The organic mercury compound ethylmercurithiosalicylate (thimerosal), an antiseptic and a preservative, has recently raised public health concern due to its presence in vaccines globally. Thimerosal dissociates in the body to thiosalicylate and ethyl mercury (EtHg), which is partly converted to inorganic mercuric mercury (Hg2+). The immunosuppressive, immunostimulatory, and de novo autoimmunogen effect of thimerosal in mice, as well as the accelerating/aggravating effect on spontaneous systemic autoimmunity including dose-response aspects were the subject of this thesis. Thimerosal perorally (590 μg Hg/kg body weight (bw)/day) to genetically susceptible (H-2s) mice caused immunosuppression during the first week with reduction of the total number of splenocytes, T- and B-cells. The suppression lasted 2 weeks for CD4+ cells, but was superseded by a strong immunostimulation/proliferation including T- as well as B-cells, and polyclonal B-cell activation (PBA). Antinuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin (AFA) appeared after 10 days, followed by renal mesangial and systemic vessel wall immune-complex (IC) deposits. The Lowest Observed Adverse Effect Level (LOAEL) was in the order AFA = glomerular and splenic vessel wall deposits < hyperimmunoglobulinemia < PBA. The LOAEL for AFA was 118 μg Hg/kg bw/day. The LOAEL for the different parameters of this thimerosal-induced systemic autoimmune condition (HgIA) was 3-11-fold higher compared with HgIA induced by HgCl2. The thimerosal-induced HgIA shared with HgCl2 a significant dose-response relationship, and requirement for: T-cells, the costimulatory factor CD28, the IFN-γ/IFN-γ-receptor pathway,but not IL-4. The mRNA expression in lymph nodes of IL-2, IFN-γ, IL-4, and IL-15 was significantly increased but not delayed compared with HgCl2. Treatment with the ubiquitous organic Hg compound methyl Hg using equimolar doses of Hg (533 μg Hg/kg bw/day) caused a transient immunosuppression, followed by a weak immunostimulation and AFA. The IgG AFA isotypes induced by the organic Hg compounds MeHg and EtHg were stable and dominated by a Th1-like pattern over a broad time- and dose range. Treatment with inorganic HgCl2 caused a dose- and time-dependent pattern of IgG AFA isotypes. Low doses favored a Th1-like pattern, a high dose a balanced or Th2-like pattern. Middle-range doses showed initially a Th1-like pattern which gradually evolved into a balanced or Th2-like pattern. The qualitative difference in IgG AFA isotypes between organic and inorganic Hg may be due to differences in activation and/or suppression of T-helper cell subsets or factors influencing the Th1/Th2-function. Speciation of the renal Hg2+ concentration and comparison with the threshold dose for induction of AFA by HgCl2 showed that even with the lowest doses of thimerosal and MeHg used in this thesis, the AFA response might from a dose threshold point of view have been caused by conversion of the organic Hg species to Hg2+. Primary treatment with inorganic Hg (HgCl2) accelerates/aggravates murine systemic autoimmunity, both spontaneous (genetic) and induced by other means. This capacity was assessed for thimerosal over a broad dose range using the (NZB X NZW)F1 hybrid mouse model. Significantly increased antinuclear antibodies (ANA) was seen after 4-7 weeks treatment (LOAEL 147 μg Hg/kg bw/day), and the response was dose-dependent up to 13 weeks. Renal mesangial and systemic vessel walls deposits similar to those in de novo HgIA were present after 7 weeks treatment. Twenty-two to 25 weeks treatment with thimerosal caused, in a dose-dependent fashion (LOAEL 295 μg Hg/kg bw/day), relocalization of the spontaneously developing glomerular IC deposits from the capillary vessel walls to the mesangium, which attenuated histological kidney damage and proteinuria, and increased survival. Thimerosal caused systemic vessel wall IC-deposits over a broad dose range: the Low Observed Adverse Effect Level (LOAEL) for renal and splenic vessel wall IC deposits was 18 and 9 μg Hg/kg bw/day, respectively. The No Observed Adverse Effect Level (NOAEL) could not be determined for the latter, since deposits were present even with the lowest dose used. Thimerosal causes in genetically susceptible mice an initial, transient immunosuppression which is superseded by a strong immunostimulation and systemic autoimmunity, sharing many characteristics with the HgIA induced by inorganic HgCl2. The IgG AFA isotype pattern is however qualitatively different, and the threshold dose substantially higher. In contrast, long-term treatment with thimerosal induces systemic vessel wall IC-deposits also using doses below those needed to induce HgIA de novo in H-2s mice.

We aimed to prospectively investigate the paternal antigen-induced cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to hormone treatment in women undergoing in vitro fertilisation (IVF) and to examine the predictive value of the cytokine secretion profile in the outcome of IVF treatment, in a pilot study. Twenty-five women were included and IVF treatment was successful for six and unsuccessful for 19 women. Blood samples were collected before IVF treatment, on four occasions during IVF and four weeks after embryo transfer. The numbers of Th1-, Th2- and Th17-associated cytokine-secreting cells and cytokine levels in cell supernatants were analysed by enzyme-linked immunospot-forming (ELISpot), enzyme-linked immune-sorbent (ELISA) or Luminex assay. None of the cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, TNF and GM-CSF) had any predictive value regarding IVF outcome. The majority of the cytokines reached their peak levels at ovum pick-up, suggesting an enhancing influence of the hormonal stimulation. Pregnancy was associated with a high number of IL-4-, IL-5- and IL-13-secreting cells four weeks after ET. In conclusion, the results do not support our hypothesis of a more pronounced peripheral Th1 and Th17 deviation towards paternal antigens in infertile women with an unsuccessful IVF outcome, although this is based on a small number of observations. A larger study is required to confirm this conclusion. Higher numbers of Th2-associated cytokine-secreting cells in pregnant women four weeks after ET do corroborate the hypothesis of a Th2 deviation during pregnancy.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disease that affects millions of people worldwide. Although the exact cause of MS is unknown, it is clear that CD4+ T helper cells play a significant role, namely T helper 1 (Th1) and T helper 17 (Th17) cells. The Notch family of proteins plays a role in the development and differentiation of T helper cells. Previous data has shown that inhibition of Notch impairs the ability of T helper cell differentiation. Additionally specific inhibition of certain Notch members inhibits specific T helper cell differentiation, for example the inhibition of Notch 1 inhibits Th1 and iTreg polarization [Samon et al., 2008]. However, the effects of the other Notch family members on CD4+ T cells are not fully studied. We propose that Notch 3 plays an extensive role in the regulation of Th1, Th2, Th17, and iTreg polarizations. In addition, we propose that Notch 3 regulates function of T helper cell function in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Data in this thesis show that Notch 3 plays a significant role in the polarization of Th1, Th17 and iTreg polarization [Karlsson et al., 2011]. We present evidence that the heterozygous and homozygous Notch 3 knockout exhibits a significant decrease in polarization toward Th1, Th17 and iTreg cell fates. Exopolysaccharide (EPS) is a compound that has been previously shown to play a protective role in other inflammatory diseases. EPS has been shown to produce anti-inflammatory macrophages. We propose that a similar anti-inflammatory effect might be possible in EAE. We found that EPS had a significant effect on EAE induction, decreasing the onset and peak disease score. EPS also reduced the concentration of IFN-γ, IL17A, and GM-CSF in the supernatants of the splenocytes after restimulation with MOG. Further experimental data is needed to prove the effects of EPS on EAE and the method by which EPS function. These data indicate that Notch 3 could be crucial in regards to EAE due to the effects on Th1 and Th17 which are instrumental in EAE induction [Raphael et al., 2015].

Over the approximate nine months of its intrauterine existence, the development of the fetus is supported by the human placenta. This transient organ is central to pregnancy success as it facilitates maternal-fetal exchange, immunological tolerance, and hormone production. Villous trophoblasts mediate placental formation by engaging in a continuous turnover process of proliferation, differentiation, fusion, and apoptosis. In doing so, cytotrophoblasts and syncytiotrophoblasts maintain the integrity of the outer placental lining known as the syncytium. Exposure to drugs, however, can compromise placental establishment, which can in turn adversely impact pregnancy and fetal health. Specifically, cannabis is widely used by women of reproductive age and during pregnancy. While maternal cannabis use is linked to poor outcomes such as preterm birth and neurodevelopmental delays in exposed children, the underlying mechanisms are not well-defined. First, we characterized a functionally relevant cell line to model differentiation and fusion. In a comparison of the BeWo and BeWo b30 cell lines, our findings demonstrated that both models similarly undergo fusion. We then explored the implications of exposure to delta-9- tetrahydrocannabinol (∆9-THC) on the immunological roles of villous trophoblasts. We observed that cytotrophoblast differentiation and fusion were associated with localized inflammation due to elevated interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) but inhibited interleukin-4 (IL-4) and interleukin-10 (IL-10) production. ∆9-THC exposure impaired this T helper 1/2 cytokine balance through decreased IL-2 and TNF-α as well as increased IL-4 and IL-10 levels. Subsequently, we investigated the effects of ∆9-THC in TNF-α- and IL-10-dominant environments, to represent inflammatory and immunomodulatory microenvironments, respectively. Coincident with inflammation, ∆9-THC attenuated trophoblast fusion and the biosynthesis of steroid hormones, progesterone and cortisol, through perturbed cytochrome P450 regulation. This thesis ultimately lays a foundation for understanding how cannabis use during pregnancy may compromise the fusogenic, immune and endocrine functions of villous trophoblasts in the placenta. / Thesis / Master of Science (MSc) / The human placenta is a pregnancy-specific organ that supports the health of the mother- to-be and fetus. Stem cells known as cytotrophoblasts undergo differentiation and fusion to support the establishment of the syncytium, which creates a boundary that separates the maternal and fetal circulations. In the case of cannabis consumption during pregnancy, its biologically active components can travel to the placenta, cross the syncytium, and enter fetal blood. Our primary objective was to determine how cannabis exposure can impact the formation and maintenance of the syncytium. While maternal use has been linked to short- and long-term consequences for child health, existing research lacks a complete understanding of the underlying mechanisms. We demonstrate that cannabis exposure alters the production of important immune and hormonal factors during cytotrophoblast fusion, which may play a role in mediating poor placental development. Ultimately, it is critical to explore the implications of cannabis use for female reproductive health due to a rising trend in its use.

Studies examining the immune response upon exposure to chitin microparticles in living models have reached drastically differing conclusions, and the reason remains unclear. One notable issue between the experiments is that they have not characterized their chitin preparations for degree of acetylation. They all use different chitin processing methods prior to administration, which could potentially be the source of the variance between studies. Chitin and chitosan preparations specified in the literature and several novel preparations were analyzed for degree acetylation (DA) using High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). It was found that autoclaving and sonication processing steps do not have a significant influence on degree of acetylation. Chitin and chitosan preparations were used to create a dose-response curve of DA compared to cytokine elicitation from THP-1 monocytes, and it was found that the initial response was dominated by TNF (similar to previous studies), though after 12 hours showed a tip toward the start of an IL-1β-dominated Th17 effector response. This study also confirmed that immunostimulatory effects can occur from chitin and chitosan particles at orparticles, which would have long residence times in air, might be implicated in initiating allergic or asthmatic processes.

We sought to evaluate the individual contributions of Notch1 and PKC-ζ to disease progression in a mouse model of immune-mediated bone marrow failure and to define a mechanism for their potential cellular cooperation. We transferred parental bulk splenocytes into F1-hybrid recipients to induce a robust immune-mediated bone marrow failure (BMF) that we could partially rescue by administering a pharmacological inhibitor of Notch activation. Transferring splenocytes from PKC--ζ-/- animals did not induce disease, and treating animals with a pharmacological inhibitor of PKC-ζ also provided full protection from disease. We found that inhibiting Notch1 resulted in PKC-ζ down-regulation, and blocking PKC-ζ reduced Notch1 activation, possibly within a positive feedback loop. Our data suggest that both Notch1 and PKC-ζ contribute to disease progression in our mouse model of immune-mediated bone marrow failure. Furthermore, additional findings from the lab demonstrated physical interactions between Notch1, members of the T cell signalosome and PKC-ζ that are essential to mediating full activation of T cells following signaling through the TCR and CD28. Notch1 and/or PKC-ζ may represent novel therapeutic targets in the treatment of bone marrow failure.

No description available.

No description available.

Les anticorps monoclonaux (AcM) ont été utilisés pour traiter des cancers dès le début des années 1980, en particulier lors du travail pionnier de l’équipe de Ronald Levy dans le traitement des lymphomes. Ces traitements ont pendant longtemps été considérés comme une sérothérapie passive à effet immédiat et à court terme. Cependant, au cours de ces dernières années, le concept d’un effet « vaccinal » des anticorps à usage thérapeutique en oncologie a peu à peu vu le jour du fait de réponses cliniques à long terme observées chez certains patients et de différentes études précliniques. En 2010, notre équipe a démontré que des souris immunocompétentes injectées avec les cellules tumorales EL4-huCD20 et traitées avec un AcM anti-huCD20 générait une réponse immunitaire anti-tumorale à long-terme par le biais de mécanismes dépendants de la région constante de l’anticorps et de lymphocytes T CD4+. Mon travail de thèse a donc porté sur l’analyse des mécanismes cellulaires et moléculaires par lesquels le traitement par un AcM anti-CD20 génère une immunité cellulaire adaptative anti-tumorale. J’ai ainsi pu montrer que le traitement des souris avec l’AcM anti-CD20 conduit à une expansion de lymphocytes Th1 producteurs d’IFN-γ, à l’apparition de lymphocytes T CD4+ effecteurs mémoires spécifiques des cellules tumorales CD20+, et au blocage de l’expansion de lymphocytes Tregs induite par les cellules tumorales. Le rôle central dans la protection anti-tumorale et la genèse d’une réponse adaptative anti-tumorale joué par l’axe IL-12/IFN-γ et leurs principales sources cellulaires, cellules dendritiques (DCs) et cellules NK, a été démontré par des expériences de neutralisation de ces cytokines, qui provoque une importante diminution du nombre de Th1 spléniques, de déplétion des cellules NK, ainsi que par des analyses phénotypiques qui ont permis d’identifier des DCs activées par le traitement - comme le montre l’expression accrue des molécules de classe II du CMH et de co-stimulation CD80 et CD86 - comme une importante source cellulaire de l’IL-12. Enfin, nous avons pu montrer qu’un variant de l’IL-2, liant préférentiellement le récepteur de l’IL-2By et faiblement le récepteur de l’IL-2aBy exprimé majoritairement par les Tregs, permettait l’obtention d’une protection anti-tumorale accrue d’animaux porteurs de tumeurs et traités par l’AcM anti-CD20. En conclusion, nous avons démontré qu’un contexte immunitaire pro-tumoral façonné par la présence d’une tumeur en développement peut être inversé par le traitement par un anticorps anti-tumoral, aboutissant à un contexte anti-tumoral. Qu’une telle réponse immunitaire adaptative cellulaire puisse être observée chez des patients atteints de lymphomes, traités par un anticorps anti-CD20, reste encore à être déterminé. / Monoclonal antibodies have been used to treat cancers since the early 1980s, in particular with the pioneer work of Ronald Levy for the treatment of lymphomas. Those treatments have been considered for a long time as a passive serotherapy with immediate and short term actions. Yet, recently, the idea of a vaccine effect of therapeutic antibodies in oncology have appeared, after preclinical studies and clinic observations suggesting a long term immune response in patients. In 2010, our team demonstrated that immunocompetent mice injected with EL4-huCD20 tumor cells and treated with anti-huCD20 monoclonal antibody generated a long term anti-tumor immune response linked with mechanisms dependent on constant part of antibodies and CD4+ T cells. My PhD work was based on the analysis of cellular and molecular mechanisms by which the treatment by an anti-CD20 mAb generates a cellar adaptive anti-tumor immunity. I could show that the treatment of mice with anti-CD20 antibody lead to the expansion of Th1 lymphocytes IFN-γ producers, to the apparition of effector memory CD4+ T cells specific for CD20 antigen, and to the blockade of the expansion of Treg cells induced by tumor cells. The key role of an adaptive anti-tumor immune response played by IL-12/IFN- γ and their main cellular sources, dendritic cells and NK cells, in the anti-tumor protection and genesis, has been demonstrated by experiments of cytokine neutralization, provoking an important decrease of splenic Th1 number, by NK depletion and by phenotypic analysis that allowed the identification of DCs activated by the treatment – as it is shown by the increased expression of MHC-II and CD80 and CD86 costimulation molecules, - as an important cellular source of IL-12. Finally, we could show that a variant of IL-2, binding preferentially IL-2By with a lower affinity for the IL-2aBy receptor mainly expressed by Tregs, could induce an increased anti-tumor protection of tumor-bearing animals treated with anti-CD20 mAb. In conclusion, we have demonstrated that a pro-tumor immune contexture affected by a growing tumor can be modified by an anti-tumor antibody leading to an anti-tumor contexture. That such cellular adaptive immune response could be observed in lymphoma patients treated with anti-CD20 still need to be determined.

Les anticorps monoclonaux (AcM) ont été utilisés pour traiter des cancers dès le début des années 1980, en particulier lors du travail pionnier de l’équipe de Ronald Levy dans le traitement des lymphomes. Ces traitements ont pendant longtemps été considérés comme une sérothérapie passive à effet immédiat et à court terme. Cependant, au cours de ces dernières années, le concept d’un effet « vaccinal » des anticorps à usage thérapeutique en oncologie a peu à peu vu le jour du fait de réponses cliniques à long terme observées chez certains patients et de différentes études précliniques. En 2010, notre équipe a démontré que des souris immunocompétentes injectées avec les cellules tumorales EL4-huCD20 et traitées avec un AcM anti-huCD20 générait une réponse immunitaire anti-tumorale à long-terme par le biais de mécanismes dépendants de la région constante de l’anticorps et de lymphocytes T CD4+. Mon travail de thèse a donc porté sur l’analyse des mécanismes cellulaires et moléculaires par lesquels le traitement par un AcM anti-CD20 génère une immunité cellulaire adaptative anti-tumorale. J’ai ainsi pu montrer que le traitement des souris avec l’AcM anti-CD20 conduit à une expansion de lymphocytes Th1 producteurs d’IFN-γ, à l’apparition de lymphocytes T CD4+ effecteurs mémoires spécifiques des cellules tumorales CD20+, et au blocage de l’expansion de lymphocytes Tregs induite par les cellules tumorales. Le rôle central dans la protection anti-tumorale et la genèse d’une réponse adaptative anti-tumorale joué par l’axe IL-12/IFN-γ et leurs principales sources cellulaires, cellules dendritiques (DCs) et cellules NK, a été démontré par des expériences de neutralisation de ces cytokines, qui provoque une importante diminution du nombre de Th1 spléniques, de déplétion des cellules NK, ainsi que par des analyses phénotypiques qui ont permis d’identifier des DCs activées par le traitement - comme le montre l’expression accrue des molécules de classe II du CMH et de co-stimulation CD80 et CD86 - comme une importante source cellulaire de l’IL-12. Enfin, nous avons pu montrer qu’un variant de l’IL-2, liant préférentiellement le récepteur de l’IL-2By et faiblement le récepteur de l’IL-2aBy exprimé majoritairement par les Tregs, permettait l’obtention d’une protection anti-tumorale accrue d’animaux porteurs de tumeurs et traités par l’AcM anti-CD20. En conclusion, nous avons démontré qu’un contexte immunitaire pro-tumoral façonné par la présence d’une tumeur en développement peut être inversé par le traitement par un anticorps anti-tumoral, aboutissant à un contexte anti-tumoral. Qu’une telle réponse immunitaire adaptative cellulaire puisse être observée chez des patients atteints de lymphomes, traités par un anticorps anti-CD20, reste encore à être déterminé. / Monoclonal antibodies have been used to treat cancers since the early 1980s, in particular with the pioneer work of Ronald Levy for the treatment of lymphomas. Those treatments have been considered for a long time as a passive serotherapy with immediate and short term actions. Yet, recently, the idea of a vaccine effect of therapeutic antibodies in oncology have appeared, after preclinical studies and clinic observations suggesting a long term immune response in patients. In 2010, our team demonstrated that immunocompetent mice injected with EL4-huCD20 tumor cells and treated with anti-huCD20 monoclonal antibody generated a long term anti-tumor immune response linked with mechanisms dependent on constant part of antibodies and CD4+ T cells. My PhD work was based on the analysis of cellular and molecular mechanisms by which the treatment by an anti-CD20 mAb generates a cellar adaptive anti-tumor immunity. I could show that the treatment of mice with anti-CD20 antibody lead to the expansion of Th1 lymphocytes IFN-γ producers, to the apparition of effector memory CD4+ T cells specific for CD20 antigen, and to the blockade of the expansion of Treg cells induced by tumor cells. The key role of an adaptive anti-tumor immune response played by IL-12/IFN- γ and their main cellular sources, dendritic cells and NK cells, in the anti-tumor protection and genesis, has been demonstrated by experiments of cytokine neutralization, provoking an important decrease of splenic Th1 number, by NK depletion and by phenotypic analysis that allowed the identification of DCs activated by the treatment – as it is shown by the increased expression of MHC-II and CD80 and CD86 costimulation molecules, - as an important cellular source of IL-12. Finally, we could show that a variant of IL-2, binding preferentially IL-2By with a lower affinity for the IL-2aBy receptor mainly expressed by Tregs, could induce an increased anti-tumor protection of tumor-bearing animals treated with anti-CD20 mAb. In conclusion, we have demonstrated that a pro-tumor immune contexture affected by a growing tumor can be modified by an anti-tumor antibody leading to an anti-tumor contexture. That such cellular adaptive immune response could be observed in lymphoma patients treated with anti-CD20 still need to be determined.