Injuries to the peripheral nervous system (PNS) are major and common source of disability, impairing the ability to move muscles and/or feel normal sensations, or resulting in painful neuropathies. Annually traumatic nerve injuries resulting from collisions, gunshot wounds, fractures, motor vehicle accidents, lacerations, and other forms of penetrating trauma, affected more than 250,000 patients just in the U.S. The clinical gold standard to bridge long non-healing nerve gaps is to use a nerve autograft- typically the patient’s own sural nerve. However, autografts are not ideal because of the need for secondary surgery to ‘source’ the nerve, loss of function at the donor site, lack of appropriate source nerve in diabetic patients, neuroma formation, and the need for multiple graft segments. Despite our best efforts, finding alternative ‘nerve bridges’ for peripheral nerve repair remains challenging – of the four nerve ‘tubes’ FDA approved for use in the clinic, none is typically used to bridge gaps longer than 10 mm due to poor outcomes. Hence, there is a compelling need to design alternatives that match or exceed the performance of autografts across critically sized nerve gaps. Here we demonstrate that early modulation of innate immune response at the site of peripheral nerve injury inside biomaterials-based conduit can favorably bias the endogenous regenerative potential after injury that obviates the need for the downstream modulation of multiple factors and has significant implications for the treatment of long peripheral nerve gaps. Moreover, our study strongly suggests that more than the extent of macrophage presence, their specific phenotype at the site of injury influence the regenerative outcomes. This research will advance our knowledge regarding peripheral nerve regeneration, and help developing technologies that are likely to improve clinical outcomes after peripheral nerve injury. The significant results presented here are complementary to a growing body of evidence showing the direct correlation between macrophage phenotype and the regeneration outcome of injured tissues.

Le Trypanosome africain, dont Trypanosoma brucei est le prototype, est un parasite sévissant en Afrique sub-tropicale. Il est responsable de la maladie du sommeil chez l’Homme et de diverses affections chez les animaux tant sauvages que domestiques. T. brucei est un parasite extracellulaire qui se développe dans le sang de son hôte mammifère. Il est donc confronté en permanence au système immunitaire de l’hôte et a en conséquence, afin de générer un environnement plus favorable à sa croissance, établit différents mécanismes d’échappement tels que la variation antigénique ou l’immunomodulation. Dans ce contexte, il a été montré que T.brucei libère des facteurs capables d’induire la voie arginase des macrophages. Cette induction peut favoriser la croissance des trypanosomes dans le sang de leur hôte de diverses manières. Premièrement, l’arginase participe à la synthèse de composés tels que les polyamines ou la trypanothione, facteurs de croissance des cellules. Deuxièmement, l’arginase partage le même substrat que la NO synthase inductible (iNOS), ces deux enzymes sont donc en compétition et l’activation de l’arginase pourrait contribuer à diminuer la quantité de NO, composé cytostatique et cytotoxique, produit par les macrophages en limitant le substrat disponible pour l’iNOS. Troisièmement, la déplétion du milieu en arginine suite à l’activation de l’arginase inhibe la prolifération de cellules du système immunitaire dont les lymphocytes T. Nous avons identifié une chaîne lourde de kinésine chez T.brucei, TbKHC1 (Trypanosoma brucei Kinesin Heavy Chain 1), appartenant à la superfamille des kinésines, comme un candidat potentiellement capable d’induire la voie arginase des macrophages. TbKHC1 est principalement exprimée au stade sanguicole du parasite et est localisée au niveau de la région endo-exocytaire. Dans un modèle d’infection murin, une invalidation de l’expression de TbKHC1 (par ARN interférence ou par knock-out) conduit à une diminution du premier pic de parasitémie et à une prolongation de la survie des souris infectées. Nous avons montré que TbKHC1 joue un rôle dans l’interaction hôte/parasite à deux niveaux indépendants : premièrement, l’induction de la voie arginase des macrophages par TbKHC1 en début d’infection favorise la croissance du parasite et son établissement au sein de son hôte. Deuxièmement, elle joue un rôle dans l’induction de la pathologie liée à l’infection.

Natural autoantibodies are defined as self-reactive antibodies present in healthy individuals in the absence of purposeful immunization or any known antigenic stimulation. While most NAAbs are of the IgM isotype, T cell dependent isotypes such as IgG and IgA NAAbs have been reported. Natural antibodies are conserved through evolution; they have been detected in sharks and humans, the phylogenetic limits of vertebrates capable of producing antibodies. The phylogenetic conservation of NAAbs suggests that they play an important, but overlooked, role in the maintenance of physiological homeostasis of the immune system.My project involved the characterization of two NAAbs. The first part of my dissertation elucidates the production and functional characterization of an anti-T Cell Receptor NAAb, OR3. OR3 is an IgM heterohybridoma made from the peripheral blood B cells of a patient with Rheumatoid Arthritis. The results show that OR3 specifically binds to the Complementarity Determining Region 1 segment of TCRV b8 regions, is specific for a subset of T cells and blocks the antigen activation of these T cells. Importantly, OR3 does not induce apoptosis or necrosis of T cells. My results support our hypothesis that anti-TCR NAAbs are immunomodulatory and indicate that autoantibodies present in humans may have significant functional activity in the regulation of T cell responses.The second part of the dissertation describes the phenomenon of NAAbs to human delta opioid receptor. I affinity purified IgG antibodies to human DOR from intravenous immunoglobulin, a therapeutic blood product that contains purified IgG isolated from the plasma of thousands of healthy donors. The anti-DOR NAAbs bind to DOR protein, and initiate an activating signaling cascade, in a manner similar to a receptor specific agonist. Interestingly, there is a significant difference in the agonistic activity of the autoantibodies compared to the synthetic DOR agonist DPDPE. Unlike DPDPE, these autoantibodies display significant immunomodulatory activity as evinced by changes in CCR5 and CXCR4 chemokine receptor expression. The presence of functional autoantibodies in IVIG shows that they are a part of the normal healthy immune repertoire in humans. This work also presents data supporting the interconnecting network between the neuroendocrine and immune systems.

Pseudomonas aeruginosa is a significant pathogen of immunocompromised individuals, and the main mechanism by which it mediates virulence is through the coordination of gene expression by an intricate quorum sensing system. One of the signalling molecules of this system, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to have immunomodulatory capabilities, discrete to its quorum sensing role. While the general focus of research in this area is on the physiological outcomes of this interaction on cell function, there is currently little concrete evidence identifying the receptor(s) for OdDHL in mammalian cells, and hence the biochemical mechanism behind the immunomodulation caused by OdDHL remains largely unknown. This study identifies peroxisome proliferator-activated receptor γ (PPARγ) as a mammalian target for OdDHL. PPARγ is a mammalian transcription factor involved in fatty acid metabolism and is heavily involved in the inflammatory response, being a negative regulator of inflammation. It is shown here that OdDHL is able to instigate signalling through PPARγ by activation of the ligand binding domain (LBD), suggesting that OdDHL may act as a PPARγ agonist. OdDHL is able to compete with the PPARγ agonist, rosiglitazone, causing a relative antagonism of PPARγ activity when given in tandem with the agonist. The bacterial signalling molecule is unable to displace the irreversible PPARγ antagonist GW9662. This effect on PPARγ is specific to OdDHL, as the smaller P. aeruginosa signalling molecule, N-butyryl-L-homoserine lactone had no significant effect on PPARγ activation. In order to confirm PPARγ as a putative receptor for OdDHL in mammalian cells, initial experiments were undertaken to optimise conditions to produce PPARγ LBD protein for binding interaction studies. The fidelity of the protein sequence was established and expression of the protein in an appropriate vector was confirmed. The protein produced was insoluble and hence not functional for binding studies, suggesting that additional optimisation of expression conditions, or manipulation and refolding of the protein is necessary before further experimentation can take place. The identification of PPARγ as a receptor for OdDHL in mammalian cells is an important step in understanding the nature and scope of the interaction between OdDHL and host cell physiology, especially the significance of this interaction during P. aeruginosa infection. Continuation of this research, in particular completion of protein-ligand binding studies between OdDHL and PPARγ has the potential to clarify the significance of the immunomodulation caused by OdDHL, while providing us with a platform from which we may exploit it.

La leishmaniose est une maladie tropicale négligée pour laquelle l’arsenal thérapeutique actuel est limité. Ce travail de thèse s’est intéressé à rechercher des nouvelles cibles thérapeutiques en ciblant la paroi des leishmanies. Le lipophosphoglycane (LPG), constituant majoritaire de la paroi, présente un motif glucidique particulier, le galactofuranose, qui semble une cible thérapeutique intéressante car il est absent des membranes de mammifères. Les galactofuranosyl-transférases sont impliquées dans le métabolisme de ce furanose, et ce travail a débuté par l’étude de ces enzymes et par la caractérisation d’une mutase, également nécessaire au métabolisme du galactofuranose. Une fois les cibles caractérisées dans les 2 stades du parasite, des analogues du galactofuranose ont été testés quant à leur capacité antiparasitaire sur les formes promastigotes et amastigotes de Leishmania donovani. Un composé s’est révélé intéressant et a été plus étudié, le n-octyl-galactofuranose (Galf). Différentes approches ont été utilisées pour caractériser son mode d’action sur les promastigotes et les amastigotes : résonance paramagnétique électronique, microscopie électronique à transmission, cytométrie en flux ou résonance magnétique nucléaire. L’observation d’une activité inductrice du métabolisme oxydatif des macrophages nous a conduits à nous intéresser aux capacités immunomodulatrices de ces analogues galacto-furanosidiques. Ainsi la dernière partie de ce travail est consacrée à l’étude de la polarisation des macrophages par les galactofuranosides, sur un modèle in vitro de macrophages humains. Nous avons pu montrer que le Galf exerçait une activation des macrophages en faveur d’une polarisation de type M1, ce qui pourrait expliquer l’effet limitateur de croissance des amastigotes. / Leishmaniasis is a neglected tropical disease for which the current therapeutic arsenal is limited. This work aimed at finding new therapeutic drugs by targeting the Leishmania cell wall. Lipophosphoglycan (LPG) is the major glycoconjugate in promastigotes cell wall, consisting of a hexasaccharide core including a galactofuranose motif. Galactofuranose is absent in mammalian membranes, thus could be a therapeutic target. First, this work studied the galactofuranosyl-transferases involved in the metabolism of this furanose, as well as a mutase, also necessary for the metabolism of galactofuranose. Once targets were identified in the two parasitic stages, galactofuranose derivatives were tested for antileishmanial activity on promastigotes and amastigotes forms of Leishmania donovani. A compound showed interesting results and has been studied further, the n-octyl-galactofuranose (Galf). Different techniques have been used to characterize its mode of action on promastigotes and amastigotes: electron paramagnetic resonance, transmission electron microscopy, nuclear magnetic resonance or flow cytometry. Infected macrophages treated with Galf were able to produce oxygen derivatives species, leading us to look at the immunomodulatory capacity of Galf derivatives. Thus, the last part of this work focused on the study of macrophage polarization by galactofuranosides on an in vitro model of human macrophages. We were able to show that Galf stimulates macrophages towards M1 polarization, which could explain the decreased growth of amastigotes inside macrophage cells.

L’ifosfamide (IFO) est un alkylant de l’ADN de la famille des Oxazaphosphorines, largement utilisé dans divers protocoles de chimiothérapie. Son utilisation en clinique est néanmoins limitée en raison d’une néphrotoxicité et une neurotoxicité liées aux métabolites tels que le chloroacétaldéhyde. En effet, l’IFO est une prodrogue présentant un profil pharmacocinétique complexe dont la voie métabolique activatrice est minoritaire et la voie toxicogène majoritaire. La pharmacomodulation chimique est une des stratégies proposées afin de contourner la voie toxicogène et ainsi améliorer son utilisation en clinique. Des analogues de l’IFO (géranyloxy-, farnesyloxy-, squalenyloxy-IFO), capables de s’auto- assembler, ont été développés et les études pharmacologiques ont démontré la preuve de concept de pré-activation de l’IFO. Des optimisations galéniques ont été testées en vue d’une application en pré-clinique puis en clinique. Par ailleurs, la proximité chimique de l’IFO et du cyclophosphamide, nous a conduit à mener des études d’immunomodulation afin d’explorer les propriétés immunomodulatrices de l’IFO et du géranyloxy-IFO et d’ouvrir de nouvelles perspectives dans l’utilisation de ces composés en association avec l’immunothérapie. / Ifosfamide (IFO) is an alkylating agent of the DNA from Oxazaphosphorine family, widely used in various chemotherapy protocols. However, its clinical use is limited due to nephrotoxicity and neurotoxicity related to metabolites such as chloroacetaldehyde. Indeed, IFO is a prodrug characterized by complex pharmacokinetic profile with a minority activating metabolic pathway and a majority toxicogenic pathway. Chemical pharmacomodulation is one of the strategies proposed to bypass the toxicogenic pathway and thus improve its clinical use. IFO analogues (geranyloxy-, farnesyloxy-, squalenyloxy-, squalenyloxy-IFO), able to self-assembly, have been developed and pharmacological studies have demonstrated the proof of concept for the pre-activation of IFO. Galenic optimizations have been tested for pre-clinical and clinical application. In addition, due to the chemical similarity of IFO and cyclophosphamide, immunomodulation studies have been conducted to investigate the immunomodulatory properties of IFO and geranyloxy-IFO with the hope of new perspectives in the use of these compounds in combination with immunotherapy.

No description available.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.

Neuromodulation of the immune system has been described to be influenced by hemispheric lateralisation (HL), the stable tendency to relatively utilise one hemisphere or its functions over another. To date there has not been a systematic review of research in this phenomenon conducted, and only one study has examined the effects of HL on the progression of a disease – Human Immunodeficiency Virus (HIV). That research was conducted on a small sample with little control for confounders. The present work sought to compile a systematic review of literature concerning HL and immunity in humans, using effect size analysis. Further, the present work also describes an empirical advancement of this earlier HIV study with stricter control over confounds in a larger sample. The findings corroborated the theory of asymmetrical immune influence by HL via the systematic review showing clear, relatively consistent and strong relationships between left-HL and immunopotentiation. The empirical prospective study extended current knowledge of this relationship in HIV to identify a moderator – HAART treatment. Specifically, left-HL predicted better immunity in HIV-1 patients independent of confounders, with further findings of the same pattern in untreated patients, but not in HAART-treated patients. Further observations were made between HL and HIV-relevant behaviours, again adding to current knowledge. The finding of left-HL being associated with fewer sexual partners in Europeans presents new information of relevance to public health. The combined findings of the present work suggest that left-HL has predictive value in illness (HIV-1) and in general immunity. The present work adds to the existing knowledge new information concerning a moderating factor of the HLimmunity relationship in HIV, and behavioural implications of HL which impact upon HIV disease. Potential explanations for moderation, proposals for neurobiological mechanisms and direction towards future, more rigourous study in the field, both in HIV and immunity, are discussed.

Despite the success of modern medicine in treating infections, infectious diseases remain a major source of morbidity and mortality worldwide. The evolution of antibiotic resistant strains of bacteria means that new innovations in therapeutics must be pursued to combat this emerging threat. A novel approach is to utilize the anti-infective properties of endogenous host defense peptides by creating smaller synthetic peptides with enhanced protective activities. Some of these peptides directly kill bacteria and many display varied immunomodulatory activities, enhancing the host innate immune response to more effectively clear an infection. Here I examined the efficacy of several synthetic peptides in a murine model of invasive bacterial infection, induced by the Gram positive bacterium Staphylococcus aureus. Several peptides were able to significantly reduce peritoneal bacterial load in vivo by up to 4-logs relative to the controls, either through direct antibacterial killing or immunomodulatory activity. The latter class was studied in more detail; in particular, the peptides IDR-1 and 1002 displayed significant immunomodulatory effects in vivo. Both peptides were able to significantly induce the proinflammatory chemokines MCP-1, RANTES and KC, as well as increased recruitment of neutrophils and monocytes to the site of infection. These effects were not dependent on live bacteria, as heat inactivated S. aureus was also able to induce chemokines and cell migration. Mice that had been depleted of macrophages did not respond to peptide treatment, indicating that macrophages are an important effector cells through which immunomodulatory peptides counter infections. These results suggest that synthetic peptides have the potential to become a viable treatment option for bacterial infections.