Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

A leishmaniose visceral causada por Leishmania infantum chagasi à caracterizada pela perda da habilidade do hospedeiro gerar uma resposta imunolÃgica eficaz. Neste estudo, foi investigado o papel da quimiocina CXCL10 no controle da infecÃÃo por L. infantum chagasi in vivo. Grupos de camundongos BALB/c foram tratados ou nÃo com CXCL10 (5 μg/kg) com 1, 3 e 7 dias de infecÃÃo e apÃs 1, 7 e 23 dias do tratamento, alguns parÃmetros foram avaliados: a carga parasitÃria, os nÃveis de IFN-, IL-4, TGF-β e IL-10, e as alteraÃÃes histopatolÃgicas no fÃgado. ApÃs 23 dias de tratamento, CXCL10 induziu, no baÃo, uma reduÃÃo expressiva no nÃmero de parasitos, quando comparado ao grupo controle. No fÃgado, a carga parasitÃria mostrou uma queda no grupo tratado, entre o 7 e 23 dia apÃs o tratamento. Entretanto, o efeito leishmanicida de CXCL10, neste trabalho, nÃo parece ser mediado por NO, uma vez que nÃo houve diferenÃa na produÃÃo de NO entre os grupos. IFN-γ foi induzida de maneira mais significativa no grupo tratado do que nos controles, e atingiu sua produÃÃo mÃxima (100 pg/mL) no 23 dia apÃs o tratamento, correlacionando-se com a queda da carga parasitÃria nos ÃrgÃos-alvo. IL-4 foi produzida em baixas concentraÃÃes, em ambos os grupos, embora os animais tratados com CXCL10 tenham mostrado nÃveis mais elevados do que os controles. Em relaÃÃo Ãs citocinas antiinflamatÃrias, apÃs 23 dias do tratamento, os nÃveis de IL-10 nos animais tratados foram menores do que os do controle. A produÃÃo de TGF-β apÃs 7 dias do tratamento foi 2 vezes menor no grupo tratado quando comparado ao controle, e apÃs 23 dias do tratamento, essa citocina continuou com nÃveis mais baixos do que aqueles observados no controle. Na anÃlise histopatolÃgica do fÃgado apÃs o 1 dia do tratamento, foram encontrados, em ambos os grupos, mais granulomas imaturos (GI), do que infiltrados nÃo granulomatosos (NG) e alguns poucos granulomas maduros (GM) apenas no grupo tratado. ApÃs 7 dias do tratamento, a quantidade de infiltrados NG estava menor e os GI ainda foram os mais encontrados, em ambos os grupos, alÃm disso, foi observado um pequeno aumento de GM no grupo tratado. Em resumo, diante dos resultados encontrados, à possÃvel sugerir um importante papel leishmanicida de CXCL10 em camundongos BALB/c infectados por L. infantum chagasi, que parece ser mediado por uma expressiva produÃÃo de IFN-g e supressÃo das citocinas imunorreguladoras, IL-10 e TGF-β, abrindo a hipÃtese se isto nÃo estaria associado a uma diminuiÃÃo na frequÃncia de cÃlulas T regulatÃrias, induzida por CXCL10, nesses animais. / Visceral leishmaniasis caused by Leishmania infantum chagasi is characterized by the loss of the ability of host to generate an effective immune response. In this study it was investigated the role of CXCL10 chemokine in controlling L. infantum chagasi infection in vivo. Groups of BALB/c mice were treated or not with recombinant CXCL10 chemokine (5 μg/kg) with 1, 3 and 7 days of infection and after 1, 7 and 23 days of treatment, some parameters were evaluated: parasite load, levels of IFN-g, IL-4, TGF-β and IL-10, and the histopathological alterations in the liver. After 23 days of treatment, CXCL10 induced in the spleen a significant reduction on the number of parasites as compared to control group. In the liver, parasite load decreased in treated group between the 7th and 23th day post treatment. However, the antileishmanial effect of CXCL10, in this work, does not seem to be mediated by NO, since there was no difference in the NO production among the groups. IFN-γ was induced most significantly in treated group than in controls, and reached its maximum production (100 pg/mL) on day 23 after treatment, correlating with the reduction in parasite burden in target organs. IL-4 was produced in low doses, in both groups, although treated animals had shown higher levels than control group. Regarding to anti-inflammatory cytokines, after the 23th day of treatment, IL-10 levels in treated animals were smaller than in control group. Production of TGF-β after 7 days of treatment was 2 times lower in treated group when compared to control, and after the 23th day of treatment, this cytokine remained with lower levels than those observed in control. In the histopathological analysis of the liver after the 1st day of treatment, were found in both groups more immature granulomas (GI) than non-granulomatous infiltrate (NG), and some few mature granulomas (GM) were only observed in treated group. After 7 days of treatment, the amount of NG infiltrates was lower, and GI were still the most frequent in both groups, besides a slight increase of GM was observed in treated group. In summary, at the light of the found results, it is possible to suggest an important leishmanicidal role to CXCL10 in BALB/c mice infected by L. infantum chagasi, which seems to be mediated by a significant IFN-g production, and suppression of immunoregulatory cytokines, IL-10 and TGF-β, opening the hypothesis that this would be associated to a decrease in the frequency of regulatory T cells induced by CXCL10 in these animals.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

No description available.

Les protéines étant au coeur d’un grand nombre de processus biologiques, elles sont des cibles thérapeutiques largement convoitées. Les foldamères, notamment les oligoamides aromatiques, présentent une structure bien définie, prévisible, stable en solution et à l’état solide. Ajouté à cela, leur taille moyenne en fait de bons candidats pour la reconnaissance de surfaces de protéines, grâce à leurs chaînes latérales protéinogènes. Cette thèse présente les différentes étapes de leur conception, de la synthèse de la brique constitutive à l’obtention d’un foldamère fonctionnalisé grâce à la synthèse en phase supportée. La stratégie d’investigation des interactions entre un foldamère et une protéine est détaillée. L’originalité réside dans le fait que le foldamère est ancré directement à la protéine et le dichroïsme circulaire sert de méthode de screening. L’analyse structurale des hits permet de générer de nouveaux foldamères dans le but d’améliorer les interactions avec la protéine : c’est une stratégie itérative. Cette approche est appliquée premièrement à l’anhydrase carbonique humaine II, protéine modèle qui sert de preuve de principe pour cette approche ; puis à des protéines d’intérêt thérapeutique plus important : l’interleukine 4 et la cyclophiline A. Enfin, une étude concernant l’introduction de flexibilité au sein de foldamères de quinolines est présentée. / Since proteins are at the basis of many biological processes, they are widely studied as therapeutic targets. Aromatic oligoamide foldamers have a very well defined structure, predictable and stable both in solution and solid state. Because of their medium size, they appear as potent candidates for protein surface recognition thanks to their proteinogenic side chains. This manuscript presents the different steps of their design, from the scaffold’s synthesis to obtaining a functionalized foldamer, thanks to solid phase synthesis. The strategy to investigate protein/foldamer interactions will be detailed. Its originality lies in the fact that the foldamer is anchored to the protein. Circular dichroism has been used as a screening method to detect foldamer/protein interactions. Structural analysis of the hits will allow the design of new foldamers with the objective of enhancing foldamer/protein interactions: it is an iterative strategy. This approach has been applied firstly to human carbonic anhydrase II (HCA). This protein is used as a model system and proof of concept before moving to more therapeutically relevant proteins; interleukin 4 and cyclophilin A. Finally, a study on introducing flexibility in quinoline foldamers is presented.

In this study, we aimed to determine the levels of cytokines IL-1β, IL-4, IL-10, IFN-γ, MIF and VEGF in tears derived from healthy subjects. We tested cytokines as potential markers of inflammation for their potential use in clinical practice. Having reliable method for measuring cytokine levels in tears would enable an early diagnosis of eye diseases. In two phases, cytokines in tears of healthy individuals were analyzed using Bio-Plex Cytokine Assay (Bio-Rad). We assessed the suitability of methods for diagnostic purposes as well as the suitability of our selected cytokines. Statistically significant positive correlations of cytokines were confirmed: IL-10 with IFN-γ (r = 0,81), MIF with VEGF (r = 0,42 / r = 0,49), IL-1β with IL-10 (r = 0,52), IL-1β with IFN-γ (r = 0,55), IL-1β with VEGF (r = 0,38), IFN-γ with VEGF (r = 0,45) and IL-4 with VEGF (r = 0,48) in healthy subjects in tears. IL-4 (r = -0,37) and IFN-γ (r = -0,42) correlate negatively with age. In healthy individuals, there seem to be no differences with regard to gender, BMI, body fat, time of meal consumption prior to tear collection, eye strain when using a computer, dry eyes. Thus, studied cytokines are suitable for diagnostic purposes. Significant differences in concentrations of four (IL-1β, IL-10, IFN-γ a VEGF) of the five...

No description available.

No description available.

<p>Food allergies are immune system-driven diseases that lead to reproducible adverse reactions which can be fatal. These severe systemic reactions are primary mediated by immunoglobulin E (IgE) that is derived from B cells which have been activated by T helper type 2 (Th2) cells. While much work has advanced the clinical and pharmacological management of patients with allergic diseases, much remains to be elucidated about how individuals initially acquire allergy. This Thesis details a mechanism linking initial gastrointestinal exposure to peanut (PN) allergen, to the generation of Th2 cells: PN allergen activates epithelial cell secretion of interleukin (IL)-33 and eosinophil degranulation of eosinophil peroxidase, which causes CD103+ dendritic cell (DC) activation and migration to mesenteric lymph nodes where DC OX40L engages naïve T cells to secrete IL-4 in an autocrine/paracrine manner to promote and consolidate Th2 cell differentiation. These events are followed by B cell activation and PN-specific IgE production, which sensitizes mast cells to be hypersensitive to PN re-exposure by causing immediate allergic reactions including anaphylaxis. This is later followed by eosinophilic inflammation that is partially mediated by innate lymphoid cells. As food allergy also serves as a unique model to better understand mechanisms of adaptive immunity, especially Th2 immunobiology, both basic science and clinical implications are discussed in this Thesis. Major themes include Th2 and disease heterogeneity, identification of ‘the original source of IL-4’, an unprecedented <em>in vivo </em>requirement for eosinophils in priming adaptive immune responses, and the need to weigh basic science findings against the human disease <em>in natura </em>litmus test. Looking forward, many questions remain to be answered in the field of food allergy research, but the findings of this Thesis may be one step towards the prevention, management or cure of a disease with growing public concern, potentially fatal consequences, and an unmet need in understanding its pathogenesis.</p> / Doctor of Philosophy (Medical Science)