(english) Natural Killers - NK cells play an important role in immune surveilance and regulation either by direct cytotoxicity towards infected, transformed or otherwise damaged cells, or by production of cytokines and chemokines. The resulting response of NK cells is given by the sum of stimulating and inhibiting signals, tranduced by a wide array of receptors. Killer Ig-like receptors KIR2DL4 and LILRB1, which recognize self HLA-G molecules in pregnancy, as well as NKR-P1 receptors, which differ in the number of isotypes, are species-dependent and reduced during phylogenesis. NKG2D, reacting to stress-inducible proteins, and adenosine receptors (AR), which supress the inflamatory reaction, remain evolutionary conserved. The aim of this work was to study the involvement of NK cells and their receptors in several immune disorders and in various species, to provide new insights into their function and posisible immune modulation. We have shown here, that the choice of species in the study of NK cell effector functions may be crucial in some cases. The reaction to glycans, using synthetic GlcNAc-terminated glycomimetics GN8P, exerted opposing effects on NK cell function in humans and C57Bl/6 mice. In humans, the glycomimetic decreased cytotoxic activity of high NKR-P1A expressing NK cells, while in...

Chickens in the poultry industry are reared under intensive conditions where they are often exposed to opportunistic pathogens. Escherichia coli strain O78 is responsible for about half of the cases of avian colisepticemia. Potential therapeutic treatments have been proposed to inhibit the hydrolases that controls the endogenous levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG). 2-AG is the full agonist at the CB2 receptors of the endocannabinoid system expressed among leukocytes and it plays a role in mediating the activation of phagocytic macrophages. It is speculated that elevating 2- AG levels could increase macrophage cytokines and promote the recruitment of immune cells at the infected tissues. The purpose of this study was to investigate the immunomodulating effect of the 2-AG hydrolase inhibitor, JZL184 in chickens. The treatments could potentially up-regulate the innate immune responses in chickens during an E. coli infection by conveying a message from the endocannabinoid system to the immune system.

Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...

Natural products have been a significant source of new drugs, especially in treating cancer, infectious diseases, hypertension, and neurological disorders. Although many natural metabolites have been screened and yielded pharmaceutically important drugs, many potential sources of natural product drug therapies still need to be investigated, including lichens. Lichens are symbiotic systems consisting of a filamentous fungus and a photosynthetic partner (an eukaryotic alga and/or cyanobacterium). Lichens produce an impressive variety of unique secondary compounds and have been used as ingredients in folk medicines for centuries. Demonstrated biological roles based on lichen chemistry include: antibiotics, anti-proliferative, antioxidants, anti-HIV, anti-cancer, immunomodulation, and anti-protozoans. Although North America is home to an impressive variety of lichen species, there is limited research to examine the biological potentials of these lichens. The core goal of this dissertation research has been to investigate some of the biological roles including, antibiotic, antioxidant, and anticancer potentials using lichen crude extracts and their metabolites collected from various locations in the United States. Antibiotic screening of crude extracts of 36 lichen species demonstrated inhibitory effects against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Methicillin-resistant S. aureus (MRSA). Generally, acetone extractions were found to be more effective than methanol extractions. It has also been shown that L. vulpina extract was bacteriocidal against MRSA with a relatively slow kill rate that disrupts cell membrane integrity and cell division as possible modes of action. Antioxidant screening of extracts from 11 lichen species, using the Oxygen Radical Absorbance Capacity (ORAC) assay, showed that lichen extracts inhibited the oxidative degradation of the fluorescent molecule (fluorescein-sodium salt) by the oxygen free radical initiator AAPH (2,2'-azobis(2-aminidopropane) dihydrochloride Acetone extracts as well as pure compounds from lichen species showed cytotoxic effects against Burkitt's lymphoma (Raji) cells and a colon cancer cell line (HT29 and SW620). They decreased proliferation, arrested cell cycle at various stages and force the cell to undergo apoptosis. The tested extracts or pure compounds were not toxic to normal cells. In colon cancer apoptosis took place independent of casapase-3. The results of this dissertation showed that lichen compounds merits for further investigation.

In recent years, nanostructured materials incorporated with inorganic particles and polymers have attracted attention for simultaneous multifunctional biomedical applications. This thesis summarized three works, which are preparation of mesoporous silica coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles (NPs) as magnetic resonance imaging T2 contrast agents, polymer grafted Fe3O4@mSiO2 NPs response to temperature change, synthesis and biocompatibility evaluation of high aspect ratio (AR) gold nanorods. Monodisperse Fe3O4@mSiO2 NPs have been prepared through a sol-gel process. The coating thickness and particle sizes can be precisely controlled by varying the synthesis parameters. Impact of surface coatings on magnetometric and relaxometric properties of Fe3O4 NPs is studied. The efficiency of these contrast agents, evaluated by MR relaxivities ratio (r2/r1), is much higher than that of the commercial ones. This coating-thickness dependent relaxation behavior is explained due to the effects of mSiO2 coatings on water exclusion. Multifunctional core-shell composite NPs have been developed by growing thermo-sensitive poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAAm-co-AAm)) on Fe3O4@mSiO2 NPs through free radical polymerization. Their phase transition behavior is studied, and their lower critical solution temperature (LCST) can be subtly tuned from ca. 34 to ca. 42 °C, suitable for further in vivo applications. A seedless surfactant-mediated protocol has been applied for synthesis of high AR gold nanorods with the additive of HNO3. A growth mechanism based on the effect of nitrate ions on surfactant micelle elongation and Ostwald ripening process is proposed. The biocompatibility of high AR nanorods was evaluated on primary human monocyte derived dendritic cells (MDDCs). Their minor effects on viability and immune regulatory markers support further development for medical applications. / QC 20110701

Problématique: Chaque année, plus de 18 millions de personnes mondialement consomment de la cocaïne. De ces consommateurs, 16% développeront un trouble de l'usage de la cocaïne (TUC). Le TUC est associé à des problèmes physiques, sociaux et psychologiques, représentant un problème majeur de santé publique. Chez les individus ayant un TUC, la consommation excessive et répétée de cocaïne induit un stress oxydatif favorisant une réponse inflammatoire systémique et une neuroinflammation. Le cannabidiol (CBD) a gagné en intérêt pour ses propriétés anti-inflammatoires dans les études précliniques ainsi que pour son profil sécuritaire et tolérable. À notre connaissance, une seule étude clinique a évalué les effets du CBD sur trois marqueurs inflammatoires spécifiques. Les propriétés anti- inflammatoires du CBD doivent être confirmées chez l’humain, et notamment chez les personnes avec un trouble lié à l'usage de substances. Objectifs: L'objectif de cette étude est de déterminer si l'administration de CBD module les concentrations plasmatiques de divers marqueurs inflammatoires comparativement au placebo chez les personnes ayant un TUC. Méthodologie: Nous avons réalisé une analyse exploratoire des données provenant d’un essai clinique randomisé contrôlé unicentrique dont l’objectif primaire était de déterminer l’effet du CBD sur la rechute et les envies intenses de consommer. 78 participants âgés de 18 à 65 ans et ayant reçu un diagnostic de TUC ont été randomisés (1:1), de manière stratifiée pour le sexe et la sévérité de la dépendance à la cocaïne, au traitement CBD ou placebo. Les participants ont reçu 800 mg/jour de leur traitement attitré pendant 92 jours. Des échantillons sanguins ont été prélevés avant le début du traitement, puis au jour 8, aux semaines 4 et 12. Ces derniers ont été analysés par immunodosage multiplexé pour déterminer les concentrations de diverses cytokines et par cytométrie de flux pour déterminer les phénotypes cellulaires. Des comparaisons entre les groupes ont été effectuées en utilisant des estimations d’équation généralisées. Résultats: Les niveaux de facteur de croissance de l'endothélium vasculaire, d’interleukine-6, de monocyte intermédiaire et de cellule tueuse naturelle faiblement cytotoxique étaient significativement inférieurs chez les participants recevant du CBD que chez ceux recevant du placebo. Comparativement au groupe placebo, les lymphocytes CD25+CD4+T étaient augmentés dans le groupe CBD. Les niveaux de lymphocytes B étaient similaires dans les deux groupes. Conclusion: Le CBD démontre certaines propriétés anti-inflammatoires chez les personnes avec un TUC. D’autres études seront nécessaires pour répliquer les résultats et pour évaluer les bénéfices cliniques de la diminution des marqueurs inflammatoires par le CBD. / Issue: More than 18 million people worldwide use cocaine each year. Of these users, 16% will develop cocaine use disorder (CUD). CUD is associated with physical, social and psychological problems, representing a major public health problem. Repeated and excessive cocaine use induces oxidative stress leading to a systemic inflammatory response and neuroinflammation. Cannabidiol (CBD) has gained interest for its anti-inflammatory properties in preclinical studies as well as for its safe and tolerable profile. To our knowledge, only one clinical study has evaluated the effects of CBD on three specific inflammatory markers. The anti-inflammatory properties of CBD need to be confirmed in humans, particularly in people with substance use disorders. Objectives: The objective of this study is to determine whether CBD administration modulates plasma concentrations of various inflammatory markers compared to placebo in individuals with CUD. Methodology: We conducted an exploratory analysis of data from a single-center randomized controlled trial whose primary objective was to determine the effect of CBD on relapse and intense cravings. 78 participants aged 18 to 65 years with a CUD diagnosis were randomized (1:1) to CBD treatment or placebo. Randomization was stratified for gender and severity of cocaine dependence. Participants received 800 mg per day of their assigned treatment for 92 days. Blood samples were taken before the start of treatment and then at day 8, week 4 and week 12. These blood samples were analyzed by multiplexed immunoassay for concentrations of various cytokines and by flow cytometry for cellular phenotypes. Comparisons between groups were made using generalized estimating equations (GEE). Results: Levels of vascular endothelial growth factor, interleukin-6, intermediate monocyte, and low cytotoxic natural killer cell were significantly lower in participants receiving CBD than in those receiving placebo. Compared to the placebo group, CD25+CD4+T lymphocytes were increased in the CBD group. B-cell levels were similar in both groups. Conclusion: CBD demonstrates some anti-inflammatory properties in people with CUD. Further studies will be needed to replicate the results and to evaluate the clinical benefits of CBDmediated reduction of inflammatory markers.

Emerging studies revealed that the Aryl hydrocarbon receptor (AhR), a receptor sensing environmental contaminants, is executing an immunomodulatory function. However, it is an open question to which extent this is achieved by its role as a transcription factor or via non-genomic signaling. We utilized a multi-post-translational modification-omics approach to examine non-genomic AhR-signaling after activation with endogenous (FICZ) or exogenous (BaP) ligand in endotoxin-activated (LPS) monocyte-derived macrophages. While AhR activation affected abundances of few proteins, regulation of ubiquitination and phosphorylation were highly pronounced. Although the number and strength of effects depended on the applied AhR-ligand, both ligands increased ubiquitination of Rac1, which participates in PI3K/AKT-pathway-dependent macrophage activation, resulting in a pro-inflammatory phenotype. In contrast, cotreatment with ligand and LPS revealed a decreased AKT activity mediating an antiinflammatory effect. Thus, our data show an immunomodulatory effect of AhR activation through a Rac1ubiquitination-dependent mechanism that attenuated AKT-signaling, resulting in a mitigated inflammatory response.

<p dir="ltr">Immunotherapy has revolutionized the field of oncology. While a lot of antibodies and small molecule inhibitors have been developed for this, a lot of targets remain undruggable in humans.</p><p dir="ltr">Targeted protein degradation has opened a new horizon in drug discovery where we can target these undruggable proteins. Proteolysis targeting chimeras using the ubiquitin-proteasomal system is one of the most popular TPD strategies that complement lysosomal degradation strategies to degrade intracellular proteins, typically using bifunctional small molecule degraders. Recently, large biomolecular and antibody conjugates have been developed for degrading membrane and extracellular proteins in cells, such as lysosomal targeting chimeras (LYTACs) and genetically encoded LYTACS, among several others. However, larger molecules have limitations in penetrating solid tumors. This dissertation work focused on the development of bifunctional small molecule degraders for programmed death-ligand 1 (PD-L1), a transmembrane protein ligand for the immune checkpoint programmed cell death 1 (PD-1). PD-L1 is highly expressed on several tumors, such as triple-negative breast cancer (TNBC), non-small cell lung carcinoma, and renal cancer, and is known to suppress cancer-killing immune cells via interaction with PD-1 on T-cells. In addition, PD-L1 is also present on macrophages in the tumor microenvironments leading to further immune suppression and acquired resistance to anti-PD-1 therapy is associated with the upregulation of alternative immune checkpoints, thereby reducing anti-tumor efficacy. We have designed and synthesized bifunctional small molecules as PD-L1 degraders with different recruiters and linkers guided by computational studies with known PD-1/PD-L1 structures to show both cell surface and total protein degradation in human TNBC cells. In a separate project, we also developed small molecule conjugates to degrade an intracellular integral membrane protein of the endoplasmic reticulum with an unknown 3D structure, namely Diglyceride acyltransferase 2 (DGAT2). Recently, our lab identified DGAT2 as a new target for combating Alzheimer’s disease. Specifically, DGAT2 catalyzes triacylglycerol (TAG) synthesis using diacylglycerol and fatty acyl CoA as substrates. The accumulation of TAGs, mechanistically linked to DGAT2, results in “fat” or lipid droplets (LDs) inside the cells. Our lab showed that microglial cells (resident immune cells in the brain) accumulate LDs in the postmortem brains of human patients and mouse models (5xFAD) of Alzheimer’s disease and that the LD accumulation is driven by amyloid-beta (Ab) – a hallmark of Alzheimer’s disease – via DGAT2 pathway. Further, these LD-laden microglia have phagocytic defects and are spared Aβ thereby affecting plaque accumulation and clearance. Inhibiting DGAT2 reduces the amount of TAG in the brain, which in turn reduces LDs and restores microglial ability to phagocytose Ab. However, commercially available DGAT2 inhibitors were unable to reduce LD load in older 5xFAD mice. Using AlphaFold’s models of DGAT2, we designed and identified sites to synthesize bifunctional DGAT2 degraders that resulted in reduced LDs in mouse primary microglial cells and enhanced phagocytosis of Aβ plaques in vivo in aged 5xFAD mice. Our approach shows a framework to develop bifunctional small molecule degraders for membrane proteins to potentially combat immune dysregulation in chronic diseases.</p>

La réaction du greffon contre l’hôte (GvH) est responsable d’un grand taux de morbidité et de mortalité chez les patients recevant des greffes de cellules souches (GCSH) allogéniques. Dans ce contexte, les cellules T régulatrices sont largement étudiées et semblent avoir un grand potentiel d’utilisation dans le domaine de la thérapie cellulaire de la GvH. Parmi les populations cellulaires T régulatrices, les lymphocytes T CD4-CD8- TCRαβ+ « Doubles-Négatifs » (DN), qui ne représentent que 1-3% des lymphocytes T, ont été décrits. Ces cellules ont des propriétés inhibitrices de la réponse immunitaire qui s’avèrent spécifiques aux antigènes auxquels elles ont préalablement été exposées. La répression de la réponse immunitaire par les cellules T DN régulatrices semble être un mécanisme important impliqué dans l’induction de la tolérance aux allo-antigènes. De plus, ces cellules confèrent une tolérance immunitaire dans des modèles de greffes allogéniques et xénogéniques. En effet, ces cellules ont la capacité d’inhiber la réaction contre un allo-antigène auquel elles ont été exposées, sans inhiber la réaction contre un allo-antigène inconnu. Les cellules T DN ont été isolées et caractérisées chez l’homme où elles ont la capacité d’interagir avec des cellules présentatrices d’antigènes (APCs) par un contact cellulaire, comme chez la souris. Cependant, leur capacité immunomodulatrice reste inconnue chez l’humain. Notre objectif consistait donc principalement à étudier le rôle et le mécanisme d’action des cellules T DN régulatrices humaines in vitro, en étudiant leur capacité à inhiber une réaction lymphocytaire mixte (MLR). Nous avons montré que les cellules T DN stimulées par un allo-antigène donné inhibent des cellules syngéniques effectrices dirigées contre ce même alloantigène mais n’inhibent pas des cellules syngéniques effectrices dirigées contre un autre alloantigène, démontrant ainsi la spécificité aux antigènes de ces cellules. De plus, les T DN non stimulées par un allo-antigène n’ont pas de rôle inhibiteur. Cependant, durant cette inhibition, nous n’observons pas de modulation de l’expression des marqueurs d’activation et d’induction de l’apoptose. Afin d’étudier le mécanisme d’action des cellules T DN, nous avons mesuré l’expression intracellulaire de la granzyme B. Les résultats démontrent que les cellules T DN stimulées expriment un niveau significativement plus élevé de granzyme B que les cellules T DN non-stimulées par l’allo-antigène. Ceci suggère que l’immunosuppression induite par les cellules T DN stimulées pourrait passer par la voie granzyme B. Le mécanisme utilisé par ces cellules reste à être confirmé par nos futures expériences. / Graft-versus-Host Disease (GvHD) is a major cause of morbidity and mortality in patients receiving an allogeneic Hematopoietic Stem Cell Transplantation (HSCT). Many regulatory T cell populations have been studied and shown to have immunosuppressive properties in GvHD. Among these populations, Double Negative CD4-CD8-TCRαβ+ regulatory T cells (DN T) have been described. These cells represent 1-3% of all T cell lymphocytes and are known to have antigen-specific inhibitory functions of the immune response. The suppression of an immune response by DN T cells seems to be an important mechanism involved in the induction of tolerance to allo-antigens. Moreover, these cells also confer immune tolerance in models of allogeneic and xenogenic grafts. DN T cells have the ability to suppress syngeneic T CD4+ and T CD8+ cells in an antigen-specific manner. Therefore, these DN T cells can inhibit the reaction caused by syngeneic effector cells against a specific alloantigen to which they have been previously exposed. However, they cannot inhibit a reaction directed against an unknown alloantigen. Human DN T cells have been isolated and characterized as cells that have the capacity to interact with APCs by cell-to-cell contact, just like in mice. However, their immunomodulatory properties are still unknown in humans. The goal of our project was to study the role and immunomodulatory functions of human DN T cells in Mixed Lymphocyte Reactions (MLR). The MLRs have allowed us to demonstrate that DN T cells, after having been stimulated by an allo-antigen, have an antigen-specific inhibitory function towards the syngeneic effector cells reacting against the same alloantigen that they have been exposed to. Interestingly, they do not inhibit the reaction of these effector cells against an unknown alloantigen. However, stimulated DN T cells did not modulate the expression of the activation markers expressed by the effector cells and did not give a death signal to these cells either. Moreover, we also wanted to study how DN T cells have an immunosuppressive activity. Therefore, we compared the expression of Granzyme B in stimulated versus non-stimulated cells. Our results suggest that DN T cells may use the Granzyme B pathway to immunosuppress the effector cells. In conclusion, our results demonstrate that DN T cells have an antigen-specific inhibitory function. The mechanism used by these DN T cells remains to be confirmed with our future experiments.

Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...