<p>In spite of the tremendous body of cancer research, cancer remains a significant health issue requiring development of better therapeutics. The elucidation of the relationship between cancer and the immune system and the identification of tumour associated antigens together suggest that novel therapeutics using the immune system to target cancer is a promising avenue of research. Since immunological tolerance is a barrier to generating immune responses to self antigen, strategies to circumvent tolerance need to be investigated to target given antigens.</p> <p>Plac1 is a novel tumour associated antigen with expression restricted to placenta, testis and many tumour cells. Initial reports concerning the expression, immunogenicity and potential tumourigenic function of Plac1 suggest that it would be an ideal tumour antigen. Initial experiments in mice indicated that generating an immune response against the murine Plac1 would be difficult and the subsequent work sought to employ strategies to facilitate anti murine Plac1 immune responses and anti tumour efficacy in Plac1 expressing tumours.</p> <p>Another more studied tumour associated antigen is gp100. Unlike Plac1, immune responses against the murine gp100 can be generated through vaccination. These responses are unable to demonstrate any anti tumour activity in gp100 expressing cells. The bulk of the gp100 studies described here sought to modify the immune:tumour interaction such that the anti tumour activity of the anti gp100 responses could be improved.</p> <p>While the specific barriers to Plac1 vaccination and efficacy and gp100 vaccination and efficacy are different, they have in common that they represent likely issues in using therapeutic cancer vaccines clinically. In both cases investigating how these barriers can be overcome is important and relevant to the understanding of these barriers to success when they appear in the clinic.</p>

<p><strong>Background:</strong> Early infancy is a critical period during which the interplay between host and environmental factors influences susceptibility to allergic sensitization, a process that can also be construed as a failure to induce tolerance. Indeed, allergic asthma emerges, in most instances, in early childhood although the specific intervals of protection or susceptibility remain to be elucidated. We found that exposure to a concentration of allergen, house dust mite (HDM), that normally induces robust airway inflammation in adult mice elicits negligible immune-inflammatory responses in infant mice.</p> <p><strong>Methods:</strong> We investigated immune-inflammatory responses in mice exposed to 25 μg of HDM intranasally for 10 consecutive days at different points in development (3, 4, 5 and 7 weeks of age). We delineated the immune cell profile in the lungs of naïve mice from birth to adulthood, focusing on markers of immune maturation and immunosuppression. Moreover, we studied the impact of T-regulatory cell (Treg) depletion with the use of α-CD25 antibodies administered intraperitoneally one day prior to the start of HDM exposures, and then again on day 6 of the above protocol.</p> <p><strong>Results:</strong> Our data show that there is a progressive acquisition of immune-inflammatory responsiveness to HDM in BALB/c mice as exposures are initiated later in development, evidenced by total cell number and eosinophilia in the BAL and serum HDM-specific IgG₁ levels. Additionally, there is an immunological shift that occurs in the infant lung during development in that the early immunosuppressive environment, defined by T-regulatory cells and immunosuppressive alveolar macrophages, subsides as the capacity to respond to ensuing immune challenges, defined by natural killer (NK) cell, dendritic cell (DC) and alveolar macrophage (AM) maturation, increases. Specifically, in regards to the immunosuppressive lung environment during infancy, we identified higher baseline levels of CD25⁺Foxp3⁺CD101⁺ and CD25⁺Foxp3^highTregs, i.e. those with more potent suppressive ability. These populations also expand following HDM exposure in both adult and infant mice. Interestingly, 2 week-old infant mice depleted of Tregs or exposed to a very high dose of HDM (125 μg) overcome the natural immunosuppressive environment resulting in the acquisition of HDM responsiveness, as manifested by robust Th2 immune-inflammatory responses, comparable to that observed in 8 week-old adult mice.</p> <p><strong>Conclusion/Implications: </strong>Together, our data suggest that the hyporesponsiveness to HDM very early in life may be explained by two connected events: a) the inherent immunosuppressive environment in the lung, and b) the immaturity of the machinery for effective immune responses. Furthermore, we demonstrate that a disruption in the homeostatic immune balance in infancy, Treg depletion in this case, may lead to the imprinting of aberrant immune-inflammatory responses, like allergen sensitization. Thus, the inherent immunosuppressive environment in infancy may have long-term implications on allergen responsiveness.</p> / Master of Science in Medical Sciences (MSMS)

<p>PPAR agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists have been shown to inhibit peripheral eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including decreased cytokine/chemoattractant (IL-5/eotaxin) release, decreased eosinophil migration and/or decreased eosinophil differentiation. This is the first study to show that PPARγ is expressed at the protein level in human airway eosinophils sampled from induced sputum, and confirms PPARγ protein expression in human peripheral blood eosinophils. We demonstrated the novel observation that peripheral blood eosinophil PPARγ protein expression, as measured by flow cytometry, is not different in eosinophils purified from asthmatic subjects compared to healthy controls and these observations suggest that the level of PPARγ expressed in human eosinophils is not related to asthmatic status. Our study also confirms, by real time PCR, the detection of mRNA for PPARγ in airway-derived leukocytes, collected from bronchial washings, increases 24hrs after whole lung allergen challenge. This increase is regulated by Symbicort® and Pulmicort® treatment in most subjects. This is the first study to show increased chemokinesis (random stimulated movement) of eosinophils <em>in vitro</em> at low concentrations of a PPARγ agonist. We have generated data to suggest this is through an effect on calcium signalling. We also observed that higher concentrations of PPAR agonists directly inhibit eotaxin-stimulated eosinophil directional migration. Finally, using methycellulose cultures of non-adherent mononuclear cells and CD34+ progenitor cells, we demonstrate that PPAR activation can inhibit the differentiation of eosinophils <em>in vitro</em>. Collectively, these data demonstrate that PPARγ is expressed consitutively on eosinophils in peripheral blood and airways, and suggest signaling through this receptor with nanomolar concentrations of agonist regulates eosinophilia through inhibition of both eosinophil migration and eosinophil differentiation.</p> / Doctor of Philosophy (PhD)

<p>Currently, there is no effective cure for Inflammatory Bowel Disease (IBD), medications are aimed solely at alleviating symptoms and not curative. Great scientific efforts have been aimed at elucidating the mechanisms underlying the pathogenesis of IBD. Macrophages—antigen presenting cells—play a chief role in the pathophysiology of IBD. It has been proposed that CD36 receptor present on the surface of macrophages, may play a role in the inflamed intestine. CD36-expressing macrophages have been implicated in a variety of human diseases; however the role of CD36+ macrophages in the intestine has been limited. The aim of this study was to decipher whether or not CD36+ F4/80+ macrophages are inflammatory in the colonic intestine. Our study discovered the proportion of CD36+ F4/80+ macrophages were markedly upregulated in active IBD patients and TNBS-induced colitis mice. CD36+ macrophages isolated from the LPMC of the small and large intestine of Balb/c treatment groups(a) 12TNBS, (b) MHS+12TNBS, and (c) MHS+PA mice confirmed these macrophages expressed some level of proinflammatory cytokine TNF-α. Two macrophage ligands, LCFA: Palmitic Acid and PGN, in conjunction or separately, appeared to be two culprits which induced MHS macrophages to produce TNF-α both in vitro and in vivo. It is possible these two ligands may work in concert, however the mouse model has yet to be examined. The precise role(s) of these CD36+ F4/80+ macrophages requires further scientific inquiry and elucidation in the context of intestinal inflammation. It is quite possible understanding the mechanisms and roles of these macrophages will greatly advance our knowledge in the pathophysiology of IBD and potential therapeutic treatments or targets.</p> / Master of Science (MSc)

<p>Cancer vaccine therapy aims at harnessing effective antigen specific immune responses to treat tumor. In particular, CD8+ T cells have the unique capacity to recognize and destroy tumor cell throughout the body. One potential approach to elicit high numbers of effector CD8+ T cells to control tumor growth is through repeated vaccination, a strategy called prime-boost vaccination. However, booster immunization is relatively inefficient during primary immune response because pre-activated effector T cells tend to impair robust antigen presentation. This phenomenon has been interpreted as a negative feedback mechanism where recently activated CD8+ T cells clear the antigen-bearing dendritic cells (DCs) and prevent memory T cells from the access of the boosting antigen. Interestingly, however, using in vitro activated B cell as a viral vector delivery system, we can boost T cell responses with the minimum viral input at a very short interval between immunizations. This B cell carrier is capable of delivering different viral vectors expressing different antigens, displaying a potential for broad application. The mechanisms behind B cell carrier-mediated efficient secondary responses are three fold: 1. Without the engagement of MHC molecules and antigen presentation, B cell carrying viral vector can bypass the killing by pre-existing effector T cells 2. B cells can delivery viruses to B cell follicles, a place separated from effector T cells, and mediate memory T cell expansion. 3. B cells can deliver antigen to both spleen and lymph node and induce antigen specific T cell expansion in multiple lymphoid organs. Our studies provide a novel boosting platform to accelerate CTL responses that has important clinical implications.</p> / Master of Science (MSc)

<p>Pulmonary infections caused by viruses, bacteria, mycobacteria and fungi, are a leading cause of death world-wide. Intracellular pathogens such as influenza virus and <em>M.tb</em> live inside host cells, making it difficult for the host to eliminate the pathogen. Adaptive T cell immune responses are required to clear intracellular pathogens. Rapid T cell priming and recruitment of effector T cells to the lung is critical to eliminating the pathogen and ultimately host survival, however, unchecked T cell responses can be detrimental. The research in this thesis examines T cell priming during pulmonary intracellular infections and examines the consequences of impaired/enhanced T cell responses at the lung mucosa.</p> <p><em>M.tb</em>, the bacterium that causes tuberculosis evades detection by the host and delays T cell priming. This delay is believed to allow <em>M.tb</em> to establish chronic infection. We show that DAP12 deficient mice prime T cell responses days sooner than wt controls, resulting in enhanced control of virulent <em>M.tb</em>. Enhanced T cell priming in DAP12 deficient mice is due to increased antigen presentation by professional antigen presenting cells in the local draining lymph nodes. These findings indicate that accelerating antigen presentation in the lymph node and T cell priming can dramatically improve host resistance to tuberculosis infection.</p> <p>Influenza infection in DAP12 deficient hosts also results in enhanced T cell activation however caused lethal lung pathology. We identified a previously unidentified role for CD4 T cells expressing FasL causing immunopathology. Furthermore, we show that DAP12 deficient antigen presenting cells are responsible for priming hyperactivated CD4 T cells and contribute to influenza mortality.</p> <p>Because T cell responses are so important in host defence against intracellular infection we examined the feasibility of using ex-vivo manipulated antigen presenting cells as a vaccine to generate T cell responses in the lung. Collectively these findings shed light on the factors which regulate host immune responses in the lung following intracellular infection and provide evidence that antigen presenting cells can be manipulated ex-vivo to elicit protective immune responses at the lung mucosa.</p> / Doctor of Philosophy (PhD)

<p>Autophagy is a cellular process in which cytoplasmic material is lysosomally degraded into its basic components. The primary functions of this process are cellular recycling and stress mitigation however it also has roles in both viral pathogenesis and tumourigenesis. Beclin1 is a key mediator of autophagy and is involved in its initiation. In an attempt to examine the effects of enhanced autophagy in the context of oncolytic VSV infection, a VSV mutant (ΔM51) expressing Beclin1 was constructed and characterized. It was determined through western blot analysis of autophagy marker LC3, that while VSV infection enhanced autophagy in infected cells, Beclin1 expression resulted in a transient increase in autophagy followed by markedly reduced levels of autophagy at mid to late time points. Still, Beclin1expression, either directly or possibly through altering the kinetics of VSV induced autophagy, enhanced the pathogenesis of VSV<em> </em>in some cell lines <em>in vitro</em>. However examination of the <em>in vivo</em> pathogenesis of VSV-Belcin1 elicited no differences from that of the parental virus. Despite enhanced pathogenesis in CT26 cells <em>in vitro</em>, VSV-Beclin1 displayed no improvement in the oncolysis of CT26 tumours <em>in vivo</em>, compared to VSV-GFP. It is hoped that the conclusions drawn from this study will help direct future research aimed at exploring the relationship between autophagy and VSV pathogenesis as well as future attempts to arm VSV with the intent of augmenting its oncolytic potential.</p> / Master of Science (MSc)

<p>Asthma is a chronic immune-inflammatory disease of the airways, characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR), and is associated with the development of airway remodeling. While our understanding of the pathophysiology of allergic asthma has increased remarkably in the last few decades, the origins of the disease remain elusive. Indeed, studies indicate that the prevalence of allergic asthma, has increased dramatically over the last 30 years. Within this context, a number of environmental factors including respiratory viral infections have been associated with the onset of this disease but causal evidence is lacking. The work presented in this thesis examines the interactions between a respiratory viral infection, specifically influenza A, and the common aeroallergen house dust mite (HDM) in an experimental murine model. To this end, we investigated the impact of an acute influenza A infection on the exposure to a subclinical dose of HDM (Chapter 2) and addressed potential underlying immune mechanisms using a global, genomic approach (Chapter 3). Our data demonstrate an enhancement of immune inflammatory responses to HDM and reveals multiple immune pathways by which influenza A may enhance the response to subsequent allergen exposure. Collectively these immune pathways are capable of lowering the threshold of HDM responsiveness. Lastly, as allergic asthma develops in most instances during infancy, we investigated the impact of an influenza A infection on allergen responses in infant mice (Chapter 4). In this setting, acute influenza A infection subverts constitutive allergen hyporesponsiveness thus resulting in sensitization, airway inflammation and, ultimately, structural and functional alterations persisting into adulthood.</p> / Doctor of Philosophy (PhD)

<p>Despite the advanced knowledge of the mechanisms of influenza infection and improved vaccines, Influenza A Virus still causes a life-threatening respiratory disease, especially during pandemics. Past investigations have proposed a synergism between Influenza A virus and a simultaneous or subsequent bacterial superinfection as the predominant cause of death. The recent development of animal models to study these heterologous infections has shed light onto the diverse mechanisms by which Influenza A Virus may increase the susceptibility to contract a secondary bacterial infection. These studies suggested an important role for the innate immune system in mediating such disease. We developed a model of heterologous infection combining Influenza A Virus and <em>Bordetella parapertussis</em> that demonstrated a critical role for MIP-2 to drive pulmonary neutrophilia in the pathology associated with bacterial superinfection of influenza. However, the origin of this increased MIP-2 production and the mechanisms underlying the immunopathology remained to be elucidated. The present studies proposed IL-1β overproduction as the upstream cause of the increased MIP-2 production observed in heterologous infection. This exaggerated IL-1β production was likely related to the increased bacterial burden observed in heterologously infected mice. This study also demonstrated that reduction in IL-1β production by blockade of the inflammasome seemed to provide an improvement in the clinical symptoms and the immunopathology of the disease. Thus, interventions to attenuate the exacerbated bacterial burden and the inflammatory responses derived from the subsequent IL-1β overproduction should be further investigate as possible therapeutic approaches to treat bacterial superinfections.</p> / Master of Science (MSc)

<p>Allergic atopic asthma is a respiratory condition that involves immune responses to specific allergens resulting in coughing, wheezing, shortness of breath and tightness in the chest. During an atopic asthmatic attack, the immune system initiates cellular infiltration of lymphocytes and eosinophils, airway hyper-responsiveness and ECM remodeling, which manifests in lung dysfunction in chronic disease. ASMC have recently been shown to play a role in the inflammatory processes of asthma through the production of inflammatory mediators. Various cytokines and chemokines serve as stimulants for these pathways and therefore require further attention to examine inflammatory signaling. OSM, a member of the gp130 family of cytokines, is secreted by inflammatory cells and has been detected in the sputum of asthmatics. Previous findings have established the potential of OSM in induction of lung inflammation, its role in increasing ECM, and its potential role in asthma. Viral or bacterial infections cause asthma exacerbations which result in increased severity of symptoms. The innate immune system relies on pattern recognition receptors including the TLRs to recognize invading pathogens and activate cells such as macrophages and natural killer cells. Although there are a number of these TLRs, this project will focus on the role of TLR3 and TLR4 in ASMC. I generally hypothesized that OSM markedly increases lung cell airway smooth muscle cell responses to external stimulae, such as products of bacteria or viruses that activate toll-like receptors. This exacerbates inflammation and extracellular matrix remodeling which contributes to pathology in asthmatic patients. Findings in this thesis have demonstrated that OSM stimulation increases the production of various cytokines and chemokines and growth factors seen in asthma. Co-stimulations with OSM and TLR-ligands augmented the production of a variety of these inflammatory mediators in comparison to ligands alone. TLR responses were shown to be associated with TLR expression, at both the mRNA and protein level, as well through the activation of the JAK-STAT and NFκB pathways. These findings implicate ASMC in immunomodulatory roles in response to TLR-ligands and OSM, and could play a role in the increased severity of asthma seen during exacerbations.</p> / Master of Science (MSc)