In the tuberculosis patients, Mycobacterium tuberculosis can stimulate production of hydrogen peroxide in the host as a result of immune response. The H2O2 accumulate in pulmonary cells, causing oxidative stress that could lead to the cancer. We select TB patients for this study which investigates the effects of quercetin as there is an increased incidence of latent TB among the migrant population in the past few years and TB can increase the risk of cancer. Sperm and lymphocytes were treated with DNA damage inducers and quercetin (10µM, 25µM and 100µM), the responses evaluated using the Comet and micronucleus techniques. The gene expressions of COX1, COX2, P53 and Bcl-2 and catalase protein expression were investigated using the qPCR and Western blot techniques. The results showed that a substantial reduction of DNA damage in lymphocytes from TB patients and sperm from healthy donors from * P ≤ 0.0283 to *** P≤0.001in the Comet assay. In the MNi assay, the effect of quercetin in lymphocytes was more significant in reduce DNA damage, whereas the DNA damage induced by a food mutagen was significant, from *p 0.0405 to ***p 0.001. The qPCR showed significance down-regulation of COX1 and Bcl-2 gene expression, rated between *p 0.045 and **p 0.0074. However, the catalase protein was up-regulated by the nano form of quercetin when using lymphocytes from TB patients and showed significant changes at *p 0.0236. In conclusion, the nano form was found to be more efficient at the reduction of DNA damage in the Comet and micronucleus assays. Also, it down-regulated COX1 and Bcl-2 and up-regulated the catalase proteins indicating a possible role for quercetin, in genoprotection to TB through its enzyme modulating effect. / Libyan Embassy

No description available.

No description available.

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

The gut-brain axis describes a paradigm wherein the trillions of microorganisms inhabiting the gastrointestinal tract engage in bidirectional communication with the host central nervous system. Adaptive immunity represents an important intermediate in this dynamic crosstalk; previous work in our lab has demonstrated that T-lymphocytes, a main class of immune effector cells, contribute to neurodevelopmental processes and behavioral outcomes across the lifespan. Parallels between the phenotype of T-cell deficient and germ free mice led us to hypothesize that bidirectional T-cell-microbe communication is critical for normal neurodevelopment, and that T-cell deficiency impacts the neural circuitry underpinning behavior via disruption of the gut-brain axis. The main objective of this thesis was to elucidate the mechanisms by which T-cells mediate developmental gut-brain signalling. The first installation examined the gut microbiome, gut metabolome, and neurochemical profile in wild-type and T-cell deficient mice from adolescence to adulthood, demonstrating that absence of T-cells impacts the developmental trajectory of functional microbiome output and levels of neuroactive molecules in the brain. Experiment two investigated the impact of T-cell deficiency on gut-brain communication through the lens of host gene expression in the parenchyma and the intestine. T-cell deficient mice showed significant changes in genes related to intestinal immunity and barrier function, in addition to decreases in microglia-related genes in the prefrontal cortex during early life. The final experiment transitioned into a wild-type model to measure the co-evolution of T-cell subsets in mucosal and central immune compartments with composition and diversity of the microbiota. We demonstrated a parallel diversification of the gut microbiome and the functional T-cell repertoire, whereby emergence and proliferation of specific T-cell subsets is linked to compositional shifts in dominant microbial communities across development. Together, our results demonstrate the importance of T-cells for normal development of the holo-organism, with implications for the developmental wiring of functional brain circuitry. / Thesis / Doctor of Philosophy (PhD) / Modern medicine has increasingly placed emphasis on the mind-body connection. This has been exemplified by a series of recent discoveries surrounding the importance of the gut microbiome in maintaining our physical and mental health. One of the key channels through which the microbiome communicates with the host is through the immune system, an equally complex network of cells and proteins that protect the body against invading pathogens. Indeed, these systems evolve alongside each other and engage in constant crosstalk throughout the lifespan, with downstream impacts on the developing brain. This thesis sought to further explore the role of T-cells, a key component of the adaptive immune system, in coordinating gut-microbiome-brain interactions across development. The first experiment examined the microbiome as well as small molecules in the gut and brain of normal mice and mice lacking T-cells. The second experiment built on this work to examine how T-cells influence the expression of different genes in the gut and brain. Finally, the third experiment mapped different populations of T-cells and microbiome composition from the first week of life to adulthood, to better understand how they interact at different stages of development. This work will offer insight into how T-cells talk to the microbiome and how they transmit signals from the gut to the brain, with implications for understanding neurodevelopmental disorders and how they arise.

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

No / Lymphocyte responses from 208 individuals: 20 with melanoma, 34 with colon cancer, and 4 with lung cancer (58), 18 with suspected melanoma, 28 with polyposis, and 10 with COPD (56), and 94 healthy volunteers were examined. The natural logarithm of the Olive tail moment (OTM) was plotted for exposure to UVA through 5 different agar depths (100 cell measurements/depth) and analyzed using a repeated measures regression model. Responses of patients with cancer plateaued after treatment with different UVA intensities, but returned toward control values for healthy volunteers. For precancerous conditions and suspected cancers, intermediate responses occurred. ROC analysis of mean log OTMs, for cancers plus precancerous/suspect conditions vs. controls, cancer vs. precancerous/suspect conditions plus controls, and cancer vs. controls, gave areas under the curve of 0.87, 0.89, and 0.93, respectively (P<0.001). Optimization allowed test sensitivity or specificity to approach 100% with acceptable complementary measures. This modified comet assay could represent a stand-alone test or an adjunct to other investigative procedures for detecting cancer.

Le diabète auto-immun résulte de la destruction des cellules bêta pancréatiques sécrétrices d’insuline par les lymphocytes T du système immunitaire. Il s’ensuit une déficience hormonale qui peut être comblée par des injections quotidiennes d’insuline d’origine exogène, toutefois il demeure à ce jour impossible de guérir les patients atteints de la maladie. De façon générale, un système immunitaire sain reconnaît une multitude d’antigènes différents et assure ainsi notre défense à l’égard de différents pathogènes ou encore de cellules tumorales. Il arrive cependant que, pour des raisons génétiques et/ou environnementales, les lymphocytes T puissent s’activer de façon aberrante suite à la reconnaissance d’antigènes provenant du soi. C’est ce bris de tolérance qui mène au développement de pathologies auto-immunes telles que le diabète auto-immun. Afin de limiter l’auto-immunité, des mécanismes de sélection stricts permettent d’éliminer la majorité des lymphocytes T présentant une forte affinité envers des antigènes du soi lors de leur développement dans le thymus. Certains de ces lymphocytes réussissent toutefois à échapper à l’apoptose et migrent en périphérie afin d’y circuler en quête d’un antigène spécifiquement reconnu. Il est alors primordial que des mécanismes périphériques assurent le maintien de la tolérance immunitaire en faisant obstacle à l’activation et à la prolifération des lymphocytes T auto-réactifs. L’une des avenues afin d’inhiber le développement de réponses immunitaires aberrantes est la génération de lymphocytes T régulateurs. Ces cellules, d’origine thymique ou périphérique, peuvent arborer différents phénotypes et agissent via de multiples mécanismes afin d’inactiver et/ou éliminer les cellules impliquées dans l’apparition de pathologies auto-immunes. L’utilisation de modèles murins transgéniques a permis la mise en évidence d’une population peu caractérisée de lymphocytes T au potentiel régulateur. En effet, la proportion de ces cellules T n’exprimant pas les corécepteurs CD4 et CD8 (double négatives, DN) a été inversement corrélée à la prédisposition à l’auto-immunité chez ces ii souris. L’objectif principal de cette thèse est de démontrer la fonction immuno-régulatrice des lymphocytes T DN, tout en investiguant les facteurs génétiques responsables du maintien de cette population cellulaire. Nous avons observé que les lymphocytes T DN exercent une activité cytotoxique à l’égard des lymphocytes B de façon spécifique à l’antigène, via la libération de granules cytolytiques contenant du granzyme B et de la perforine. Par ailleurs, nous avons établi qu’un unique transfert adoptif de ces cellules est suffisant afin d’inhiber le développement du diabète auto-immun chez des hôtes transgéniques prédisposés à la maladie. Le recours à des souris déficientes pour l’expression du gène CD47 a permis de constater que la voie de signalisation CD47-Sirp est essentielle dans le maintien de la proportion des lymphocytes T DN. De plus, le locus murin de prédisposition au diabète auto-immun Idd13, qui contient le gène Sirp, a été identifié pour son rôle dans la régulation de la proportion de ces cellules. Finalement, une analyse génétique a révélé que d’autres intervalles génétiques sont impliqués dans le contrôle de la population des lymphocytes T DN. Parmi ceux-ci, un locus situé en région proximale du chromosome 12 a été validé grâce à la création de souris congéniques. Grâce aux résultats présentés dans cette thèse, notre compréhension de la biologie ainsi que de la régulation des lymphocytes T DN est approfondie. Ces connaissances constituent un pas important vers la création de thérapies cellulaires novatrices permettant de prévenir et de guérir diverses pathologies auto-immunes. / Autoimmune diabetes results from the destruction of the insulin-secreting pancreatic beta cells by the T lymphocytes of the immune system. This leads to a hormonal deficiency that can be regulated with daily injections of exogenous insulin. However, to date, there is no cure for autoimmune diabetes. A healthy immune system generally recognizes a multitude of antigens in order to ensure our defence against different pathogens and tumor cells. Yet, depending on genetic and/or environmental factors, individuals may develop T cells that are aberrantly activated following the recognition of self-antigens. This break in tolerance leads to the development of autoimmune pathologies, such as autoimmune diabetes. In order to limit autoimmunity, rigorous selection mechanisms eliminate the vast majority of the T lymphocytes that present a high affinity for self-antigens during their thymic development. However, some of these auto-reactive lymphocytes escape from the elimination processes and migrate to the periphery where they might encounter a self-antigen. It is then essential that peripheral mechanisms maintain the immune tolerance by abrogating the activation and the proliferation of these self-specific T lymphocytes. One of the means to inhibit aberrant immune responses is the generation of regulatory T lymphocytes. These cells, which can be of thymus or peripheral origin, display various phenotypes and can mediate their action through several mechanisms in order to inactivate and/or eliminate the cells that are implicated in the development of autoimmune diseases. The use of transgenic mouse models made it possible to identify a poorly characterized population of T lymphocytes that exhibit a regulatory potential, namely CD4-CD8- (double negative, DN T cells). Indeed, the proportion of DN T cells in lymphoid organs is inversely correlated to autoimmune predisposition. The main objective of this thesis is to determine the immunoregulatory function of the DN T cells, as well as to reveal the genetic factors underlying the regulation of the proportion of DN T cells. iv We observed that through the release of cytolytic granules containing granzyme B and perforin, DN T lymphocytes exert a cytotoxic activity towards B cells in an antigen-specific manner. In addition, we have established that a single injection of those DN T cells is sufficient to inhibit the development of autoimmune diabetes in highly susceptible transgenic mice. The use of CD47 deficient mice also demonstrated that the CD47-Sirp pathway is essential to maintain DN T cell proportion. Also, we identified that the autoimmune diabetes susceptibility locus Idd13, which contains Sirp participates in defining the proportion of DN T cells. Finally, a genetic analysis revealed that other loci are implicated in the control of the DN T cell population. Among those, the role of a locus situated in the proximal region of chromosome 12 has been validated through to the generation of congenic mice. The results presented in this thesis have allowed us to enhance our understanding of the biology and genetic regulation of DN T lymphocytes. This knowledge constitutes an important step towards the creation of innovative cellular therapies that may prevent and cure a diversity of autoimmune pathologies.

La mémoire immunologique est le mécanisme biologique fondamental à la base du développement de la vaccination. La compréhension de ce mécanisme ainsi que de ses interactions avec les différents acteurs du système immunitaire a permis l’élaboration de vaccins qui sont aujourd’hui les garants d’une protection accrue face à l’émergence de maladies infectieuses potentiellement mortelles. La voie d’injection et le mode de transfert de ces vaccins sont des paramètres majeurs à prendre en considération car ils définissent une modulation des réponses immunitaires et de leurs spécificités d’action. De nos jours, seule la voie intramusculaire demeure la voie majoritaire d’administration de vaccins lors de la prophylaxie primaire en santé humaine. Au cours de notre étude, nous nous sommes intéressés à comparer l’injection d’un antigène (l’ovalbumine) selon deux voies d’administration : la voie intramusculaire et la voie intradermique. Nous nous sommes également appuyés sur une technologie du laboratoire qui consiste à transférer des gènes par des vecteurs AAV2/1 recombinants. Nous disposions de deux constructions de ces vecteurs ayant une spécificité pour cibler les cellules musculaires et permettant l’apport d’un effet auxiliaire par les lymphocytes T CD4+ lors d’injections dans des souris femelles. De plus, une de ces constructions nous permettait d’éviter la voie de présentation directe de l’antigène par les cellules dendritiques (DCs) aux lymphocytes T CD8+. Les capacités modulatrices de ces vecteurs nous permirent de montrer pour la première fois que le vecteur AAV2/1 recombinant était capable de faire exprimer un transgène au sein de la peau et d’y générer une réponse cellulaire forte. Nous avons également montré qu’il existait une synergie d’action entre l’effet auxiliaire et la voie intradermique qui améliorait considérablement les réponses cellulaires issues de la présentation croisée d’antigène. Enfin, nous avons pu démontrer que les lymphocytes T CD8+ générés suite à cette synergie d’action présentaient un profil phénotypique de cellules mémoires polyfonctionnelles et capables de protéger l’hôte face à un challenge pathogénique. / Immunological memory is the fundamental biological mechanism at the beginning of the development of vaccination. Understanding this mechanism and its interactions with the various players of the immune system has allowed the development of vaccines that are today the most effective barrier against the emergence of life-threatening infectious diseases. Route of injection and the nature of carriers of these vaccines are key parameters to be taken into consideration because they define a modulation of immune responses and their specific features. Nowadays, only the intramuscular injection route remains the major route of vaccines injection in the context of primary prophylaxis in human health. During our study, we were interested in comparing the injection of antigen (ovalbumin) following two routes of administration: intramuscular and intradermal routes. We also relied on a technology in the laboratory that involves the transfer of genes by rAAV2/1 vectors. We had two constructs of these vectors having specificity to target skeletal muscle cells and allowing us to provide a helper effect from CD4+ T cells during injections into female mice recipients. Moreover, one of these constructs enabled us to avoid the direct presentation of antigens by dendritic cells (DCs) to CD8+ T cells. The capacity of modulation of these vectors allowed us to show for the first time that the rAAV2/1 vector was able to trigger the expression of a transgene in the skin, and there to generate a strong cellular response. We have also shown that CD4+ T cell help and the intradermal route of immunization synergize to improve greatly cellular responses from the cross-presentation of antigens. Finally, we have demonstrated that CD8+ T cells generated following this synergism exhibited a phenotypic profile of polyfunctional memory cells and able to protect the host against a pathogenic challenge.