Influenza is one of the most common diseases worldwide, yet the vaccines against influenza are only 35% effective at protecting against infection. Creating a more effective vaccine requires an understanding of the foundation and the factors that contribute to a strong and protective adaptive immune response. T-bet [TBX21] is a transcription factor that plays an instrumental role in the orchestration of the type 1 immune response, which is the specialized response used by the immune system for a cell-mediated response against intracellular pathogens, such as influenza. It has yet to be explored in an influenza setting on the role T-bet in the production of antibodies. The aim of this study is to understand T-bet's role in production of antibody isotypes and identify whether expression of T-bet is more important for antibody production in T cells or B cells. We expected T-bet knockout (KO) mice to have IgG2a and that T-bet expression would be more important in T cells for antibody production. An enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of virus-specific antibody in T-bet KO versus wild type (WT) mice infected with influenza. The results show that the T-bet KO and WT mice have relatively the same amount of IgG and IgG1, but the T-bet KO have a significantly lower level of IgG2a, confirming T-bet's importance for its production. To distinguish the importance of T-bet expression while T-bet expression in T cells was constant, a model was developed to allow us to control expression of T-bet in B cells. The results however were inconclusive, and the experiment will have to be repeated to make a firm conclusion on the roles of lymphocytes in the control of IgG isotypes. Overall, these results indicate that the manipulation of T-bet expression can be used as a vector to control IgG antibody levels, which holds potential for the improvement of vaccines.

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No description available.

Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa protein's gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence. / Master of Science

Objetivos: Determinar a reatividade dos soros e determinação dos isotipos IgG e IgE a proteínas de sementes da dieta de crianças com alergia ao leite de vaca IgE mediada. Métodos: Foram avaliados soros de três grupos de crianças: alérgicas ao leite de vaca IgE mediada, crianças tolerantes ao leite e um grupo controle com crianças não atópicas. Foram usados extratos protéicos de diferentes tipos de sementes utilizando o teste de ELISA para análise da reatividade dos isotipos IgG e IgE. Resultados: Comparando as concentrações séricas de IgG dos diferentes grupos, observou-se concentrações mais elevadas e estatisticamente significante no grupo alérgico em relação aos grupos tolerante e controle, exceto para as sementes de soja e feijão roxinho. Em relação ao isotipo IgE observou-se os mesmos padrões de reatividade mostradas nas analises para IgG, com diferença significante do grupo alérgico em relação ao controle, exceto para milho. Observou-se que para a soja houve grande dispersão das concentrações séricas tanto no grupo alérgico quanto no tolerante, em valores superiores ao do grupo controle. Conclusão: A comparação entre os diversos grupos avaliados mostra que pacientes alérgicos ao leite e os tolerantes apresentam concentrações mais elevadas de IgG e IgE a outros alimentos que as crianças do grupo controle, o que pode sugerir possível alteração de permeabilidade da mucosa intestinal nestes grupos, mesmo na ausência de sintomatologia gastrintestinal / Objective: To determine the reactivity of serum and determination of IgG and IgE isotypes to seed proteins included in the diet of children with cow\'s milk allergy IgE mediated. Methods: We evaluated sera from three groups of children: cow\'s milk allergic patients, tolerant children and a control group with non-atopic children. It was used protein extracts from different types of seeds using an ELISA assay to analyze the reactivity of IgG and IgE isotypes. Results: Comparing the IgG serum from different groups, it was observed higher concentrations and statistically significant in the allergic group compared to the tolerant and control groups, except for soybeans and kidney beans. To the IgE isotype it was observed the same patterns of reactivity shown in the analysis for IgG, with significant difference in the allergic group compared to control, except for corn. It was observed that for soybeans there were values of serum, both in the allergic and tolerant group higher than in the control group. Conclusion: Our study showed that the allergic and tolerant groups of CMA patients presented higher IgG and IgE concentrations to many seeds than the control group. These findings may suggest possible changes in permeability intestinal mucosa in these groups

Objetivos: Determinar a reatividade dos soros e determinação dos isotipos IgG e IgE a proteínas de sementes da dieta de crianças com alergia ao leite de vaca IgE mediada. Métodos: Foram avaliados soros de três grupos de crianças: alérgicas ao leite de vaca IgE mediada, crianças tolerantes ao leite e um grupo controle com crianças não atópicas. Foram usados extratos protéicos de diferentes tipos de sementes utilizando o teste de ELISA para análise da reatividade dos isotipos IgG e IgE. Resultados: Comparando as concentrações séricas de IgG dos diferentes grupos, observou-se concentrações mais elevadas e estatisticamente significante no grupo alérgico em relação aos grupos tolerante e controle, exceto para as sementes de soja e feijão roxinho. Em relação ao isotipo IgE observou-se os mesmos padrões de reatividade mostradas nas analises para IgG, com diferença significante do grupo alérgico em relação ao controle, exceto para milho. Observou-se que para a soja houve grande dispersão das concentrações séricas tanto no grupo alérgico quanto no tolerante, em valores superiores ao do grupo controle. Conclusão: A comparação entre os diversos grupos avaliados mostra que pacientes alérgicos ao leite e os tolerantes apresentam concentrações mais elevadas de IgG e IgE a outros alimentos que as crianças do grupo controle, o que pode sugerir possível alteração de permeabilidade da mucosa intestinal nestes grupos, mesmo na ausência de sintomatologia gastrintestinal / Objective: To determine the reactivity of serum and determination of IgG and IgE isotypes to seed proteins included in the diet of children with cow\'s milk allergy IgE mediated. Methods: We evaluated sera from three groups of children: cow\'s milk allergic patients, tolerant children and a control group with non-atopic children. It was used protein extracts from different types of seeds using an ELISA assay to analyze the reactivity of IgG and IgE isotypes. Results: Comparing the IgG serum from different groups, it was observed higher concentrations and statistically significant in the allergic group compared to the tolerant and control groups, except for soybeans and kidney beans. To the IgE isotype it was observed the same patterns of reactivity shown in the analysis for IgG, with significant difference in the allergic group compared to control, except for corn. It was observed that for soybeans there were values of serum, both in the allergic and tolerant group higher than in the control group. Conclusion: Our study showed that the allergic and tolerant groups of CMA patients presented higher IgG and IgE concentrations to many seeds than the control group. These findings may suggest possible changes in permeability intestinal mucosa in these groups

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T20:51:05Z : No. of bitstreams: 1 rocha_tg_me_jabo.pdf: 837046 bytes, checksum: f72346486eb135d1df17c2bfd41fd6ee (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do estudo foi avaliar a transferência de imunidade passiva (TIP) de vacas Canchim aos seus bezerros. Um grupo experimental foi constituído por 13 vacas primíparas e seus bezerros, e o outro por 13 vacas pluríparas e seus bezerros. Foram coletadas amostras de sangue venoso dos bezerros e secreções lácteas das vacas até 1 hora e 1, 2, 7, 15 e 30 dias após o nascimento/parto. No hemograma, notaram-se alterações características do período neonatal, como redução na contagem de hemácias, teor de hemoglobina e volume globular após a ingestão do colostro, além de aumento na contagem de linfócitos e redução da contagem de neutrófilos segmentados. Os exames bioquímicos séricos revelaram aumento nas atividades de GGT (até 3.746 U/L) e ALP (até 1.030 U/L) e nos teores de proteína total (até 7,77 g/dL), globulinas (até 6,01 g/dL), IgA (até 322 mg/dL) e IgG (até 2.918 mg/dL) após a ingestão do colostro, seguidos por redução gradual nestes parâmetros até os 30 dias de idade. As avaliações bioquímicas das secreções lácteas revelaram alta concentração de todos os componentes colostrais analisados, com redução gradual nos seus teores no decorrer do período experimental, à exceção dos minerais, cujas concentrações oscilaram pouco entre os momentos. A TIP foi eficiente em ambos os grupos de bezerros, e a qualidade das secreções lácteas, embora diferentes entre vacas primíparas e pluríparas, não interferiu na passagem de imunoglobulinas da vaca para o bezerro. / The aim of this study was to evaluate the passive immunity transfer from Canchim cows to its calves. One experimental group comprised 13 primiparous cows and its calves and the other group, 13 multiparous cows and its calves. Samples of calves’ venous blood and cow`s lacteal secretions were collected until 1 hour and 1, 2, 7, 15 and 30 days after birth/parturition. In the hemogram, characteristic alterations of this period, such as reduction in the erytocyte count, hemoglobin concentration and corpuscular volume after colostrum intake and rise in the lymphocyte count and reduction in the neutrophil count were noticed. The serum biochemistry revealed augmentation in the GGT (up to 3,746 U/L) and ALP (up to 1,030 U/L) activities and in the total protein (up to 7.77 g/dL), globulin (up to 6.01 g/dL), IgA (up to 322 mg/dL) and IgG (up to 2,918 mg/dL) concentrations after colostrum intake, followed by a gradual reduction in these parameters until 30 days of age. The biochemical evaluation of the lacteal secretions revealed high concentrations of all the colostral components analysed, with gradual reduction in their concentrations along the experimental period, except for the minerals, which showed little oscillation between the moments. The passive immunity transfer was efficient in both groups of calves, and the quality of the lacteal secretions, although different between primiparous and multiparous cows, did not interfere in the transference of immunoglobulins from the cows to the calves.

Les cellules dendritiques classiques (cDC) jouent un rôle crucial dans l’efficacité des réponses immunitaires précoces conduisant au contrôle ou à la persistance virale. A cet égard, il a été montré que l’infection par le VIH induit des dysfonctions des cDC caractérisées par une inhibition de leur capacité à stimuler les cellules T et associées à la progression de la maladie. Parmi les mécanismes moléculaires impliqués dans ces dysfonctions, des études in vitro ont mis en évidence le rôle du récepteur inhibiteur LILRB2. Néanmoins, la dynamique d’expression de LILRB2 ainsi que son rôle dès les premiers stades de l'infection restent à démontrer. Chez des patients en primo-infection VIH-1, nous observons une augmentation de l’expression de LILRB2 et de ses ligands HLA-I à la surface des cDC. Par ailleurs, la cinétique d’expression de LILRB2 et CMH-I au cours de l’infection de macaques cynomolgus par le SIVmac251 montre une augmentation transitoire de l'expression de LILRB2 et du CMH-I sur les cDC du sang et des ganglions lymphatiques dès les premiers jours de l’infection. Parmi les mécanismes qui pourraient être impliqués dans la régulation de l’expression de LILRB2, nos résultats indiquent que la réplication du VIH-1, l'activation de voies TLR7/8 ainsi que la présence d’IL-10 et d’IFN-I induisent une forte expression de LILRB2. Enfin, cette expression exacerbée de LILRB2 sur les cDC semble être spécifique à l'infection par le VIH/SIV. En effet, l’infection de macaques cynomolgus par le virus chikungunya, qui est caractérisée par une réponse immunitaire antivirale robuste aboutissant à un contrôle de la virémie, est associée à une expression diminuée de LILRB2 sur les cDC dès les premiers jours de l’infection. L’ensemble de nos données suggèrent un rôle majeur de l’axe inhibiteur LILRB2/MHC-I dans les mécanismes de dérégulations des cDC qui pourrait participerait à l’inefficacité des réponses immunitaires adaptatives et à la persistance du VIH/SIV. / Conventional dendritic cells (cDCs) play a crucial role in setting up early immune responses leading to viral control or persistence. In this regard, it has been shown that HIV-1 infection induces cDC dysfunctions characterized by inhibitions in their ability to stimulate T-cells and associated with disease progression. In vitro studies have shown the implication of LILRB2 inhibitory receptor in cDC dysfunctions. However, the dynamic of LILRB2 expression and its role in the early stages of infection are yet to be characterized. In primary HIV-1 infected patients, we observe an increased expression of LILRB2 and its ligands, HLA-I, on the surface of cDCs. Kinetics of LILRB2 and MHC-I expressions during SIV infection of Cynomolgus macaques shows a transient increase in LILRB2 and MHC-I expressions on blood and lymph node cDCs during the first days of infection. We also show that HIV replication, activation of TLR7/8 pathways, and presence of IL-10 and IFN-I drive upregulated expression of LILRB2. Finally, this strong induced LILIRB2 expression seems specific to HIV/SIV infections. Indeed, chikungunya virus infection of cynomolgus macaques, which characterized by a robust antiviral immune response leading to viral control, is associated with decreased expression of LILRB2 on cDCs in the first days of infection. Taken together, our data suggest a major role of the LILRB2/HLA-I inhibitory axis, mediating cDC dysfunctions and thus contributing to inefficient adaptive immune responses and viral persistence.