these may leave the horses at greater risk for infection or disease (Nesse et al ., 2002; Robson et al ., 2003 ; Stull et al ., 2004 ). Transportation has been reported to increase cortisol, neutrophil and WBC counts while decreasing lymphocyte counts and proliferative responses to ConA (Stull et al ., 2004). As stated earlier, this is the same chain of events that is triggered by exercise stress. Se lenium supplementation has received limited investigation in performance horses in regards to antioxidant benefits, but even less attention h to the best of my knowledge the study presented in this thesis is the first to investigate
43 whether Se supplementation could enhance cell mediated immune function following prolon ged exercise.
44 CHAPTER 2 INTRODUCTION Upper respiratory tract infections are a common occurrence among race and sport horses alike ( Burrell et al., 1996 ). Strenuous exercise has been shown to induce a T helper lymphocyte (T h ) 2 type response from the immun e system (Smith, 2003), whereby cell mediated immunity is down regulated, potentially making the host more susceptible to intracellular pathogens and viral infections such as influenza and Equine Herpes Virus 1. Additional stress caused by rigorous trainin g schedules, traveling, crowded stabling areas, and competition may increase exposure to pathogens and increase susceptibility to infections. Selenium (Se) deficiency or supplementation has been shown to influence both innate and adaptive immune responses in humans and animals (Desowitz and Barnwell, 1980; Stable et al., 1991 ; Israel et al ., 1992; Sappey et al ., 1994; Beck, 2007; Hoffman n 2007; Kamada et al., 2007; Kumar et al., 2009 ) When supplemented, Se is thought to drive a Th1 or cell mediated immun e response (Broome et al ., 2004; Hoffman n 2007). Selenium has been shown to increase IL 2 receptor expression by T helper cells (a Th1 response) and enhance proliferation and differentiation of lymphocytes in vitro and in vivo in mice (Roy et al., 1993). Based on these studies, as well as numerous others, there appears to be sufficient evidence that Se status affects the Th1/Th2 balance of immune responses towards the Th1 pathway following an immune challenge. Exercise has been shown to produce a Th2 cytok ine profile both during and after exercise in humans (Pedersen and Hoffman Goetz, 2000). The prevailing cytokine pattern following exercise in horses is still controversial but does appear to lean towards
45 a suppression of Th1 and an enhancement of a Th2 pa thway (Folsom et al ., 2001; Davis et al., 2005; Davis et al., 2007). C urrent understanding of how Se interacts with the equine immune system is immunosuppression and oxidative stress following strenuous exercise in horses. The current NRC (2007) requirement for Se in equine diets is 0.1 mg/kg DM, regardless of physiological state This requirement is based on a small number of studies that documented no further incre ase in seru m Se concentrations or whole blood glutathione peroxidase (GPx) activity when levels higher than 0.1 mg/kg were fed (Stowe, 1967; Shellow et al. 1985). However, Janicki et al (2001) found greater antibody titers to influenza in foals from mare s receiving 3 mg Se/d compared to 1 mg Se/d, suggesting that Se intake necessary for optimum immune function could be greater than that needed to modify serum Se or GPx activity. The objectives of the current study were to evaluate the effects of a prolon ged bout of exercise on lymphocyte proliferation and viability and determine if inclusion of Se in the diet above the NRC (2007) requirement could modify exercise induced immune responses in horses. We hypothesize that a higher level of dietary Se will pro tect lymphocytes from oxidative insult in vitro and modify the exercise induced suppression of lymphocyte responses to mitogens.
46 CHAPTER 3 METHODS AND MATERIAL S Horses Twelve mature, untrained Thoroughbred horses (6 mares and 6 geldings) with a mean SE age of 11.1 1.1 yr and body weight (BW) of 564.6 11.0 kg were utilized in this study. Horses were housed in 6 m x 18 m paddocks at the Institute of Food and Agricultural Sciences (IFAS) Equine Sciences Center in Ocala, FL. Paddocks were devoid of vege tation to facilitate control of Se intake. All animal handling and data collection procedures were reviewed and approved by the IFAS Animal Care and Use Committee at the University of Florida. Dietary Treatments Horses were blocked by age and gender, and r andomly assigned to one of two dietary treatments: 0.1 mg Se/kg DM (NRC Se, n = 6) or 0.3 mg Se/kg DM (HIGH Se, n = 6). To facilitate exercise testing and sample collection, horses were further divided into three groups of four horses each with equal dieta ry treatment representation in each group. Dietary treatments were initiated in one group of horses per day, over three consecutive days. As intended, this arrangement staggered data collection and exercise testing over 4 consecutive days. All horses were fed individually and received their respective dietary treatments for 36 d. The basal diet consisted of whole oats offered at 0.4% BW/d (20% of total diet) Coastal bermudagrass hay fed at 1.6% BW/d (80% of total diet) and a vitamin/mineral premix contain ing no Se (custom mixture, Lakeland Nutrition Group, Lakeland, FL) fed at a rate of 0.009% BW/d. Sodium selenite served as the supplemental Se source and was hand mixed into the oats daily in quantities to provide either 0.1 or 0.3 mg Se/kg
47 DM in the total diet. Sodium selenite accounted for approximately 38% and 79% of the total daily Se intake for the NRC Se and HIGH Se treatments, respectively. The horses were fed once a day at approximately 0900 h. At each feeding, a small quantity of molasses (about 30 mL) was mixed into the oats to help with adhesion of the vitamin/mineral premix. Sodium selenite was mixed separately with a small amount of applesauce (30 g) and top dressed on the oat mixture to ensure the horses would consume the selenite. Diets were f ormulated to maintain BW and meet or exceed NRC (2007) requirements for horses in light work. Nutrient analysis was performed on all feeds prior to the start of the study. Feeds were analyzed for Se by Olson Biochemistry Laboratories (South Dakota State U niversity, Brookings, SD) using the fluorometric method (974.15) outlined by AOAC (2000) with an ultraviolet detector (Turner filter, fluorometer, model 112, Unipath, Mountain View, CA). All other nutrient analyses were performed by Dairy One ( Ithaca, NY ) using standard analytical methods. The nutrient composition of all dietary ingredients is presented in Table 3 1. Throughout the study, BW of horses was monitored weekly using a livestock scale accurate to 0.5 kg (MP800, Tru Test, Inc., Mineral Wells, TX ). Feeding regimes were adjusted as necessary, based on changes in BW. Exercise Test In order to acclimate the horses to the outdoor free stall exerciser used for exercise testing, horses underwent 10 min of walking and trotting on three separate occasions Otherwise, horses remained sedentary prior to exercise testing. On d 35 at 0800 h, horses were subjected to a 120 min bout of submaximal exercise on the free stall exerciser. Horses were fitted with onboard heart rate monitors equipped with
48 telemetry sig naling for remote monitoring (Polar Equine, Polar Electro Inc., Lake Success, NY). The exercise bout consisted of 7 replications of walking (1.51 m/sec; for a total of 21 min), jogging (4.09 m/sec; for a total of 41 min), and extended trotting or cantering (4.83 m/sec; for a total of 43 min). Midway through the exercise bout, horses were removed from the exerciser, hand walked for 15 min, offered water, then returned to the free stall exerciser for the remaining 45 min of exercise. The total distance travel ed was 25.8 km with a mean heart rate of 135 39 bpm at the fastest speed of 4.83 m/s. Mean ambient temperature during exercise testing was 20.0 1.7C with 93.8 1.2% relative humidity. Blood Collection Blood samples were obtained before (d 0) and aft er 3 4 d of supplementation to determine serum Se concentrations. Additional blood samples were obtained at d 0, before exercise (pre ex) immediately after exercise (0 h post ex) and at 6 (6 h post ex) and 24 h post exercise ( 24 h post ex). Basic complete blood count ( CBC ) panels were conducted at all sample points. Peripheral blood mononuclear cells (PBMC) were isolated for immune assays from samples obtained at pre ex, 6 h post ex and 24 h post ex At each sampling interval, approximately 48 mL of blood was collected from each horse by jugular venipuncture into evacuated tubes (Vacutainer, Becton Dickinson Co., Franklin Lakes, NJ) containing either no anticoagulant for harvesting of serum or in tubes containing ethylenediaminetetraacetic acid (EDTA) or so dium heparin for CBC test or harvesting of PBMC, respectively. Serum was harvested and stored in polypropylene cryogenic vials in 0.05 2.0 mL aliquots at performed.
49 PBMC Isolation Reagents for PBMC isolation include d phosphate buffer ed saline (PBS; MediaTech Inc, Manassas, VA), lymphocyte separation medium (LSM, MP Biomedicals, Solon, OH ), trypan blue (TB 0.4% Sigma Aldri ch, St Louis, MO), dimethyl sulfoxide (DMSO, MediaTech Inc, Manassas, VA ), and fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA). Blood samples collected for PBMC isolation were immediately transported to the laboratory and processed within 2 h of collection. During transport and until processing, blood tubes were continually mixed by gentle inversion at room temperature. Using sterile technique, 25 mL of whole blood was diluted in 10 mL of PBS and gently inverted. The dilute d blood was slowly layered over 15 mL of LSM while maintaining a sharp interface. Vials were centrifuged at 400 g for 25 min at 22 C (room temperature). Plasma was aspirated off to within 0.5 cm above the PBMC buffy coat and discarded. The buffy coat (cont aining PBMC) and approximately half the LSM (approximately 10 12 mL) was removed and placed in a separate vial. PBMC vials were brought up to a volume of 40 mL with PBS and gently inverted. The vials were then centrifuged at 100 g for 10 min at 22 C (room temperature). The supernatant was suctioned off and discarded, leaving the PBMC pellet undisturbed. The PBMC pellet was then broken up and rinsed with 10 12 mL of PBS. The vials were centrifuged again for 10 min at 100 g The supernatant was suctioned off and discarded and the PBMC pellet was re suspended in 1 mL of PBS. Using trypan blue exclusion, live cells were counted using light microscopy at 40x magnification by loading a hemacytometer with 10 L of a mixture containing 90 L trypan blue and 10 L PB MC. A maximum of 5x10 6 live cells per vial were added to freezing media and frozen in 1 mL aliquots in cryogenic vials.
50 Freezing media consisted of 10% DMSO and 90% FBS. Cryogenic vials were placed in a Nalgene Mr. Frosty Cryo 1 C Freezing Container and i nitially placed in a 80 C freezer for 24 h. The vials were then removed and stored in liquid nitrogen until analyses were performed. Complete Blood Count Blood tubes used for CBC analysis were placed on ice immediately after collection and delivered to th e University of Florida Clinical Pathology Diagnostic Lab for analysis. Hematology automated system was used to determine the number and percent of total leukocytes neutrophils, lymphocytes, monocytes, eosinophils, and basophils. The Advia Hematology system uses a combination of light scatter, cytochemical staining and nuclear density on two independent channels to measure the total and differential leukocyte counts. Serum Se Serum was analyzed for Se concentration by Olson Biochemis try Laboratories (South Dakota State University, Brookings, SD) using the fluorometric method (974.15) outlined by AOAC (2000) with an ultraviolet detector (Turner filter, fluorometer, model 112, Unipath, Mountain View, CA). Lymphocyte Proliferation Assay Reagents for lymphocyte proliferation included Roswell Park Memorial Institute 1640 medium (RPMI, Hyclone Laboratories Inc, Logan UT), fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA), 2 mercaptoethanol (Fisher Scientific, Fairlawn, NJ), ge ntamycin (MediaTech Inc, Manassas, VA), GlutaMax 100x (Gibco Invitrogen cell culture, Grand Island, NY), HEPES (MediaTech Inc, Manassas, VA), trypan blue (TB 0.4% Sigma Aldrich, St Louis, MO), phosphate buffered saline ( PBS; MediaTech Inc,
51 Manassas, VA) C oncanavalin A (ConA, Sigma Aldrich, St Louis, MO), p hytohemmaglutinin (PHA, Sigma Aldrich, St Louis, MO ), and pokeweed mitogen (PWM, Sigma Aldrich, St Louis, MO ). Each mitogen for this assay was chosen based on the ability to stimulate a different populat ion of immune cells. PHA and ConA predominately stimulate the T cell population, while PWM predominately stimulates B cells (Bell et al., 2001). In addition, two concentrations of each mitogen were evaluated to show a possible titration effect. The tritiat ed [ 3 H] thymidine incorporation method was used to assess lymphoproliferative responses. To maintain cell viability, three samples were removed from liquid nitrogen at a time and partially thawed in at 56 C water bath for 1 2 min. The ice chunk created fro m partial thawing was immediately emptied and dissolved into 13 mL of lymphocyte culture medium in an effort to limit PBMC exposure to the DMSO in the freezing medium. Lymphocyte culture medium consisted of 86% RPMI 1640 medium, 10% FBS, 0.1% 2 mercaptoeth anol (50 mM), 0.1% gentamycin (50 mg/m L ), 1% L glutamine (200 mM), and 2.5% HEPES (25 mM). The same batch of culture medium and mitogen preparations were used for all samples. Vials with thawed PBMC and medium were centrifuged for 10 min at 250 g at 10 C. Supernatant was removed and the cell pellet was re suspended in an amount of PBS appropriate for cell pellet size (approximately 400 L of PBS). In preparation for the hydrogen peroxide viability assay performed on the same day (see below), cells were re suspended in PBS instead of culture medium. Based on preliminary experiments, the amount of PBS contained in the wells after plating is negligible and produces no harmful effects (data not shown). Using trypan blue exclusion, live cells were counted using light microscopy at 40x
52 magnification by loading a hemacytometer with 10 L of a mixture of a 90 L trypan blue and 10 L PBMC. Viability of cells varied from 36 80 % and greatly depended on collection time of the original sample with lower viability consi stently observed in the 24 h post ex samples Aliquots of 50 L of the cell suspension (7x10 4 live cells/well) were pipetted into 96 well clear, round bottom plates (Corning Inc., Corning, NY). Most s amples were analyzed in triplicate for each mitogen conc entration; however a small number of samples were analyzed in duplicate or with only a single mitogen concentration due to limited cell numbers. PBMC samples obtained from a donor horse that was not part of this study were included on each plate to serve as an interassay control. Cells in separate wells were stimulated with 50 L of either 0.5 g/mL ConA, 1 g/mL ConA, 0.5 g/mL PWM, 1 g/mL PWM, 5 g/mL PHA, 10 g/mL PHA, or culture medium (no mitogen control). Optimal concentrations of cells, mitogen conc entrations and incubation times were determined prior to the start of this study (data not shown). The cells were incubated at 37C for 96 h in 6% CO 2 Sixty hours into the incubation period, 25 L of [ 3 H] thymidine (0.25 Ci/well; PerkinElmer, Boston MA) was added to each well and then the plate was returned to the incubator. Eighteen hours after the [ 3 H] thymidine was added, wells were harvested using a FilterMate Har vester (PerkinElmer, Turku, Fin land) onto printed Filtermat A glass fiber filter paper (size 90 x 120 mm; Wallac Oy, Waltham, MA) and dried in a 900 W household microwave oven (Westing House, Lake Forest, IL) for 90 sec The filter paper was then seal ed in a plastic bag with 3.5 mL of scintillation fluid (Betaplate Scint, PerkinElmer Life Sciences, Waltham, MA). The scintillation fluid was evenly distributed over the filter paper to ensure saturation. [ 3 H] thymidine incorporation in PBMC DNA was measur ed
53 using a MicroBeta Jet 1450 LSC & Luminescence Counter (PerkinElmer Precisely, Turku, Finland) using standard parameters for tritium. Inter assay variation for stimulated cells and non stimulated cells was 11.3% and 5.37% respectively. Data were analyz ed as counts per minute (CPM) and as the ratio of stimulated culture CPM to non stimulated culture CPM (stimulation index (SI)) Lymphocyte Viability Assay Reagents used to assess lymphocyte viability included 30% hydrogen peroxide (Sigma Aldrich, St. Lou is, MO), phosphate buffered saline ( PBS; MediaTech Inc, Manassas, VA) Ethidium homodimer 1 (Ethd, Invitrogen Life Technologies, Carlsbad, CA), and 100% methanol (Fischer Scientific, Fairlawn, NJ). Aliquots of 50 L of cell suspension (1x10 5 live cells/wel l) were placed in each well of a 96 well black plate with flat, clear bottom wells (Corning Inc., Corning, NY). Cells were incubated with either 10 mM of hydrogen peroxide (25 L H 2 O 2 and 50 L PBS) or with 75 L PBS as a blank control (no hydrogen peroxid e). Cells were challenged with hydrogen peroxide for 4 h at 37 C in 6% CO 2 The optimal concentration of hydrogen peroxide and incubation time were determined prior to the start of the study (data not shown) Ethd 1 (2 mM) was used to identify cells with p oor membrane integrity. Ethd 1 is a high affinity nucleic acid stain that is impermeable to live cells and only weakly fluorescent until bound to DNA. The dye emits a red fluorescence excited at 530 12.5 nm. The optimal concentration of Ethd 1 and dye lo ading time was determined by a thorough optimization procedure prior to the start of the study. Optimization demonstrated that Ethd 1 saturation of 1x10 5 cells clearly occurred at a concentration of 4 M Below a cell concentration of 1x10 5 cells, there wa s no clear linear relationship between dye concentration and fluorescence emitted (data not shown). After the 4 h
54 incubation with hydrogen peroxide, 100 L of 4 M of Ethd 1 was added to each well. Plates were placed back into the incubator and dye was all owed to incorporate for 1 h. Cell free wells containing only PBS or 10 mM hydrogen peroxide and wells containing all dead cells were included on each plate. A stock of all dead cells was made by exposing the cells to 70% methanol (1 2 mL) for 5 min. The ce lls with methanol were diluted with PBS (10 12 mL) and centrifuged for 10 min at 250 g at 10 C. The supernatant was removed and the dead cells were re suspended in PBS (approximately 400 L). A dead cell count was obtained by trypan blue exclusion, as desc ribed previously. PBMC samples obtained from a donor horse that was not part of this study were included on each plate to serve as an interassay control. F luorescence was determined with a microplate reader (Synergy HT, BioTek Instruments Inc., Winooski, V T) using an excitation wavelength of 5 40 nm and emission was measured at 645 nm. A normalization factor (NF) for each plate was obtained by the following equation: NF = U / A = NF where A = the individual mean fluorescence of wells containing all dead cell s on a single plate; and U = the mean fluorescence of all wells containing all dead cells on all plates. The percentage of dead cells was determined by the following equation: % Dead cells = [(NF*H) B] / A where H = the mean fluorescence of hydrogen pe roxide exposed wells; and B = the background fluorescence of cell free wells with or without hydrogen peroxide. The inter assay variation for the cells exposed to hydrogen peroxide and the blank wells were 23% and 12%, respectively.
55 Statistical Analysis In sufficient PBMC were obtained for one HIGH Se horse at the 6 h post ex sampling; thus, lymphocyte viability and proliferation data from this horse were excluded from analysis. During isolations for the 6 h post ex sample, we were unable to obtain enough ce lls to freeze from one HIGH Se horse. Differences in serum Se were analyzed using the PROC MIXED procedure of SAS (Version 9.2 SAS Institute Inc., Cary, NC). Serum Se concentration on d 34 was analyzed using serum Se on d 0 as a covariate. Differences in CBC, lymphocyte proliferation and lymphocyte viability were analyzed using the PROC MIXED procedure of SAS with repeated measures. The effects of dietary Se treatment, sex, time and treatment x time interaction were evaluated as fixed effects H orse with in treatment served as a random effect If sex was not significant, it was removed from the model. The PDIFF option of the LS MEANS statement of PROC MIXED was used to compare treatment means. All data are expressed as the mean SE. Differences were consi dered significant at and trends for significance were recognized at P<0.10.
56 Table 3 1. Nutrient composition of basal diet feeds and sodium selenite supplement a Basal Diet Nutrient Oats Bermudagrass Hay Vitamin/Mineral Premix b Sodium Selenite DE c (Mcal/kg) 3.40 1. 94 0 0 Crude fat (%) 5.8 1.80 0 0 Crude protein (%) 11.7 10.2 0 0 NDF b (%) 25.5 71.8 0 0 ADF b (%) 11.8 36.8 0 0 Ca (%) 0.07 0.34 12.0 28.0 P (%) 0.37 0.25 8.0 0 Zn (ppm) 27 27 9287 0 Cu (ppm) 5 7 2388 0 Se (ppm) 0.24 0.02 0 10,000 Vitamin A d (IU/ kg) 31 6,904 455,040 0 Vitamin E (IU/kg) 15 12 19,001 0 a Values presented on a 100% dry matter basis. b Vitamin/mineral premix include d : d icalcium phosphate, calcium carbonate, copper sulfate, zinc sulfate, manganese sulfate, retinyl palmitate and DL tocopherol acetate. c DE = digestible energy; NDF = neutral detergent fiber; ADF = acid detergent fiber d Measured as caroteins and converted to vitamin A equivalents.
57 CHAPTER 4 RESULTS Serum Se Serum Se concentrations were similar between treatments pr ior to the initiation of Se supplementation, reflecting that all horses began the study with similar Se status. Serum Se remained unchanged in NRC Se horses, but increased (P=0.01) in horses receiving HIGH Se after 34 d of supplementation. As a result, ser um Se was higher (P= 0.0 2) at d 3 4 in horses receiv ing HIGH Se than NRC S e (Figure 4 1). Complete Blood Count The total number of leukocytes was affected by time (P=0.001), demonstrated by an increase in total leukocytes immediately after the completion of a prolonged exercise bout that persisted through 24 h post exercise (Table 4 1). Total leukocytes were not affected by Se treatment or the interaction of treatment and time. The specific populations of leukocytes were not altered by Se supplementation or t he interaction of Se treatment and time, but were influenced by time. Therefore, data for each cell population were combined for all treatments and are presented by time in Table 4 1 Prolonged exercise resulted in an increase in both the number (P<0.0001) and the percentage (P<0.0001) of neutrophils and a decrease in the number (P<0.0001) and the percentage (P<0.0001) of lymphocytes and eosinophils. With the exception of the number of lymphocytes, the numbers and percentages of these cell populations rema ined altered through 24 h post exercise. The number and percentage of monocytes showed a tran sient decrease (P=0.001) following exercise. Both the number (P=0.01) and percent (P=0.01) of basophils were increased immediately after exercise, but had returned to pre exercise levels by 6 h post exercise. The ratios of
58 monocytes to lymphocytes (P=0.02) and neutrophils to lymphocytes (P<0.0001) were affected during recovery from exercise, each showing an increase at 6 h post exercise, but a return to pre exercise values by 24 h post exercise. In addition to the changes in leukocyte populations observed in response to prolonged exercise, it is worth noting that the number of eosinophils (P<0.0 001 ) and lymphocytes (P= 0.0 2 ) and the number (P=0.001) and percentage of monocytes (P=0.02), exhibited a decrease during the 34 d supplementation period when horses were sedentary (i.e., d 0 ( baseline) to d 34). Lymphocyte Proliferation Background lymphocyte proliferation (without mitogen stimulation) was not altered by Se supp lementation, but was influenced by exercise Therefore, data from both treatments were combined and are presented in Figure 4 2 Background l ymphocyte proliferation was lower (P<0.01) at 6 and 24 h post exercise compared to that observed before exercise. L ymphoproliferative responses to mitogen stimulation were not altered by Se supplementation, but were influenced by exercise (Table 4 2). For all three mitogens used and at all mitogen concentrations evaluated, lymphocyte proliferation was lower at 6 (P<0. 01 ) and 24 h (P<0 .0 5 ) post exercise compared to pre exercise. With the exception of ConA, no titration effect of the different concentrations of mitogens was observed (Table 4 2). Similarly, lymphocyte stimulation indices (SI) were altered during recovery from exercise (time effect ), but were not altered by Se supplementation or the interaction of time*treatment. Therefore, SI data were combined for all treatments and are presented in Figure 4 3 A significant effect of time on SI was observed for both conc entrations of ConA (P<0.01) and PHA (P<0.05), with a trend for a time effect observed for PWM at
59 the higher mitogen concentration (P=0.06), but not the lower concentration. For all mitogens, the SI was higher (P<0.05) at 24 h post exercise compared to 6 h post exercise (Figure 4 3 ). Lymphocyte Viability The effects of hydrogen peroxide on PBMC viabi lity are presented in Figure 4 4 Cell viabilit y was affected by time (P=0.0003 ), where exposure to hydrogen peroxide resulted in a greater number of dead PBMC in samples obtained 24 h post exercise c ompared to pre exercise (P=0.003 ) and 6 h post exercise (P <0.0001 ). Although there was no overall eff ect of dietary treatment (P=0.46 ) or t he interaction of time* treatment (P= 0.51) numerically there were more PBMC dead after hydrogen peroxide exposure in NRC Se horses compared to HIGH Se horses at 24 hours post exercise (Figure 4 4 ). Lymphocyte viability, even without exposure to hydrogen peroxide, was affected by exercise (P=0.00 08) but there was no dietary treatm ent (P=0.14) or time* t reatment interaction (P=0.61) (Figure 4 5 ) In blank wells that were not exposed to hydrogen peroxide, a greater percentage of PBMC were identified as dead with Ethd 1 staining at 24 h post exercise, c ompared to pre exercise (P=0.001 8 ) and 6 h post exercise (P=0.0005). PBMC viability was similar between dietary treatments before and 6 h after a prolonged bout of exercise. Although it was not significant (P=0.11) PBMC from horses fed NRC Se had numerically reduced viability (greater p ercentage of dead cells) compared to PBMC from horses fed HIGH Se a t 24 h post exercise (Figure 4 5 ). It may be worth noting that similar effects of sampling time (relative to exercise) on cell viability were observed via trypan blue exclusion when thawing and preparing cells for the lymphocyte proliferation and cell viability assays.
60 Figure 4 1. Serum Se concentrations in horses receiving 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se) for 34 d. Overall effects of time (P=0.3), treatment (P=0.02), and time *treatment (P=0.14). a,b Bars with different letters differ (P<0.05).
61 Figure 4 2 Time effect on background lymphocyte proliferation without mitogen stimulati on in s amples obtained from horses before (Pre Ex) and 6 and 24 h after prolonged exercise. Data shown represent the mean s of dietary treatments combined. Overall effects of time (P=0.01), treatment (P=0.39), and time*treatment (P=0.77). a,b Bars with diff erent letters differ (P<0.01).
62 Figure 4 3 Lymphocyte stimulation index (SI) for c oncanavalin A ( ConA ) p okeweed ( PWM ) and phytohemmaglutinin ( PHA ) mitogens in samples obtained from horses before (Pre Ex) and 6 and 24 h afte r prolonged exercise. SI was calculated as the ratio of CPM from stimulated PBMC to non stimulated PBMC. Data shown represent the mean s of dietary treatments combined. Overall effect of time: ConA P=0.008; PWM P=0.06; PHA P=0.02. a,b Within a mitogen, bars with different letters differ (P<0.05).
63 Figure 4 4 Percentage of dead perpheral blood mononuclear cells ( PBMC ) after exposure to 10 mM H 2 O 2 for 4 h in samples obtained before (Pre Ex) and 6 and 24 h after prolonged exer cise from horses fed 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se). Overall effect of time (P=0.000 3 ), treatment (P=0. 46 ), and time*treatment ( P=0. 51 ). a,b,c Bars with different letters differ (P<0.05).
64 Figure 4 5 Perc entage of dead perpheral blood mononuclear cells ( PBMC ) not exposed to H 2 O 2 in samples obtained before (Pre Ex) and 6 and 24 h after prolonged exercise from horses fed 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se). Overall effect of time (P=0.0008 ) treatment (P=0. 14 ), and time*treatment ( P=0. 61 ). a,b,c Bars with different letters differ (P<0.05)
65 Table 4 1. Mean circulating leukocyte populations on d 0 (baseline), pre exercise (d 34), immediately after (0 h) and 6 h after exercise (d 35), and 24 h after exercise (d 36). Each cell population is presented as cell numbers and as the percentage of total leukocytes. All data represent both dietary treatments combined. Sampling Time P values Leukocyte Population Baseline Pre Ex 0 h Post ex 6 h Post e x 24 h Post Ex SEM Time Trt Time*Trt Cell numbers ( x 10 3 / L) Total Leukocytes 7.75 c,d 7.17 d 9.05 a,b 10.45 a 8.30 b,c 0.57 < 0.00 0 1 0. 31 0.20 Neutrophils 4.50 c 4.50 c 6.57 b 8.35 a 5.67 b 0.72 <0.0001 0.50 0.64 Lymphocytes 2.54 a 2.25 b 2.11 b 1.77 c 2.2 3 b 0.12 <0.0001 0.43 0.58 Monocytes 0.39 a 0.27 b 0.21 b 0.28 c 0.28 c 0.03 0. 0003 0. 12 0.2 8 Eosinophils 0.28 a 0.11 b 0.11 b 0.03 c 0.07 b 0.04 <0.0001 0.50 0.95 Basophils 0.02 b 0.02 b 0.04 a 0.02 b 0.03 a,b 0.004 0.01 0.88 0.60 M:L Ratio 1 0.15 a,b 0.12 a 0.10 a 0.1 6 b 0. 13 a 0.01 0. 02 0. 77 0.3 1 N:L Ratio 2 1.8 c 2.0 b,c 3.1 b 4.8 a 2.6 b,c 0.54 <0.0001 0.64 0.56 Percentage of Leukocytes Neutrophils 58.1 c 61.7 c 72.3 b 79.8 a 67.6 b 3.84 <0.0001 0.62 0.93 Lymphocytes 32.8 a 32.2 a 23.6 b 16.9 c 27.2 b 2.95 <0.0001 0.69 0 .95 Monocytes 5.0 a 3.8 b 2.4 c 2.7 c 3.8 a.b 0.46 0.001 0.74 0.43 Eosinophils 3.5 a 1.6 b 1.2 b,c 0.3 d 0.9 c 0.56 <0.0001 0.60 0.98 Basophils 0.3 a,b 0.3 a,b 0.4 a 0.2 b 0.4 a 0.05 0.01 0.37 0.71 M:L Ratio 1 0.16 a,b 0.12 b 0.10 b 0.21 a 0.14 b 0.02 0.02 0.71 0.31 N:L R atio 2 1.9 c 2.3 b,c 3.4 b 5.7 a 2.7 b,c 0.67 <0.0001 0.63 0.62 1 M:L Ratio = monocyte to lymphocyte ratio 2 N:L Ratio = neutrophil to lymphocyte ratio a,b,c,d Means in the same row with different superscripts differ (P<0.05)
66 Table 4 2. Lymphoproliferative res ponses (CPM) to two concentrations of three different mitogens from samples obtained before exercise (Pre Ex) and 6 and 24 h post exercise in horses supplemented with 0.1 mg Se/kg DM (NRC Se) or 0.3 mg Se/kg DM (HIGH Se). Sampling Time and Dietary Treatme nt Mitogen 1 Pre exercise 6 h Post exercise 24 h Post exercise P values NRC Se HIGH Se NRC Se HIGH Se NRC Se HIGH Se SEM Time Trt Time* Trt ConA 0.5 g/mL 3,637 a 4,248 a 1,235 b 1,071 b 1,170 b 1,125 b 658 0.01 0.84 0.92 1.0 g/mL 8,736 a 9,225 a 2,031 b 1,758 b 2,717 b 1,995 b 1,452 0.01 1.0 0.97 PWM 0.5 g/mL 12,652 a 12,537 a 2,586 b 2,097 b 1,685 b 2,045 b 2,215 0.001 0.98 0.99 1.0 g/mL 13,45 9 a 13,052 a 2,885 b 2,448 b 2,590 b 2,288 b 2,258 0.002 1.0 0.99 PHA 5 g/mL 14,132 a 14,007 a 2,784 b 2,865 b 2,235 b 2,555 b 2,418 0.002 0.97 1.0 10 g/mL 15,442 a 14,631 a 2,938 b 3,292 b 3,660 b 2,849 b 2,503 0.003 0.91 0.98 1 ConA = concanavalin A; PWM = pokeweed mitogen; PHA = phytohemmaglutinin a,b Means in the same row with differ ent superscripts differ (P<0.05)
67 CHAPTER 5 DISCUSSION In the current study, a prolonged bout of exercise had a significant impact on lymphocyte function, which agrees with others who have documented changes in immune responses in horses following exercise (Kurcz et al. 1988; Keadle et al. 1993; Folsom et al. 2001; Robson et al. 2003). Feeding unconditioned horses three times the NRC requirement for selenium increased s erum Se and presumably Se availability to lymphocytes. H owever, a higher level of dietary Se did not appear to mitigate exercise induced decreases in lymphocyte viability or proliferation. In the current study, serum Se respond ed to a higher level of dieta ry Se intake after only 34 d of supplementation. This finding is consistent with Calamari et al. (2009) who reported greater whole blood and plasma Se levels in horses fed 0. 2, 0. 3, and 0. 4 mg Se/kg DM compared to control horses consuming a basal diet with about 0. 1 mg Se/kg DM. Shellow et al. (1985) measured Se concentration in whole blood or plasma after supplementation and found plasma Se plateaued at 0.14 g/mL and whole blood Se plateaued at 0.21 g/mL. No further increase in plasma or whole blood Se l evel was achieved when the horses received more than 0.1 mg Se/kg diet. The c hanges in circulating immune cell populations in response to prolonged, submaximal exercise in the current study agree with reports where horses were performing similar types of exercise, but differ somewhat from studies where horses were exercising at high intensity for short duration. For example, Wong et al (1992) reported significant increases in total leukocytes, neutrophils, and the ratio of neutrophils to lymphocytes 6 h a fter horses performed a single bout of high speed
68 exercise, whereas the number of lymphocytes remained stable or increased slightly after exercise. In the current study, the lymphocyte population was reduced after exercise and the percentage of lymphocytes remained lower through 24 h post exercise. This difference in lymphocyte status was most likely related to the lower intensity and longer duration of exercise compared to the high speed exercise used by Wong et al. (1992). Hines et al (1996) reported an increase in neutrophils and a decrease in lymphocytes in horses competing in an endurance race. Robson et al. (2003) observed similar changes in the number of neutrophils and lymphocytes in endurance horses that persisted up to 24 h after completion of the race. These findings agree with the current study, where the percentage and number of neutrophils remained elevated and lymphocytes reduced at 24 h post exercise. Neutrophils are the primary phagocytes and are mobilized quickly in the event of inflammatio n or infection. An increase in the circulating neutrophil population implies recruitment of neutrophils during exercise recovery and suggests the prolonged exercise bout was sufficient in producing a t least some level of inflammation in the horses Lymphoc ytopenia is the condition of having abnormally low levels of lymphocytes circulating in the blood and has been shown to occur following exercise (Tanimura et al. 2008). The underlying cause of this is not fully understood although it has been suggested th at increased cortisol levels or DNA damage induced apoptosis following exercise may contribute. High levels of cortisol are thought to inhibit lymphocytes from entering the blood from tissues (Tanimura et al. 2008) However, Green et al (2003) found no i ncrease in cortisol level after high intensity exercise and no correlation between cortisol level and lymphocyte apoptosis in humans. The present study did not
69 measure cortisol levels but an increase in plasma concentration following exercise and a positiv e correlation between duration of exercise and cortisol level, has been previously reported in horses (Horohov et at., 1999; Nagata et al., 1999). Exercise induced ROS production can cause oxidative damage to lymphocyte DNA and may lead to post exercise ap optosis as well as necrosis (Tanimura et al. 2008 ). Additionally, exhaustive exercise in humans is more likely to induce apoptosis compared to moderate intensity exercise (Mooren et al., 2002; Wang and Huang, 2005). These negative side effects of ROS prod uction could account for the lymphocytopenia seen in the current study. Another possible mechanism of lymphocytopenia involves the availability of glutamine following exercise. Prolonged physical activity has been shown to directly affect plasma glutamine levels (Nieman and Pedersen, 1999). This nonessential amino acid is an important fuel for many types of cells, including lymphocytes. Decreased availability of glutamine after exercise could restrict the growth of lymphocytes. Robson et al. (2003) found n o change in serum glutamine concentration in horses after an 80 k m endurance race and suggested that the rate of glutamine release from the muscle had increased to match the demand for glutamine uptake by tissues, resulting in no net change of serum glutam ine. Abnormally low levels of circulating lymphocytes, as seen after exercise, may lead to an overall reduction in response to an immune challenge which requires T cells (Pedersen and Hoffman Goetz, 2000). The cause of lymphocytopenia following prolonged e xercise is likely multi factorial and deserves further investigation in horses. In addition to exercise induced changes in neutrophils and lymphocytes, other cell populations also appeared to be transiently affected during recovery from prolonged
70 exercise in the current study. T he number and percentage of monocytes was reduced immediately following exercise. By comparison, Robson et al. (2003) reported a significant increase in the number of monocytes immediately following an 80 km endurance race. Eosinoph ils and basophils were also significantly altered by prolonged exercise in the current study, but because these immune cells make up such a small population of leukocytes, the changes observed following exercise are probably inconsequential. Unexpectedly, the percentage and number of eosinophils and monocytes as well as the number of lymphocytes showed a significant decrease from baseline samples obtained prior to the start of the study (d 0) and samples obtained before exercise (d 34). Because there was n o treatment effect on these populations, these differences cannot be attributed to dietary Se level. Several factors, like anthelmintic administration before the study began or stress associated with relocating from a familiar pasture environment to dry lo t paddocks for the study could have been responsible for the noted declines in these cell populations. Similar to leukocyte population dynamics, lymphoproliferative responses were not altered by Se supplementation, but were influenced by exercise. Lymphoc yte function in response to exercise was assessed by measuring the proliferative response of these cells to mitogens ConA, PWM and PHA. With the exception on ConA, no titration effect of the different concentrations of mitogens was observed suggesting that the cells were maximally stimulated at the lower concentration of PWM and PHA. Each mitogen was chosen based on the ability to stimulate a different population of immune cells. PHA and ConA predominately stimulate the T cell population, while PWM predomin ately stimulates B cells (Bell et al., 2001). Here we report that all three mitogens produced a
71 similar pattern of proliferation (as SI and CPM) following exercise, suggesting that both T and B cells are affected by prolonged exercise In the present stud y, exercise proved to have an effect on both mitogen and non mitogen stimulated proliferation of lymphocytes in vitro Non mitogen stimulated ackground proliferation was evaluated from the negative control wells in the lymphocyte proliferation assay a nd is reflective of the activity of lymphocytes at the time of sampling. Background proliferation was high prior to exercise, indicating that lymphocytes possessed sufficient activity before exercise occurred. In contrast, background proliferation was sign ificantly depressed at 6 and 24 h post exercise, indicating that lymphocyte activity was greatly influenced by exercise. Additionally, the data illustrate that lymphocytes were unable to regain normal proliferative function within 24 h of exercise. The eff ects of prolonged exercise on basal ability to proliferate were mirrored by proliferation in response to mitogen stimulation in vitro Regardless of the mitogen used to stimulate cells or the mitogen concentration, lymphocyte proliferation was depressed at 6 and 24 h post exercise. This response deficit to mitogen stimulation is consistent with previous studies conducted in exercised horses ( Kurcz, et al., 1988; Keadle et al ., 1993 ). In humans, the extent of proliferative suppression generally increases wit h the exercise intensity or duration (Hines et al., 1996). Unconditioned horses had a reduced lymphoproliferative response to PWM immediately after a high intensity exercise test (Keadle et al ., 1993). Similarly, Kurcz et al. (1988) also reported a decreas ed response to ConA and PHA in conditioned horses following high intensity exercise, but by 24 h post exercise the response had returned to pre exercise values.
72 One possible explanation for an exercise induced decrease in lymphoproliferative response is t he increase in cortisol following exercise, which has been well documented in humans and horses (Horohov et al., 1999; Nagata et al., 1999 ; Robson et al ., 2003 ) Furthermore, cortisol is thought to contribute to post exercise lympho cyto penia by affecting l ymphocyte migration patterns or by increasing lymphocyte apoptosis (Kr ger et al., 2011). Hoffman Goetz and Zajchowski (1999 ) induced apoptosis in murine thymocytes and necrosis in both murine thymocytes and splenocytes after in vitro exposure to cortisol at physiological concentrations such as those that would follow moderate exercise. The mechanisms by which cortisol causes programmed cell death are still largely unknown. However, thymocyte apoptosis is mediated via a mitochondrial pathway, which can be i nhibited by the anti apoptotic protein Bcl 2 (Kr ger et al ., 2011). This intracellular protein is distributed in membranes and plays a role in the maintenance of mitochondrial integrity by preventing the release of pro apoptotic proteins into the cytosol ( Kr ger et al ., 2011). Peripheral lymphocyte concentrations of Bcl 2 have previously been shown to be altered by exercise (Sureda et al., 2008). It is theorized that cellular stressors, such as the exercise induced increase in ROS, are responsible for the m odulation of Bcl 2 (Kr ger et al ., 2011). Another possible explanation for the reduction in lymphocyte proliferation in response to exercise is oxidative induced DNA damage to the cells. A erobic metabolism used to generate ATP in the presence of oxygen re sults in an increased production of stress. Oxidative damage by ROS can cause DNA base damage or deletion, single strand breaks, or damage to DNA protein cross links (Bayi r, 2005). The decreased
73 proliferative response seen in the current study may be due to lymphocyte DNA damage which prevented proliferation. While preparing PBMC for the proliferation and viability assays, cells were evaluated under light microscopy using t rypan blue exclusion. Using this staining procedure, the difference between a healthy cell and an unhealthy cell is quite apparent. It was consistently noted that samples obtained at 24 h post exercise contained cells that appeared to have poor membrane in tegrity. In contrast, PBMC obtained before exercise were more numerous and appeared to be in good health. The average viability was 73% and 51% for the pre exercise and 24 h post exercise samples, respectively. Cell membrane fluidity and integrity are impo rtant functional requirements of a cell. Cellular lipids which make up the phospholipid membrane can be damaged by excess ROS (Bayir, 2005). The visual appearance of numerous unhealthy cells at 24 h post exercise in the current study indicates that damage acquired prior to freezing (i.e., damage incurred during prolonged exercise) hindered the ability of the cells to survive the freeze/thaw process. It is also possible that the methods used to process and thaw PBMC affected proliferation. However, all sampl es were handled similarly, including those obtained from the internal, non exercised control horse, and only samples obtained at 24 h post exercise consistently exhibited poor integrity when examined under trypan blue exclusion; thus, human error seems unl ikely. The lymphocyte stimulation index provides a means to normalize data and permits the comparison of lymphocyte activity between animals that possess different levels of background (non mitogen stimulated) proliferation. The stimulation index measures how many times greater the lymphocyte response is to a mitogen compared to the
74 background proliferation. In the current study, stimulation indices were not altered by dietary Se level but were affected following prolonged exercise. Across all three mitog ens used to stimulate PBMC, the stimulation index was lower at 6 h compared to 24 h post exercise. The stimulation index at 24 h post exercise was greater than that observed before exercise when cells were stimulated with ConA, and was numerically greater at 24 h when PWM and PHA served as the mitogen. Thus, lymphoproliferative responses to mitogen before exercise may be seen as less virulent because the background proliferation was already high. Ultimately, the decreased lymphoproliferative response to mit ogen observed after exercise may not, in and of itself, be as dramatic when viewed in terms of the simultaneous decrease in background proliferation. Nonetheless, the reduction in lymphocyte proliferation by mitogen stimulated and un stimulated cells follo wing prolonged exercise is significant. Direct application of lymphocyte activity observed in vitro cannot be made to in vivo lymphocyte function; however, it can be suggested that as a result of prolonged exercise in unconditioned horses, lymphocyte funct ion may be inadequate at least up to 24 h post susceptibility following exhaustive exercise. During this time it is possible for bacteria or viruses to gain a foothold, thereby increasin g the risk of infection (Nieman and Pedersen, 1999). This could be one explanation for the high prevalence of upper respiratory tract infections reported in the sport horse industry. High levels of ROS, such as hydrogen peroxide produced during prolonged exercise are toxic to cellular lipids and can therefore cause a loss of membrane integrity. The propagation of lipid peroxidation can be prevented by lipid soluble
75 antioxidants ( e.g. Vit E) or the reduction of lipid hydroperoxides by Se containing GPx. Wi thin the cell, the major role of GPx is to maintain low levels of hydrogen peroxide and therefore decrease the potential of free radical damage (Tapiero et al., 2003). During the pentose phosphate shunt, hydrogen peroxide is degraded to water by GPx and CA T. In vitro Se supplementation of human endothelial cells was shown to increase GPx and TrxR activity and decrease the amount of hydrogen peroxide within the extracellular medium (Tolando et al ., 2000). In the current study, t he hydrogen peroxide challenge served two purposes: first to determine if PBMC, which were already affected by a bout of prolonged exercise, could withstand an additional oxidative assault in vitro and secondly, to determine if a higher level of dietary Se could provide PBMC an extra measure of protection against oxidative damage. Hypothetically, if more Se is available to support GPx and TrxR activity (e.g., horses fed HIGH Se ) PBMC should be able to reduce more hydrogen peroxide and therefore have increased viability after the chall enge. Fisher et al. (2011) exposed human lymphocytes to 1 mM of hydrogen peroxide and found a significant decrease in viability at 3 h post high intensity exercise. Viability had returned to pre exercise values by 24 h post exercise. The authors concluded that lymphocytes may be more vulnerable to cytotoxic agents during recovery from exercise. The Ethd 1 dye used in the cell viability assay in the present study is a high affinity nucleic acid stain that is impermeable to live cells with intact plasma memb ranes and only weakly fluorescent until bound to DNA. Any loss of cellular membrane integrity will allow the dye to enter the cell, bind to DNA and fluoresce. Ethd 1 has been used in
76 previous studies and been shown to accurately determine cell viability af ter hydrogen peroxide exposure ( Viarengo et al., 1999 ; Neuss et al., 2001 ). Although the current study showed no overall improvement in cell viability after exposure t o hydrogen peroxide when horses received a higher level of dietary Se PBMC from horses fed 0.3 mg Se/kg DM had numerically greater viability at 24 h post exercise than cells from horses fed 0.1 mg Se/kg DM. A higher level of Se intake prior to exercise could fortify lymphocytes, such that they show greater resistance to subseque nt episodes o f oxidative stress. Under the additional stress of hydrogen peroxide, the HIGH Se PBMC were able to survive whereas the NRC Se PBMC experienced a significant loss of membrane integrity allowing Ethd 1 to bind to DNA. Because of the high variability of this assay, a larger number of horses will be needed to confirm that a higher level of dietary Se can be cytoprotective against hydrogen peroxide insult. Level of Se in the diet had no overall affect on the viability of unchallenged PBMC. However, PBMC from h orses fed HIGH Se again had numerically greater viabilit y compared to horses fed NRC Se at 24 h post exercise. Because these cells were not exposed to the additional oxidative insult of hydrogen peroxide, this might be interpreted to mean that PBMC from HI GH Se horses were better able to withstand the exercise challenge, as well as t he freeze/thaw process. In previous experiments (d ata not shown), the recovery of frozen PBMC is between 60 85%. In support of this, PBMC obtained before exercise, as well as PB MC obtained from the non exercised internal control horse all had good viability after thawing (67 85%). As described earlier, it is a fair assumption that the ROS generated during exercise compromised the cell
77 membrane. Disruptions of the cell membrane ar e common in vivo and an overwhelming number of cells would die if they were not sealed rapidly (McNeil and Steinhardt, 1997). However a break in the plasma membrane does immediately comprise the cell and can result in cell death (McNeil and Steinhardt, 19 97). The lymphocytes in the viability assay were suspended in PBS instead of culture medium, because of culture medium interferes with fluorescence readings on the plate reader. As a result, none of the substrates normally found in culture medium were avai lable for use by the cells to repair damaged membranes. Because Ethd 1 is not permeable to intact membranes, the lymphocytes stained by this dye must have had a compromise to their phospholipid membrane. Under ideal or in vivo conditions, the cells might h ave been able to recuperate from an oxidative challenge. Since PBMC from HIGH Se horses appeared to have recovered slightly better from exercise by 24 h post ex, this could mean an overall improvement in lymphocyte function. Again, because of the high vari ability of the assay, a larger number of horses will be needed to strengthen the power to detect a difference due to dietary Se. PBMC were most vulnerable to hydrogen peroxide exposure and the freeze/thaw process at 24 h post ex ercise. Reduced cell viabi lity, coupled with observations of reduced lymphocyte proliferation in un stimulated and mitogen stimulated cells at 6 and 24 h post exercise suggest that lymphocytes may be more vulnerable to cytotoxic agents during recovery from exercise. In addition, su bjective observations of a loss of membrane integrity and an increased proportion of dead cells recovered after freezing and thawing in the 24 h post exercise samples were noted during the initial evaluation
78 of cells using trypan blue exclusion. Collective ly, these data demonstrate that prolonged CONCLUSIONS The current study demonstrated that prolonged exercise produces changes in immune cell numbers and functional activity i n unconditioned horses that could lead to an open window of susceptibility to infections or intracellular parasites. Although these observations may not be directly transferable to horses that are physically fit, the direct effects of exercise on immune fu nction, coupled with the stress of hectic training schedules, long distance travel, confinement to stables with poor air quality, and exposure to unfamiliar horses could contribute to the recent prevalence of EHV 1 infections and high incidence of influenz a among equine athletes. Supplementing the diets of performance horses with Se in hopes of improving performance and or recovery time has become a common industry practice. However, support for the use of higher levels of Se as an antioxidant or for purpos es of reducing risk of infection is lacking in the literature. In the current study, feeding horses a diet containing 3X the current NRC (2007) requirement for Se improved Se availability to lymphocytes, but failed to mitigate decreases in cell viability a nd lymphocyte proliferation in response to prolonged exercise. Because exercise can cause oxidative stress, which may continue to impact the immune system for a prolonged period after exercise, dietary strategies are needed to boost antioxidant defenses. The number of horses utilized in the present study likely limited our ability to effectively evaluate whether a higher level of dietary Se would be beneficial for performance horses Se lenium supplementation for a longer period o f time and/or at a higher d ose may show a greater enhancement of immune function in
79 exercised horses Additional bouts of exercise, possibly mimicking a competition setting, could be used to assess if supplementary Se in the diets of equine athletes could help with overall recovery time and therefore decreasing immunosuppression which occurs after prolonged exercise or further environmental stressors. Finally, a vaccine or viral challenge following prolonged exercise, might also be useful for evaluating the effect of dietary Se on in vivo immune responses.
80 APPENDIX A PROTOCOL FOR PBMC IS OLATION NOTE: rinse pipette with PBS before drawing cells into clean pipette (cells are sticky). Use sterile technique; spray all unsterilized instruments, counter top, & gloves with 70% Ethanol 1. Label all conical (50 mL) with horse # and step/stage of PBMC isolation One for Whole blood, LSM and WBC. 2. Dilute 25 mL of whole blood in 10 mL PBS in 50 mL conical vial Add PBS first to 50 mL conical vial, then blood, then invert. Can use same pipette wi thin a horse; but use clean pipette for each different horse. 2 mL). 3. SLOWLY layer 35 mL diluted blood (from step 2) over 15 mL LSM. **Maintain sharp interface** First, pipette 15 mL LSM into a newly labeled 50 mL conical vial. Invert diluted blood before layering over LSM. To layer, tilt conical vial and dribble diluted blood in slowly along the edge of the conical vial. Once a few mL of diluted blood have been added, you can go a bit faster. i.e. 4. Centrifuge at 1500 rpm (400 x g ) for 25 min at room temperature. 5. Aspirate plasma to within ~1/2 cm above PBMC buffy coat and discard. Use 25 mL pipette Can save plasma if running fatty acids. Ca n discard plasma in any beaker. 6. Remove PBMCs and approximately half of the LSM below them, place in new 50 mL conical vial. DO NOT DISTURB RBC (can save RBC for fatty acid analysis) U se 10 mL pipette Take fuzzy interface (8 12 mL) stick pipette through plasma to fuzzy area (buffy coat). Tilt conical vial slightly and suction in a circular motion at the fuzzy layer.
81 Will take about half the LSM layer below with it. Try not to get too much LSM (contamination with platelets); suctioning more plasma is better than suctioning more LSM. 7. Bring all conical vials to a volume of 40 mL with PBS; cap and then mix by gentle inversion. Some conical vials will get more PBS than others (essentially just balancing for centrifuge). 8. Centrifuge at 740 rpm (100 x g ) for 10 min at room temperature. If successful, should see small cell pellet at the bottom of the vial. 9. Suction off and discard supernatant, being careful not to disturb cell pellet. Use 25 m L pipette Can suction off until ~1/2 cm above cell pellet. 10. Bring volume up to 10 15 mL with PBS. Cap and mix by gentle inversion. 11. Cen trifuge at 740 rpm (100 x g ) for 10 min at room temperature. 12. Suction off and discard supernatant as close to cell pellet as you can get without disturbing it. 13. Resuspend cells in 1 mL PBS. Mix gently. 14. Count live cells.
82 APPENDIX B PROTOCOL FOR CELL CO UNTING 1. Add 10 L of resuspended cell pellet (from PBMC isolation) to 90 L trypan blue. Flick vial with finger to mix. Pipette trypan blue into microcentrifuge vial first. This dilution (10:1) of trypan blue and cells is im portant to maintain. 2. Clean and load hemacytometer To clean, rinse with deionized water; wipe dry (mirror and cover slip ) with kimwipes. 10 L of cell trypan mixture goes into hemacytometer uch it to cover slip ); pipette mixture in slowly. Should appear as a square shape under the cover slip 3. Count LIVE cells Count at 40X magnification Trypan only stains the dead cells (will appear as dark dots or disturbed/exploded dots); live cells will b e clear, round, uniform. Count cells present in all 25 squares or equivalent (will be # cells/10 L) If ~50/square, count cells in 5 squares, find average, then multiply by 25 to get total. If ~5 cells/square, count cells individually in all 25 squares to get total. See diagram below 4. Calculate concentration of cells counted Cells lying on edge COUNTED
83 Total # cells counted in 25 squares x 10 (dilution factor with trypan blue and cells) x 10,000 (converts concentration from cells/ L to cells/mL) Should yield some value of mill ions/mL; ideally would like at least 15 x 10 6 cells/mL 5. Calculate concentration of cells to freeze in suspension (desired final concentration)(desired final volume) = 1.0 mL (or less) of isolated (concentration of cells counted) cell suspension Concentration of cells counted ideally, want 15 million (15 x 10 6 ) or more, because would like to store in aliquots of 5 million/mL; if less, will need to play with numbers, either storing fewer cells in 3 vials or 2 vials. Desired final conce ntration and desired final volume will need to be played with. Goal is to optimize the number of cells stored, based on the actual concentration of cells counted and the limited 1 mL volume (technically, 990 L) of cell suspension (from isolation procedure ). Ideally, would like 3 vials of 5 x 10 6 cells/mL Freeze a concentration no less than 2 x 10 6 cells/mL If cell count 10 x 10 6 cells/mL Store in 3 cryogenic vials Final volume = 3 .1 (or higher) Cell suspension = 1 mL (or less) Desired final concentrati on = 3.0 to 5.0 x 10 6 cells/mL (3mL=minimum; 5mL =max) If cell count 6.3 to 9.9 x 10 6 cells/mL Store in 2 cryogenic vials Final volume = 2.1 Cell suspension = 1 mL (or less) Desired final concentration = 3.0 x 10 6 cells/mL If cell count < 6.3 x 10 6 cells/ mL Store in 2 cryogenic vials Final volume = 2.1 Cell suspension = 1 mL (or less) Desired final concentration = try to get as close to 3.0 x 10 6 cells/mL but no less than 2.0 x 10 6 cells/mL
84 6. Calculate concentration of freezing media to add to cell suspens ion for freezing From equation above: Final volume mL of cell suspension = mL of freezing media Can also be done on L basis (to aid in pipetting) Example: Say you have calculated final volume = 2.1 mL and cell suspension = 1.0 mL 2.1 1.0 = 1.1 m L (or 1100 L) of freezing media needed 7. 6). Mix gently. Use 15 mL vial From example in step 6, 1.0 m L of cell and 1.1 m L freezing media in clean vial to then be distribute d by step 8 8. Pipette 1 mL of cell s + freezing media mix into 2 or 3 cryogenic vials (based on your calculations) 9. Place cryogenic vials in Mr Frosty (filled with 70% ethanol ); place in 80C for 24 hr; then place in liquid N storage tank.
85 APPENDIX C PRO TOCOL FOR LYMPHOCYTE PROLIFERATION Thawing Cells from Liquid Nitrogen 1. Remove medium from fridge, sterilize outside of bottle and place under sterile hood; a. Medium should remain cold until use 2. Pour ~11 13 mL of medium into sterile 15 mL conical vial; Do not touch vial edge to medium container as you are pouring 3. Remove cyro vial from liquid nitrogen 4. Swirl vial in a 56C water bath to quickly thaw the outside of the vial until the ice chunk in vial is barely mobile and able to be poured, 1 2 min 5. Pour ice chunk into 15 mL conical vial with medium 6. Ri nse cyro vial twice with ~1 2 mL of medium and pour into 15 mL conical vial 7. Look to make sure ice chunk dissolved in medium before spinning vial 8. Centrifuge 15 mL vial of stock cell solution with program 4 or centrifuge. a. Program g 5 min 10C b. x g 10 min, 10C c. To start centrifuge program, press Rcl, #5 enter, start 9. While waiting for centrifuge, pipette 90 L of trypan blue in 1 2 separate 1 m L micro vials; does not n eed to be under hood 10. Use pipette man to remove most of the medium, Do not disturb cell pellet 11. Re suspend cells in appropriate amount of medium depending on pellet size a. 200 1000 L media 12. Mix cells by gently drawing liquid up into pipette a few times, tr y not to get bubbles 13. Make up trypan blue dilution(s); See below for LP cell counting procedure
86 14. Place stock cell solution on ice while counting live cells 15. Fill out plate map 16. Calculate/make working cell solution and working mitogen solution; see below 17. Plate 50 L of working cell solution on a 96 well plate; start with lowest concentration of cells if more than one Only touch pipette tip to bottom of well if necessary. 18. Plate 50 L of working mitogen solution, start with lowest concentration of mitoge n if more than one Only touch pipette tip to upper sidewall of well if necessary. Total volume per well is 100 L 19. Put cover on plate and place in incub ator for specified time (~96 h ) Cell Counting Protocol for L ymphocyte P roliferation NOTE: This co unting procedure was found to be easier for calculations of total number of cells per m L 1. Make up trypan blue + cell dilution(s), mix by tapping vial with finger Option if large cell pellet Make 2 st dilution (1:100) a. 1:10 1 st dilution (90 L trypan blue : 10 L stock cells) b. 1:100 2 nd dilution (optional; 90 L trypan blue : 10 L 1 st dilution) 2. If few cells, FIRST mix and load 10 L from 1 st dilution (1:10) into one side of hemocytometer 3. If many cells, only load 10 L from 2 nd dilution into opposite side o f hemocytometer 4. Count live cells in 4 outer quadrants under 40 x power a. Live cells will be clear, but do not need to be perfectly round as long as they do not absorb any dye 5. If cells lay on a line, only count if on the bold lines in each quadrant or squa re within quadrant
87 6. Count 2 nd dilution (1:100) first if you loaded both dilutions; if counts from two 7. Average the counts of the quadrants 8. Average of counts is how many million PBM Cs per m L (AVG x 10 6 PBMC/m L ) 9. If few cells, also count cells in 1 st dilution (1:10) 10. Average all cell counts (from both dilutions) a. Example: cell count from 2 nd is 9, 11 and 1 st dilution is 109, 112. Average 9, 11, 10.9 and 11.2 to adjust for dilution dif ference 11. Rinse hemocytometer and cover slip with distilled water; Dry with Kim wipes Calculations of Cells and Mitogens for L ymphocyte Proliferation P late 1. Calculate working solution of cells + medium a. (live count x 10 6 PBMC/m L ) (x m L ) = (2x desired working solution/m L )( L total volume of working solution needed per run) b. X= L of cells needed from stock solution c. Total volume of working solution needed per run x = L of medium to add to working solution of cells
88 2. Calculate working solution of mitogens a. ( g stock solution of mitogen/m L ) (x L ) = ( g 2x desired working concentration of mitogen/m L ) (Total volume of working solution needed per run m L or L ) b. X= L needed from stock solution of mitogen c. Total volume of working solution needed per run x = L or m L of medium to add to working solution of mitogen Addition of Tritiated T hymidine to L ymphocyte P roliferation Plate 20. Sterilize and place beige tray, with lab paper, under hood a. Wear lab coat 21. 60 h into incubation, remove plate from incubator and place unde r hood 22. Remove working solution of 3 H Thymidine from fridge and place under hood 23. Use digital auto pipette to dispense 25 L /well 3 H Thymidine a. Use auto pipette tip AND additional regular pipette tip b. When drawing up the radioactive liquid, do not touch side o f vial 24. Dispense first 2 aliquots back into working solution of 3 H Thymidine 25. Dispense 3 H Thymidine into wells a. Only touch pipette to side of wells, opposite of side mitogen pipette touched 26. Put extra 3 H Thymidine back into working solution a. Leave pipettes un der hood until swiped 27. 28. Return plate to incubator 29. Wrap up pipette tips in lab paper and pull gloves over to create a package 30. Dispose of radioactive package in dry radioactive waste container
89 31. Perform swipes, use correct swipe sheet for locations to be swiped (9 areas) 32. Place each swipe in correct scintillation vial, add 2 m L of scintillation fluid to each vial a. Cap vials with lids 33. Place divider top on plate holding vials 34. Place plate in Microbeta machine below s top plate, close door a. Open Microbeta program on computer b. Follow instructions on side of Microbeta to set up swipe program c. d. Make sure it is set to Excel 4 e. Green light on program means you can run the swipes f. Good to let vials sit because touching swipes with gloves causes static that could read as radioactivity 35. Fill out swipe sheet with CPM numbers from machine, DPM numbers x2 a. If any numbers above 100, area correlating to that swipe must be decontaminated with Fan tastik, re swiped and re run b. If clean, fill out swipe sheet as Net CPM <50, DPM <100 36. Throw scintillation vials in radioactive dry waste container L ymphocyte P roliferation Cell Harvest 37. Remove plate from incubator after 18 h with 3 H T hymidine 38. Turn on power strip and then compressor 39. Label filter paper with pencil and place into harvest machine 40. Wet filter paper using blank plate by pushing the wash/vacuum buttons a. Black handle does lock into place, push slowly to lock b. break 41. Remove blank plate and put harvest plate in machine 42. Vacuum out wells of plate, wash wells 2x
90 43. Carefully remove filter paper, place on paper to wel, place in the microwave to 90 sec 44. Turn off harvest machine, make sure vacuum in locked ON 45. Place dry fil ter paper in plastic bag 46. Seal bag very close to top edges of paper 47. Cut corner of sealed bag opened and add ~3 m L of scintillation fluid into bag 48. Use roller to evenly distribute fluid completely over filter paper 49. Re seal opened corner of bag a. Use sealer wit h no heat to press air bubble to edge of bag and double seal b. Cut in between double seal to remove air bubble 50. any black when you put top on reader plate 51. Place reader pl ate into Microbeta machine below stop plate 52. Select protocols, general then 3H filter map on computer program; select program, output, file 1, name file 53. Name file (note 3 day difference between plate set up and harvest date) 54. Dispose of filter paper in dry r adioactive container 55. Empty liquid radioactive waste container from harvest machine 56. Perform swipes (19 areas)
91 APPENDIX D PROTOCOL FOR H 2 O 2 LYMPHOCYTE VIABILITY 1. Following cell thawing protocol 2. If cells thawed in media, rinse media off cells with PBS and spin 3. Following cell counting protocol to obtain number of live cells 4. Make appropriate dilutions of cell concentrations with PBS (not media) a. 5x10 4 /well (x 20 D ilution F actor 5. Plate 50 L of each cell concentrations in duplicate, 96 well flat bottom p late 6. Plate 50 L of PBS in each well 7. Make up appropriate H 2 O 2 dilutions from stock solution of 30% H 2 O 2 (9.79M) a. Filter 30% H 2 O 2 before using, 0.22 m syringe filters b. Always keep H 2 O 2 solutions wrapped in tin foil and in fridge c. H 2 O 2 30% + PBS for working so lutions d. May have to make multiple dilutions to get accurate working solution 8. Plate 25 L of appropriate H 2 O 2 dilution into wells 9. For Blank wells, add 25 L PBS instead of H 2 O 2 10. Incubate plate for 4 h 11. Make up EthD 4 M dilution 12. Add 125 L PBS to duplicate em pty wells 13. Add 100 L EthD to each well 14. Incubate plate for 1 h 15. Read plate on fluorescent plate reader
92 APPENDIX E PROTOCOL TO MAXIMIZE CELLS DURING PBMC IS OLATION Note: This protocol was not used for isolations of study samples however it was used for sub sequent and interassay control horse isolations. 1. From vaccutainer tubes, transfer whole blood into 50 mL conical vial a. About 4 vaccutainer tubes per 50 mL conical b. Optional add HBSS or PBS EDTA 2 mM to help with clumping (0.5 m L /1 L PBS), not usually a problem with equine cells c. Can wash out vaccutainer tubes with PBS 2. Spin 50 m L x g 30 min at 18 C 3. Collect WBC layer that forms on top of the red blood cells from each 50 mL conical into new sep arate 50 m L conicals a. To ensure full collection, suck up some RBCs too 4. Bring WBCs volume up to 35 m L with PBS ( optional EDTA) 5. Invert gently 6. Carefully layer the 35 m L on top of 15 m L LSM, keep the sharp interface 7. x g 30 min at 18 C 8. Continue with normal PBMC isolation protocol Do NOT need to invert vaccutainer blood tubes after original heparin is mixed in All spins in isolation procedure can be done at 18 or 10 C
93 LITERATURE CITED Ainsworth, D.M., J.A. Appleton, S.W. Eicker, R Luce, M.J. Falminio, and D.F. Antczak. 2003. The effect of strenuous exercise on mRNA concentrations of interleikin 12, interferon gamma and interleukin 4 in equine pulmonary and peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 91: 61 71. Albers, R., M. Bol, R. Bleumink, A.A Willems, and R.H. Pieters. 2003. Effects of supplementation with vitamins A, C, and E, selenium, and zinc on immune function in murine sensitization model. Nutrition. 19: 940 946. AOAC. 2000. Official Methods of Analysi s.17 th ed. Assoc. Off. Anal. Chem., Arlington, VA. Baalsrud, K.J., and G. Overnes. 1986. Influence of vitamin E and selenium supplement on antibody production in horses. Equine Vet. J. 18(6): 472 474. Baker, S.S., and H.J. Cohen. 1983. Altered oxidative me tabolism in selenium deficient rat granulocytes. J. Immunol. 130: 2856 2860. Baum, M.K., M.J Miguez Burbano, and A. Campa. 2000. Selenium and interleukins in persons infected with human immunodeficiency virus type 1. J. Infec. Dis. 182(Suppl 1): S69 S73. B ayir, H. 2005. Reactive oxygen species. Crit. Care Med. 33:12(Suppl.) S498 S500. Beck, M.A. 2007. Selenium and Vitamin E status: Impact on viral pathology. J. Nutr. 137: 1338 1340. Beck, M.A., H.K. Nelson, Q. Shi, P. Van Dael, E. Schiffrin, S. Blum, D. Bar clay, O.A. Levander. 2001. Selenium deficiency increases the pathology of an influenza virus infection. FASEB J. 15: 1481 1483. Bell, S.C., C. Savidge, P. Taylor, D.C. Knottenbelt, and S.D. Carter. 2001. An immunodeficiency in Fell ponies: A preliminary st udy into cellular responses. Equine Vet. J. 33(7): 687 692. Broome, C.S., F. McArdle, J. AM Kyle, F. Andrews, N. M. Lowe, C. A. Hart, J. R. Arthur, and M.J. Jackson. 2004. An increase in selenium intake improves immune function and poliovirus handling in a dults with marginal selenium status. Am. J. Clin. Nutr. 80: 154 162. Brummer, M., S. Hayes, S.M. McCown, A.A. Adams, D.W. Horohov, and L.M. Lawrence. 2011. Selenium depletion reduces vaccination response in horses. Proc. Equine Science Soc. Symp. Murfreesb oro, TN. 31(5 6): 266 (Abstr)
94 Bruunsgaard, H., A. Hartkopp, T. Mohr, H. Konradsen, I. Heron, C.H. Mordhorst, and B.K. Pedersen. 1997. In vivo cell mediated immunity and vaccination response following prolonged intense exercise. Med. & Sci. in Sports and E x. 29(9): 1176 1181. Burrell, M.H., J.L.N. Wood, K.E. Whitwell, N. Chanter, M.E. Mackintosh, and J.A. Mumford. 1996. Respiratory disease in thoroughbred horses in training: The relationship between disease and viruses, bacte ria and environment. Vet. Rec 1 39: 308 313. Calamari, L., A. Ferrari, and G. Bertin. 2009. Effect of selenium source and dose on selenium status of mature horses. J. Anim. Sci. 87: 167 178. Calder, P.C. 2007. Immunological parameters: What do they mean? J. Nutr. 137: 773S 780S. Carrick, J.B., and A.P. Begg. 2008. Peripheral blood leukocytes. Vet. Clin. Equine. 24: 239 259. Cases, N., A. Sureda, I. Maestre, P. Tauler, A. Aguil, A. Crdova, E. Roche, J.A. Tur, and A. Pons. 2006. Response of antioxidant defenses to oxidative stress induced by prolonged exercise: A ntioxidant enzyme gene expression in lymphocytes. Eur. J. Appl. Physiol. 98: 263 269. Colahan, P.T., C. Kollias Baker, C.M. Leutenegger, and J.H. Jones. 2002. Does training affect mRNA transcription for cytokine production in circu lating leucocytes? Equine Vet. J. Suppl. 34: 154 158. Covey, T.L., N. E. Elam, J. A. Carroll, D. B. Wester, M. A. Ballou, D. M. Hallford, and M. L. Galyean. 2010. Supplemental selenium source on Holstein steers challenged with intranasal bovine infectious rhinotracheitis virus: Blood metabolites, hormones, and cytokines. Professional Anim. Sc 26: 93 102. Davis, M.S., J.R Malayer, L. Vandeventer, C.M. Royer, E.C. McKenzie, and K.K. Williamson. 2005. Cold weather exercise and airway cytokine expression. J. A ppl. Physiol. 98: 2132 2136. Davis, M.S., C.C. Williams, J.H. Meinkoth, J.R Malayer, C.M. Royer, K.K Williamson, and E.C. McKenzie. 2007. Influx of neutrophils and persistence of cytokine expression in airways of horses after performing exercise while brea thing cold air. Am. J. Vet. Res. 68: 185 189. Desowitz, R.S., and J.W. Barnwell. 1980. Effects of selenium and dimethyl dioctadecyl ammonium bromide on the vaccine induced immunity of Swiss Webster mice against malaria (Plasmodium berghei). Infect. Immun. 27(1): 87 89.
95 Donovan, D.C., C. A. Jackson, P.T. Colahan, N. Norton, and D.J. Hurley. 2007. Exercise induced alterations in pro in horses. Vet. Immunol. Immunopath. 118: 263 269. Edwards, K.M., V.E. Burns, T. Re ynolds, D. Carroll, M. Drayson, and C. Ring. 2006. Acute stress exposure prior to influenza vaccination enhances antibody response in women. Brain Behav. Immun. 20: 159 168. Escribano, B.M., E.I Agera, R. Vivo, R. Santisteban, F.M. Castejn, and M.D. Rubi o. 2002. Benefits of moderate training to the nonspecific immune response of colts. Equine Vet. J. Suppl. 34: 182 185. Escribano, B.M., F.M. Castejn, R. Vivo, R. Santisteban, E.I Agera, and M.D. Rubio. 2005. Effects of training on phagocytic and oxidativ e metabolism of peripheral neutrophils in horses exercised in the aerobic anaerobic transition area. Vet. Res. Comm. 29: 149 158. Ferrer, M.D., P. Tauler, A. Sureda, J.A. Tur, and A. Pons. 2009. Antioxidant regulatory mechanisms in neutrophils and lymphocy tes after inte nse exercise. J. Sports Sciences 27(1): 49 58. Field, C.J, R. Gougenon and E.B. Marliss. 1991. Circulating mononuclear cell numbers and function during intense exercise and recovery. J. App. Physiol. 71(3): 1089 1097. Finkel T. 1998. Oxygen r adicals and signaling. Current Opinion in Cell Biology. 10: 248 253. Fisher, G., D.D. Schwartz, J. Quindry, M.D. Barberio, E.B. Foster, K.W. Jones, and D.D. Pascoe. 2011. Lymphocyte enzymatic antioxidant responses to oxidative stress following high intensi ty interval exercise. J. Appl. Physiol. 110: 730 737. Folsom, R.W., M.A Littlefield Chabaud, D.D. French, S.S. Pourciau, L. Mistric, and D.W. Horohov. 2001. Exercise alters the immune response to equine influenza virus and increases susceptibility to infec tion. Equine Vet. J. 33(7): 664 669. Giraldo, E., J.J. Garcia, M.D. Hinchado, and E. Ortega. 2009. Exercise intensity dependent changes in the inflammatory response in sedentary women: Role of neuroendocrine parameters in the neutrophil phagocytic process and the pro /anti inflammatory cytokine balance. Neuroimmunomodulation 16: 237 244. Gomez Cabrera, M.C., E. Domenech, L.L. Ji, and J. Via. 2006. Exercise as an antioxidant: It up regulates important enzymes for cell adaptations to exercise. Science & Sports. 21: 85 89. Green, K.J., S.J. Croaker, and D.G. Rowbottom. 2003. Carbohydrate sup plementation and exercise induced changes in T lymphocyte function. J. Appl. Physiol. 95: 1216 1223.
96 Hines, M.T., H.C. Schott II, W. M. Bayly, and A. J. Leroux. 1996. Exercise and immunity: A review with emphasis on the horse. J. Vet. Intern. Med. 10(5): 2 80 289. Hoffman Goetz, L., and S. Zajchowski. 1999. In vitro apoptosis of lymphocytes after exposure to levels of corticosterone observed following submaximal exercise. J. Sports Med. Phys. Fitness. 39(4): 269 274. Hoffmann, P.R. 2007. Mechanism by which s elenium influences immune response. Arch. Immunol. Ther. Exp. 55: 289 297. Horohov, D.W., A. Dimock, P. Guirnalda, R.W. Folsom, K.H. McKeever, and K. Malinoswki. 1999. Effects of exercise on the immune responses of young and old horses. Am. J. Vet. Res. 60 (5): 643 647. Israel, N., and M.A. Gougerot Pocidalo. 1997. Oxidative stress in human immunodeficiency virus infection. Cell. Mol. Life Sci. 53: 864 870. Israel, N., M.A. Gougerot Pocidalo, F. Aillet, and J.L. Virelizier. 1992. Redox status of cells influe nces constitutive or induced NF kappa B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149: 3386 3393. Janicki, K.M., L.M Lawrence, T. Barnes, and C.J. Stine. 2001. The effect of dietary selenium source and level on selenium concentration, glutathione peroxidase activity and influenza titers in broodmares and their foals. Proc. 17th Equine Nut. Physiol. Soc. Symp., Lexington, KY. 43 44. Kamada, H., I. Nonaka, Y. Ueda, and M. Murai. 2007. Selenium additio n to colostrum increases immunoglobulin G absorption by newborn calves. J Diary Sci. 90(12): 5665 5670. Karren, B.J., J.F. Thorson, C.A. Cavinder, C.J. Hammer, and J.A. Coverdale. 2010. Effect of selenium supplementation and plane of nutrition on mares and their foals: Selenium concentrations and glutathione peroxidase. J. Anim. Sci. 88: 991 997. Keadle, T.L., S.S Pourciau, P.A. Melrose, S.G. Kammerling, and D.W. Horohov. 1993. Acute exercise stress modulates immune function in unfit horses. J. Equine Vet. Sci. 13(4): 226 231. Kiremidjian Schumacher, L., and G. Stotzky. 1987. Review: Selenium and immune responses. Environ. Res. 42: 277 303. Knight, D.A., and W.J Tyznik. 1990. The effect of dietary selenium on humoral immunocompetence of ponies. J. Anim. Sci. 68: 1311 1317.
97 Kohut, M.L., B.A. Arnston, W. Lee, K. Rozeboom, K.J. Yoon, J.E. Cunnick, and J. McElhaney. 2004. Moderate exercise improves antibody response to influenza immunization in older adults. Vaccine 22: 2298 2306. Kr ger, K., S. Agnischock, A. Le chtermann, S. Tiwari, M. Mishra, C. Pilat, A. Wagner, C. Tweddell, I. Gramlich, and F. Mooren. 2011. Intensive resistance exercise induces lymphocyte apoptosis via cortisol and glucocorticoid receptor dependent pathways. J. Appl. Physiol. 110:1226 1232. Ku mar, N., A.K. Garg, R.S. Dass, V.K. Chaturvedi, V. Mudgal, and V.P. Varshney. 2009. Selenium supplementation influences growth performance, antioxidant status and immune response in lambs. Anim. Feed. Sci. Tech. 153(1 2): 77 87. Kurcz, E.V., L.M. Lawrence, K.W. Kelley, and P.A. Miller. 1988. The effect of intense exercise on the cell mediated immune response of horses. Equine Nutr. Physio. Soc. 8(3): 237 239. Liburt, N.R., A.A. Adams, A. Betancourt, D.W. Horohov, and K.H. McKeever. 2010a. Exercise induced i ncreases in inflammatory cytokines in muscle and blood of horses. Equine Vet. J. 42(Suppl 38): 280 288. Liburt, N.R., K.H. McKeever, J.M. Streltsova, W.C. Franke, M.E. Gordon, H.C. Manso Filho, D.W. Horohov, R.T. Rosen, C.T. Ho, A.P. Singh, and N. Vorsa. 2 010b. Effects of ginger and cranberry extracts on the physiological response to exercise and markers of inflammation in horses. Comp. Ex. Physiol. 6(4): 157 169. Lowder, T., D.A. Padgett, and J.A. Woods. 2006. Moderate exercise early after influenza virus infection reduces the Th1 inflammatory response in lungs of mice. Exerc. Immunol. Rev. 12:97 111. Maylin, G.A., D.S. Rubin, and D.H. Lein. 1980. Selenium and vitamin E in horses. Cornell Vet. 70(3): 272 289. McNeil, P.L., and R.A. Steinhardt. 1997. Loss, r estoration, and maintenance of plasma membrane integrity. J. Cell Bio. 137(1): 1 4. Mooren, F.C., D. Bloming, A. Lechtermann, M.M. Lerch, and K. Volker. 2002. Lymphocyte apoptosis after exhaustive and moderate exercise. J. Appl. Physiol. 93: 147 153. Morel AP., and T.B., Oriss. 1998. Crossregulation between Th1 and Th2 cells. Crit. Rev. Immunol. 18: 275 303. Mns, G. 1994. Effect of long distance running on polymononuclear neutrophils phagocytic function of the upper airways. Int. J. Sports Med. 15(2): 96 99. Garland Science, Taylor and Francis Group, LLC. New York, NY.
98 Nagata, S., F. Takeda, M. Kurosawa, K. Mima, A. Hiraga, M. Kai, and K. Taya. 1999. Plasma adrenocorticotr opin, cortisol and catecholamines response to various exercise. Equine. Vet. J. 31(S30): 570 574. Nelson, H.K., Q. Shi, P. Van Dael, E.J. Schiffrin, S. Blum, D. Barclay, O.A. Levander, and M.A. Beck. 2001. Host nutritional selenium status as a driving forc e for influenza virus mutations. FASEB J. 15: 1846 1848. Nesse, L.L., G.I. Johansen, and A.K. Blom. 2002. Effects of racing on lymphocyte proliferation in horses. Am. J. Vet. Res. 63(4): 528 530. Neuss, M., R. Monticone, M.S. Lundberg, A.T. Chesley, E. Fle ck, and M.T. Crow. 2001. The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress mediated cell death by preserving mitochondrial function. J. Bio. Chem. 276(36): 33915 33922. Nieman, D.C. 1997. Imm une response to heavy exertion. J. Appl. Physiol. 82: 1385 1394. Neiman, D.C. 2008. Regular moderate exercise boosts immunity. Agro. Food Industry Hi Tech. 19(3): 8 10. Nieman, D.C., and B.K. Pedersen. 1999. Exercise and immune function: Recent development s. Sports Med. 27(2): 73 80. Nieman, D.C., D.A. Henson, G. Gusewitch, J.B. Warren, R.C. Dotson, D.E. Butterworth, and S.L. Nehlsen Cannarella. 1993. Physical activity and immune function in elderly women Medicine and Sci ence 25(7): 823 831 Nieman, D.C., D.A Henson, L.L. Smith, A.C. Utter, D.M. Vinci, J.M. Davis, D.E. Kaminsky, and M. Shute. 2001. Cytokine changes after a marathon race. J. Appl. Physiol. 91: 109 114. Nieman, D.C., S.L. Nehlsen Cannarella, P.A. Markoff, A.J. Balk Lamberton, H. Yang, D.B. C hritton, J.W. Lee, and K. Arabatzis. 1990. The effects of moderate exercise training on natural killer cells and acute respiratory tract infections. Int. J. Sports Med. 11(6): 467 473. NRC. 2007. Nutrient Requirements of Horses, 6th Revised Ed. National Re search Council, National Academies Press, Washington, DC. Pedersen, B.K., and L. Hoffman Goetz. 2000. Exercise and the immune system: Regulation, integration and adaptation. Physiological Rev. 80(3): 1055 1081. Pedersen, B.K., N. Tvede, L.D. Christensen, K Klarlund, S. Kragbak, and J. Halkjr Kristensen. 1989. Natural killer cell activity in peripheral blood of highly trained and untrained persons. Int. J. Sports Med. 10(2): 129 131.
99 Richardson, S.M., P.D. Siciliano, T.E. Engle, C.K. Larson, and T.L. Ward. 2006. Effect of selenium supplementation and source on the selenium status of horses. J. Anim. Sci. 84: 1742 1748. Robson, P. J., T.D. Alston, and K.H. Myburgh. 2003. Prolonged suppression of the innate immune system in the horse following an 80 km endura nce race. Equine Vet. J. 35(2): 133 137. Roneus, B.O., and B. Lindholm. 1983. Glutathione peroxidase activity in blood of health y horses given different selenium supplementation. Nord. Veterinaermed. 35: 337. Roy, M., L. Kiremidjian Schumacher H.I. Wishe M.W. Cohen and G. Stotzky 1993. Selenium supplementation enhanced the expression of interleukin 2 receptor subunits and internalization of interleukin 2. Proc. Soc. Exp. Biol. Med. 202: 295 301. Roy, M., L. Kiremidjian Schumacher H.I. Wishe M.W. Cohen and G. Stotzky 1994. Supplementation with selenium and human immune cell functions. I. Effect on lymphocyte proliferation and interleukin 2 receptor expression. Biol. Trace Elem. Res. 41: 103 114. Safir, N., A. Wendel, R. Saile, and L. Chabraoui. 2003. The effect of selenium on immune functions of J774.1 cells. Clin. Chem. Lab. Med. 41(8): 1005 1011. Sappey, C., S. Legrand Poels, M. Best Belpomme, A. Favier, B Rentier, and J. Piette. 1994. Stimulation of glutathione peroxidase activity decreases HIV typ e 1 activation after oxidative stress. AIDS Res. Hum. Retroviruses. 10: 1451 1461. Shellow, J.S., S.G. Jackson, J.P. Baker, and A.H Cantor. 1985. The influence of dietary selenium levels on blood levels of selenium and glutathione peroxidase activity in th e horse. J. Anim. Sci. 61(3): 590 594. Smith, L.L. 2003. Overtraining, excessive exercise, and altered immunity: Is this a T helper 1 versus T helper 2 lymphocyte response? Sports Med. 33(5): 347 364. Snow, D., and R. Rose. 1981. Hormonal changes associate d with long distance exercise. Equine Vet. J. 13: 195 1 97. Stable, J.R., T.A. Reinhardt, and B.J. Nonnecke. 1991. Effect of selenium and reducing agents on in vitro immunoglobulin M synthesis by bovine lymphocytes. J. Dairy Sci. 74(8): 2501 2506. Stowe, H. D. 1967. Serum selenium and related parameters of naturally and experimentally fed horses. J. Nutr. 93: 60 64.
100 Streltsova, J.M., K.H. McKeever, N.R. Liburt, M.E. Gordon, H.M. Filho, D.W. Horohov, R.T. Rosen, and W. Franke. 2006. Effect of orange peel and b lack tea extracts on markers of performance and cytokine markers of inflammation in horses. Equine and Comp. Ex. Physiol. 3(3): 121 130. Stull, C.L., S.J. Spier, B.M. Aldridge, M. Blanchard, and J.L Stott. 2004. Immunological response to long term transpor t stress in mature horses and effect of adaptogenic dietary supplementation as an immunomodulator. Equine Vet. J. 36(7): 583 589. Sureda, A., P. Tauler, A. Aguil N. Cases, I. Llompart, J.A. Tur, and A. Pons. 2008. Influence of an antioxidant vitamin enri ched drink on pre and post exercise lymphocyte antioxidant systems. Ann. Nutr. Metab. 52: 233 240. Tapiero, H., D.M. Townsend, and K.D. Tew. 2003. Antioxidant role of selenium and seleno compounds. Biomedicine & Pharmacotherapy 57: 134 144. Tanimura, Y., K. Shimizu, K. Tanabe, T. Otsuki, R. Yammauchi, Y. Matsubara, M. Iemitsu, S. Maeda, and R. Ajisaka. 2008. Exercise induced oxidative DNA damage and lymphocytopenia in sedentary young males. Med. Sci. Sports Ex. 40(8): 1455 1462. Tauler, P., A. Sureda, N. C ases, A. Aguil, J.A. Rodrguez Marroyo, G. Villa, J.A. Tur, and A. Pons. 2006. Increased lymphocyte antioxidant defenses in response to exhaustive exercise do not prevent oxidative damage. J. Nutr. Biochem. 17: 665 671. Tinggi, U. 2008. Selenium: Its role as antioxidant in human health. Environ Health Prev. Med. 13: 102 108. Tolando, R., A. Jovanovic, R. Brigelius Floh, F. Ursini, and M. Maiorino. 2000. Reactive oxygen species and proinflammatory cytokine signaling in endothelial cells: Effect of selenium supplementation. Free Radical Bio. and Med. 28(6): 979 986. Tvede, N., J. Steensberg, B. Baslund, J. Halkr Kristensen, and B.K. Pedersen. 1991. Cellular immunity in highly trained elite racing cyclists during periods of training with high and low intensi ty. Scand. J. Sports Med. 1(3): 163 166. Viarengo, A., B. Burlando, M. Cavaletto, B. Marchi, E. Ponzano, and J. Blasco. 1999. Role of metallothionein against oxidative stress in the mussel Mytilus galloprovincialis. Am. J. Physiol. Integr. Comp. Physiol. 2 77: R 1612 R1619. Via, J., C. Borras, M.C. Gomez Cabrera, and W. C. Orr. 2006. Role of reactive oxygen species and (phyto) estrogens in the modulation of adaptive response to stress. Free Radical Research. 40(2): 111 119.
101 Via, J., M.C. Gomez Cabrera, A. L loret, R. Marquez, J.B. Miana, F.V. Pallard, and J. Sastre. 2000. Free radicals in exhaustive physical exercise: Mechanism of production, and protection by antioxidants. Life. 50: 271 277. Wang, J.S., and Y.H. Huang. 2005. Effects of exercise intensity o n lymphocyte apoptosis induced by oxidative stress in men. Eur. J. Appl. Physiol. 95: 290 297. Wichert, B., T. Frank, and E. Kienzle. 2002. Zinc, copper and selenium intake and status of horses in Bavaria. J. Nutr. 132: 1776S 1777S. Woods, J.A., M.A. Ceddi a, C. Kozak, and B.W. Wolters. 1997. Effects of exercise on the macrophage MHC II response to inflammation. Int. J. Sports Med. 18(6): 483 488. Wong, C.W., S.E. Smith, Y.H. Thong, J.P. Opdebeeck, and J.R. Thornton. 1992. Effects of exercise stress on vari ous immune functions in horses. Am. J. Vet. Res. 53(8): 1414 1417.
102 BIOGRAPHICAL SKETCH Jill Bobel is an Ohio native but spent her developing years in Chicago, Illinois. seems to have never come down. Throughout middle school and high school, she worked at stables and took lessons regularly. At the age of 15, her parents were gracious enough to buy her a horse. She started three day eventing at the age of 16 and has since been on the list of top ten riders in the nation four times. She considers accomplish together. They are currently competing at the preliminary level of three day eventing. Gro wing up, Jill had always wanted to be a large animal veterinarian. However, during high school, she got her first taste of animal nutrition and was captivated. After high school, Jill moved to Florida and attended the University of Florida. In 2008, she ea s of science in Animal Sciences with an emphasis on animal industry equine and a minor in Management and Sales in Agribusiness and was proud meet an amazi ng professor of equine nutrition, Dr. Lori Warren. From here, her path in life became clear: she wanted to get her PhD in Equine Nutrition. In the summer of 2008, s he received a paid internship by the Florida Agriculture Experiment Station to work in Dr. W arren and it was here that she discovered her love of research and lab work. After her internship ended, she remained employed by Dr. Warren until August 2009. During this time, Jill dually worked in the ruminant nutrition lab at the universit y under Dr. Adeg bola Adesogan.
103 Upon completion of her M.S. program, Jill plans to pursue a Doctor of Philosophy at the University of Florida in animal sciences with a focus on equine nutrition. She hopes to make the field of equine/animal nutritional rese arch a career.