The present study is an investigation of the basis of the enhanced antilisterial resistance exhibited by splenectomized mice. The response of the mononuclear phagocyte system of these hosts has been extensively investigated as a result of the finding that enhanced antilisterial resistance in these hosts is abolished by treatment with silica. Splenectomized mice demonstrate a quantitatively greater influx of immature macrophage precursors in response to listerial infection and intraperitoneal stimuli. Despite the higher responsiveness of immature macrophage precursors in the splenectomized host, radiation studies have determined that antilisterial resistance in splenectomized mice is provided by the mature mononuclear phagocytes of the liver, the Kupffer cells. However, Kupffer cells of splenectomized mice exhibit the same level of function associated with listericidal activity as non-splenectomized animals. An examination of the cellular composition of the spleen at early time intervals following infection with Listeria has led to the conclusion that enhanced antilisterial resistance in splenectomized mice may be explained on the basis of the loss of a storage function of the spleen. In the absence of the spleen, leucocytes are made available for migration to the liver upon infection of the host. The early destruction of Listeria which presumably results, is responsible for the lower growth of the organism measured at 48 hours post-infection.

The immunobiology and the control of experimental visceral leishmaniasis were studied in susceptible (C57BL/6J) and resistant (C57L/J) strains of mice. It was found that the resistant/susceptibility phenotype for infection with L. donovani is expressed only by liver macrophage in vitro; the resistant/susceptible phenotypes were transfered reciprocally by bone marrow radiation chimeras. It was also found that the phagocytic activity of macrophages is reduced by the infection and that liver and peritoneal macrophages reflect their specific resistance/susceptibility phenotype following protective or curative treatment with lymphokines: macrophages from resistant C57L/J mice responded better to lymphokine activation. The production of IL-1 by spleen and peritoneal macrophages is inhibited by the infection. Immunosuppression with cyclosporin A (CsA) exacerbated the infection, without affecting phenotype; both CsA-treated strains of mice heal the infection in the absence of IL-1 and IL-2. There were more infected liver macrophages in normal or CsA-treated susceptible C57BL/6J mice and a greater number of amastigotes per cell than in normal or CsA-treated resistant C57L/J mice. CsA treatment did not affect the responsiveness of macrophages to lymphokine activation. IL-2-treated spleen and blood leucocytes from infected animals reduced infection in macrophages infected in vitro. Adoptive immunotherapy in vivo with IL-2-treated spleen cells from infected animals showed significant specific reduction of the parasite load in the liver; daily injections of IL-2 enhanced cure. T lymphocytes are the cells involved in cure; cure is mediated by soluble factors produced by the treated cells, and is specific to Leishmania infection.

Control of protein synthesis in eukaryotic cells was studied by using two models: the c-myc oncogene and the translational transactivation of the Human Immunodeficiency Virus (HIV) mRNAs. These two models are examples of control of expression of specific genes at the translation level. It was shown that the presence of the 5$ sp prime$ non-coding regions of the human and murine c-myc oncogenes strongly inhibits the in vitro translation of their respective mRNAs. The presence of the 5$ sp prime$ non-coding region of the mouse c-myc gene is sufficient by itself to reduce the translational efficiencies of messages since, when it is placed at the 5$ sp prime$ end of coding sequences, it also reduces the translational level of these messages to similar levels observed with c-myc. Secondary structure at the level of the 5$ sp prime$ end non-coding region might be the origin of the observed inhibition since this has been shown to be a determinant in translational efficiency. There was however no correlation between the results obtained from in vitro translation systems and observations made in cells transfected with genes containing the inhibitory region at their 5$ sp prime$ end. The presence of the HIV mRNAs TAR sequence at the 5$ sp prime$ end of the hybrid CAT message also reduces the in vitro translational efficiency of this mRNA. No transactivational activity in the presence of the tat III protein could be observed in the in vitro translation systems that were used in these studies. However, the cross-linking of an 80 kDa polypeptide (most likely eIF-4B) to the mRNA cap structure was enhanced by the presence of the TAR region at the 5$ sp prime$ end of hybrid messages. Moreover, cross-linking of a novel set of proteins to the cap structure of hybrid mRNA with the TAR sequence at the 5$ sp prime$ end was detected in HeLa cells transfected with the tat III gene and expressing the transactivation activity in vivo. These results suggest that the c-myc oncogene and the

A mouse model of surgically induced renal failure was utilized to investigate the pathogenesis of Staphylococcus epidermidis peritonitis which is a frequent and serious complication of continuous ambulatory peritoneal dialysis (CAPD). Compared to sham-operated controls, chronically uremic mice were more susceptible to intraperitoneal S. epidermidis inoculation, presenting decreased survival time and survival (10$ sp9$ cfu, 10$ sp8$ cfu), delayed bacterial clearance and attenuated peritoneal inflammatory response (10$ sp6$ cfu). In mice bearing a peritoneal catheter implant, the catheter was a preferred site for peritoneal bacterial persistence up to one month after intracatheter inoculation. Despite in vitro cytotoxicity of commercial peritoneal dialysis solutions toward peritoneal leucocytes, repeated peritoneal instillation of dialysis solutions did not influence S. epidermidis recoveries following inoculation. Although the mouse preparation did not undergo peritoneal dialysis, these studies nevertheless demonstrate that chronic uremia and the peritoneal catheter may be important etiological factors in the development and persistence of CAPD peritonitis.

The primary muscle of respiration is the diaphragm. Diaphragm muscle dysfunction and ventilatory pump failure are well documented phenomena in animal models of sepsis. However, the primary cellular mechanisms underlying respiratory muscle dysfunction in sepsis are poorly understood. In addition, most investigations of respiratory muscle dysfunction in sepsis have been performed in models involving high doses of bacterial endotoxin and these investigations have been criticized on the basis of questionable relevance to human sepsis. Therefore, the objective in the first study of this thesis was to study respiratory muscle dysfunction in a more clinically relevant animal model, namely, the Pseudomonas aeruginosa pulmonary infection model. Remote inflammatory processes in different diseases, such as cancer, arthritis, sepsis, and cystic fibrosis are known to contribute to muscle wasting and weakness through more widespread systemic effects. In keeping with the above notion, we hypothesized that sustained P. aeruginosa lung infection would cause diaphragmatic and limb muscle weakness. In this thesis, we demonstrate for the first time that persistent pulmonary infection with P. aeruginosa induces significant dose- and time-dependent contractile dysfunction of the diaphragm. By comparison, prototypical slow- and fast-twitch hindlimb muscles were not influenced by pulmonary P. aeruginosa infection. / Because skeletal muscles can express a variety of immune modulating molecules such as cytokines, chemokines, adhesion molecules, and major histocompatibility molecules, the objective of the second study in this thesis was to study the possible role of pro-inflammatory cytokines in diaphragm muscle dysfunction in our animal model. Our results indicate for the first time that intra-diaphragmatic pro-inflammatory cytokine gene expression (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-18) is highly up-regulated in infected animals and the magnitude of such upregulation is dependent upon the dose of P. aeruginosa lung infection. Parallel to the absence of muscle contractile dysfunction in hindlimb muscle under the same conditions, P. aeruginosa infection did not alter the levels of pro-inflammatory gene expression within the hindlimb muscle. To further address the involvement of muscle-derived pro-inflammatory cytokines in diaphragmatic contractile dysfunction, we have employed recombinant adenovirus (Ad) as a vehicle for systemic delivery of the anti-inflammatory cytokine IL-10, in order to shift the balance between pro- and anti-inflammatory cytokines within the diaphragm toward a more anti-inflammatory profile. We report here that systemic delivery of Ad-IL-10 suppresses pro-inflammatory gene expression and improves force generating capacity of the diaphragm in P. aeruginosa infected animals. This finding emphasizes the role of anti-inflammatory cytokines as beneficial immune modulators in respiratory muscle failure caused by pro-inflammatory cytokines. / P. aeruginosa lung infection is a major cause of morbidity and mortality among cystic fibrosis (CF) patients and many patients with CF have weak peripheral and respiratory muscles. Although the role of pro-inflammatory cytokines has been extensively studied within the lungs of CF patients, the involvement of these cytokines in skeletal muscle dysfunction in animal models of CF or in human CF patients has not been studied. Therefore, in the third study of this thesis we have used mice sharing the same genetic defect as CF patients (Cftr knockout mice), in combination with our model of P. aeruginosa lung infection, to address several fundamental questions related to muscle function in CF. Our first objective in this portion of the thesis was to determine if diaphragmatic skeletal muscle cells express the CFTR mRNA. Our second objective was to ascertain whether intrinsic differences between CF and wild-type muscle cells could be detected in vitro, which might differentially affect the regulation of pro-inflammatory mediators in the setting of infection/inflammation. Our third objective was to evaluate possible differences in the ability of respiratory muscles to generate force prior to and after P. aeruginosa lung infection in Cftr knockout mice, as compared to their wild-type littermates. Finally, we aimed to determine if the absence of CFTR expression would predispose to muscle dysfunction triggered by up-regulation of intra-diaphragmatic pro-inflammatory gene expression. Our major results indicate that: First, in vitro stimulation with pro-inflammatory cytokines (TNF-alpha, IL-1alpha, and IFN-gamma) and LPS (extracted from Pseudomonas aeruginosa) triggered increased expression of pro-inflammatory mediators (iNOS, RANTES, MIP-1alpha, MIP-1beta, MIP-2 and KC) in both Cftr -/- and wild-type diaphragmatic myotubes, but the magnitude of cytokine/chemokine upregulation was significantly greater in CF than in wild-type diaphragm muscle cells. Sec / In the final study of this thesis, we sought to test the hypothesis that increased diaphragm muscle activation would lead to increased production of intra-diaphragmatic cytokine expression, since this could possibly explain the greater susceptibility of the diaphragm to express pro-inflammatory cytokines in response to pulmonary P. aeruginosa infection as compared with the hindlimb muscle. To test this hypothesis, we subjected rats to inspiratory resistive loading (IRL), corresponding to 45-50% of the maximum inspiratory pressure, and described that mRNA levels of IL-1beta, IL-6, and to a lesser extent, IL-4, IL-10, TNF-alpha, and IFN-gamma were all significantly increased in a time-dependent fashion in the diaphragm but not hindlimb muscle (gastrocnemius) of loaded animals. In addition, elevated protein levels of IL-1beta and IL-6 in response to loading were confirmed with immunoblotting and immunostaining. We also detected significant IL-6 protein to be localized inside diaphragmatic muscle fibers of loaded animals. We conclude that increased diaphragm muscle activity during resistive loading induces upregulation of pro-inflammatory cytokine gene expression in the diaphragm, which could also provide an explanation for the greater cytokine expression observed in the diaphragms of animals with P. aeruginosa lung infection.

Auto-immune diseases stem from an inability to eliminate immune cells that react to "self" peptides. In particular, when T lymphocytes react to self peptides, or become activated, they undergo elimination in a process of programmed cell death or apoptosis. In T lymphocytes one of the key effectors of apoptosis is a membrane protein, Fas ligand, since it engages Fas, the caspase cascade, and ultimately cell death. Therefore the biosynthesis of Fas ligand is tightly controlled to maintain immune homeostasis. HuR is an RNA binding protein that has been shown to have an important role in all aspects of T lymphocyte maturation and function. HuR is known to be important in the posttranscriptional regulation of many cytokines by stabilizing, relocalizing and promoting the translation of their mRNAs. The effects of HuR are mediated through binding to AU rich elements in the untranslated regions of these mRNAs. We find that HuR posttranscriptionally regulates Fas ligand through AU rich and U rich motifs in its mRNA and explore the contexts in which this regulation is important. / Les maladies auto-immunes découlent d'une incapacité d'éliminer les lymphocytes T qui reconnaissent l'hôte. En général, l'apoptose suit lors qu'un lymphocyte T devient activé ou répond aux peptides dérivés de l'hôte. Une des molécules principales qui induit l'apoptose dans les lymphocytes T est une protéine membranaire nommée Fas ligand qui se lie au récepteur Fas pour activer la cascade de caspases ce qui entraine la mort cellulaire. En conséquence, la production de Fas ligand est strictement contrôlée pour entretenir l'homéostase immunitaire. HuR est une protéine qui lie l'ARN qui est important pour les fonctions des lymphocytes T. HuR agit par des effets post-transcriptionels sur les ARNm de plusieurs cytokines par stabilisation, relocalisation et l'efficacité de traduction. HuR promeut ses fonctions en liant des régions riches en AU dans les parties non-traduites des ARNm. Nous démontrons que Fas ligand est régulé de façon post-transcriptionel par HuR via des régions AU riches et U riches, et nous examinons dans quels contextes cette régulation est importante.

The genus Mycobacterium comprises a variety of highly successful intracellular pathogens. Mycobacterium tuberculosis is the causative agent of tuberculosis (TB) in humans and currently infects one third of the world's population. Mycobacterium avium ssp. paratuberculosis is the established cause of Johne's disease in ruminants and is epidemiologically associated with Crohn's disease (CD) in humans. Both TB and CD are complex genetic diseases for which immunological pathways associated with disease susceptibility or resistance have been identified based on human genetic studies. Common polymorphisms in NOD2 have recently been described to predispose to CD. NOD2 encodes a receptor of the Nod-like receptor (NLR) family involved in mediating innate immunity upon recognition of fragments of bacterial peptidoglycan (PGN), a structural component of most bacterial cell wall. CD-associated NOD2 polymorphisms were shown to abrogate this response. While recent studies have uncovered an important role of NOD2 for the recognition of mycobacterial species, the consequences and significance of this recognition remain obscure. The first part of the work presented in this thesis investigates the consequences of NOD2-mediated recognition on innate responses, adaptive immunity and resistance to mycobacterial infection. Using Nod2-deficient mice as a model to study CD-associated NOD2 mutations in humans, we show that the NOD2 pathway is critical for both innate and acquired anti-mycobacterial immunity. Impaired mycobacterial recognition at early time points following infection altered the immunopathology in the lungs and resulted in decreased survival of Nod2-deficient mice when virulent M. tuberculosis was given by aerosol. The second part of this thesis focuses on the biochemical basis of mycobacterial recognition by NOD2. The bacterial N-acetylmuramic acid hydroxylase (NamH) enzyme introduces a specific modification in PGN. We correlated the presence of this enzyme in mycobacteri / Le genus Mycobacterium comprend une variété de bactéries pathogènes intracellulaires. Mycobacterium tuberculosis cause la tuberculose (TB) chez les humains et infecte présentement près d'un tier de la population mondiale. Mycobacterium avium ssp. paratuberculosis cause la maladie de Johne chez les ruminants et est associé à la maladie de Crohn chez les humains. La TB et la maladie de Crohn sont toutes deux des maladies génétiques complexes pour lesquelles des aspects clés de la réponse immunitaire associés à la résistance ou à la susceptibilité à la maladie ont émergés d'études génétiques humaines. Il a récemment été démontré que des polymorphismes communs dans le gène NOD2 prédisposaient à la maladie de Crohn. NOD2 encode un récepteur de la famille des Nod-like receptor (NLR) impliqué dans la réponse immunitaire innée lors de la détection de fragments de peptidoglycan (PGN) bactérien, une composante structurelle de la membrane de la plupart des bactéries. De plus, il a été démontré que les polymorphismes de NOD2 associés à la maladie de Crohn empêchaient cette réponse. Bien que certaines études récentes rapportent un rôle important de NOD2 pour la détection d'espèces mycobactériennes, les conséquences et les raisons associées à cette détection demeurent obscures. La première partie du travail présenté dans cette thèse explore les conséquences du rôle de NOD2 sur la réponse immunitaire innée, la réponse immunitaire adaptative et la résistance à une infection mycobacterienne. Des souris déficientes du gène Nod2 sont utilisées comme modèle d'étude des mutations de NOD2 associées à la maladie de Crohn's. À l'aide de ces souris, nous démontrons que la voie de signalisation NOD2 est critique pour la réponse immunitaire anti-mycobactérienne à la fois innée et adaptative. Un défaut de détection mycobactérienne dans les semaines suivant l'infection a mené à une altération de l'immunopat

Cystic fibrosis (CF) female patients have a worse prognosis compared to their male counterparts. CF patients infected with Pseudomonas aeruginosa have also been shown to have dysregulated cytokine profiles. Therefore, we studied the importance of sex and interleukin-10 in the susceptibility of C57BL/6 mice to pulmonary infection with P. aeruginosa. The results clearly demonstrate that both wildtype and interleukin-10 knockout (KO) female mice are more susceptible to P. aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs. / Several animal models of CF show most of the CF symptoms; however, only a few of these display the CF lung phenotype. The cystic fibrosis transmembrane conductance regulator (Cftr)-KO mice that we developed in collaboration with Drs. Tsui and Kent represent a unique model of spontaneously occurring lung disease. We studied the characteristics of this model and analyzed the differences between the lungs of wildtype and Cftr-KO mice by assessing their histopathological status, gene and protein expression and fatty acid profiles. / We recently developed a novel non-invasive method of lung infection. The studies described contain major improvements for lung infection techniques employing P. aeruginosa bacteria embedded in agar beads. This novel and less invasive technique is crucially important in studying the host response to bacterial infection using the Cftr-KO mouse model. / CF lung disease is also characterized by imbalanced lipid profiles. Interestingly, docosahexanoic acid (DHA) has been shown to have antiinflammatory properties and to reverse intestinal and pancreatic pathologies in a CF mouse model. We have therefore treated our Cftr-KO mice developing spontaneous lung disease with DHA and observed a reduction in lung inflammation in the CF-affected organs compared to the untreated Cftr-KO mice. / It has also been demonstrated that ceramide is crucially important for P. aeruginosa internalization. Fenretinide is a synthetic retinoid inducing the cellular level of ceramide. Using our Cftr-KO mouse model, we tested the effect of fenretinide treatment during the course of lung infection with P. aeruginosa. Interestingly, we observed major decrease in the bacterial burden of Cftr-KO mice that were treated with fenretinide.

Different individuals react differently to infection with similar pathogens and weakly pathogenic organisms can cause life-threatening infections in some, while highly virulent microbes may go undetected in others. The basis of these differences lies within the genetic makeup of each individual, which determine their response to infection. Unraveling the genetic determinants of susceptibility to infection brings a much clearer understanding of the pathogenesis of diseases and paves the way to potential prophylactic and therapeutic interventions urgently needed in the context of increasing antimicrobial resistance, globalization of infectious diseases, and emerging or re-emerging pathogens. / Salmonella spp are highly successful pathogens that have co-evolved with countless host species. Even today, they continue to threaten public health throughout the world. Their zoonotic nature, their propensity to establish long-term carrier states and the emergence of antimicrobial resistant, highly virulent strains greatly complicate the fight against this pathogen. As for other infectious diseases, the host response to Salmonella is genetically controlled. In order to genetically dissect this response, a mouse model was developed and allowed identification of a few genes having a strong impact on the outcome of Salmonella infection. The mouse response to Salmonella is, however, complex and several additional genetic variants influencing the response to infection remain to be identified. / Here, we present a series of experiments, which contribute to our understanding of the host response to acute Salmonella Typhimurium infection in mice. First, we investigated the impact of Tlr4 expression during Salmonella infection by comparing host responses in mice carrying 1, 2 and 3 copies of Tlr4 on the same genetic background. We show for the first time, in this narrow range of Tlr4 expression, an incremental protective effect against Salmonella due to improved control of bacterial growth and increased expression of important downstream immune genes. Second, using a set of reciprocal A/J and C57BL/6J recombinant congenic strains, we identified five novels QTL influencing the outcome of Salmonella Typhimurium infection in mice. Finally, we present evidence for the genetic basis for one of the newly identified QTL and describe a role for anemia and iron balance in the mouse response to Salmonella.

Two RNA trafficking cis sequences (A2RE-1 and A2RE-2) were identified in HIV-1 RNA. Their activity is assessed here during HIV-1 gene expression. Single point mutations in each A2RE reduced the levels of bound hnRNP A2, but also resulted in a marked accumulation of viral genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation in progeny virions, with the strongest phenotype observed for the A2RE-2 mutant. Immunofluorescence analyses revealed marked changes in viral gene expression patterns for pr55Gae and Vpr, as well as a significant reduction in Vpr incorporation levels. Viral infections were markedly compromised in the A2RE-2 mutant but this virus quickly reverted in culture. These data point to the importance of the HIV-1 A2RE determinants and the trans factor hnRNP A2 in the control HIV-1 gene expression patterns. This also provides further details on the implication of host factors in RNA trafficking during HIV-1 replication.