Les macrophages associés aux tumeurs (TAM) orchestrent l'inflammation nécessaire à la croissance tumorale (propriétés de type M1) et favorisent les métastases et l'angiogenèse (caractéristiques de type M2). Cependant, la nature des facteurs capables de conférer des propriétés M1 et M2 aux macrophages humains demeure inconnue. L'acide lactique (AL) est un métabolite produit par les cellules tumorales connu pour moduler les fonctions des cellules présentes dans le microenvironnement tumoral. Dans cette étude, nous avons analysé son impact sur la différenciation des monocytes humains. Les résultats montrent que l’AL induit la différentiation des monocytes (cultivées en présence de GM-CSF) en macrophages présentant un phénotype atypique (CD14high CD163high IL-10low IL-12low) et, de manière intéressante, des caractéristiques phénotypiques M1 (production de cytokines inflammatoires) et M2 (production de facteurs de croissance, expression de gènes prototypiques M2). Un profil similaire est obtenu lorsque les monocytes sont cultivés avec des cellules cancéreuses primaires glycolytiques. Ces effets de l'AL sur la polarisation des macrophages nécessitent l'entrée du lactate dans les cellules (via le transporteur MCT-1) et son oxydation en pyruvate et sont médiés par une stabilisation de HIF-1α et une consommation autocrine de M-CSF.Ces résultats (i) identifient l'AL comme un médiateur induisant la génération de macrophages humains présentant des caractéristiques M2 et des propriétés inflammatoires et (ii) renforcent l'intérêt de l’utilisation des inhibiteurs de la glycolyse aérobie pour moduler les fonctions des TAM. / In established tumors, tumor-associated macrophages (TAM) orchestrate unresolving cancer-related inflammation (M1-related properties) and favor tumor development, metastasis and angiogenesis (M2-like properties). However, to date, the nature of the polarization factor(s) able to confer M1 and M2 functional properties to human macrophages remains unknown.Lactic acid (LA), a metabolite produced at high levels in most established tumors, can impact the phenotype and functions of cells present in the tumor microenvironment. In this study, we analyzed the impact of LA on the human monocyte differentiation. Results showed that LA skews monocytes (differentiated in the presence of GM-CSF) into macrophages (GM+LA-Mφ) exhibiting an atypical CD14high CD163high IL-10low IL-12low phenotype. Interestingly they harbor M1 and M2 phenotypic features, as assessed the production of a wide variety of inflammatory and growth factors and the expression of prototypic M2-like genes. A similar profile is induced by culturing monocytes with glycolytic human primary cancer cells. These effects of LA on macrophage polarization require the entry of lactate into the cells (via the monocarboxylate transporter 1) and its oxidation into pyruvate and are mediated via HIF-1α stabilization and autocrine M-CSF consumption by differentiating cells. These results identify tumor-derived LA as a missing link reconciling the M2-like features of TAM with their inflammatory properties. They also reinforce the interest of aerobic glycolysis inhibitors to modulate the functions of TAM.

No description available.

Interferon (IFN)τ is the luteotrophic signal in ruminants and is secreted by bovine blastocysts both in vivo and in vitro. IFNτ secretion is highly variable and its control is only partly understood. Most studies on the effects of environmental factors on IFNτ production have evaluated IFNτ production during the time of embryo elongation and attachment. There is less knowledge of how IFNτ production at the blastocyst stage is modulated. Therefore, the hypothesis of this thesis was that the amounts of IFNτ expressed and/or secreted by bovine blastocysts produced in vitro were modulated by environmental factors. In the first set of experiments, bovine embryos were incubated with a cytokine (granulocyte macrophage colony stimulating factor, GM-CSF). GM-CSF had been shown previously to promote embryo viability in a range of species and to modulate IFNτ secretion by ovine blastocysts and thus was classified as a beneficial environmental factor. Three experiments were conducted to test whether GM-CSF stimulated bovine blastocyst development and IFNτ secretion. Embryos were incubated with a range of different concentrations of GM-CSF (2, 5, 10 and 50 ng mL-1) and at different stages of development (1 to 3 and 1 to 9 days post-insemination). Bovine embryos were unresponsive to GM-CSF in terms of IFNτ secretion, pyruvate oxidation, rate of development, blastocyst yield, morphological quality and apoptotic index, irrespective of timing of exposure and/or concentration of GM-CSF. In the second part of the thesis, bovine blastocysts were exposed to a mild heat treatment (42°C for four h) to determine whether heat stress affected IFNτ expression by bovine blastocysts. A novel multiplex reverse-transcription polymerase chain reaction methodology was validated to detect IFNτ and heat shock protein (HSP)70 mRNA in individual bovine embryos relative to an endogenous gene (YWHAZ) and an exogenous mRNA (α-globin) and results were expressed both in absolute terms and in relation to the endogenous control. Heat treatment upregulated IFNτ mRNA expression, suggesting that detrimental environmental factors may influence IFNτ expression. Heat treatment also caused an increase in HSP70 mRNA expression but did not affect blastocyst morphology, suggesting that the level of stress caused by the heat treatment was great enough to activate the cellular stress response, but mild enough not to cause a change in morphology. In addition, the positive correlation between HSP70 and IFNτ transcript levels and the higher IFNτ expression by embryos which showed signs of degeneration and collapse compared to those which progressed in development suggested that IFNτ expression may be indicative of stress. The relationship between IFNτ expression and secretion in vitro with morphology, pyruvate metabolism, apoptotic index and cell number was inconsistent, suggesting that IFNτ production did not correlate with ‘quality’ (defined as an index of viability). Blastocyst yield, day of blastulation and change in morphology index did account for at least part of the variation in IFNτ production, suggesting that some intrinsic factors may regulate IFNτ secretion. These intrinsic factors, however, did not explain all the variation in IFNτ secretion between blastocysts. Therefore, the amount of IFNτ secreted by bovine blastocysts is modulated by both intrinsic and environmental factors. A model was proposed where different levels of stress affect survivability to different extents, and the ability to respond to mild levels of stress may be indicative of improved survivability.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

<p>In this thesis, two different vaccines were evaluated as adjuvant therapy for patients with colorectal cancer. The ability of the two candidate vaccines to generate antigen-specific cellular and humoral responses, respectively, was studied. The effectiveness of granulocyte colony stimulating factor (GM-CSF) as a cytokine adjuvant to augment the immune response was also examined.</p><p>The first vaccination strategy involved immunization with the recombinant tumor-associated protein, carcinoembryonic antigen (CEA). Recombinant CEA was administered at 4 different dose levels 7 times during one year. Peripheral blood samples were regularly analyzed during 36 months. This vaccination regimen induced a strong immunoglobulin 1 (IgG1) and IgG4 response, a moderate IgG2 response and a weak IgG3 response against CEA. GM-CSF markedly augmented the effect on IgG1 and IgG4 as well as the T cell response. In contrast, dose of rCEA had no or modest effect on induced immune responses. The response gradually increased during the 12 months immunization period. Responses of all three IgG subclasses and of T cells were protracted up to 36 months. The anti-CEA IgG titers related significantly to survival. Functional HLA-DR epitopes of CEA could be defined. These major histocompatibility class II epitopes may serve as putative components of a peptide-based vaccination strategy. </p><p>The other vaccine strategy consisted of the tumor-associated antigen epithelial cell adhesion molecule (Ep-Cam) expressed as a transgene in a viral vector, ALVAC. Patients were immunized subcutaneously/intradermally 3 times over 6 weeks and monitored for immune responses for 46 weeks. No anti-Ep-Cam specific humoral response was induced, but Ep-Cam specific type 1 T cells (interpheron-gamma production) were induced, mainly in the GM-CSF group. The cytotoxic cellular response appeared late, or a few months after the last immunization.</p><p>Both vaccines were well tolerated. Since GM-CSF was an important component for both regimens, immungenicity of this cytokine was assessed. Multiple immunizations with low dose GM-CSF were associated with a low incidence of GM-CSF antibodies that did not neutralize the biological effect of GM-CSF. </p><p>In conclusion, both vaccines are promising candidate vaccines. GM-CSF is necessary to induce a strong humoral and cellular immune response. Large clinical trials are urgently warranted to evaluate the clinical efficacy.</p>

Macrophages (monocytes/microglia) could play a critical role in central nervous system repair. We have previously found a synchronism between the regression of spontaneous axonal regeneration and the deactivation of macrophages 34 wk after a compression-injury of rat spinal cord. To explore whether reactivation of endogenous macrophages might be beneficial for spinal cord repair, we have studied the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) in the same paraplegia model and in cell cultures. There is significant, though transient, improvement of locomotor recovery after a single delayed intraperitoneal injection of 2g GM-CSF. This improvement is associated with an increased expression of 5HT at the level of the CPG (T13-L2). At longer survival delays, axonal regeneration is significantly enhanced in GM-CSF-treated rats. We then studied the effects of GM-CSF on brain-derived neurotrophic factor (BDNF)secretion by macrophages/microglia, inflammatory reaction and phagocytosis by macrophages/microglia. In vivo, at short post-treatment delays, we found that GM-CSF increases significantly the expression of Cr3 and BDNF by macrophages at the lesion site. In vitro, BV2 microglial cells expressed higher levels of BDNF in the presence of GM-CSF and neurons cocultured with microglial cells activated by GM-CSF generated more neurites, an effect blocked by a BDNF antibody. In vivo, we showed that GM-CSF treatment (either immediate or delayed) does not increase IL-6 expression by macrophages/microglia or astrocytes. We showed that a delayed GM-CSF treatment down regulates IL-1 expression by astrocytes. In vivo, we showed that a delayed GM-CSF treatment can decrease MAG expression at the lesion site. These experiments suggest that GM-CSF could be an interesting treatment option for spinal cord injury and that its beneficial effects might be mediated by BDNF.

In this thesis, two different vaccines were evaluated as adjuvant therapy for patients with colorectal cancer. The ability of the two candidate vaccines to generate antigen-specific cellular and humoral responses, respectively, was studied. The effectiveness of granulocyte colony stimulating factor (GM-CSF) as a cytokine adjuvant to augment the immune response was also examined. The first vaccination strategy involved immunization with the recombinant tumor-associated protein, carcinoembryonic antigen (CEA). Recombinant CEA was administered at 4 different dose levels 7 times during one year. Peripheral blood samples were regularly analyzed during 36 months. This vaccination regimen induced a strong immunoglobulin 1 (IgG1) and IgG4 response, a moderate IgG2 response and a weak IgG3 response against CEA. GM-CSF markedly augmented the effect on IgG1 and IgG4 as well as the T cell response. In contrast, dose of rCEA had no or modest effect on induced immune responses. The response gradually increased during the 12 months immunization period. Responses of all three IgG subclasses and of T cells were protracted up to 36 months. The anti-CEA IgG titers related significantly to survival. Functional HLA-DR epitopes of CEA could be defined. These major histocompatibility class II epitopes may serve as putative components of a peptide-based vaccination strategy. The other vaccine strategy consisted of the tumor-associated antigen epithelial cell adhesion molecule (Ep-Cam) expressed as a transgene in a viral vector, ALVAC. Patients were immunized subcutaneously/intradermally 3 times over 6 weeks and monitored for immune responses for 46 weeks. No anti-Ep-Cam specific humoral response was induced, but Ep-Cam specific type 1 T cells (interpheron-gamma production) were induced, mainly in the GM-CSF group. The cytotoxic cellular response appeared late, or a few months after the last immunization. Both vaccines were well tolerated. Since GM-CSF was an important component for both regimens, immungenicity of this cytokine was assessed. Multiple immunizations with low dose GM-CSF were associated with a low incidence of GM-CSF antibodies that did not neutralize the biological effect of GM-CSF. In conclusion, both vaccines are promising candidate vaccines. GM-CSF is necessary to induce a strong humoral and cellular immune response. Large clinical trials are urgently warranted to evaluate the clinical efficacy.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Dendritic cells (DCs) function mainly as antigen presenting cells (APCs) and as such they play a significant role in activating the adaptive immune system. Dendritic cells express toll-like receptors (TLR), and when these receptors are engaged by their cognate agonists, they promote DC maturation, which is critical in the induction of potent T helper (Th) cell -1 responses. Due to the multifunctional abilities of DCs, they have been explored as vaccine carriers, largely in cancer immunotherapy and some infectious diseases including hepatitis C. Previous studies showed that DCs loaded with mRNA of hepatitis C virus (HCV) antigen(s) induced strong immune responses but immune protection was not complete. Therefore, I expected that adoptive transfer of DCs transfected with HCV NS3/4A and/or NS5A mRNA and further treated with TLR agonist(s) ex vivo would induce HCV-specific immunity in vivo. Bone marrow-derived DCs generated with Flt3L (FL-DCs) or GM-CSF (GM-DCs), and loaded with HCV NS3/4A and/or NS5A mRNA showed maturation characteristics and produced substantial amounts of IL-12 after ex vivo activation with CpG ODN or CpG ODN plus Poly I:C, when compared to their untreated counterparts. Treatment with a combination of CpG ODN and Poly I:C synergized to augment IL-12 production in comparison with stimulation with CpG ODN alone. IL-12 secretion by DCs is pivotal in directing immune responses towards a Th1-bias response, which is needed to eliminate HCV. However, the ex vivo responses of stimulated DCs bearing HCV antigen(s) were not efficiently translated in mice to potentiate vigorous antigen-specific T cell responses. This resulted in a lack of protection after challenge with recombinant vaccinia virus expressing HCV NS3/NS4/NS5 in immunized mice. In contrast, both antigen-specific humoral and cell-mediated immune responses were induced in mice vaccinated with HCV recombinant NS3 or NS5A protein co-formulated with CpG ODN, host defense peptide and polyphosphazene. These responses, however, did not mediate viral clearance, as vaccinated mice remained unprotected from infection with recombinant vaccinia virus expressing HCV antigens. Taken together, these results suggest HCV recombinant protein co-formulated with triple adjuvant to be a better vaccine candidate than the DC-based vaccine.