Equine Protozoal Myeloencephalitis (EPM) is a serious neurologic disease of horses in the United States. The primary etiologic agent is Sarcocystis neurona (S. neurona). Currently, there is limited knowledge regarding the protective or pathologic immune response to infection to the intracellular protozoa S. neurona. The objective of these studies was to determine the effects of S. neurona infection on the immune response of horses that had EPM due to natural infection (experiment 1) and experimental infection (experiment 2). In experiment 1, twenty-two horses with naturally occurring cases of EPM, which were confirmed positive based on detection of antibodies in the serum and/or CSF and clinical signs, and 20 clinically normal horses were included to determine whether S. neurona altered the immune responses, as measured by immune cell subsets (CD4, CD8, B-cell, monocytes, and neutrophils) and leukocyte proliferation (antigen specific and non-specific mitogens). Our results demonstrated that naturally infected horses had significantly higher percentages of CD4 and neutrophils (PMN) in peripheral blood mononuclear cells (PBMCs) than clinically normal horses. Leukocytes from naturally infected EPM horses had a significantly lower proliferation response, as measured by thymidine incorporation, to a non-antigen specific mitogen phorbol 12-myristate 13-acetate (PMA) / ionomycin (I) than did clinically normal horses (p=0.04). The implications of these findings will be discussed. In experiment 2, 13 horses were randomly divided into two groups. Baseline neurologic examinations were performed and all horses were confirmed negative for S. neurona antibodies in the CSF and serum. Then, one group with 8 clinically normal seronegative horses was inoculated intravenously with approximately 6000 S. neurona infected autologous leukocytes daily for 14 days. All the challenged horses showed neurologic signs consistent with EPM. PBMCs were isolated from the control and infected horses to determine how S. neurona alters the immune responses based on changes in immune cell subsets and immune function. There were no significant differences in the percentage of CD4 cells in peripheral blood lymphocytes or IFN-γ production by CD4 and/or CD8 cells. PMA/I stimulated proliferation responses in PBMCs appeared suppressed compared to that of uninfected controls. Additional studies are necessary to determine the role of CD4 and CD8 cells in disease and protection to S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. This project was funded by Patricia Stuart Equine grants and paramutual racing funds from Virginia Tech. / Master of Science

Equine Protozoal Myeloencephalitis is the most frequently diagnosed neurologic disorder of horses in the united states, which is caused by the protozoan organism Sarcocystis neurona. The disease has a profound impact on the American Horse Industry. This impact includes prolonged and expensive treatment without a guaranteed return to a previous level of use for the individual horse. Poor respponse to and prolonged duration of treatment may suggest an immune mediated impariement of host response. There is limited information about the direct interaction between the pathogen and the host. In two in vitro experiments we investigated a) whether the presence of the protozoan organism can influence mitogen-stimulated peripheral blood mononuclear cells (PBMCs), suggesting a direct influence of the protozoan organism on cells of the immune system, and b) if cerebrospinal fluid (CSF) from horses with EPM has an effect on mitogen-stimulated PBMCs, suggesting that the microenvironment of the site of infection influences the course of disease. Experiment 1: Mitogen simulated PBMCs from EPM affected and control horses were co-cultured with fragments of freeze thawed bovine turbinate cells that were infected with S. neurona merozoites. Compared to controls PBMCs co-cultured with S. neurona fragments were the only cells that showed a decreased proliferation (p<0.05). A difference between EPM affected and control horses could not be detected (p>0.05). These results may imply that the persistence of S. neurona infection in the horses CNS is, in part, due to a pathogen-derived mechanism that attentuates the hosts immune response. Experiment 2: Mitogen stimulated PBMCs from a horse affected with EPM and a control were co-cultured n the presence of CSF from EPM affected and uninfected controls. Prior to co-culture the CSF was fractionated by a filtration process over two microfilter units. An identical volume of NaCl (0.9%) served as a control for the volume of CSF that was added. The proliferation assay revealed a deviation of the response depending on cell donor and CSF fraction used. The effect was independant of the protein concentration of the CSF fraction, and a decrease in lymphocyte proliferation was not caused by increased cellular death. This suggests the presence of subsets within the CSF which have a stimulatory of suppressive influence on the cells in culture. The effect was cell donor dependant which implies a difference in lymphocyte subsets between the two horses that were used. / Master of Science

Previous studies have demonstrated differences in CD4+, CD8+ and B-cell populations between EPM affected and normal horses. The overall goal of our project was to further define the immune deficiencies associated with S. neurona infection. We hypothesized that PMA/I stimulated suppression in EPM horses is due to decreased proliferation of monocytes, CD4+ and CD8+ cells. Our objectives were 1) to determine whether S. neurona infection causes an increase in apoptosis of a particular immune subset, and 2) to determine whether S. neurona causes a decrease in the number of cellular divisions (proliferation) of a particular immune cell subset. For this study, nine S. neurona antibody negative, immunocompetent horses were obtained. Baseline neurologic examinations, SnSAG1 (S. neurona Surface Antigen 1) ELISAs on cerebrospinal fluid (CSF) and serum, and baseline immune function assays were performed. Horses were randomly divided into groups. Five horses were challenged for ten days via intravenous injection of autologous lymphocytes infected with S. neurona. Neurologic parameters of all horses were assessed for 70 days following infection. Immune function was based on proliferation responses to mitogens, as assessed through thymidine incorporation. Enumeration of cellular subsets, degree of apoptosis and number of cellular divisions were assessed through flow cytometry. SnSAG1 ELISA of serum and CSF samples performed post-infection confirmed infection and disease. All infected horses displayed moderate neurologic signs on clinical examination. Some significant differences in cellular activities were noted. Additionally, this is the first time the method using S. neurona infected lymphocytes has been reproduced successfully by different investigators. / Master of Science

<p>As the human population increases and the demand for aquaculture increases, aquaculturists are coming up with new ways to mitigate stress in fish to increase their production. Cannabidiol (CBD) is an up and coming nutraceutical that may have potential to reduce stress in not only humans but also other vertebrates such as fish. In this project the effect of CBD on the stress physiology and immunology of Nile tilapia was evaluated both <i>in-vitro</i> and <i>in-vivo</i>. In the <i>in-vitro</i> study, spleen cell proliferation was conducted to observe the effect of CBD on fish T and B cells and were compared to mouse T and B cell proliferation. In the <i>in-vivo</i> study, the fish were reared in a recirculating aquaculture system. The effect of CBD on the stress physiology of the fish in short term and long term were evaluated. Based on the short-term acute study, a longer chronic study was designed where tilapia were fed with and without CBD (0.001% of feed weight) and with and without hydrocortisone stress hormone (0.01% of body weight) every day for four weeks. This experiment compared the various growth and feed utilization parameters as well as physiological and immunological parameters such as, plasma cortisol, blood glucose and protein levels, liver and spleen somatic indices (HSI and SSI, respectively), and lysozyme activity of the fish. From our current research, CBD shows potential in stress modulation and in immune modulation. It may have different effects based on the species, whether they need to enhance their immune response or reduce inflammation to be healthy. It also seems to have had different effect on different parts of the immune system. Hematological parameters were not significantly affected by acute stress. CBD did not make any substantial difference in growth. However, in the presence of stress, CBD was able to lower lysozyme activity down to the normal control levels. By administering the proper dosage of CBD on a case by case basis, health benefits can be achieved. Further investigation into the matter may not just be useful in stress mediation in aquatic organisms but may also have implications in human medicine as well.</p>

La prévalence des allergies alimentaires IgE-médiées aurait triplé au cours de la dernière décennie avec des études Nord-Américaines atteignant les 8% chez les enfants. Quoiqu’il n’y ait à ce jour aucun traitement curatif pour les allergies alimentaires, l’immunothérapie oral (OIT) constitue une nouvelle approche expérimentale prometteuse. Cette dernière consiste en l’administration de doses progressive d’allergènes par voie orale sur une période prolongée dans le but d’instaurer un état de désensibilisation et possiblement une tolérance orale soutenue. Cette approche a été démontrée sécuritaire et permettrait la désensibilisation à haute dose de plus de 80% des participants allergiques aux arachides, lait ou œufs. Dans cette thèse, nous présentons 2 études de phase 1 portant sur des protocoles d’OIT, destinés à optimiser l’efficience du traitement chez les sujets avec allergies alimentaires multiples. Près de 30% des enfants avec allergie alimentaire sont allergiques à plus d’un aliment, une proportion qui augmente à 70% lorsqu’on considère les cas les plus sévères. Ces enfants sont à risque augmenté de réactions accidentelles et souffrent d’un impact plus grand sur leur qualité de vie. Dans la première étude, en créant un mélange individualisé avec un ratio stochiométrique 1:1 entre les protéines des aliments allergiques de l’enfant, nous démontrons qu’il est possible de désensibiliser jusqu’à 5 aliments simultanément avec un profil d’innocuité similaire à une monothérapie. Dans la seconde étude, nous utilisons un traitement à l’omalizumab, un anticorps monoclonal anti-IgE, pour permettre une désensibilisation orale multi-allergénique fortement accélérée. Lorsque comparé à l’approche sans omalizumab, ce protocole s’associe à une nette diminution du temps requis pour atteindre les doses d’entretien, passant d’une médiane de 21 à 4 mois, sans affecter le profil d’innocuité. Alors que ces études fournissent des approches cliniques raisonnables pour désensibiliser la population multi-allergique, plusieurs questions persistent, notamment en ce qui a trait à l’induction de tolérance permanente. Une barrière majeure à cet égard réside dans notre piètre compréhension des mécanismes sous-jacents à l’immunothérapie. Prenant avantage d’échantillons cliniques bien caractérisés provenant des essais cliniques ci-haut mentionnés, nous utilisons les nouvelles technologies de séquençage TCR pour suivre la distribution clonale des lymphocytes T spécifiques aux arachides durant une immunothérapie orale. Nous démontrons que l’OIT s’associe à des changements significatifs dans les fréquences des clones spécifiques, suggérant un processus d’épuisement clonal et de remplacement. Nous démontrons par ailleurs que le test de prolifération lymphocytaire, traditionnellement utilisé pour évaluer la réponse cellulaire allergique, est dominé par une distribution polyclonale hautement non-spécifique. Cette observation a des implications majeures considérant que la plupart de la littérature actuelle sur la réponse T se base sur cette technique. En somme, cette thèse jette les bases pour des programmes de recherche translationnelle pour optimiser et personnaliser les protocoles cliniques actuels et développer de nouvelles avenues d’investigation et de traitement pour améliorer la prise en charge des sujets avec allergies alimentaires. / The prevalence of IgE-mediated food allergies has tripled over the last decade with prospective studies indicating that up to 8% of children may be affected in North America. There is currently no cure for food allergy but oral immunotherapy (OIT) is an experimental approach to treat food allergies. It consists in the progressive administration from minute to large amounts of the allergenic food by the mouth over a prolonged period of time to induce a state of desensitization and possibly sustained tolerance. This approach has been shown to be safe and to allow desensitization to high doses in over 80% of participants allergic to peanuts, milk or egg. In this thesis, we present two phase 1 trials on OIT protocols designed to efficiently treat multiple foods allergies. About 30% of children with food allergy are allergic to more than one food. This proportion increases to 70% when considering the most severe cases. Children with multiple food allergies are at higher risk of accidental reactions and suffer from greater impact on quality of life than those with single food allergies. By creating a customized treatment mix with a 1:1 stoichiometric ratio for the child’s relevant food proteins, we were first able to safely desensitize up to 5 foods simultaneously with a safety profile similar to single allergen therapy and a minimal increase in time to maintenance. Then, taking advantage of recent evidence showing that omalizumab, an anti-IgE receptor monoclonal antibody, can significantly raise reaction thresholds in food allergic subjects, we used short courses of omalizumab to allow very rapid oral desensitization to various foods in a second phase 1 study. When compared to “standard” multi-OIT, the omalizumab-enabled rush protocol resulted in a decreased time to maintenance from a median of 21 to 4 months. While these studies provide reasonable clinical approaches to this population, many questions remain, especially with regards to long term tolerance. A major limit to our progress in improving these protocols stems from our lack of understanding of the underlying immune mechanisms of oral immunotherapy. Taking advantage of well phenotyped samples from the afore-mentioned trials, we used next-generation high-throughput TCR sequencing to follow clonal distribution of peanut specific T cells during oral immunotherapy. We found that OIT is associated with significant changes in food-specific clonal frequencies, suggesting clonal exhaustion and replacement as an underlying mechanism of OIT. In addition, we show that the proliferation assay which is traditionally used to assess the cellular response is dominated by a highly non-specific polyclonal distribution. This observation has important implications considering most of the current literature on T cell response to immunotherapy is based on this assay. This highlights the need for the development of new tools to assess the cellular allergic response. Overall this thesis lays the ground for further comprehensive translational research programs on the treatment of food allergy.

M. Tech. (Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology / Tulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol vi extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.

Introduction: Le mélanome est le type de cancer de la peau le plus fréquent et les métastases du système nerveux central en sont une complication fréquente et grave. Les cellules de mélanome interagissent avec une grande variété de types de cellules dans le microenvironnement tumoral (MET), ce qui peut entraîner des effets pro- ou antitumoral. Plusieurs voies immunosuppressives ont été récemment découvertes comme des cibles médicamenteuses prometteuses, notamment la voie de l'adénosine. L'adénosine extracellulaire s'accumule dans le MET suite à l'hydrolyse de l'ATP par les ectonucléotidases CD39 et CD73. Les principaux régulateurs de la voie de l'adénosine sont CD39, CD73, et les récepteurs A2a et A2b. Matériel et Méthodes: Pour caractériser spatialement le MET des métastases cérébrales du mélanome (MCM), nous avons quantifié l'expression de 35 marqueurs protéiques à l'aide du time of flight (CyTOF) Imaging Mass Cytometry (IMC) dans 21 MCM, et segmenté et classé plus de 130 000 cellules. Ensuite, pour évaluer les effets du ciblage du récepteur A2b et du CD73 dans la voie de l'adénosine sur le développement du mélanome, nous avons utilisé les tests de prolifération IncuCyte et MTS pour évaluer la prolifération des cellules de mélanome. Résultats: Dans notre ensemble de données, les caractéristiques immunitaires du MET étaient hétérogènes dans tous les échantillons et le type de cellule le plus courant après les cellules cancéreuses du mélanome était les macrophages dérivés de la moelle osseuse (MDMO). Les échantillons à propagation leptoméningée avaient significativement moins de neutrophiles, de MDMO de type M1, d'autres cellules T et plus de cellules cancéreuses dans leur microenvironnement. Nous avons observé que la stimulation du récepteur A2b a un effet antiprolifératif sur les cellules cancéreuses du mélanome. Conclusion: Cette recherche met en évidence le rôle du MET dans la progression du mélanome et l'importance du MET comme base pour le développement de nouvelles thérapies pour les patients atteints de cancer. / Background: Melanoma is the most frequent type of skin cancer and metastasis to the central nervous system is a common and serious complication of it. Melanoma cells interact with a wide variety of cell types in the tumor microenvironment (TME) which can lead to tumor-promoting or tumor suppressive effects. Several immunosuppressive pathways have emerged as promising drug targets, including the adenosine pathway. The extracellular adenosine accumulates in the TME as the result of ATP hydrolysis by the ectonucleotidases CD39 and CD73. Key regulators of the adenosine pathway are CD39, CD73, A2a and A2b receptor. Methods: To spatially characterize the TME of melanoma brain metastases (MBM), we quantified the expression of 35 protein markers using time of flight (CyTOF) Imaging Mass Cytometry (IMC) in 21 MBMs, and segmented and classified over 130 000 cells. Then, to evaluate the effects of targeting the A2b receptor and CD73 in the adenosine pathway on the development of melanoma, we used the IncuCyte and MTS proliferation assays to assess the proliferation of melanoma cells. Results: In our dataset, the immune landscape of the TME was heterogeneous across all samples and the most common cell type after melanoma cancer cells were bone marrow derived macrophages (BMDM). Samples with leptomeningeal spread had significantly less neutrophils, M1-like BMDM, T other cells and more cancer cells in their microenvironment. We observed that stimulation of the A2b receptor has an antiproliferative effect on melanoma cancer cells. Conclusion: This research highlights the role of the TME in the progression of melanoma and the importance of the TME as grounds for development of new therapies for cancer patients.