A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

Le macrophage joue un rôle clé dans l'athérogenèse, intervenant notamment, dans la captation de cholestérol à l'origine de la formation des stries lipidiques. Sa capacité à éliminer le cholestérol en excès est donc critique pour l'évolution de la pathologie. Il intervient aussi dans la captation des cellules apoptotiques présentes au sein de la lésion, processus déterminant en termes d'inflammation et de stabilité de la plaque. Mes travaux ont consisté à évaluer le rôle du transporteur ATP-Binding-Cassette G1 (ABCG1), dans ces mécanismes clés de l'athérogenèse. Ainsi, nous avons donc montré qu'ABCG1 est à la fois impliqué dans les processus d'efflux au sein des cellules spumeuses chargées en cholestérol libre mais aussi qu'il participe au mécanisme de phagocytose des corps apoptotiques. Ainsi, grâce à ces travaux ABCG1 pourrait être envisagé comme une nouvelle cible thérapeutique de l'athérosclérose.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.

A degradable/polar/hydrophobic/ionic (D-PHI) polyurethane with properties intended to promote tissue regeneration in a small diameter peripheral artery vascular graft was evaluated for cell biocompatibility and growth. Films were cast in polypropylene 96 well plates for monocyte/endothelial cell (EC) co-culture in vitro studies and porous scaffold discs were implanted in an in vivo subcutaneous mouse model. After 7 days in culture the co-culture demonstrated cell adhesion and growth, low esterase activity (a measure of degradative potential and cell activation), no detectable release of pro-inflammatory cytokine (tumour necrosis factor -α) but measurable anti-inflammatory interleukin (IL)-10. The EC and the co-culture expressed the EC biomarker CD31, whereas the monocyte monoculture did not. Cytokine array analysis of the in vivo characterization of D-PH supported an anti-inflammatory phenotype of cells at the site of the implant. Levels of IL-6 significantly decreased over time while IL-10 was significantly higher at 6 weeks post implant. TNF-α levels did not change significantly from 24 hours onwards, however the trend was towards lesser amounts following the initial time point. Histological analysis of the explanted scaffolds showed excellent tissue ingrowth and vascularization. A live/dead stain showed that the cells infiltrating the scaffolds were viable. Both the in vitro and in vivo results of this thesis indicate that D-PHI is a good candidate material for tissue engineering a peripheral artery vascular graft.

Elevated Thyroid-Stimulating Hormone (TSH) is associated with an increased risk of cardiovascular disease (CVD). We hypothesized that TSH-stimulated FA release from adipocytes contributes to macrophage inflammation. 3T3-L1 and human subcutaneous differentiated adipocytes were treated with TSH for 4 hours under various conditions and lipolysis assessed via glycerol secretion. Optimal conditions were determined and protein expression of ATGL, HSL and perilipin remained stable. TSH-stimulated 3T3-L1 or human adipocyte-conditioned medium (T-ACM) was placed on murine J774 or human THP-1 macrophages, respectively, and macrophage cytokine mRNA levels (IL-1β, IL-6, MCP-1, and TNFα) were measured by real-time RT-PCR. T-ACM did not change cytokine mRNA expression in J774 macrophages or THP-1 macrophages when compared to ACM. Absence of BSA in the medium may have hindered release of FA from differentiated adipocytes into the medium, BSA may be required to permit adequate FA accumulation in the medium to then evaluate the effect of T-ACM on macrophages. Further investigation is required to determine the effect of FA on J774 and THP-1 inflammatory response.

La malaria, maladie causée par le protozoaire Plasmodium, tue chaque année plus d'un million de personnes. L'émergence de parasites résistants à plusieurs traitements anti-malariques ainsi que l'absence de vaccin efficace rendent la lutte contre le Plasmodium de plus en plus difficile. Afin de pouvoir identifier de nouvelles cibles thérapeutiques et d'optimiser l'efficacité des vaccins dans les zones endémiques, une meilleure compréhension des interactions hôte/parasite est nécessaire. Lors de la malaria, un état d'immunosuppression systémique est induit par le parasite. Cet état de suppression immunitaire pourrait être à l'origine de la persistance du parasite au sein de l'hôte ainsi que de la difficulté à obtenir une protection efficace contre la réinfection décrite dans les zones endémiques. Mieux comprendre comment le Plasmodium interfère avec la réponse immunitaire permettra de mettre en place des thérapies favorisant la résolution des infections par ce parasite. Dans ce sens, notre étude a pour but d'évaluer l'activation des cellules T régulatrices naturelles (TregN) ainsi que la modulation de la physiologie des macrophages (MP) lors des infections par Plasmodium. Une étude antérieure a suggéré que l'activation des cellules TregN lors des infections par Plasmodium yoelii l7XL (PyL) était impliquée dans le développement de l'immunosuppression et permettait au parasite d'échapper au système immunitaire. En utilisant un modèle alternatif d'infection par Plasmodium, nous avons démontré que l'activation des cellules TregN pouvait aussi représenter un mécanisme protecteur pour l'hôte. Lors des infections par Plasmodium chabaudi adami (Pca) chez les souris BALB/c, l'activation des cellules TregN permet de tempérer la réponse immunitaire afin d'éviter le développement d'une inflammation excessive sans toutefois affecter le contrôle du parasite. Ainsi, il semblerait que le rôle des cellules TregN lors des infections par Plasmodium dépende de l'espèce du parasite considérée. Les infections par Plasmodium sont associées à une diminution de la sécrétion d'interleukine 12 (IL-12) et de la viabilité des MP ayant vraisemblablement des répercussions sur le développement de la pathologie et le contrôle du parasite. Notre étude a démontré que l'hémine (HE), produite suite à l'oxydation de l'hème provenant de l'hémoglobine (Hb), pourrait être impliquée dans ces deux phénomènes. Lors des infections par Plasmodium, les MP entrent en contact avec et internalisent des quantités importantes de ce composé ferrique via la phagocytose de globules rouges (GR) parasités ou abimés ainsi que via l'ingestion d'Hb et/ou d'HE libérées dans le sang suite à la lyse des érythrocytes. In vitro, des doses physiologiques d'HE diminuent de manière significative la quantité d'IL-12 sécrétée par les MP dérivés de la moelle osseuse en réponse à une stimulation avec l'interféron gamma (IFN-y) et le lipopolysaccharide (LPS). L'HE agit principalement sur l'effet synergique de l'IFN-y et ceci d'une manière dépendante de l'interleukine 10 (IL-10), de la MAPK (de l'anglais "mitogen activated protein kinase") p38 et du stress oxydatif. D'autre part, l'utilisation de GR de souris infectées par le Pca ou de GR oxydés, permettant de stimuler une forte érythrophagocytose in vitro, a permis de démontrer que l'internalisation d'un nombre important de GR induisait l'apoptose des MP de la lignée J774A.1. La phagocytose des GR conduit à l'internalisation d'une quantité importante d'HE induisant un stress oxydatif impliqué dans la diminution de la viabilité des MP. Bien que l'activation des cellules TregN et l'inhibition des MP à la suite de l'ingestion d'HE puissent représenter des mécanismes permettant au Plasmodium d'échapper au système immunitaire, ces mécanismes immunosuppresseurs pourraient aussi permettre de limiter l'inflammation et la pathologie favorisant ainsi la survie de l’hôte lors de la malaria. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Plasmodium, immunosuppression, macrophages, interleukine 12, érythrophagocytose.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.