<p>Prenatal life events have been long observed to be able to influence disease into adulthood in both epidemiological and animal studies. Prenatal stress (maternal stress during gestation) is one of such factors that has been shown to impact cognition and behaviour of the offspring. However, the effects of prenatal stress on the immune system are not understood. This study has evaluated the effects of prenatal stress on a murine model. Prenatal stress increased allergic airway inflammation in male, but not female offspring following sensitization and challenge with cockroach extract. This corresponded with stress-induced changes in the immune environment of non-sensitized animals. These changes included a decrease in regulatory T cells at baseline in males compared to non-stressed controls and increased splenic dendritic cell percentage and cytokine, particularly IFN-γ, secretion compared to prenatally stressed females. In females, prenatal stress decreased allergic inflammation, which corresponded to a decreased percentage of dendritic cells in the lung and mesenteric lymph node. Prenatal stress did not affect dendritic cell antigen presentation in ether male or female offspring. There was no evidence to suggest a prenatal stress induced change in glucocorticoid sensitivity of dendritic cells. In order to explore the possibility of prenatal stress induced decrease of parasympathetic output, a vagotomy model was used as a proof of concept in naïve animals not exposed to prenatal stress. Vagal modulation of dendritic cell phenotype and function was assessed. While there was some evidence that vagotomy may indirectly modulate dendritic cell function, its effects on the immune system were different then the changes caused by prenatal stress and thus it is a role of reduced parasympathetic output was not supported. Overall this data indicates a role of prenatal stress on the immune system with clear sex differences, but the mechanism for how this occurs is currently unknown. Further research is needed to investigate the role of TLRs and IFN-γ in this model, as well as other possible mediators of prenatal stress such as the changes to the parasympathetic nervous system that may in turn mediate alterations to the immune system. Differences in when the effects of prenatal stress are expressed during postnatal life are discussed.</p> / Master of Science (MSc)

Nesta tese, foi apresentado um estudo sistemático para o desenvolvimento de uma nova formulação do veneno de abelha (BV) encapsulado em microesferas (MS) de PLGA para VIT. Estudou-se a estabilidade das proteínas do BV durante o processo de microencapsulação. Houve uma correlação entre as diferentes soluções salinas e as alterações na solubilidade e nas conformações estruturais do BV. Foram preparadas formulações BV-MS-PLGA estáveis com atividade biológica preservada. As MS tiveram eficiência de encapsulação entre 49-75% e perfis trifásicos de liberação do BV. Houve mudanças estruturais no BV liberado, mas seu reconhecimento imunológico foi mantido. Ensaios ex vivo e in vivo mostraram que as formulações evitaram as reações inflamatórias causadas pelo contato direto do BV com o organismo, aumentando a fagocitose das partículas com BV, o entregando diretamente ao meio intracelular. As formulações BV-MS-PLGAs estimularam a produção de anticorpos anti-BV equivalentes à VIT tradicional com a redução do número de injeções necessárias para se produzir o mesmo efeito imunológico. Portanto, as MS-PLGA contendo BV são seguras e eficazmente superiores à VIT tradicional. / In this thesis, it was presented a systematic study to develop a new formulation of bee venom (BV) encapsulated within PLGA-MS for VIT. It was studied the stability of BV proteins during the microencapsulation process. There was a correlation between the different salt solutions and changes in the solubility and structural conformations of BV. It was prepared stable BV-PLGA-MS formulations with preserved biological activity. The MS showed encapsulation efficiencies between 49-75%, and a triphasic release profiles of BV. Although structural changes were observed in the released BV, its immunological recognition was maintained. Ex vivo and in vivo assays showed that formulations avoided the inflammatory reactions caused by direct contact of BV and organism leading to increases in phagocytosis rate of particles containing BV, providing its delivery directly to the intracellular environment. These formulations could stimulate the production of anti-BV antibodies comparable to traditional VIT with reduced number of injections needed to produce the same immunological effect. Therefore, PLGA-MS containing BV are safe and effectively superior to the VIT traditional.

Natural killer (NK) cells are non-specific killer cells of the innate immune system that eliminate target cells based on discrimination between self and non-self. Activation is carefully regulated through integration of signals received through both activating and inhibitory receptors. During the course of a herpes simplex virus type-1 (HSV-1) infection, NK cells can influence host susceptibility to infection with severe infections occurring in individuals with genetic defects in the NK cell response. In response to HSV infection, NK cells are recruited to the inflammatory tissue where ensuing reciprocal interactions with accessory cells and proinflammatory cytokines induce NK cell activation, cytolytic activity, and cytokine production, contributing to innate immune response and ultimately influencing the adaptive immune response. The objective of this study was to elucidate the multiple roles of NK cells during the numerous steps in anti-HSV immune induction. Accordingly, we have demonstrated that NK cells are novel helpers that assist and influence an anti-HSV immune response via the secretion of cytokines that enhance HSV-specific CD8+ T cell effector function and cytokine production. Taken together, data from this study presented the critical importance of NK cells in mounting an essential and efficient anti-HSV immunity. The key findings of our study were: 1. In the absence of NK cells, dendritic cells have decreased capacity to prime HSV-specific T cells. 2. HSV infected NK cells can be directly activated via toll-like receptor (TLR) in a MyD88-dependent mechanism; however, interaction with HSV infected dendritic cells yields optimal NK cell activation and function (CD69 and IFNγ). 3. TRAIL-expressing NK cells eliminate antigen-bearing immature dermal DCs (CD11c+CD8α-DR5+), that migrate to draining lymphoid organs, to facilitate antigen transfer to lymphoid resident CD8α+ DC for T cell cross priming. 4. 'Helpless' CD8+ T cell function, generated in the absence of CD4+ T cells, can be partially restored to wild-type levels by NK cell supplementation. 5. Treatment of NK cells with anti-CD69 antibody results in a heightened NK activated state and augments the adaptive immune response, without increasing NK cell numbers. These findings may contribute to the potential revelation of avenues to manipulate NK cells for anti-viral therapies.

Cholangiocarcinoma (CCA) is a fatal primary malignancy that is formed in the bile ducts. Cancer-associated myofibroblasts play a crucial role in CCA proliferation and invasion. Furthermore, there is a growing interest in the role of the exosome in the interaction between the cancer-associated myofibroblasts and cholangiocarcinoma which lead to CCA growth. However how cholangiocarcinoma-derived exosome affect the cancer-associated myofibroblasts in the tumor microenvironment remain unknown. In this study, we examined whether exosome produced by cholangiocarcinoma could involve in the prompt of CCA cells growth by regulation of myofibroblast. We found that cholangiocarcinoma-derived exosome could prompt elevated α-smooth muscle actin and stromal cell-derived factor one expression that induces myofibroblast proliferation. We then demonstrated that cholangiocarcinoma-derived exosome upregulated periostin expression that plays an important role in cancer metastasis. In 3D organotypic rat CCA coculture model, TCA and S1P considerably increase the growth of CCA cell. Conclusion: cholangiocarcinoma-derived exosome trigger cancer-associated myofibroblasts proliferation in the tumor microenvironment that leads to prompt CCA growth.

Herpes simplex virus (HSV) infection of the cornea leads to a blinding immuno-inflammatory condition of the eye also called stromal keratitis (SK). SK immunopathology is characterized by the infiltration of CD4+ T cells of Th1 phenotype as well as the development of new blood vessels into the normally avascular cornea. Studies in mouse models of SK have firmly established the role of CD4+ T cells, and particularly of Th1 phenotype, as the principal mediators of SK immunopathology. However, with the recent discovery of IL-17A and Th17 cells, the role of this cytokine as well as Th17 cells remains to be further defined. Recently it was shown that the normal cornea expresses VEGF-A, however its biological activity is impeded by its binding to a soluble form of VEGF-A receptor-1 (sVEGFR-1). Past studies have implicated the role of vascular endothelial growth factor-A (VEGF-A) in HSV induced corneal angiogenesis, however the source of VEGF-A as well as molecular mechanisms, particularly in the context of VEGF-A/sVEGFR-1 balance during HSV infection, are poorly understood. The first part of this dissertation (I) reviews past literature on HSV induced corneal SK immunopathology. It focuses on the understanding of HSV-1 induced events that particularly results in corneal angiogenesis as well as tissue damage mediated by different type of cells as well as their secreted products. The next three parts (II-IV) focus on the mechanisms of HSV induced corneal angiogenesis as well as the relative role of Th1 and Th17 cells in SK immunopathology. Results in part II focuses on the relative role of IFN-γ/IL-17 as well as Th1/Th17 cells in HSV induced corneal immunopathology. The third section evaluate the significance of VEGF-A/sVEGFR-1 balance in HSV induced corneal neovascularization. Results in part IV focus on the role of IL-17A in altering the balance between VEGF-A and sVEGFR-1 post ocular HSV infection and subsequent corneal angiogenesis. Collectively these studies identified novel mechanisms by which HSV infection of the cornea leads to the development of angiogenesis as well as corneal tissue damage and subsequent SK immunopathology, the most common cause of infectious blindness in the Western World.

Sepsis claims over 215,000 lives in the US annually. Inducible glucocorticoids (iGC) is produced during sepsis. However, the precise effects of iGC in sepsis remain unclear due to a lack of appropriate animal models. Glucocorticoid (GC) insufficiency is associated with a marked increase in mortality and occurs in 60% of severe septic patients. Yet the conclusion of GC therapy on septic patients is still controversial. Scavenger receptor class B type I (SR-BI) in the adrenal mediates the selective uptake of cholesteryl ester from lipoproteins for GC synthesis. SR-BI-/- mice completely lack iGC during sepsis and are highly susceptible to septic death, which presents SR-BI-/- mice as a GC insufficient model. However, SR-BI-/- mice display multiple defects contributing to septic death, making it difficult to study iGC by using these mice. Therefore, we utilized adrenal-specific SR-BI-/- mice (ADR-T SR-BI-/-) generated by adrenal transplantation. As expected, the ADR-T SR-BI-/- mice failed to generate iGC under cecal ligation and puncture (CLP)-induced sepsis and showed a significantly higher mortality than the control mice, demonstrating that iGC is essential for preventing septic death. High blood urea nitrogen (BUN) was observed in the ADR-T SR-BI-/- mice but not in the control mice in CLP, indicating that iGC protects kidney injury in sepsis. Plasma IL-6 was remarkably higher in the ADR-T SR-BI-/- mice than the control mice, demonstrating an anti-inflammatory effect of iGC in sepsis. The ADR-T SR-BI-/- mice also displayed significantly lower phagocytic activity of monocytes and neutrophils in the blood and lower activation of T cells in the spleen compared to the control mice in CLP, suggesting that iGC is immunomodulatory in sepsis. Low-dose GC supplementation significantly improved the survival of SR-BI-/- mice in CLP, but did not increase the survival rate of SR-BI+/+ mice in CLP, indicating that GC supplementation improves the survival specifically in mice with adrenal insufficiency. Overall, we revealed that iGC is essential for sepsis survival. iGC prevents kidney damage, modulates inflammatory responses and exerts immunomodulatory functions in sepsis. GC supplementation specifically improves survival of individuals with adrenal insufficiency in sepsis.

Nesta tese, foi apresentado um estudo sistemático para o desenvolvimento de uma nova formulação do veneno de abelha (BV) encapsulado em microesferas (MS) de PLGA para VIT. Estudou-se a estabilidade das proteínas do BV durante o processo de microencapsulação. Houve uma correlação entre as diferentes soluções salinas e as alterações na solubilidade e nas conformações estruturais do BV. Foram preparadas formulações BV-MS-PLGA estáveis com atividade biológica preservada. As MS tiveram eficiência de encapsulação entre 49-75% e perfis trifásicos de liberação do BV. Houve mudanças estruturais no BV liberado, mas seu reconhecimento imunológico foi mantido. Ensaios ex vivo e in vivo mostraram que as formulações evitaram as reações inflamatórias causadas pelo contato direto do BV com o organismo, aumentando a fagocitose das partículas com BV, o entregando diretamente ao meio intracelular. As formulações BV-MS-PLGAs estimularam a produção de anticorpos anti-BV equivalentes à VIT tradicional com a redução do número de injeções necessárias para se produzir o mesmo efeito imunológico. Portanto, as MS-PLGA contendo BV são seguras e eficazmente superiores à VIT tradicional. / In this thesis, it was presented a systematic study to develop a new formulation of bee venom (BV) encapsulated within PLGA-MS for VIT. It was studied the stability of BV proteins during the microencapsulation process. There was a correlation between the different salt solutions and changes in the solubility and structural conformations of BV. It was prepared stable BV-PLGA-MS formulations with preserved biological activity. The MS showed encapsulation efficiencies between 49-75%, and a triphasic release profiles of BV. Although structural changes were observed in the released BV, its immunological recognition was maintained. Ex vivo and in vivo assays showed that formulations avoided the inflammatory reactions caused by direct contact of BV and organism leading to increases in phagocytosis rate of particles containing BV, providing its delivery directly to the intracellular environment. These formulations could stimulate the production of anti-BV antibodies comparable to traditional VIT with reduced number of injections needed to produce the same immunological effect. Therefore, PLGA-MS containing BV are safe and effectively superior to the VIT traditional.

Glucans are fungal cell wall PAMPs that promote survival in polymicrobial and candidal sepsis. Dectin-1 is the primary PRR for glucans. The goals of the present study were to characterize 1) the effects of fungal infection on Dectin-1; 2) the effects of polymicrobial sepsis in the presence and absence of glucan on Dectin-1; 3) the effects of systemic administration of glucans on Dectin-1; and 4) the intracellular trafficking of glucans. Mice were either systemically infected with Candida albicans, or made septic by CLP with and without glucan phosphate (GP) injection, or injected with GP. Flow cytometry was performed to assess cell surface Dectin-1 expression. C. albicans sepsis resulted in an increase in the percentage of Dectin-1 positive (Dectin+) blood and splenic leukocytes by increasing the percentage of neutrophils. C. albicans infection increased the percentage of Dectin+ splenic T cells. CLP decreased the percentage of highly Dectin-1 positive leukocytes in the blood by decreasing the percentage of Dectin+ neutrophils. GP treatment in sepsis further decreased the percentages of Dectinhigh blood leukocytes and Dectin+ neutrophils. CLP decreased the percentage of Dectin+ splenic leukocytes by decreasing the percentage of splenic macrophages. GP administration to CLP mice further decreased the percentage of Dectin+ splenocytes by decreasing the percentage of Dectin+ macrophages. Administration of GP resulted in a prolonged decrease in the percentage of Dectinhigh blood leukocytes. The changes in Dectin-1 expression with GP were because of decreases in the percentage of Dectin+ neutrophils and monocytes. In the trafficking studies, macrophages were incubated with fluorescent labeled glucans and then stained for intracellular organelles and signal transduction molecules. Cells were imaged using confocal microscopy. GP is internalized by clathrin and trafficked to the Golgi apparatus. GP internalization is regulated but not dependent on caveolin-1. GP co-localized with SRA, TLR2, and PI3K/p85. The trafficking of laminarin and particulate glucan is similar. We speculate that loss of cell surface Dectin-1 may be important in the protection conferred by glucans in sepsis. Additionally, intracellular trafficking and interaction with signaling components may be important steps in modulation of cellular function by glucan-pattern recognition receptor complexes.

<p>Inflammatory Bowel Disease (IBD) is a chronic intestinal inflammation that has caused many challenges for healthcare providers in treating the disease and also altered the quality of life of the patients. The cure for IBD is still symptomatic-based; the causes mechanism and pathogenesis of IBD are to be further investigated. Currently, IBD has been considered as an excessive immune response to commensal flora that in normal condition is tolerable to the host. Antigen presenting cells (APCs) play an important role in the pathogenesis of IBD. Macrophage is one of the professional APCs that present antigen information to T cells and induce the T cell subtype proliferation. Aside from this role, macrophages also phagocytose pathogens and clean cell debris in thebody.</p> <p>T cell immunoglobulin and mucin domain (TIM)-4 is a glycoprotein expressed on the surface of macrophage, which recognizes phosphatidylserine (PS) that is expressed mainly on the surface of the early apoptotic cell phospholipid membrane; the latter is a negatively charged molecule that can bind to the TIM-4 to enhance the phagocytosing activity. In IBD, the loss of intestinal epithelial cells (IECs) due to apoptosis is prominent in the site of inflammation especially in ulcerative colitis (UC).</p> <p>The aim of this study is to elucidate whether there is an increase of TIM-4 expression in colitis mice model after exposure to excessive number of apoptotic IECs and whether TIM-4 plays a role in the development of colitis in mice.</p> <p>The expression of TIM-4 is measured with several tests; including flow cytometry, immunohistochemistry, western blotting and real time RT-PCR. In the first step, we tried to see if there is a difference in the TIM-4 expression in colitis mice and ethanol control mice. After the association was established, we further observed the role of TIM-4 in the pathogenesis of IBD by injecting TIM-4+ macrophages into the mice prior to inducing a mild colitis in the mice and finally injected neutralizing anti TIM-4 antibodies to block the available TIM-4 receptors.</p> <p>We found that TIM-4 expression was higher in a colitis mouse model compared to the control. Also by injecting TIM-4+ macrophages into the mice, the frequency of intestinal T regulatory (Treg) cells was decreased significantly. Finally in the group treated with anti-TIM-4 neutralizing antibodies prior to colitis induction, the frequency of intestinal Treg cells increased significantly and the inflammation response was less severe than the colitis control group. This study revealed, for the first time in the world, that TIM-4 expression in the colon of colitis mice was significantly increased, which suppressed Tregs and promoted T effector cells.</p> / Master of Science in Medical Sciences (MSMS)

<p>AMR as a cause of graft rejection has been long recognized and the presence of pre formed antibodies against donor HLA is a risk factor for increased graft rejection. FlowXM is the current clinical gold standard for detecting harmful DSA in the recipients and a positive FlowXM is considered a strong contraindication to transplantation. However, newer techniques such as SAB provide with a highly sensitive and specific method for DSA detection that is unattainable by FlowXM. But due to the intrinsic limitations associated with SAB assays, the clinical relevance of DSA detected on SAB has been highly disputable. Therefore, the overall aim of this study was to investigate the utility of SAB in predicting harmful DSA levels, by establishing a fluorescence range on SAB that correlated to positive FlowXM. This was done by retrospectively testing the highest serum dilutions on FlowPRA SAB that produced positive B or T cell FlowXM from 15 variably sensitized patients. Thus, a very narrow MFI range on SAB was established, for B and T cells separately, that correlated to positive FlowXM. On B cells this correlate ranged from 2780-7772 MFI (Mean MFI =5641), whereas T cell range was 1089-6731 (Mean MFI= 3226). In order to test these ranges for prediction of positive FlowXM, B and T cell FlowXM tests were carried out using various serum/cell combinations. DSA MFI of >3000 on SAB resulted in a significantly higher T cell positive FlowXM (p</p> / Master of Science (MSc)