Patients with peritonitis or malignant obstructive jaundice (HPB+) have an increased morbidity and mortality due to sepsis. An altered cell-mediated immunity in the intestinal mucosa might promote gut barrier failure, increased endotoxin and cytokine release and bacterial translocation (BT) in these conditions. A clinically relevant rat model of polymicrobial peritonitis induced sepsis by cecal ligation and puncture (CLP) was used. Septic animals demonstrated a superficial injury in the small intestinal mucosa, and a significant reduction in T lymphocytes in the villi, as well as increased number of macrophages in the villi and in the MLNs as compared to sham. CLP caused increased concentration of TNF-α and IL-6 in ascitic fluid. CLP + the immunomodulator Linomide decreased the TNF-α level, reduced mucosal damage and attenuated the changes in T lymphocytes and macrophages observed following CLP. CLP + selective cyclooxygenase (COX)-2 inhibitor (SC-236) or nonselective COX inhibitor (indometacin) decreased the amount of macrophages in the mucosa and the MLNs compared to untreated CLP. CLP + indometacin decreased T lymphocytes in the villi and MLNs. SC-236 + CLP reduced mucosal injury and cytokine release as compared to indometacin. An increased rate of apoptosis in both the mucosa and MLNs was seen following CLP; COX inhibitors enhanced this phenomenon in the MLNs. BT occurred infrequently in patients with acute peritonitis and in HPB+ there was no evidence of BT. Peritonitis and HPB+ causes significant inflammatory cellular reactions as increased endotoxin and cytokine plasma levels and an altered immune cell distribution in MLNs, in HPB+ a high rate of apoptosis in MLNs was observed. An altered pattern of immunocompetent cells within the mucosa and in MLNs was found in experimental and clinical peritonitis as in HPB+. Lymphocyte depletion may be a result of increased apoptosis, which could reduce the ability of septic or jaundice patients to eradicate infection.

Candida albicans est le premier pathogène fongique chez l'homme (avec 78 % des infections). Nous avons choisi d'étudier, chez cette levure diploïde, les protéines de surface ancrées à la membrane et/ou à la paroi par une ancre Glycosylphosphatidylinositol (GPI). Des expériences antérieures au laboratoire ont montré que l'absence de Gpi7p (une enzyme de la voie de biosynthèse de l'ancre GPI) affectait l'ancrage des protéines à ancre GPI (GpiP) pariétales, l'intégrité de la paroi et la virulence de C. albicans. Outre le fait d'être localisées à la surface, plus de 60 % des 104 GpiP n'ont pas d'orthologue connu. Toutes ces données nous laissent penser que cette classe de protéines pourrait jouer un rôle clé dans les interactions de C. albicans avec son environnement. L'étude, présentée dans cette thèse, a été menée selon deux approches. La première consistait à interrompre un maximum de gènes codant des GpiP, puis à étudier le phénotype associé à ces invalidations pour ensuite cribler ces mutants GpiP pour les interactions macrophage-C. albicans in vitro. Cette approche globale nous a permis de construire une banque de mutants relativement importante ; son étude phénotypique montre que les GpiP sont impliquées dans trois grandes catégories fonctionnelles : (i) la morphogenèse, (ii) la réponse au stress et (iii) l'intégrité de la paroi avec la sensibilité à la caspofungine. Le crible pour des mutants atteints dans leur résistance à la phagocytose ne nous a pas permis d'obtenir des résultats clairs, probablement à cause des conditions testées qui n'étaient pas assez stringentes. La seconde approche consistait à étudier l'impact de la mauvaise localisation de certaines protéines pariétales, en utilisant le mutant gpi7-/-, sur la réponse innée de l'hôte face à C. albicans. Nos expériences montrent très clairement que le mutant gpi7-/- induit une très forte production de TNFΑ par les macrophages accompagnée d'un recrutement accru de neutrophiles au site de l'infection. Cette étude suggère qu'en l'absence de Gpi7p, la paroi de C. albicans serait mieux reconnue par les phagocytes mais que cette reconnaissance n'impliquerait pas les récepteurs TLR2 et TLR4.

Ο ρινικός πολύποδας (ΡΠ) είναι μια χρόνια φλεγμονώδης νόσος του ρινικού βλεννογόνου, η οποία χαρακτηρίζεται από διήθηση φλεγμονωδών κυττάρων, όπως ηωσινόφιλα, λεμφοκύτταρα και πλασμοκύτταρα, τροποποιήσεις στη διαφοροποίηση του επιθηλίου και ανάπλαση ιστού, που περιλαμβάνει υπερπλασία της βασικής μεμβράνης, συσσώρευση εξωκυττάριου υλικού και οίδημα. Ο παράγοντας αναστολής της μετανάστευσης μακροφάγων (MIF) είναι μία μοναδική κυτταροκίνη με σημαντικό ρόλο στο σηπτικό σόκ και στις χρόνιες φλεγμονώδεις και αυτοάνοσες ασθένειες.Παράγεται από ενεργοποιημένα Τ-κύτταρα, μακροφάγα αλλά και από ποικιλία άλλων κυττάρων. Εκτός των άλλων, έχει βρεθεί ότι επάγει την έκφραση της IL-6 από διάφορα κύτταρα και ανταγωνίζεται την κατασταλτική επίδραση των γλυκοκορτικοειδών στην έκφρασή της. Απαντάται σε αυξημένα επίπεδα στο ρινικό πολύποδα. Ο αυξητικός παράγοντας μετασχηματισμού-β1 (TGF-β1) θεωρείται ένας αντιφλεγμονώδης παράγοντες, ο οποίος ανταγωνίζεται τη δράση της IL-1β και του TNF-α, στην έκφραση της ΜΜΡ-1 και ΜΜΡ-3. Διεγείρει επίσης την έκφραση του ΤΙΜΡ-1, του κολλαγόνου τύπου Ι και της IL-6 και λόγω των δράσεών του αυτών εμπλέκεται ισχυρά στις διαδικασίες ίνωσης. Απαντάται σε σημαντικά επίπεδα στο ρινικό πολύποδα. Η IL-6 είναι μία Th2 πολυλειτουργική κυτταροκίνη η οποία εμπλέκεται σε ποικίλες φλεγμονώδεις καταστάσεις. Διεγείρει την ανάπτυξη των ινοβλαστών, αυξάνει τη σύνθεση και εναπόθεση του κολλαγόνου και μειώνει την αποικοδόμησή του. Απαντάται σε αυξημένα επίπεδα στο ρινικό πολύποδα και θεωρείται σημαντικός παθαγενετικός παράγοντας μέσω της επαγωγής του σχηματισμού των πλασμοκυττάρων και της σύνθεσης συστατικών του στρώματος, και της προαγωγής της σύνθεσης και εναπόθεσης κολλαγόνου και της ανάπλασης ιστού. Σκοπός της παρούσης εργασίας είναι η μελέτη της συμβολής του MIF και του TGF-β1 στη παραγωγή της IL-6 από ινοβλάστες ρινικού πολύποδα, των σηματοδοτικών μονοπατιών που εμπλέκονται, και η διερεύνηση της πιθανής συνεργειακής δράσης τους στην έκφραση της κυτταροκίνης. Τόσο ο TGF-β1 (0,01-1 ng/ml) όσο και ο MIF (1-100 ng/ml) προκάλεσαν διέγερση της έκφραση της IL-6 σε ινοβλάστες ρινικού πολύποδα κατά δοσοεξαρτώμενο τρόπο, η οποία κατεστάλλει σημαντικά από αναστολέις των ΜΑΡ κινασών και της ΡΙ-3 κινάσης. Ο TGF-β1 (1 ng/ml) προκάλεσε επίσης χρονοεξαρτώμενη αύξηση στα επίπεδα της IL-6 γιά χρόνο επώασης μέχρι 2 ώρες, τα οποιά έμεινα σταθερά στη συνέχεια μέχρι τις 72 ώρες επώασης, επαγωγή της παραγωγής ενδοκυτταρικών ενεργών ειδών οξυγόνου (ROS) με μέγιστο σε δύο χρόνους επώασης 20 και 180 λεπτά, και ενεργοποίηση των ERK κινασών από 15-60 λεπτά επώασης, με μέγιστη ενεργοποίηση στα 30 λεπτά, και στη συνέχεια πτώση στα επίπεδα του μάρτυρα. Ενώ ο MIF σε συγκέντρωση 1-100 ng/ml προκάλεσε μικρή μείωση στην έκφραση της φωσφατάσης-1 των ΜΑΡΚ (ΜΚΡ-1), ο TGF-β1 αντίθετα, σε συγκέντρωση 1 ng/ml προκάλεσε αύξηση στην έκφραση της ΜΚΡ-1 σε δύο χρόνους επώασης 0,5 και 24 ώρες. Μετά από επώαση των ινοβλαστών ρινικού πολύποδα παρουσία TGF-β1 (1 ng/ml) και MIF (100 ng/ml) μαζί, δεν παρατηρήθηκε συνεργειακή επίδραση στην έκφραση της IL-6. Ενώ τόσο ο TGF-β1 όσο και ο MIF προκάλεσαν διέγερση στην έκφραση της IL-6 από ινοβλάστες πνεύμονα, δεν παρατηρήθηκε και πάλι συνεργειακή επίδρασή τους στην έκφραση της κυτταροκίνης αυτής. Συμπερασματικά, φαίνεται ότι ο MIF, ενώ ανταγωνίζεται την κατασταλτική επίδραση των γλυκοκορτικοειδών στην έκφραση της IL-6 μέσω της ρύθμισης των επιπέδων της ΜΚΡ-1, δεν έχει την ίδια επίδραση στην έκφραση της IL-6 από τον TGF-β1, μέσω ρύθμισης της έκφρασης της φωσφατάσης αυτής. Από την άλλη μεριά η επαγωγή της έκφρασης της ΜΚΡ-1 από τον TGF-β1 φαίνεται να μην επηρεάζει την παραγωγή της IL-6 για μικρούς χρόνους επώασης, μέχρι 2 ώρες, πιθανόν λόγω της ανασταλτικής επίδρασης των ROS, που επάγονται από τον TGF-β1, στη δράση της ΜΚΡ-1, ενώ την επηρεάζει για μεγάλους χρόνους επώασης , εξ ού και τα σταθερά επίπεδα της IL-6 μέχρι και 72 ώρες επώασης. / Nasal polyp (NP) is a chronic inflammatory condition of nasal mucosa, characterized by infiltration of inflammatory cells such as eosinophils, lymphocytes and plasma cells, alterations in epithelial differentiation and tissue reconstruction, involving hyperplasia of basal membrane, accumulation of extracellular material and edema. Macrophage migration inhibitory factor (MIF) is a unique cytokine, the role of which in chronic inflammatory and autoimmune diseases and septic shock pathogenesis is very important. MIF is produced by activated T-lymphocytes, macrophages and a plethora of other cells. MIF appears to antagonize the suppressive effect of glucocorticoids as well as induce the expression of IL-6 in multiple cells. High levels of MIF are detected in nasal polyps. Transforming growth factor- β1 (TGF-β1) is an anti-inflammatory factor antagonizing the positive effect of IL-1β and TNF-α on the expression of MMP-1 and MMP-3, TGF-β1 also stimulates the expression of TIMP-1, collagen type I and IL-6; because of these effects, TGF-β1 is involved in the process of fibrosis. TGF-β1 levels in nasal polyps are significantly elevated. IL-6 is a cytokine participating in Th2 response and consequently is involved in a subset of inflammatory reactions. IL-6 stimulates the growth of fibroblasts, increases the production and deposition of collagen and it decreases its degradation. IL-6 is found in nasal polyps at elevated levels and it is thought to be an important pathogenic factor acting mainly in tissue reconstruction, stimulation of plasma cell differentiation, production of stromal material, promotion of collagen synthesis and deposition. The purpose of this paper is to study the effect of MIF and TGF-β1 in IL-6 production by fibroblasts isolated from nasal polyps, dissect the signaling pathways involved, and investigate their synergistic effect on the production of IL-6. Both TGF-β1 (0, 01-1 ng/ml) and MIF (1-100 ng/ml) induced IL-6 expression in nasal polyp fibroblasts in a dose-dependent manner. This effect was significantly suppressed by inhibitors of MAP and PI-3 kinase pathways. TGF-β1 (1 ng/ml) also induced IL-6 expression within 2 hours of administration. Elevated IL-6 levels remained unchanged for 72h further. TGF-β1 also promoted the production of intracellular reactive oxygen species (ROS), which peaked in 20 and 180 minutes and the activation of ERK kinase, peaked in 30 minutes. While MIF, at a concentration of 1-100 ng/ml, caused a slight decrease in the expression of phospatase-1 of MAPK (MKP-1), TGF-β1, at a concentration of 1 ng/ml, increased the expression of MKP-1. No synergistic effect on IL-6 expression was detected after incubating nasal polyp and lung fibroblasts together with TGF-β1 (1 ng/ml) and MIF (100 ng/ml). In conclusion, while MIF antagonizes the suppressive effect of glucocorticoids on the expression of IL-6 by regulating the levels of MKP-1, it fails to antagonize the TGF-β1 inducing effect on IL-6 via MKP-1. The induction of MKP-1expression by TGF-β1 is not affecting the production of IL-6 after short incubation periods; this effect can be explained by the inhibitory effect of TGF-β1 induced ROS on MKP-1. After prolong incubation with TGF-β1 (up to 72 hours), IL-6 levels remain elevated.

Gold nanoparticles exhibit a combination of physical, chemical, optical, and electronic properties unique from all other nanotechnologies. These structures can provide a highly multifunctional platform with which to diagnose and treat diseases and can dramatically enhance a variety of photonic and electronic processes and devices. The work herein highlights some newly emerging applications of these phenomena as they relate to the targeted diagnosis and treatment of cancer, improved charge carrier generation in photovoltaic device materials, and strategies for enhanced spectrochemical analysis and detection. Chapter 1 introduces the reader to the design, synthesis, and molecular functionalization of gold nanotechnologies, and provides a framework from which to discuss the unique photophysical properties and applications of these nanoscale materials and their physiological interactions in Chapter 2. Chapter 3 discusses ongoing preclinical research in our lab investigating the use of near-infrared absorbing gold nanorods as photothermal contrast agents for laser ablation therapy of solid tumors. In Chapter 4, we present recent work developing a novel strategy for the targeted treatment of hormone-dependent breast and prostate tumors using multivalent gold nanoparticles that function as highly selective and potent endocrine receptor antagonist chemotherapeutics. In Chapter 5, we discuss a newly-emerging tumor-targeting strategy for nanoscale drug carriers which relies on their selective delivery to immune cells that exhibit high accumulation and infiltration into breast and brain tumors. Using this platform, we further investigate the interactions of nanoscale drug carriers and imaging agents to a transmembrane protein considered to be the single most prevalent and single most important contributor to drug resistance and the failure of chemotherapy. Chapter 6 presents work from a series of studies exploring enhanced charge carrier generation and relaxation in a hybrid electronic system exhibiting resonant interactions between photovoltaic device materials and plasmonic gold nanoparticles. Chapter 7 concludes by presenting studies investigating the contributions from so-called “dark” plasmon modes to the spectrochemical diagnostic method known as surface enhanced Raman scattering.

The societal burden of autoimmune diseases and donor organ transplant rejection in developed countries reflects the lack of effective immune suppressive drugs. The main objective of my thesis was to develop novel fusion proteins targeting receptors linked to autoimmunity; strategies that will allow the suppression of autoreactive cells while sparing resting lymphocytes. Interleukin (IL) 15 has been demonstrated to exert its effects mainly on activated T-cells triggered via their T-cell receptor (TCR). Since we found that the fusion of granulocyte-macrophage colony stimulating factor (GMCSF) to IL15 - aka GIFT15 - paradoxically leads to aberrant signalling downstream of the IL15R and blocks interferon (IFN)-gamma secretion in a mixed lymphocyte reaction (MLR), we hypothesized to use this fusokine in proof-of-principle cell transplantation models and shown that GIFT15 can indeed block the rejection of allogeneic and xenogeneic cells in immunocompetent mice. Additionally, we found that ex vivo GIFT15 treatment of mouse splenocytes lead to the generation of regulatory B-cells (Bregs). These Bregs express high levels of MHCII, IL10 and are capable to block antigen (Ag)-presentation in vitro as third party bystander cells. Moreover, a single injection of these GIFT15-generated Bregs in mice with pre-developed experimental autoimmune encephalomyelitis (EAE) leads to long lasting remission of disease. / Along those lines, we also found that mesenchymal stromal cells (MSCs) lead to the paracrine conversion of CCL2 to an antagonist form capable of specifically inhibiting plasma cells and activated Th17 cells. This mechanistic insight informed the design of a second class of suppression fusokine. Namely, the fusing of antagonist CCL2 to GMCSF - aka GMME1. We tested its potential use in autoimmune diseases such as EAE and rheumatoid arthritis (RA). We demonstrated that GMME1 leads to asymmetrical signalling and inhibition of plasma cells as well as Th17 EAE/RA-reactive CD4 T-cells. The net outcome of these pharmacological effects is the selective depletion of CCR2-reactive T-cells as demonstrated both in vitro and in vivo. / Overall, our data support the use of our fusion proteins as part of a powerful and specific immunosuppressive strategy either as directly injectable protein biopharmaceuticals or through the ex vivo generation of autologous Bregs in the case of GIFT15.

Invariant natural killer T (iNKT) cells are a versatile subclass of T lymphocytes which recognize glycolipid antigens. iNKT cells are capable of rapidly producing a broad array of cytokines in response to stimulation; thus, they play an important role in the early regulation of a variety of immune responses. It was hypothesized that iNKT cells express functional Toll-like receptors (TLRs) and that stimulation of TLRs by their ligands modulates iNKT cells responses. In the first objective, it was revealed that upon stimulation with anti-CD3 monoclonal antibody and interferon (IFN)-α, expression of TLRs was enhanced in iNKT cells. Furthermore, stimulation of iNKT cells with TLR ligands led to a significant increase in the expression of several cytokines. In the second objective, the mechanisms behind the modulatory effects of the TLR9 ligand (CpG-ODN) on iNKT cells were determined. Altogether, these findings suggest a direct role for TLRs in iNKT cell activation. / Ontario Graduate Scholarship

Les maladies cardiovasculaires (MCV) sont la principale cause de décès dans les pays occidentaux et constituent la principale complication associée au diabète. La lipoprotéine lipase (LPL) est une enzyme clé du métabolisme des lipides et est responsable de l'hydrolyse des lipoprotéines riches en triglycérides (TG). Plusieurs études ont démontré que la LPL sécrétée par les macrophages dans la paroi artérielle est pro-athérogénique. La dysfonction endothéliale caractérise les stades précoces du processus athérosclérotique. Il a été observé qu’un récepteur nouvellement identifié des lipoprotéines de basse densité oxydées (LDLox), le récepteur de type lectine des LDLox (LOX-1), est fortement exprimé dans les lésions athérosclérotiques humaines et dans l’aorte de rats diabétiques, suggérant un rôle clé de LOX-1 dans la pathogénèse de l’athérosclérose diabétique. Au vu du rôle potentiel de la LPL macrophagique et du LOX-1 dans l’athérosclérose associée au diabète de type 2, nous avons évalué la régulation de ces deux molécules pro-athérogéniques par des facteurs métaboliques et inflammatoires augmentés dans le diabète, soit la leptine, l’acide linoléique (LA) et la protéine C-réactive (CRP). Nos résultats démontrent que : 1) Dans les cellules endothéliales aortiques humaines (HAECs), LA augmente l’expression protéique de LOX-1 de façon temps- et dose-dépendante; 2) La pré-incubation de HAECs avec des antioxydants et des inhibiteurs de la NADPH oxydase, de la protéine kinase C (PKC) et du facteur nucléaire-kappa B (NF-kB), inhibe l’effet stimulant de LA sur l’expression protéique de LOX-1; 3) Dans les HAECs traitées avec LA, on observe une augmentation d’expression des isoformes classiques de la PKC; 4) LA augmente de manière significative l’expression génique de LOX-1 ainsi que la liaison des protéines nucléaires extraites des HAECs à la séquence régulatrice NF-kB présente dans le promoteur du gène de LOX-1; 5) LA augmente, via LOX-1, la captation des LDLox par les cellules endothéliales. Pris dans leur ensemble, ces résultats démontrent que LA augmente l’expression endothéliale de LOX-1 in vitro et appuient le rôle clé de LA dans la dysfonction endothéliale associée au diabète. Au vu de nos études antérieures démontrant qu’une expression accrue de LPL macrophagique chez les patients diabétiques de type 2 et que l’augmentation de facteurs métaboliques dans cette maladie, soit l’homocystéine (Hcys), les acides gras et les produits terminaux de glycation (AGE), accroissent l’expression de la LPL macrophagique, nous avons par la suite déterminé l’effet, in vitro, de deux autres facteurs métaboliques et inflammatoires surexprimés dans le diabète, soit la leptine et la CRP, sur l’expression de la LPL macrophagique. Les concentrations plasmatiques de leptine sont élevées chez les patients diabétiques et sont associées à un accroissement des risques cardiovasculaires. Nous avons démontré que : 1) Dans les macrophages humains, la leptine augmente l’expression de la LPL, tant au niveau génique que protéique; 2) L’effet stimulant de la leptine sur la LPL est aboli par la pré-incubation avec un anticorps dirigé contre les récepteurs à la leptine (Ob-R), des inhibiteurs de la PKC et des antioxydants; 3) La leptine augmente l’expression membranaire des isoformes classiques de la PKC et la diminution de l’expression endogène de la PKC, abolit l’effet de la leptine sur l’expression de la LPL macrophagique; 4) Dans les macrophages murins, la leptine augmente le taux de synthèse de la LPL et augmente la liaison de protéines nucléaires à la séquence protéine activée-1 (AP-1) du promoteur du gène de la LPL. Ces observations supportent la possibilité que la leptine puisse représenter un facteur stimulant de la LPL macrophagique dans le diabète. Finalement, nous avons déterminé, in vitro, l’effet de la CRP sur l’expression de la LPL macrophagique. La CRP est une molécule inflammatoire et un puissant prédicteur d’événements cardiovasculaires. Des concentrations élevées de CRP sérique sont documentées chez les patients diabétiques de type 2. Nous avons démontré que : 1) Dans les macrophages humains, la CRP augmente l’expression de la LPL au niveau génique et protéique et la liaison de la CRP aux récepteurs CD32 est nécessaire pour médier ses effets; 2) La pré-incubation de macrophages humains avec des antioxydants, des inhibiteurs de la PKC et de la protéine kinase mitogénique activée (MAPK), prévient l’induction de la LPL par la CRP; 3) La CRP augmente l’activité de la LPL, la génération intracellulaire d’espèces radicalaires oxygénées (ROS), l’expression d’isoformes classiques de la PKC et la phosphorylation des kinases extracellulaires régulées 1/2 (ERK 1/2); 4) Les macrophages murins traités avec la CRP démontrent une augmentation de la liaison des protéines nucléaires à la séquence AP-1 du promoteur du gène de la LPL. Ces données suggèrent que la LPL puisse représenter un nouveau facteur médiant les effets délétères de la CRP dans la vasculopathie diabétique. Dans l’ensemble nos études démontrent le rôle clé de facteurs métaboliques et inflammatoires dans la régulation vasculaire de la LPL et du LOX-1 dans le diabète. Nos données suggèrent que la LPL et le LOX-1 puissent représenter des contributeurs clé de l’athérogénèse accélérée associée au diabète chez l’humain. Mots-clés : athérosclérose, maladies cardiovasculaires, diabète de type 2, macrophage, LPL, cellules endothéliales, LOX-1, stress oxydatif, leptine, LA, CRP. / Atherosclerotic cardiovascular disease is the leading cause of death in western countries and is the major complication of diabetes. Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism, responsible for the hydrolysis of triglyceride (TG) rich lipoproteins. Many studies have shown that LPL secreted by macrophages in the arterial wall is proatherogenic. Endothelial dysfunction is a characteristic feature of early-stage atherosclerosis. The observation that lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a newly identified receptor for oxidized LDL (oxLDL), is highly expressed in human atherosclerotic lesions and upregulated in the aorta of diabetic rats, suggests a key role for LOX-1 in the pathogenesis of diabetic atherosclerosis. Based on the role of macrophage LPL and LOX-1 in atherosclerosis, we sought to investigate the regulation of these two proatherogenic molecules by metabolic and inflammatory factors dysregulated in diabetes namely leptin, linoleic acid (LA) and C-reactive protein (CRP). Our results demonstrated that: 1) In human aortic endothelial cells (HAECs), LA increased LOX-1 protein expression in a time- and dose-dependent manner; 2) Pretreatment of HAECs with antioxidants and inhibitors of NADPH oxidase, protein kinase C (PKC), and nuclear factor-kB (NF-kB) inhibited the stimulatory effect of LA on LOX-1 protein expression; 3) Increased expression of classic PKC isoforms was observed in LA-treated HAECs; 4) LA led to a significant increase in LOX-1 gene expression and enhanced the binding of nuclear proteins extracted from HAECs to the NF-kB regulatory element of the LOX-1 gene promoter; 5) LA enhanced, through LOX-1, oxLDL uptake by endothelial cells. Overall, these results demonstrated that LA enhances endothelial LOX-1 expression in vitro and support a key role for LA in endothelial dysfunction associated with diabetes. Based on our previous studies showing that macrophage LPL expression is increased in patients with type 2 diabetes and that metabolic factors dysregulated in this disease such as glucose, homocysteine (Hcys), fatty acids and advanced glycation end products (AGE), increased macrophage LPL expression, we next determined the effect of two other metabolic and inflammatory factors dysregulated in diabetes, namely leptin and CRP, on macrophage LPL expression in vitro. Leptin levels are elevated in diabetic patients and are associated with greater cardiovascular risks. We found that: 1) In human macrophages, leptin increased LPL expression, at both the gene and protein levels; 2) The stimulatory effect of leptin on LPL was abolished by pre-treatment with anti-leptin receptor (Ob-R) antibody, PKC inhibitors and antioxidants; 3) Leptin increased the membrane expression of conventional PKC isoforms and downregulation of endogenous PKC expression abolished the effects of leptin on macrophage LPL expression; 4) In murine macrophages, leptin raised LPL synthetic rate and enhanced the binding of nuclear proteins to the activated protein-1 (AP-1) sequence of the LPL gene promoter. These observations support the possibility that leptin may represent a macrophage LPL stimulatory factor in diabetes. Finally, we sought to determine the effect of CRP on macrophage LPL expression in vitro. CRP is an inflammatory molecule and a strong predictor of cardiovascular events and high serum levels of CRP are observed in type 2 diabetic patients. We found that: 1) In human macrophages, CRP increased LPL expression at the gene and protein levels and CRP binding to CD32 receptors is required for these effects; 2) Preincubation of human macrophages with antioxidants, PKC and mitogen-activated protein kinase (MAPK) inhibitors, prevented the CRP-induced LPL expression; 3) CRP increased LPL activity, intracellular reactive oxygen species (ROS) generation, classic PKC isozymes expression and extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation; 4) CRP-treated murine macrophages demonstrated increased binding of nuclear proteins to the AP-1 sequence of the LPL gene promoter. These data suggest that LPL might represent a novel factor underlying the adverse effect of CRP on the diabetic vasculature. Overall, our studies indicate a key role of metabolic and inflammatory factors in the regulation of vascular LPL and LOX-1 in diabetes. Our data suggest that LPL and LOX-1 are key contributors to the accelerated atherogenesis associated with human diabetes. Keywords : atherosclerosis, cardiovascular diseases, type 2 diabetes, macrophage, LPL, endothelial cells, LOX-1, oxidative stress, leptin, LA, CRP.

Seit 20 Jahren unterteilt man Monozyten in eine klassische CD14++/CD16-Population und eine proinflammatorische CD16+ Population. Letztere macht 10-20 % der peripheren Blutmonozyten aus und ist unter verschiedenen pathologischen Zuständen drastisch erhöht. Seit Kurzem wird die CD16+ Fraktion in zwei weitere Untergruppen aufgeteilt, die CD14++/CD16+ und die CD14+/CD16+ Monozyten. In der hier vorliegenden Arbeit wurde versucht, die relativ neue und wenig charakterisierte Subpopulation der CD14++/CD16+ Monozyten näher zu beschreiben. Es sollte die Frage beantwortet werden, ob diese sich von den übrigen Subpopulationen abzugrenzen lässt und damit als eigenständige Zellgruppe anzusehen ist. Die von gesunden Spendern gewonnen peripheren mononukleären Blutzellen (PBMCs) wurden mithilfe einer Magnetsäulenvorseparation von einem Großteil der T-, B- und NK-Zellen befreit. Die restlichen Zellen wurden mit Anti-CD14-FITC und Anti-CD16-PE markiert. In einem Hochgeschwindigkeitssortierer wurden diese, anhand ihres Fluoreszenzmusters, in die drei Subpopulationen CD14++/CD16- (Sub1), CD14++/CD16+ (Sub2) und CD14+/CD16+ (Sub3) aufgeteilt. Durch dieses Verfahren konnte ein hoher Reinheitsgrad erreicht werden. Die erhaltenen Subpopulationen wurden mit heterologen CD4+ T-Lymphozyten zusammen gebracht. Die Reaktion dieser T-Zellen auf die verschiedenen Subpopulationen wurde im Proliferations- und Sekretionsassay (IFN-γ, IL-4) studiert. Des Weiteren wurde die Zytokinsekretion der Monozytenarten nach definierter Stimulierung (LPS, Zymosan, aktivierte T-Zellen) analysiert. In einem zweiten Versuchskomplex wurden aus den jeweiligen Subpopulationen unreife dendritische Zellen (Sub-DCs) differenziert und diese auf bestimmte Oberflächenmarker bzw. deren Verhalten mit heterologen CD4+ T-Zellen im Proliferations-/Sekretionsassay (IFN-γ, IL-4) untersucht. Nach 7 Tagen Inkubation fanden sich im Sub2- und Sub3-Ansatz ca. 10 % mehr proliferierende T-Zellen als im Sub1-Ansatz. Dabei lag der Anteil der CD25+ T-Zellen in diesen beiden Versuchsansätzen ebenfalls um 10 % höher. Der Proliferationsassay über 14 Tage zeigte für die Sub3 ca. 10-15 % mehr proliferierende T-Zellen als für die anderen Populationen. Das Niveau der CD25-Expression der T-Zellen war hierbei in allen drei Ansätzen gleich. Im Sekretionsassay induzierten alle drei Subpopulationen eine Th1-Antwort, jedoch mit einem leicht höheren Anteil IFN-γ-positiver T-Zellen für die CD16+ Monozyten. Durch LPS-Gabe produzierten die CD14+/CD16+ Monozyten am meisten TNF-α, gefolgt von den CD14++/CD16+. Bei den CD14++/CD16- fanden sich die geringsten Mengen an TNF-α. Zusammen mit aktivierten T-Zellen demonstrierten die CD14++/CD16+ Monozyten die stärkste TNF-α-Sekretion unter allen anderen Subpopulationen. Für die beiden CD16+ Populationen wurden nach LPS-Stimulation höhere Werte für IL-1β bestimmt als für die klassischen Monozyten. Sowohl im LPS- als auch im Zymosanansatz lag die IL-6-Sekretion der CD14++/CD16- Subpopulation über der der anderen zwei Fraktionen. Unter allen drei Stimuli wurden die höchsten Werte für IL-8 bei den CD14++/CD16- Monozyten detektiert, gefolgt von den CD14++/CD16+. Am niedrigsten lag die IL-8-Produktion bei den CD14+/CD16+. Die IL-10-Sekretion im LPS- und im Zymosanansatz der CD14++/CD16- und CD14++/CD16+ Monozyten war gegenüber der CD14+/CD16+ Subpopulation erhöht. Nach Entwicklung der unreifen dendritischen Zellen aus den entsprechenden Monozytenarten weisen diese eine differenzierte Morphologie auf. Die DCs der CD14+/CD16+ Monozyten hatten eine stärkere HLA-DR-Expression in der mittleren Fluoreszenzintensität als die anderen beiden Sub-DC-Populationen, wobei sich keine Unterschiede in der HLA-DRExpressionsintensität zwischen Sub1- und Sub2-DCs feststellen ließen. Der Anteil der CD11c positiven Zellen war bei den Sub1-DCs deutlich größer als bei den Sub2-DCs. Dagegen exprimierten die Sub3-DCs praktisch kein CD11c. Im Proliferationsassay über 7 Tage ergaben sich keine signifikanten Differenzen zwischen den Subpopulationen. Allerdings zeigte sich eine Tendenz zu geringeren Werten im Anteil proliferierender bzw. CD25-positiver T-Lymphozyten für den Sub2-DC-Ansatz. Nach 14 Tagen im Assay war der Anteil der proliferierenden T-Zellen bei den Sub1-DCs um 10 % höher als bei den Sub2-DCs. Es fanden sich ca. 10 % mehr CD25+ T-Zellen im Sub1-DC-Ansatz als in den anderen beiden Ansätzen. Der Sekretionsassay erbrachte für alle Sub-DCs eine Th1-Induktion der T-Zellen. Dabei ließen sich keine Abweichungen im Anteil IFN-γ-positiver T-Zellen zwischen den einzelnen Sub-DC-Kulturen nachweisen. Die gewonnen Ergebnisse dieser Arbeit verdeutlichen auf der einen Seite das proinflammatorische Potential (z.B. TNF-α-Sekretion, erhöhte Proliferation), auf der anderen Seite die antiinflammatorische Komponente (IL-10-Sekretion) der CD14++/CD16+ Monozyten. Eine Rolle in der Regulation von entzündlichen und infektiologischen Erkrankungen erscheint für diese Subpopulation denkbar. Die Subpopulation 2 kann dabei als eigenständige Monozytenfraktion betrachtet werden. Die funktionellen Unterschiede zwischen den analysierten Monozytensubpopulationen zeigten sich auch nach deren Differenzierung zu unreifen dendritischen Zellen. In Anbetracht des erhöhten Anteils der CD16+ Monozyten im Rahmen diverser autoimmuner Krankheiten und der immer klarer werdenden Unterteilung in eine CD14++/CD16+ und eine CD14+/CD16+ Subpopulation, sollten weitere Untersuchungen zur klinischen Relevanz dieser Monozytengruppen durchgeführt werden. Die in der vorliegenden Arbeit erzielten Ergebnisse könnten bei der Zuordnung und Interpretation in diese zukünftigen klinischen Befunde behilflich sein. / For more than 20 years monocytes were subdivided in the classical CD14++/CD16- population and the proinflammatory CD16+ populations. The latter one includes about 10-20 % of the peripheral blood monocytes and is dramatically expanded under different pathological circumstances. Recently, the CD16+ fraction was further subdivided into two subpopulations: The CD14++/CD16+ and CD14+/CD16+ monocytes. In the present paper, the subpopulation of CD14++/CD16+ monocytes was characterized in order to answer the question if this subset can be distinguished as an independent cell population. T cells, B cells and NK cells were depleted from peripheral mononuclear blood cells (PBMCs) from healthy donors using immunomagnetic cell separation. Subsequently, the pre-separated cells were stained with anti-CD14-FITC and anti-CD16-PE and separated by fluorescence activated cell sorting (FACS) in three subpopulations: The CD14++/CD16- (Sub1), the CD14++/CD16+ (Sub2) and the CD14+/CD16+ (Sub3). The achieved purity was sufficient for the subsequent experiments. The obtained subpopulations were co-cultured together with heterologous CD4+ T lymphocytes. The reaction of these T cells to the different subpopulations was studied in the proliferation and secretion assay (IFN-γ, IL-4). Furthermore, the cytokine secretion of the monocyte subsets after defined stimulation (LPS, Zymosan, activated T cells) was analysed. In a second set of experiments, immature dendritic cells were differentiated from the monocyte subpopulations (Sub-DCs) and phenotypically and functionally characterized according to the expression of cell surface markers and the response to heterologous CD4+ T cells in the proliferation/secretion assay (IFN-γ, IL-4). After 7 days of incubation, the Sub2 and Sub3 of the monocytes induced approximately 10 % higher proliferation rates of T cells than the Sub1. The resulting frequency of CD25+ T cells in these two co-culture settings was also 10 % higher. The proliferation analysis after 14 days again showed for the Sub3 ca. 10-15 % more proliferating T cells than for the other populations. At this, the frequency of CD25 expression was equal in all co-cultures. In the secretion assay all three subpopulations induced a Th1 response, but the range of IFN-γ-positive T cells was somewhat higher for the CD16+ monocytes. Under LPS stimulation the CD14+/CD16+ monocytes produced the highest amounts of TNF-α followed by the CD14++/CD16+. The CD14++/CD16- showed the lowest amounts of TNF-α. In co-culture with activated T cells the CD14++/CD16+ monocytes demonstrated the strongest TNF-α secretion from all subpopulations. After LPS stimulation, a higher level of IL-1β was measured for both CD16+ populations than for the classical monocytes. In the LPS and the Zymosan stimulated cultures, the IL-6 secretion of the CD14++/CD16- subpopulation was higher than in the two other fractions. Under all three stimuli the highest levels of IL-8 were detected for the CD14++/CD16- monocytes followed by the CD14++/CD16+. The lowest IL-8 production was found by the CD14+/CD16+. The IL-10 secretion of the CD14++/CD16- and CD14++/CD16+ monocytes was increased compared to the CD14+/CD16+ subpopulation after LPS and Zymosan stimulation. The in vitro generated immature dendritic cells from the different monocyte subsets showed a differentiated morphology. The DCs of the CD14+/CD16+ monocytes had the strongest HLA-DR expression compared to the other two Sub-DC populations. No differences in the HLA-DR intensity were found between the Sub1- and Sub2-DCs. The rate of CD11c-positive cells was significantly higher in the Sub1-DCs than in the Sub2-DCs. However, the Sub3-DCs expressed no CD11c altogether. The proliferation assay over 7 days showed no significant differences between the subpopulations. Nevertheless, a tendency for lower levels of proliferating or CD25-positive T lymphocytes was seen in T cells co-cultured with the Sub2-DC. After 14 days, the ratio of proliferating T cells was 10 % higher with the Sub1-DCs than with the Sub2-DCs. The Sub1-DC co-culture yielded ca. 10 % more CD25+ T cells than the other two. The secretion assay revealed for all Sub-DCs a Th1 response of the T cells, with no differences in the amount of IFN-γ-positive T cells between the Sub-DC cultures. The results illustrate on one hand the proinflammatory potential (e.g. TNF-α secretion, higher proliferation), on the other hand the antiinflammatory effect (IL-10 secretion) of CD14++/CD16+ monocytes. A role in the regulation of inflammatory and infectious diseases seems to be possible for this subpopulation. The subpopulation 2 can be regarded as an independent fraction of monocytes. The functional differences between the analyzed monocyte subpopulations are further underscored following differentiation into immature dendritic cells. Considering the increased proportion of CD16+ monocytes in various autoimmune diseases and their clear subdivision in a CD14++/CD16+ and a CD14+/CD16+ subpopulation, new investigations about the clinical relevance are warranted. The findings obtained in the work presented could be in the basis for these future clinical studies.

We comparatively analyzed the natural killer (NK) cell response against HCMV-infected pro-inflammatory (M1) and anti-inflammatory (M2) M[Fi] derived from autologous monocytes. M1 M[Fi] were more resistant to infection, secreting TNF-[alfa], IL-6, IL-12 and type I IFN. By contrast, in HCMV-infected M2 M[Fi] the production of proinflammatory cytokines, type I IFN and IL-10 was limited, and IL-12 undetectable. NK cell degranulation was triggered by interaction with HCMV-infected M1 and M2 M[Fi] and was partially inhibited by specific anti-NKp46, anti-DNAM-1 and anti-2B4 mAbs, thus supporting a dominant role of these activating receptors. By contrast, only HCMV-infected M1 M[Fi] efficiently promoted NK cell-mediated IFN-[gamma] secretion, an effect partially related to IL-12 production. These observations reveal differences in the NK cell response triggered by distinct HCMV-infected monocyte-derived cell types, which may be relevant in the pathogenesis of this viral infection. HCMV infection has been proposed to contribute to the development of atherosclerosis, a chronic inflammatory process in which M[Fi] play a key role. The contribution of HCMV to vascular disease may depend on features of the immune response not reflected by the detection of specific antibodies. Persistent HCMV infection in healthy blood donors has been associated with changes in the distribution of NK cell receptors (NKR). The putative relationship among HCMV infection, NKR distribution, subclinical atherosclerosis and coronary heart disease was assessed. An association of overt and subclinical atherosclerotic disease with LILRB1+ NK and T cells was observed, likely reflecting a relationship between the immune challenge by infections and cardiovascular disease risk, without attributing a dominant role for HCMV. / Hem analitzat la resposta de la cèl•lula NK als macròfags proinflamatoris (M1) i antiinflamatoris (M2) derivats de monòcits autòlegs infectats pel citomegalovirus humà (HCMV). Els macròfags M1 son més reistents a la infecció i secreten TNF-[alfa], IL-6, IL-12 i IFN de tipus I. Per altra banda, en els macròfags M2 infectats per HCMV la producció de citoquines proinflamatories, IFN de tipus I i IL-10 es limitada i la IL-12 indetectable. La cèl•lula NK degranula al interaccionar amb els macròfags M1 i M2 infectats. Aquesta degranulació s’inhibeix parcialment al bloquejar amb anticossos específics anti-NKp46, anti-DNAM-1 i anti-2B4, això indica que aquests receptors tenen un rol important en el procés. En canvi, només els macròfags M1 infectats amb HCMV promouen de manera eficient la producció d’IFN-[gamma] per part de la cèl•lula NK, degut parcialment a la producció de IL-12. Aquestes observacions posen de manifest diferències en la resposta de la cèl•lula NK a diferents tipus de macròfags infectats per HCMV que pot ser relevant en la patogènesis d’aquesta infecció viral. S’ha proposat que la infecció per HCMV contribueix al desenvolupament de l’aterosclerosis, un procés inflamatori crònic en el que els macròfags tenen un paper clau. La contribució del HCMV a la malaltia cardiovascular pot dependre de la resposta immune. La infecció per HCMV en donants de sang sans s’ha associat a canvis en la distribució dels receptors de les cèl•lules NK. S’ha evaluat la possible relació entre la infecció per HCMV, la distribució dels receptors de les cèl•lules NK i l’infart agut de miocardi. S’ha observat una associació de l’infart agut de miocardi i l’aterosclerosi subclínica tant amb les cèl•lules NK LILRB1+ com amb les cèl•lules T LILRB1+. Això possiblement reflexa la relació entre la pressió que les infeccions exerceixen en el sistema immunitari i el risc cardiovascular sense atribuir un paper principal al HCMV.

The TGFβ family of cytokines are potent signalling molecules that regulate tissue development, inflammation and immunity. Previous studies in mice with a null mutation in the Tgfb1 gene (TGFβ1-/- mice) implicate a key role for TGFβ1 in male reproductive function. These mice show profound infertility due to an inability to copulate successfully, associated with reduced testosterone and sperm production. The focus of this project was to 1) further characterize mechanisms underpinning reproductive deficiency in male TGFβ1-/- mice, 2) identify a reliable physiological marker of TGFβ1 availability in vivo, and 3) to determine whether exogenous TGFβ1 administration influences TGFβ1 availability and restores fertility. To investigate the causes of unsuccessful copulation by TGFβ1-/- mice, penis morphometry was examined. Penile organ structure, as assessed by scanning electron microscopy, was comparable between genotypes however a superfluous epidermal covering that impeded penile spine protrusion was evident in TGFβ1-/- mice. The epidermal covering was not due to increased epithelial cell proliferation, as measured by Brdu labelling and immunohistology. Behavioural observations of erectile activity showed that TGFβ1-/- mice achieved spontaneous erections albeit at reduced frequency compared to TGFβ1+/+ mice. The efficacy of exogenous TGFβ1 replacement was evaluated by first identifying measures of in vivo TGFβ1 availability and/or function and selecting an effective route of administration. Serum TGFβ1 and testosterone levels were reliable discriminators of TGFβ1 genotype. Gene expression and phagocytic function of peritoneal macrophages revealed no differences between genotypes. Exogenous sources of TGFβ1 for replacement studies included colostrum, naturally occurring in breast milk and recombinant human latent TGFβ1 (rhLTGFβ1). Colostrum did not increase circulating levels and rhTGFβ1 injection caused only transient elevation of serum levels. Thus mini-osmotic pumps were used to deliver a constant supply of cytokine to TGFβ1-/- mice. The fertility status of TGFβ1-/- mice receiving exogenous TGFβ1 was investigated. Reproductive behaviour in response to normal receptive female mice was assessed twice during treatment, on day 7 and day 14. Blood, liver and reproductive tissues were collected at sacrifice. Circulating TGFβ1 was increased in TGFβ1 treated TGFβ1-/- mice above TGFβ1-/- control levels, although this did not affect circulating testosterone. Erectile activity and sperm production were unchanged. Videotaping behaviour with estrous females revealed that the TGFβ1+/+ mice successfully mounted and intromitted, unlike the TGFβ1-/- controls. The TGFβ1-/- mice receiving exogenous TGFβ1 displayed moderately enhanced mounting and intromission behaviour although this remained less frequent than in the TGFβ1+/+ controls. Ejaculation behaviour was not observed in any TGFβ1-/- mice regardless of TGFβ1 replacement, compared to TGFβ1+/+ controls where >90% mice displayed ejaculated. Modest improvement in the copulation activity of the TGFβ1-/- mice receiving exogenous TGFβ1 suggests that systemic TGFβ1 availability can influence reproductive performance in male TGFβ1-/- mice. However since fertility was not restored, locally produced TGFβ1 in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility. These findings advance our knowledge of the role of the TGFβ1 cytokine in male reproductive physiology and may have relevance for devising new treatments for infertility and erectile dysfunction in men. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008