Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Physiology. Title from pdf file main screen (viewed on October 11, 2009). Includes bibliographical references.

<p>Amb a 1 is the major allergen found in ragweed. Our observations have suggested that Amb a 1 may bind lipopolysaccharide (LPS), which would likely contribute to the allergenicity of Amb a 1. In order to assess whether Amb a 1 can bind LPS, peptide sequences from Amb a 1 were assayed for their ability to bind to LPS using an ELISA based LPS binding assay. A 15 amino acid sequence in the β- chain of Amb a 1 demonstrated affinity for biotin labeled <em>E. coli </em>LPS. The sequence also bound to <em>P.</em> <em>aeruginosa</em> LPS, which is structurally disparate in the lipid A region, indicating that the sequence has flexibility in recognizing different lipid A moieties, or that the binding site may not include the lipid A portion of the LPS molecule. An IL-10 ELISA was also used to determine whether the LPS bound to the peptides induced an immunological response in leukocytes. Peptides containing the LPS-binding sequence were able to bind to LPS and induce IL-10 production, suggesting the interaction between Amb a 1 and LPS may have immunological consequences. We have identified a sequence within the major ragweed allergen Amb a 1 that has the potential to bind to LPS. This indicates that the allergen may provide its own adjuvant when encountered by the immune system, leading to an enhanced immunological response to an otherwise innocuous environmental protein.</p> / Master of Science (MSc)

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Industrial and Manufacturing Systems Engineering / Master / Master of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Samples from 93 of 115 consecutive cadaveric renal transplants were selected to define polymorphisms in both IFN-γ and IL-10. A 12 CA repeat IFN-γ polymorphic allele was found in 73 patients (70 in patients analysed further). This polymorphism was associated with high IFN-γ production in vitro. According to the presence or not of the 12 CA repeat allele patients were separated in high and low producer genotype groups. The incidence of acute rejection was 54.3% in this high IFN-γ genotype group, contrasting with 44.4% in the low IFN-γ. Requirement for ATG therapy was greater in the high IFN-γ group (odds ratio [OR]=2.5). Among HLA-DR-mismatched patients, IFN-γ high producer genotype was more strongly associated with rejection (OR=1.6). In the cyclosporine monotherapy subgroup, 11 out of 14 patients with IFN-γ high genotype (78%) had acute rejection (OR=2.88, p=0.09). Graft survival was similar between the two IFN-γ groups. When the analysis was controlled for the presence of delayed graft function, 40.5% of the high IFN-γ genotype patients had serum creatinine levels above 200 micromoles/L contrasting with only 14.3% of the low IFN-γ genotype recipients at 5 years after transplantation (p=0.05). In a regression model of creatinine at 1 year the significant variables were the presence of DGF, donor age greater than 50, greater than two rejection episodes, DR mismatch, donor female to male recipient sex, IL-10 high genotype, and IFN-γ high genotype. Conclusion: The 12 CA repeat IFN-γ polymorphic allele is associated with high IFN-γ production. We have shown that this high producer genotype for IFN-γ influences acute rejection in kidney transplantation, particularly in high-risk groups; it is also associated with worse long-term graft function.

Aquaculture is growing rapidly worldwide and is projected to become the major source of fish used for human consumption. A major factor that limits aquaculture reaching its full potential is an adequate supply of the raw materials necessary for formulated fish feeds. The dependence of modern aquaculture on fishmeal obtained from wild fisheries is not environmentally sustainable and replacements for fishmeal must be found. Some animal by-products are viable replacements for fishmeal, and can provide sufficent nutrition for high growth rates, but little is known about the potential of animal by-products to adversely affect fish health. The objectives of these experiments were to determine if animal by-products used in fish feeds impair immune response or alter bone physiology in cultured juvenile rainbow trout. Four animal by-product containing diets (poultry meat meal (PMM)/ PMM plus feathermeal / PMM plus bloodmeal) and two reference diets (fishmeal or soya) were evaluated to determine their effect on innate immune response, the ability of fish to cope with normal husbandry stressors, and bone physiology. PMM was then selected due to its favourable amino acid profile and high digestibility and assessed to determine if the high levels of fishmeal replacement that may be required in the future, impact the health of rainbow trout. Due to the lack of reliable indicators of bone quality and quantity in salmonids the effects of exercise and phosphorus deficiency in rainbow trout were also examined. Relative to the fishmeal control diet, fish fed diets with PMM [(PMM) 50% crude protein, by substitution], PMM plus two percent blood meal, or PMM plus five percent feather meal, did not have an impaired innate immunity (lysozyme, alternative complement, phagocytosis, intracellular respiratory burst, differential counts of peripheral blood leukocytes) or changes in bone physiology as assessed by dynamic bone histomorphometry. Higher levels of PMM (0-70% digestible protein, by substitution) caused a reduction in apparent net mineral retention of phosphorus and calcium (P<0.001), a lower vertebral bone mineral content (P<0.001) and reduced vertebral mechanical properties (compressive extension (P=0.04), Young’s Modulus (P=0.03)), but fish growth was not affected. Exercise influenced bone modelling, with exercised animals having a reduced bone area and trabecular thickness (P=0.01), increased autocentrum width (P=0.04), and higher bone mineral content (P= 0.02); however, bone mechanical properties were unaffected. Induction of genes (receptor activator nuclear factor kappa beta and osteoprotogenerin), involved in the resorption of mineralised tissue, was not observed in fish fed phosphorus deficient diets although scales were evidenced to be an important source of labile minerals. Overall our results indicate that low level replacement of fish meal by poultry meat meal, and blends of poultry meat meal with blood or feathermeal do not affect fish innate immune response, bone physiology, or growth however the greatly elevated levels of poultry meat meal that may be required in future salmonid aquafeeds could increase the risk of spinal malformations. Thus the category 3 animal by products tested are valuable fishmeal replacements for aquaculture based on the endpoints measured in this study.

Type 2 (TH2) immune responses evolved to respond to helminth parasite infections by the production of TH2 cytokines, which stimulate anti-helminth immunity. Macrophage migration inhibitor factor (MIF) is a pleiotropic cytokine, which is produced by many cell types. We demonstrate that mice deficient in MIF have enhanced clearance of a helminth parasite. MIF deficiency in CD4+ T cells was found to be the most important for mediating parasite clearance. We mimicked MIF deficiency by administering an inhibitor of the MIF tautomerase activity, sulforaphane, and this also increased parasite clearance (Section I). TH2 immune responses underlie allergy and allergic asthma, in which the same cytokines that help expel parasites are released in response to innocuous substances. Integral to the initiation of adaptive TH2 immunity are dendritic cells (DCs), which take up antigen and stimulate antigen-specific CD4+ T cell responses. We found that DC expression of ADAM10, a zinc-dependent metalloproteinase, is critical for the development of TH2 immune responses and IgE production from B cells. This effect is demonstrated in both allergic airway inflammation and anaphylaxis models. ADAM10-deficient DCs are unable to cleave Notch1 receptors, resulting in reduced IL-6 production and this ultimately results in decreased TH2 activity. ADAM17 is closely related to ADAM10 in both structure and function. Interestingly, mice from which ADAM10 and 17 are removed from DCs (ADAM10/17DC-/-) have a distinct phenotype from both ADAM10DC-/- and ADAM17DC-/- mice in models of allergic airway inflammation (Section I). We also examined another effect of TH2 cytokines on the interaction between mast cells and myeloid derived suppressor cells (MDSCs). We sought to understand how histamine and IL-13, mediators made by mast cells, affect the immunoregulatory function of MDSCs. MDSCs in IL-13-deficient mice with tumor are more prevalent in circulation rather than in tumor or organs, which could be due to changes in CCL2/CCR2 chemotaxis. In addition, MDSC function after treatment with the DNA methyltransferase inhibitor, decitabine was examined. This treatment reduced their suppressive function and increased the expression of molecules needed for antigen presentation. Overall, TH2 immunity has multifaceted roles in anti-parasite immunity, allergic asthma, and MDSC function (Section II).

Em bovinos, o desenvolvimento embrionário pré implantacional depende das funções do endométrio bovino que tem suas funções mediadas por uma complexa interação da ação e dos efeitos dos hormônios esteroides ovarianos E2 e P4. Estes hormônios regulam a expressão gênica e controlam o ambiente uterino modulando, entre outros, a expressão de microRNAs e a rede de citocinas relacionadas ao sistema imune. Os objetivos do presente trabalho foram abordados em dois capítulos, sendo (I) comparar os efeitos dos distintos ambientes endócrinos peri-ovulatórios sobre a expressão de microRNAs (II) e na modulação do sistema IL1/TLR no endométrio bovino nos dias 4 e 7 após a indução da ovulação. Para isso, controlou-se farmacologicamente o crescimento do folículo objetivando induzir a ovulação de folículos de maior diâmetro (grupo folículo grande-CL grande, FG-CLG) ou de menor diâmetro (grupo folículo pequeno-CL Pequeno, FP-CLP). Vinte e duas vacas multíparas nelore, foram pré-sincronizadas, metade destes animais foram destinados para o grupo FG-CLG e receberam uma dose de prostaglandina F2&#945; (PGF) e um dispositivo de progesterona, juntamente com benzoato de estradiol no D10. No momento da retirada dos dispositivos de progesterona (entre D1,75 e D2,5) todos os animas receberam uma dose de PGF. A ovulação foi induzida com acetato de buserelina (D0). O que diferiu entre os tratamentos foi que os animais do grupo FP-CLP não receberam uma dose de PGF no D10 e o momento da retirada dos dispositivos foi entre D1,25 e o D1,5. No capítulo I, o a expressão de microRNAs foi determinada por qPCR nos dias 4 e 7. Dos 90 microRNAs testados, 21 apresentaram se up-regulated e dois down-regulated no grupo FG-CLG (P<0.1) no D4. No D7, quatro microRNAs foram diferentemente expressos, sendo um up-regulated e três down-regulated no grupo FG-CLG (P<0.1) no D7. Para os microRNAs diferentemente expressos determinou-se mRNA-alvos preditos. Uma análise de ontologia demonstrou que os mRNAs-alvos apresentaram enriquecimento funcional na via dos receptores de hormônios esteroides, entre outras. No capítulo II, o sistema IL1/TLR foi avaliado quanto a abundância de transcriptos envolvidos neste sistema, do microRNA bta-mir-155 e das proteínas IL1&#946; e IL1R1. A abundância relativa de mRNA apresentou diferença (P<0.1) na abundância dos mRNAs de IL1R1, TAB1 e FOXP3, das proteínas IL1&#946; e IL1R1, sendo essas moléculas up-regulated no grupo FG-CLG. O microRNA bta-mir-155 foi down-regulated no grupo FG-CLG (P<0.1). Diante disto, pode-se concluir que o ambiente endócrino peri-ovulatório determina o perfil de expressão de microRNAs e modula o sistema IL1/TLR no endométrio bovino / In cattle, the pre implantation embryo development depends on the functions of the bovine endometrium that has its functions mediated by a complex interaction of action and the effects of ovarian steroid hormones E2 and P4. These hormones regulate gene expression and control the modulating uterine environment among others, the expression of microRNAs and the network of cytokines related to the immune system. The objectives of this study were discussed in two chapters, (I) to compare the effects of different peri-ovulatory endocrine environment on the expression of microRNAs (II) and modulation of the IL-1 system / TLR in bovine endometrium on days 4 and 7 after induction of ovulation. For this, it was controlled pharmacologically follicle growth aiming to induce ovulation of follicles larger diameter (great grand-CL follicle group, FG-CLG) or smaller in diameter (small-CL Small follicle group, FP-PLC). Twenty two nelore multiparous cows were pre-sync, half of these animals were used for the FG-NCG group and received a dose of F2á prostaglandin (PGF) and progesterone device along with oestradiol benzoate in D-10. Upon withdrawal of progesterone devices (between 1.75 and D-D-2,5) all animas received a dose of PGF. Ovulation was induced with buserelin acetate (D0). What differed between treatments was that animals FP-CLP group did not receive a dose of PGF in the D-10 and the time of removal of the devices was between D-1,25 and D-1.5. In Chapter I, the expression of microRNAs was determined by qPCR on 4 and 7. Of the 90 microRNAs tested, 21 showed was up-regulated and down-regulated in two FG-CLG group (P <0.1) in the D4. In D7 four microRNAs were differently expressed, one up-regulated and down-regulated in three FG-CLG group (P <0.1) at D7. For differently expressed microRNAs was determined predicted mRNA-target. An ontology analysis showed that the mRNA-targets had functional enrichment in via the steroid hormone receptors, among others. In Chapter II, the IL-1 / TLR system was evaluated as the abundance of transcripts involved in this system, the bta-mir-155 microRNA and IL1&#946; and IL1R1 proteins. The relative abundance of mRNA was different (P <0.1) in the abundance of mRNAs IL1R1, TAB1 and FOXP3, the IL1&#946; and IL1R1 proteins, and these up-regulated molecules in the FG-CLG group. The bta-mir-155 microRNA was down-regulated in the FG-CLG group (P <0.1). Given this, we can conclude that the peri-ovulatory endocrine milieu determines the profile of microRNA expression and modulates the IL1 / TLR system in bovine endometrium

O tumor de Ehrlich (TE) foi descrito inicialmente, como adenocarcinoma mamário de camundongos, desenvolvendo-se na forma ascítica ou sólida a partir de sucessivos transplantes no peritônio ou tecido subcutâneo destes animais. O tumor ascítico de Ehrlich (TAE) tem sido utilizado como um tumor transplantável para o desenvolvimento de pesquisas relacionadas à oncologia. Estudos tem demonstrado que o desenvolvimento de TAE resulta no estímulo de células citotóxicas, como os linfócitos T e células Natural Killer (NK), mediadas principalmente por macrófagos. Os macrófagos e as células dendríticas (DCs), podem induzir a síntese da enzima indoleamina 2,3 dioxigenase (IDO) em tecidos tumorais, via ligação das moléculas co-estimuladoras B7-1 e B7-2, presentes nestas células, com a molécula CTLA-4 (antígeno 4 associado a linfócitos T citotóxicos), presente em linfócitos T reguladores CD4+ CD25+. A IDO é uma enzima que degrada o aminoácido essencial triptofano, processo que além de levar à privação do mesmo no microambiente celular, gera metabólitos que impedem a ativação e proliferação de linfócitos T e, consequentemente, mecanismos a eles associados como os de rejeição podem ser seriamente comprometidos. Mediante o exposto e pela presença de células imunológicas que expressam IDO no microambiente do tumor ascítico de Ehrlich, este trabalho teve como objetivo verificar a expressão da IDO após o bloqueio da interação B7/CTLA-4 por meio da citometria de fluxo. De acordo com as análises realizadas, os resultados demostraram que houve redução de 4,9% para 2,53% na expressão da enzima IDO. Em face dos resultados, parece plausível sugerir que o bloqueio desta via de ligação foi eficaz na redução dos níveis de atividade da IDO, o que poderia restaurar a capacidade de resposta dos linfócitos T contra as células tumorais. Nesta perspectiva sobre a IDO como mediadora no controle do escape imune feito pelas células tumorais, tais resultados podem colaborar para modulação desta enzima no microambiente tumoral. / The Ehrlich tumor (TE) was first described, as breast adenocarcinoma of mice. The TE develops in ascitic or solid form from successive transplantations in the peritoneum or subcutaneous tissue of these animals. The Ehrlich ascites tumor (TAE) has been used as a transplantable tumor for the development of research related to oncology. Studies have shown that the development of TAE results in stimulation of cytotoxic cells, such as T lymphocytes and Natural Killer cells (NK), mediated mainly by macrophages. Macrophages and dendritic cells (DCs) in tumor tissues, via co-stimulation of molecules B7-1 and B7-2, present in these cells, toghether with the CTLA-4 (Cytotoxic T-Lymphocyte-Associated antigen 4) molecule, present on regulatory T CD4 + CD25 + lymphocytes , may induce the synthesis of indoleamine 2, 3 dioxygenase (IDO). IDO is an enzyme that catabolizes the essential amino acid tryptophan, impairing activation and proliferation of T lymphocytes and consequently, compromising mechanisms associated with them such as rejection. Considering the presence of immune cells in the tumor microenvironment Ehrlich ascites that express IDO, this study aimed to verify the expression of IDO after the blockade of interaction B7/CTLA-4 by flow cytometry. Results demonstrated that there was a reduction of 4.9% to 2.53%in the expression of IDO. Given the results, it seems plausible to suggest that blocking this binding via was effective in reducing the levels of expression of IDO, which could restore the responsiveness of T cells against tumor cells. In this perspective on the IDO as a mediator in the control of immune escape made by tumor cells, these results may collaborate for modulation of this enzyme in the microenvironment.