Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

Innate immune memory has become an increasingly popular area of research in the last decade. However, much of the work done on innate immune memory using inflammatory agents such as BCG, C. albicans, and β-glucan has been pursued through systemic administration, which has been shown to induce training in circulating monocytes. In addition, little is known about whether microbial ligands can induce training. Here, we show that local mucosal exposure to an acute dose of LPS induces long-lasting phenotypic changes in airway macrophage populations. LPS-exposed macrophages display increased glycolytic metabolism and differential cytokine expression upon restimulation, whereas circulating monocytes are not affected. Finally, we show that LPS exposure provides long-lasting protection against Streptococcus pneumoniae in the lung, likely due to the higher acquisition of CD11b, which is indicative of macrophage activation and phagocytosis. As much of the work on innate immune memory has been done through systemic administration of training agents, this project aims to fill existing knowledge gaps in the induction of innate immune memory upon local mucosal exposure to inflammatory agents. / Thesis / Master of Science in Medical Sciences (MSMS) / The innate immune system is one of the first defenders in our bodies that fight against a variety of pathogens. In the last decade, the innate immune system was found to be capable of having memory, meaning it reacts faster or at a heightened magnitude in response to a wide range of subsequent pathogens after it is trained by an agent. This project explores the effect a bacteria wall component, LPS, has on the lung environment and examines if it will induce memory in the lung. Our findings show that intranasal exposure to LPS changes the cellular landscape in the lung. LPS-exposed airway innate immune cells become more activated and provide subsequent protection against bacterial infections. This work has implications for using LPS as a vaccine adjuvant in order to provide protection against a variety of pathogens in addition to specific protection brought by the vaccine.

Deux grandes classes de cellules composent le pool de lymphocytes T (LT) CD8 mémoires. D'une part, les LT CD8 mémoires conventionnels sont générés via la reconnaissance spécifique d'antigènes dérivés de pathogènes ou de tumeurs. D'autre part, les LT CD8 mémoires innés sont générés via différents mécanismes impliquant de fortes stimulations par des cytokines γc indépendamment de la reconnaissance d'antigènes du non soi. Le phénotype extrêmement similaire de ces deux populations cellulaires ne permet pas de les distinguer in vivo. En conséquence, la population de LT CD8 mémoires innés est relativement peu caractérisée. Mon travail de thèse comportait donc deux objectifs majeurs : 1 / Identifier des marqueurs permettant de distinguer in vivo ces deux classes de LT CD8 mémoires. 2/ Caractériser la population de LT CD8 mémoires innés. Dans cette étude, nous démontrons qu'au sein du pool de LT CD8 mémoires, seules les cellules conventionnelles expriment la chimiokine CCL5 et le récepteur NKG2D. Ces deux biomarqueurs permettent ainsi pour la première fois de distinguer les LT CD8 mémoires innés et conventionnels in vivo, à la fois chez la souris et chez l'homme. Grâce à l'expression de NKG2D, nous démontrons que ces LT CD8 mémoires innés possèdent des caractéristiques typiques de cellules mémoires, notamment une réactivité augmentée ainsi qu'un programme génétique comparable à celui des LT CD8 mémoires conventionnels. Néanmoins, cette population cellulaire conserve certaines caractéristiques de cellules naïves. Ainsi, le répertoire TCR diversifié de cette population cellulaire permet à ces cellules de participer à des réponses immunitaires primaires contre différents pathogènes. Enfin, dans un contexte inflammatoire, les LT CD8 mémoires innés présentent un défaut d'accès au tissu pulmonaire comparé aux LT CD8 mémoires conventionnels. Ceci corrèle avec un déficit d'expression de certaines intégrines par les LT CD8 mémoires innés. L'ensemble de nos résultats démontre que les LT CD8 mémoires innés, caractérisés par l'absence d'expression de CCL5 et NKG2D, constituent une population cellulaire hybride, à la frontière entre cellules naïves et cellules mémoires conventionnelles / The pool of memory CD8 T cells is composed of two major cell classes. On one hand, conventional memory CD8 T cells are generated consequently to the specific recognition of pathogen or tumor derived antigens. On the other hand, innate memory CD8 T cells are generated through several mechanisms involving strong yc cytokine stimulation in the absence of cognate antigen recognition. However, these cell classes harbor a very similar phenotype. As a consequence, innate memory CD8 T cell population remains poorly characterized. This PhD has two main objectives : 1 / Identify new biomarkers that enable the discrimination between memory CD8 T cell classes 2/ Characterize the population of innate memory CD8 T cells in physiological condition Our results show that among the pool of memory CD8 T cells, only the conventional ones express the chemokine CCL5 and the NK receptor NKG2D. These two biomarkers enable for the first time the discrimination of memory CD8 T cell classes in physiological settings, in both mouse and human. Thanks to these new tools, we show that innate memory CD8 T cells hold typical memory features, such as an increased reactivity compared to naïve cells and a genetic program similar to the one of conventional memory cells. Nevertheless, this cell population also retains some features typical of naïve cells. The diversified TCR repertoire of this cell population allows it to participate to primary immune responses against various intracellular pathogens. Moreover, like naïve cells, innate memory CD8 T cells fail to access peripheral tissues upon local inflammation, which correlate with an absence of expression of some integrins. Altogether, these results demonstrate that innate memory CD8 T cells, characterized by the absence of expression of CCL5 and NKG2D, represent a hybrid cell population, at the boundary between naïve cells and conventional memory cells

Le clade des invertébrés cristallise en 2017 de grandes problématiques sociétales à la fois économiques et sanitaires. En effet un certain nombre des organismes présent dans ce groupe phylétique sont des vecteurs des grandes pandémies infectieuses telles que le paludisme (Anopheles sp), Zika, Chinkungunya, Fièvre jaune, etc (Aedes sp), Chagas (Triatoma sp) ou encore la bilharziose (Biomphalaria sp, Bulinus sp). La compréhension du système immunitaire de ces organismes vecteurs doit aider la communauté scientifique à proposer des solutions pour réduire la transmission de toutes ces maladies sur le terrain. Biomphalaria glabrata est le vecteur unique de la Bilharziose intestinale (Schistosomamansoni) en Amérique Latine. Depuis un premier cas de résistance induite par une première infection en 1998, de nombreux travaux ont exploré la réponse immunitaire mémoire innée de cet escargot tropical d’eau douce. Dans le cadre de ce travail de thèse, différents aspects de cette immunité (également appelé priming, résistance acquise) ont été explorés, de la mise en place phénotypiques, aux bases moléculaires et cellulaires. En premier lieu,nous avons pu démontrer qu’elle était dépendante d’une bascule phénotypique (d’une réponse cellulaire d’encapsulation à une réponse humorale) et transcriptomique qui lui permet de mieux répondre lors d’une seconde infection. La spécificité de cette réponse est portée par la production de répertoire complexe de récepteurs et d’effecteurs immunitaire spécifiques qui sont capables de différencier jusqu’aux différents stades de développement parasitaire d’une même espèce de parasite. Nous avons également pu montrer que cette interaction dépendait de microARN circulants ainsi que de Biomphalysines, des ß-PFT acquises par transferts horizontaux depuis le monde bactérien. Enfin, cette résistance semble posséder une proximité avec l’immunité mémoire entraînée des cellules immunitaires innées des vertébrés en particulier sur la base des mécanismes moléculaires sous-jacent qui seraient liés chez Biomphalaria comme chez les Vertébrés à unereprogrammation épigénétique des cellules du système immunitaire innée. / Invertebrates focus in 2017 among the major economical and societal issuesacross Earth. Some members are vectors of important infectious pandemic as malaria(Anopheles sp), Zika, Chinkungunya, Yellow fever, etc (Aedes sp), Chagas (Triatoma sp) andtrematodes (Biomphalaria sp, Bulinus sp). Comprehension of immune system of thesevectors has to help scientist to decrease transmission on endemic area. Biomphalariaexposed first failure to be reinfected following first infection as soon as 1998. In my thesiswe explore this immune priming (innate immune memory) and describe an immune shiftfrom cellular to humoral immune response both in phenotype and transcriptomic response.A specificity is handle by specific immune receptor and effector repertoire to distinguish upto different developmental stage of same parasite species. This interaction is alsodependent of mRNAs and Biomphalysin, a ß-PFT coming from bacterial kingdom. Finally,this resistance seems to look alike the trained immune memory of innate cells in vertebrates.

Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

In the last decade, the potential of β-glucan, a fungal cell wall component, to induce epigenetic and functional modification of innate immune cells, signified as trained innate immunity (TII) has been demonstrated in several pre-clinical and clinical studies. Parenteral administration of β-glucan has resulted in centrally induced TII in the bone marrow/circulating monocytes. Such trained innate immune cells play a critical role in protection against secondary infections. However, there are now indications that inducing local long-lasting immunity at mucosal barrier tissues such as the lung is warranted for protective immunity against respiratory pathogens. Currently, it remains unclear whether respiratory mucosal administration of β-glucan will induce long-lasting resident-memory macrophages and TII and if so, what are the underlying mechanisms of development and maintenance of memory macrophages at respiratory mucosa. To address this, and kinetics of immune responses in the lung were studied. Profound changes in airway macrophage (AM) pools were observed starting from 3 days post-exposure, which was associated with monocyte recruitment, and this was followed by a series of phenotypic shifts in AMs. The altered AM phenotype profile persisted for up to 8 weeks post-exposure. Importantly, β-glucan-trained AMs demonstrated heightened MHC II expression, enhanced responses to secondary stimulation and improved capacity to perform bacterial phagocytosis. Furthermore, mice with, β-glucan-trained AMs displayed higher rates of survival and improved bacterial control, in the lung and periphery, following a lethal S. pneumoniae infection. Our findings together indicate that a single intranasal delivery of β-glucan is able to train AMs. Further work into epigenetics, metabolism, and the contribution of AMs in protection is needed. / Thesis / Master of Health Sciences (MSc) / The immune system has been classically divided into two major compartments known as the innate and adaptive immune system. For decades, the predominant consensus amongst the field was that only the adaptive immune system can form memory against any pathogens encountered. It has been well established that plants and invertebrates only possess an innate immune system and still show boosted responses and enhanced protection against previously encountered as well as new pathogens. Recently, such capacity for innate immune memory has also been demonstrated in humans and pre-clinical animal models. Innate immune memory provides non-specific, broad- spectrum protection whereas adaptive memory is specific to a singular pathogen. Inducing broad-spectrum protection can be crucial for the future of human medicine. Activation of both adaptive and innate immune arms could prove to be extremely beneficial in vaccination strategies. Through the use of a pre-clinical model, we have found that administering β-glucan, a component of fungal cell wall, directly into the lung significantly alters the phenotype and functionality of lung immune cells, and also provides enhanced protection against a heterologous infection.

Mon travail de Thèse a porté sur la description de l’immunité antibactérienne de la planaire, et plus particulièrement la mémoire immunitaire innée.La mémoire immunitaire innée constitue une ligne de défense de l’hôte à la réinfection qui ne fait intervenir que des composants de l’immunité innée. Présente chez les vertébrés et les invertébrés, ces derniers constituent un modèle de choix car dépourvus d’immunité acquise. La planaire dispose d’une mémoire immunitaire innée envers S. aureus, qui, suite à une réinfection, se traduit par une élimination exacerbée. La déplétion des planaires en cellules souches et la greffe tissulaire ont permis de mettre en avant les cellules souches comme acteurs principaux de cette réponse immunitaire. Un criblage RNAi associé à un profilage transcriptomique ont fait ressortir des gènes en les hiérarchisant au sein d’une voie de signalisation impliquant un récepteur au peptidoglycane (pgrp-2), une histone méthyltransférase (setd8.1), et un mécanisme effecteur dans l’élimination bactérienne (p38 et morn2). Setd8.1, histone méthyltransférase, se placerait au cœur du processus en déposant des marques épi-génétiques sur des loci de l’ADN, garantissant l’expression accrue des gènes effecteurs suite à la réinfection. Ce mécanisme, décrit chez l’Homme, n’avait jusqu’alors jamais impliqué des cellules souches, ni ce type d’histone méthyltransférase comme acteurs dans la mémoire immunitaire innée.Collectivement, l’investigation du système immunitaire de la planaire a permis la découverte de mécanismes de défense antibactérienne inédits, dont le transfert à l’Homme pourrait compléter l’approche actuelle du traitement des maladies infectieuses. / My Thesis work has focused on the description of the planarian antibacterial immunity, and more precisely the innate immune memory.The innate immune memory forms a host defense line to the reinfection which only involves components from innate immunity. Present in vertebrates and invertebrates, invertebrates are a model of choice because devoid of acquired immunity. The planarian has an innate immune memory against S. aureus, which, after a reinfection, displays an exacerbated elimination. The depletion of stem cells from planarians and tissue graft highlighted stem cells as the main actors of this immune response. An RNAi screening combined with a transcriptomic profiling brought out genes and classified them within a signaling pathway involving a peptido-glycan receptor (pgrp-2), a histone methyltransferase (setd8.1), and an effector mechanism of the bacterial elimination (p38 and morn2). Setd8.1, histone methyltransferase, would be the core of the process putting epigenetic marks on DNA loci, ensuring the increased expression of effector genes after reinfection. This mechanism, described in humans, has neither involved stem cells, nor this type of histone methyltransferase as actors in the innate immune memory.Collectively, the investigation of the planarian immune system allowed the discovery of new antibacterial defense mechanisms, and transferring it to humans could complete the actual approach of the infectious disease treatment.

Il est maintenant connu que de nombreux invertébrés peuvent moduler leur réponse immunitaire en fonction de leur expérience immunologique. Ce phénomène est appelé priming immunitaire. Si les mécanismes du priming immunitaire restent encore assez méconnus, il a pour conséquence d’apporter un bénéfice aux individus lors d’une seconde rencontre avec un agent pathogène, via une élévation de leur immunocompétence. Une caractéristique assez étonnante du priming immunitaire est qu’il peut se manifester chez la descendance. Ce transfert trans-générationnel d’immunité (TTGI), ainsi que le priming immunitaire, doivent avoir évolués à la suite de challenges répétitifs par les mêmes agents pathogènes durant la vie des individus et au fil des générations. Ainsi, le priming et le TTGI doivent être plus efficaces et moins coûteux vis à vis des parasites exposant l’hôte à la plus grande probabilité de réinfection. De plus, il est maintenant prouvé que la réponse immunitaire chez les insectes est génétiquement variable. Pour comprendre l’évolution du TTGI et de son potentiel de réponse à la sélection, il convient d’étudier la composante génétique de sa variabilité. Au cours de cette thèse, j’ai associé l’expression du priming et du TTGI chez un insecte à un type de bactéries, qui a du agir comme la principale pression de sélection sur le système immunitaire de cette espèce hôte. Cela s’est fait via l’identification de différents coûts et bénéfices, qui ont également mis en exergue certains mécanismes possibles dans la réalisation de ces phénomènes immunitaires. Pour ce faire, j’ai utilisé comme organisme modèle le ténébrion meunier, Tenebrio molitor.Dans le premier chapitre, nous avons étudié la survie d’individus adultes de T. molitor face à une infection bactérienne, en fonction de leur propre expérience immunitaire ou de celle de leur mère. Nous avons constaté que le priming et le TTGI étaient plus efficaces et moins coûteux vis à vis des bactéries à Gram-positif. Cetté étude a également révélé que, contrairement à ce que de précédentes recherches suggérent, les hémocytes ne jouent pas nécessairement un rôle majeur dans le priming immunitaire et le TTGI.Dans le deuxième chapitre, nous avons stimulé le système immunitaire de femelles adultes de T. molitor avec deux bactéries Gram-positives. Nous avons mis en évidence que la protection transmise aux oeufs pouvait résulter d’un transfert maternel de peptides antibactérien, ou que ces peptides pouvaient être produits par l’œuf lui-même, en fonction de la bactérie utilisé pour stimuler la mère. Il s’avère que quel que soit le mécanisme, le TTGI améliore le taux d’éclosion des œufs et peut même s’accompagner d’un bénéfice en survie pour les jeunes larves.Dans le troisième chapitre, nous avons stimulé le système immunitaire de femelles de lignées consanguines afin de quantifier la variation génétique de l'investissement maternel à la protection des œufs et mesuré d’autres traits associés à la valeur sélective des mères et de la descendance. Malheureusement, du fait d’un nombre trop faible de lignées et d’individus utilisés au sein de nos lignées, il nous a été impossible de conclure quant à l’existence de bases génétiques associées au TTGI.Dans le quatrième chapitre, nous avons passé en revue l’ensemble des études concernant le TTGI. Cela nous a permis de mettre en exergue les principales caractéristiques et les mécanismes identifiés, en fonction de l’écologie et de l’évolution du phénomène.Les bénéfices et les coûts associés au priming ainsi qu’au TTGI suggèrent que les bactéries à Gram-positif ont été la principale pression de sélection ayant contraint l’évolution du système immunitaire de T. molitor. En ce qui concerne le TTGI, de plus amples recherches sont nécessaires afin de trancher quant à l’existence de bases génétiques associées au phénomène. / Many organisms can improve their immune response as a function of their immunological experience, a phenomenon called immune priming. While the mechanisms through which immune priming is achieved remain unknown, individuals that survived to a given parasite are better protected against subsequent exposures. This immune priming can cross generations (trans-generational immune priming – TGIP), preparing offspring for prevailing parasite environment. Both individual and trans-generational immune priming might be adaptive and may have evolved from repeated challenges by the same pathogens during the host lifetime or across generation. While protection could be cross-reactive, a certain level of specificity may exist in response to the range of pathogens from which immue priming may have evolved. Thus, immune priming and TGIP should be more efficient and less costly with respect to pathogens exposing the host to the greatest probability of re-infection. Moreover, it is now known that insect immune response is genetically variable. To understand the evolution of TGIP and its impact on life history evolution, we need to explore its quantitative genetics. During my thesis, I found that the expression of individual immune priming and TGIP in the mealworm beetle, Tenebrio molitor, is dependent of a range of pathogens that might have been a major selective pressure on the immune system of this insect species. This was done through the characterisation of costs and benefits of the expression of immune priming in response to challenges with a large range of bacterial pathogens. This work also highlighted potential mechanisms through which these immune phenomena could be achieved.In a first chapter of this thesis, we examined the survival of individuals to infection with different bacteria according to their own immunological experience or that of their mother with these bacteria. We found that priming response to Gram-positive bacteria was particularly more efficient and less costly than priming response to Gram-negative bacteria. This study also shows that, contrary to what is currently believed, the cellular component of the T. molitor immune system does not necessarly play a major role in providing immune protection through individual immune priming or TGIP.In a second chapter, we have stimulated the immune system of adult females with two Gram-positive bacteria to study maternal transfer of immunity to the eggs. We found that the process throght which eggs are protected is dependent on the bacterial pathogen used to immune challenge the mother. Indeed, depending of the bacterial pathogen that immune challenged the mother, antibacterial activity in the eggs are either transfeered by the mother or produced by the egg itself, Furthermore, whatever the mechanism through which egg protection was achieved, primed eggs exhibited enhanced hatching rate and the resulting larvae even showed improved early survival to food privation.In a third chapter, we used inbred lines of T. molitor to study the quantitative genetics of TGIP. The aim of this work was to test whether TGIP could be heritable and whether its expression is genetically associated to other fintness traits of mothers and offspring. Unfortunately, due to a low number of inbred lines available and a low number of samples within some of these lines, it was impossible to conclude about the genetic basis associated to TGIP.In a fourth chapter, we produced a review on TGIP. This allowed us to highlight the main characteristics and mechanisms curently identified, and the ecology and the evolution of the phenomenon.Costs and benefits associated to immune priming and TGIP suggest that Gram-positive bacteria might have been a major selective pressure at the origin of these phenomena in T. molitor. Whether TGIP has genetic basis still required further research.

Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 JoaoFRN_DISSERT.pdf: 3516278 bytes, checksum: 9fe0338a2547517c2f45d91b7860116c (MD5) Previous issue date: 2010-12-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Visceral leishmaniasis (VL) in Brazil is a disease caused by Leishmania infantum chagasi (L.i.chagasi). The clinical evolution post-infection depends on the vertebrate host immune response, which is genetically mediated. This study aimed to evaluate the immune response of individuals living in endemic area for VL in the state of the Rio Grande do Norte, considering individuals with VL under treatment (n = 9), recovered VL <1 year post treatment (n = 10), > 10 years posttreatment (n = 9), uninfected individuals living in endemic areas (n = 7), individuals that lost DTH response (n=6) and asymptomatic individuals for VL (n=9). Peripheral blood cells were evaluated in the presence and absence of soluble Leishmania antigens (SLA) and ex vivo, to determine activation, presence of regulatory cells and memory cells. The Leishmania parasitemia and anti-Leishmania antibodies were determined respectively by qPCR and ELISA. Cells from individuals with VL under treatment showed less cell activation after stimulation with SLA for the markers CD4/CD69, CD8/CD69 and CD8/CD25 compared with VL post treatment treatment (p <0.001). Apparently uninfected individuals have a higher cell activation than symptomatic VL (p <0.001), with the exception of CD8/CD25 marker (p = 0.6662). On the other hand, in the ex-vivo group, significant differences were observed for CD4/CD69, CD8/CD69 and CD8/CD25 between the 4 groups due to increased cell activation present in cells of individuals symptomatic LV (p <0.001). VL cells under treatment, ex vivo, have a lower percentage of memory cells (CD4/CD45RO and CD8/CD45RO) than individuals VL post-treatment or control group (p = <0.01). Likewise, individuals with symptomatic VL have fewer regulatory cells when stimulated by SLA [CD4/CD25 (p = 0.0022) and CD4/FOXP3 (p = 0.0016)] and in the ex-vivo group (p = 0.0017). Finally, DNA isolated from recovered VL contained Leishmania DNA, supporting the hypothesis of non-sterile clinical cure for Leishmania infection. Recovered VL, even 10 years after treatment have high levels of memory cells, which may be due to the presence of stimulation, either by reexposure to Leishmania or non-sterile cure / A Leishmaniose visceral (LV) nas Am?ricas ? uma doen?a causada pela esp?cie Leishmania infantum chagasi (L.i.chagasi). A forma cl?nica evolutiva p?s-infec??o depende da resposta imune do hospedeiro vertebrado, que ? geneticamente mediada. Este estudo teve como objetivo avaliar a resposta imune de indiv?duos residentes em ?rea end?mica para LV no estado do Rio Grande do Norte, considerando indiv?duos com LV em tratamento (n=9), indiv?duos curados de LV < 1 ano (n=10) e > 10 anos p?s-tratamento (n=9), indiv?duos residentes em ?reas end?micas (n=7) aparentemente n?o infectados, indiv?duos que perderam a resposta DTH (n=6) e indiv?duos assintom?ticos para LV (n=9). C?lulas de sangue perif?rico foram avaliadas em presen?a e na aus?ncia de ant?genos sol?veis de Leishmania (SLA) e ex-vivo, para determina??o da ativa??o, da presen?a de c?lulas regulat?rias e de c?lulas de mem?ria. A parasitemia e anticorpo anti-Leishmania foram determinadas, respectivamente, por qPCR e ELISA. C?lulas oriundas de indiv?duos com LV em tratamento apresentaram menor ativa??o celular p?s-est?mulo com SLA para os marcadores CD4/CD69, CD8/CD69 e para CD8/CD25 quando comparado com LV p?s-tratamento (p<0,001). Indiv?duos aparentemente n?o infectados apresentam maior ativa??o celular que LV sintom?tico (p<0.001), com exce??o do marcador CD8/CD25 (p=0,6662). Por outro lado, na condi??o ex-vivo, diferen?as significativas foram observadas para CD4/CD69, CD8/CD69 e CD8/CD25 devido a uma maior ativa??o celular presente em c?lulas de indiv?duos LV sintom?ticos (p<0,001). Indiv?duos LV sintom?ticos, ex vivo, apresentam um menor percentual de c?lulas de mem?ria (CD4/CD45RO CD8/CD45RO) do que indiv?duos com LV p?stratamento ou controles (p=<0.01). Da mesma forma, indiv?duos com LV sintom?ticos apresentam uma menor quantidade de c?lulas regulat?rias quando estimuladas por SLA [CD4/CD25 (p= 0,0022) e CD4/FOXP3 (p= 0,0016)] e na condi??o ex-vivo (p=0.0017). Finalmente, pacientes com LV clinicamente recuperados permaneceram com parasitemia, determinado por qPCR, dando suporte ? hip?tese de cura cl?nica n?o est?ril para infec??o por Leishmania. Pacientes com LV recuperado, mesmo 10 anos p?s-tratamento mant?m n?veis elevados de c?lulas de mem?ria, que pode ser devido ? presen?a de ant?genos de Leishmania, seja devido ? re-exposi??o ? Leishmania ou por uma prov?vel cura n?o est?ril