<p>Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants.</p><p>In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values.</p><p>Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation.</p><p>Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.</p>

Les différents agents pathogènes ont développé de multiples stratégies pour contourner ou modifier les mécanismes de défense de l'hôte. La bactérie intracellulaire Brucella n'est pas une exception à la règle, car elle a développé des mécanismes qui lui permettent d'échapper à la surveillance immunitaire, persister pendant de longues périodes dans l'hôte et établir une infection chronique. En effet, Brucella responsable de la brucellose ou fièvre de Malte. La brucellose est une zoonose en réémergence ; dans cette maladie l'homme infecté représente une impasse épidémiologique. Il est indispensable de mieux connaître l'immunité développée contre Brucella qui est un excellent modèle d'étude d'autres maladies chroniques bactériennes. Le projet de thèse a été centré sur le développement et la modulation des réponses immunes par Brucella et les molécules bactériennes. Dans la première partie de cette thèse, nous avons analysé les réponses immunitaires innées et adaptatives lors de l'infection avec Brucella. D'une part, nous avons étudié l'influence des neutrophiles dans la réponse immunitaire lors de l'infection, en étudiant le cours de brucellose dans les modèles de souris neutropéniques. D'autre part, nous avons identifié et caractérisé in vivo une population des cellules CD4+ cytotoxiques au cours des infections avec Brucella. Dans la deuxième partie, nous nous sommes intéressés dans les modèles murins et humains aux propriétés immunomodulatrices des différentes molécules bactériennes telles que les glucans cycliques et des lipopolysaccharides (LPS). / Various successful pathogens have evolved multiple mechanisms to overcome or alter many normally very effective host defense mechanisms, including both innate and acquired immunity. The intracellular pathogen Brucella is not an exception to the rule as it displays mechanisms that allow it to evade immune surveillance and that are required to establish persistent infections in mammals. In this work, we studied the induction and modulation of immune responses by the intracellular bacteria Brucella and bacterial components. In the first part of this thesis, we have performed a systemic analysis of the innate and adaptive immune responses upon Brucella infection. On the one hand, we investigated the influence of polymorphonuclear neutrophils (PMNs) in the immune response during Brucella infection by exploring the course of brucellosis in antibody neutropenic mouse models. On the other hand, we identified and characterized in vivo a cytotoxic CD4+ T cell population upon Brucella abortus and Salmonella thyphimurium infections. In the second part, we focused on the immunomodulatory properties of various bacterial components such as cyclic glucans and lipopolysaccharides (LPS) both in mouse and human models.

La vigne est sujette à de nombreuses contraintes biotiques et abiotiques face auxquelles elle devra optimiser ses stratégies de défense, en favorisant parfois des interconnections entre les réponses adaptatives au stress abiotique et la gestion de la réponse immune face à un pathogène. Dans cette étude nous avons évalué l'effet du stress hydrique (par privation d'eau) sur différentes réactions adaptatives au stress, mais aussi sur des réponses de défense et sur la sensibilité des feuilles et des baies de la vigne à Botrytis cinerea. Ces réactions ont été suivies en utilisant des boutures végétatives de deux cépages : cv. Meski (MSK), tolérant à la sécheresse et cv. Chardonnay (CHR), sensible. La relation entre les réponses au stress hydrique et la réponse immune a également été recherchée au moyen de feuilles de vitroplants et de baies isolées de Chardonnay exposées à des osmotica : le polyéthylène glycol (PEG) et une forte concentration en saccahrose (SUC). Les résultats montrent que le stress hydrique/osmotique conduit à des modifications physiologiques et biochimiques importantes dans les feuilles et les baies mûres de vigne. L'amélioration de la tolérance chez MSK est associée à une faible inhibition de l'activité photosynthétique, une altération du profil des acides aminés et un catabolisme actif des polyamines (PAs) comparé au CHR. Ces résultats suggèrent un rôle potentiel des déviations métaboliques observées dans les processus de tolérance de la vigne au stress osmotique. La tolérance du MSK au déficit hydrique est également corrélée à une forte induction des réponses de défense accumulation de resvératrol et e-viniférine, expression de certains gènes de défense dont STS, Gluc (PR-2), Chit-4c (PR-3) et PR-5 dans les feuilles, ainsi qu'à une faible sensibilité à B. cinerea.Ces résultats suggèrent un lien étroit entre la tolérance au déficit hydrique et la capacité de la vigne à exprimer davantage ses mécanismes de défense et à résister mieux à B. cinerea. Des expériences pharmacologiques ont montré qu'en situation de stress hydrique/osmotique, le catabolisme des PAs, via des diamine- et PA-oxydases, est impliqué dans la régulation de l'homéostasie des PAs et de l'expression des réactions de défense. L'application du stress osmotique avant l'infection des feuilles par B. cinerea potentialise l'accumulation des PAs en réduisant fortement leur dégradation. Ces effets sont corrélés à une réduction de l'amplitude des réponses de défense après infection, et à une sensibilité accrue à B. cinerea. Ces résultats rendent compte (1) de l'importance des stress abiotiques dans la régulation de la réponse immune chez la vigne et sa résistance à B. cinerea et (2) du fait que le niveau des réponses de défense osmo-induites et la résistance au pathogène sont tributaires, au moins en partie, de certains mécanismes adaptatifs au stress, comme c'est le cas ici des voies de dégradation des polyamines. / Grapevine is often exposed to both biotic and abiotic stresses, and it will optimize defense strategies by favoing sometimes the cross-talk between adaptive responses to abiotic stress and immune response against pathogen challenge. In this study we evaluated the effect of water stress (by withholding water) on different adaptive responses, and also on defense responses and sensitivity of grapevine leaves or berries to Botrytis cinerea. These reactions were monitored using vegetative cuttings of two varieties: Meski (MSK) a drought tolerant cultivar and Chardonnay (CHR) as a sensitive one. The relationship between the responses to water stress and the immune response was also assessed using detached leaves from vitroplantlets and detached ripe berries from Chardonnay exposed to osmotic agents: polyethlene glycole (PEG) and a high concentration of sucrose (SUC). The results show that water/osmotic stress leads to significant physiological and biochemical changes in grapevine leaves and ripe berries. The improved tolerance of MSK to drought is associated with a weak inhibition of photosynthetic activity, altered amino acid profile and an activation of polyamine (PA) catabolism, compared to the sensitive plant CHR. These results suggest a potential role of metabolic deviations observed in the process of osmotic stress olerance. MSK tolerance to water deficit is also correlated with a strong induction of defense responses, such as accumulation of resveratrol and e-viniferin, enhanced expression of defense-related genes, including STS , Gluc (PR-2), Chit-4c (PR-3) and PR-5, and a low susceptibility of leaves to B. cinerea. These results suggest a close connection between water stress tolerance and the ability of grapevine to express more their defense mechanisms and then to resist better to the pathogen B. cinerea. Pharmacological experiments showed that experiencing water/osmotic stress, PA oxidation through diamine- and PA-oxidases is involved in the regulation of PA homeostasis and the expression of defense reactions in both leaves and berries. The application of osmotic stress before leaf infection by B. cinerea potentiates PA accumulation probably by reducing PA degradation. These effects are correlated with a reduction of defense responses after B. cinerea infection, as well as to an increased susceptibility to B. cinerea.These results highlight (1) the importance of abiotic stress in regulating the immune response in grapevine plants and resistance to B. cinerea and (2) that the level of defense responses induced by osmotic and the resistance of grapevine to the pathogen are dependent, at least in part, on some adaptive mechanisms to stress, as it is the case here for polyamine degradation pathways.

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants. In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values. Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation. Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

<p>The urinary tract is one of the most intractable mucosal surfaces for pathogens to colonize. In addition to the natural barriers at this site, potential pathogens have to contend with the vigorous local innate immune response that is initiated by engagement of surveillance molecule TLRs. TLR4 appears to be not only exclusively expressed on superficial BECs but also critical to triggering robust local innate immune responses. TLR4 recognizes Gram-negative bacterial component LPS and initiates a series of intracellular NF-kappaB associated signaling events resulting in a cytokine response. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by E. coli and isolated LPS, and observed that, in addition to the classical NF-kappaB associated pathway, BEC TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, AC3 generated cAMP, and the transcriptional factor CREB. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract. </p><p>Here, we also report two additional distinct TLR4-mediated defense mechanisms in BECs. First, BEC TLR4 inhibits bacterial invasion, a necessary step for successful infection. TLR4-mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Additionally, we found that BECs continue to fight UPEC even after bacterial invasion by triggering bacterial exocytosis through a distinct TLR4-mediated mechanism following activation by LPS. In addition, we reveal that Caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment and that they form a complex involved in the exocytosis of bacteria. The ability of TLR4 to mediate the rapid cytokine response, the inhibition of bacterial invasion, and the expulsion of intracellular bacteria from infected cells represents three previously unrecognized functions for this innate immune receptor.</p> / Dissertation

Orientador: Rosangela Zacarias Machado / Banca: Solange Maria Gennari / Banca: Aramis Augusto Pinto / Banca: Deise Aparecida de Oliveira Silva / Banca: Ana Patricia Yatsuda Natsui / Resumo: Neospora caninum é um protozoário do Filo Apicomplexa, que foi primeiramente descrito como causa de encefalomielite em filhotes caninos sorologicamente negativos para Toxoplasma gondii. Estudos anteriores neste importante hospedeiro da cadeia epidemiológica de N. caninum demonstram que as respostas de anticorpos IgG são tardiamente detectadas e que a infecção clínica é de difícil indução. Desta forma, este trabalho objetivou o estudo da imunidade de cães frente à infecção oral por N. caninum. Os resultados obtidos a partir de análises de diversos animais experimentalmente infectados indicam que os cães apresentam uma prolongada fase aguda da infecção, com eliminação de oocistos associado à queda nos níveis de linfócitos T CD4+ e CD8+ e diminuição de MHC de classe II por células apresentadoras de antígeno. Adicionalmente, os animais apresentam soroconversão instável durante o mesmo período, sendo que somente IgG1 e IgG3 foram detectados em adultos e filhotes, respectivamente, entre o 2o e 3o mês de infecção. De forma concomitante, observa-se uma predominância da expressão de citocinas imunomoduladoras como TGF 1, IL-4 e IL-10. Após dois meses de infecção, o perfil da resposta se inverte, sendo observado picos de produção dos marcadores CD4 e CD8 de linfócitos T e citocinas próinflamatórias como IFN , IL-6 e IL-12, além do aumento nos títulos de anticorpos, principalmente IgG1 e IgG4 nos cães jovens. Com base nestes resultados, conclui-se que os cães apresentam uma relação de equilíbrio com N. caninum, a qual induz nesta espécie uma modulação da resposta imunológica durante a fase de merogonia. / Abstract: Neospora caninum is an Apicomplexan parasite firstly described as the cause of encephalomyelitis in puppies serologically negative to Toxoplasma gondii. Previous reports on the parasite’s definitive host indicate a late IgG antibody response and that clinical disease is difficult to be induced. The aim of this study was to investigate canine immunity during N. caninum oral infection. The results obtained from the analysis of infected animal’s samples indicate that dogs present a protracted acute phase, with oocyst shedding correlated to a drop in CD4+ and CD8+ T cell levels, and low MHC class II expression by antigen presenting cells. Additionally, the dogs presented an unstable seroconversion pattern in the same period, with only IgG1 and IgG3 being detected in adult dogs and puppies, respectively, between the second and third months of infection. Concomitantly, dominant Th2 cytokine expression was observed, with peak expression levels of TGF 1, IL-4 and IL-10. After 2 months of infection, the immunity profile shifts towards a Th1 response, with high levels of CD4 and CD8 lymphocytary marker production and pro-inflammatory cytokine expression (IFN , IL-6 and IL-12), besides of the raise in antibody levels, especially IgG1 and IgG4 in puppies. Based in the results presented herein, we may conclude that dogs present a balanced host-parasite relationship, modulating the host immune response during N. caninum merogony. / Doutor

Les cellules dendritiques classiques (cDC) jouent un rôle crucial dans l’efficacité des réponses immunitaires précoces conduisant au contrôle ou à la persistance virale. A cet égard, il a été montré que l’infection par le VIH induit des dysfonctions des cDC caractérisées par une inhibition de leur capacité à stimuler les cellules T et associées à la progression de la maladie. Parmi les mécanismes moléculaires impliqués dans ces dysfonctions, des études in vitro ont mis en évidence le rôle du récepteur inhibiteur LILRB2. Néanmoins, la dynamique d’expression de LILRB2 ainsi que son rôle dès les premiers stades de l'infection restent à démontrer. Chez des patients en primo-infection VIH-1, nous observons une augmentation de l’expression de LILRB2 et de ses ligands HLA-I à la surface des cDC. Par ailleurs, la cinétique d’expression de LILRB2 et CMH-I au cours de l’infection de macaques cynomolgus par le SIVmac251 montre une augmentation transitoire de l'expression de LILRB2 et du CMH-I sur les cDC du sang et des ganglions lymphatiques dès les premiers jours de l’infection. Parmi les mécanismes qui pourraient être impliqués dans la régulation de l’expression de LILRB2, nos résultats indiquent que la réplication du VIH-1, l'activation de voies TLR7/8 ainsi que la présence d’IL-10 et d’IFN-I induisent une forte expression de LILRB2. Enfin, cette expression exacerbée de LILRB2 sur les cDC semble être spécifique à l'infection par le VIH/SIV. En effet, l’infection de macaques cynomolgus par le virus chikungunya, qui est caractérisée par une réponse immunitaire antivirale robuste aboutissant à un contrôle de la virémie, est associée à une expression diminuée de LILRB2 sur les cDC dès les premiers jours de l’infection. L’ensemble de nos données suggèrent un rôle majeur de l’axe inhibiteur LILRB2/MHC-I dans les mécanismes de dérégulations des cDC qui pourrait participerait à l’inefficacité des réponses immunitaires adaptatives et à la persistance du VIH/SIV. / Conventional dendritic cells (cDCs) play a crucial role in setting up early immune responses leading to viral control or persistence. In this regard, it has been shown that HIV-1 infection induces cDC dysfunctions characterized by inhibitions in their ability to stimulate T-cells and associated with disease progression. In vitro studies have shown the implication of LILRB2 inhibitory receptor in cDC dysfunctions. However, the dynamic of LILRB2 expression and its role in the early stages of infection are yet to be characterized. In primary HIV-1 infected patients, we observe an increased expression of LILRB2 and its ligands, HLA-I, on the surface of cDCs. Kinetics of LILRB2 and MHC-I expressions during SIV infection of Cynomolgus macaques shows a transient increase in LILRB2 and MHC-I expressions on blood and lymph node cDCs during the first days of infection. We also show that HIV replication, activation of TLR7/8 pathways, and presence of IL-10 and IFN-I drive upregulated expression of LILRB2. Finally, this strong induced LILIRB2 expression seems specific to HIV/SIV infections. Indeed, chikungunya virus infection of cynomolgus macaques, which characterized by a robust antiviral immune response leading to viral control, is associated with decreased expression of LILRB2 on cDCs in the first days of infection. Taken together, our data suggest a major role of the LILRB2/HLA-I inhibitory axis, mediating cDC dysfunctions and thus contributing to inefficient adaptive immune responses and viral persistence.

No description available.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a devastating disease in the channel catfish industry. Our research group has developed several E. ictaluri live attenuated vaccine (LAV) candidates (EiΔevpB, EiΔevpBΔfur, EiΔevpBΔhfq, EiΔevpBΔfurΔhfq), which were able to stimulate an immune response in vaccinated channel catfish and reduce ESC. However, innate, and adaptive immune responses in the lymphoid tissues of channel catfish to these LAVs are not known well. The overall goal of the project is to determine the role of adaptive and innate immune responses in catfish after vaccination with LAVs. Analysis of innate and adaptive immune-related gene expressions showed that the LAVs induced expression of adaptive immune-related genes in lymphoid tissues with less inflammation compared to wild type control. Also, the LAVs induced the expression of IgM in the sera of catfish.

Das Gen für die Expression von Helicobacter pylori Urease wurde in das Genom von Salmonella typhi Ty21a (Typhoral() integriert. Der resultierende Stamm erhielt die Bezeichnung Salmonella typhi Ty21a(pDB1). Neun gesunden Probanden wurde Salmonella typhi Ty21a(pDB1) verabreicht. In der Kontrollgruppe erhielten drei gesunde Probanden Salmonella typhi Ty21a. Schwerwiegende Nebenwirkungen wurden bei keinem der Probanden beobachtet. Zehn von 12 Probanden zeigten eine humorale Immunantwort gegen Antigene von Salmonella typhi, nachgewiesen über die Detektion spezifischer Antikörper produzierender Zellen, aber bei nur zwei Probanden ließ sich eine Serokonversion nachweisen. Eine zelluläre Immunantwort gegen das H-Antigen von Salmonella typhi konnte bei insgesamt fünf Probanden nachgewiesen werden. Insgesamt sechs Probanden zeigten eine zelluläre Immunantwort gegen Urease von Helicobacter pylori in zumindest einem von drei der zur Anwendung gebrachten Testsysteme (Proliferation, T-cell-Elispot, IFN-gamma Elisa). Eine humorale Immunantwort gegen Urease von Helicobacter pylori konnte bei keinem der Probanden nachgewiesen werden. Ty21a(pDB1) ist somit ein geeigneter Prototyp zur Optimierung einer Salmonella basierte Impfung zur Induktion einer zellulären Immunantwort, die zu einer protektiven Immunität gegenüber Helicobacter pylori führen könnte. / Helicobacter pylori urease was expressed in the common live typhoid vaccine Salmonella typhi Ty21a (Typhoral() yielding Salmonella typhi Ty21a(pDB1). Nine volunteers received Salmonella typhi Ty21a(pDB1) and three control volunteers received Salmonella typhi Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed cellular responses to the carrier. Six out of nine volunteers that had received Salmonella typhi Ty21a(pDB1) showed a T-cell response to Helicobacter urease in at least one of the three assays for detection of cellular response (Proliferation, T-cell-Elispot, IFN-gamma Elisa). No volunteer had detectable humoral responses to urease. Salmonella typhi Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.