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PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.

PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2013-02-15T17:56:02Z No. of bitstreams: 1 thatiane_s_simone_ioc_bcm_0011_2010.pdf: 9522536 bytes, checksum: fa6d5ea74828d496e73423373aedb60d (MD5) / Made available in DSpace on 2013-02-15T17:56:02Z (GMT). No. of bitstreams: 1 thatiane_s_simone_ioc_bcm_0011_2010.pdf: 9522536 bytes, checksum: fa6d5ea74828d496e73423373aedb60d (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / Este estudo apresenta os resultados da identificação de epitopos consecutivos responsáveis pelo desencadeamento da resposta imune humoral em todas as proteínas estruturais (C, prM / M e E) e não-estruturais (NS1, NS2a, NS2b, NS3, NS4a, NS4b e NS5) dos vírus dengue (DENV) tipos 1,2 e 3 circulantes no Brasil. A metodologia de síntese paralela de peptídeos (do inglês Spot synthesis) foi utilizada para a construção de uma biblioteca composta por 2007 peptídeos e, a tiragem utilizando pool de soros de pacientes com dengue, previamente confirmados através de técnicas laboratoriais, permitiu a identificação de 96 epitopos para os DENV-1, 103 epitopos para os DENV-2 e 106 epitopos para os DENV-3. Tais resultados foram comparados com métodos computacionais que permitem a predição de regiões imunogênicas (DNASTAR e IEDB) e demonstrou que, apesar de serem considerados bons indicadores de antigenecidade de uma proteína, os métodos computacionais requerem mais do que um parâmetro de predição para a confiabilidade dos resultados. Por sua vez, a síntese paralela provou ser altamente sensível e eficiente no mapeamento de epitopos consecutivos, permitindo a síntese em nano-escala de um grande número de peptídeos, de forma simultânea e reprodutível.. A fim de avaliar a capacidade em discriminar as infecções causadas pelos sorotipos de dengue daqueles negativos para esta patologia, foi realizado um teste de reação cruzada dos peptídeos identificados com pool de soros de pacientes com DENV-1, DENV-2 ou DENV-3, seguido pela avaliação utilizando pool de soros negativos para dengue, pool de soro de voluntários previamente vacinados para a febre amarela e pool de soros de pacientes com rubéola, sarampo, leptospirose, malária varíola e indicaram a existência de um total de 195 epitopos comuns ao grupo dengue e 3 epitopos específicos para os DENV-1, 9 para os DENV-2 e 11 para os DENV-3. A modelagem molecular das proteínas dos DENV permitiu a confirmação da localização dos epitopos na superfície da molécula. Esses resultados constituem a primeira descrição completa de epitopos contínuos dos DENV e poderão contribuir para a compreensão da interação anticorpo-epítopo em nível molecular e, conseqüentemente, a patogenicidade do dengue, bem como servir de base para a construção racional de vacinas preventivas e para o desenvolvimento de testes de diagnóstico sorológicas vírus-específicos. / This study shows the results of identification of epitopes responsible for consecutive triggering of humoral immune response in all structural proteins (C, prM / M, and E) and non-structural (NS1, NS2a, NS2b, NS3, NS4A, NS4B and NS5) of dengue virus (DENV) types 1,2 and 3 circulating in Brazil. The methodology for parallel synthesis of peptides (English Spot synthesis) was used to construct a library comprising peptides and 2007, using the pull pool of sera from patients with dengue previously confirmed by laboratory techniques allowed the identification of 96 epitopes for DENV-1, 103 epitopes for DENV-2 and 106 epitopes for DENV-3. These results were compared with computational methods which allow predicting immunogenic regions (DNASTAR and iedb) and demonstrated that, although they are considered good predictors of antigenicity of a protein, computational methods require more than one parameter for predicting the reliability of results. In turn, the parallel synthesis proved to be highly sensitive and efficient in the epitope mapping consecutive allowing the nano-scale synthesis of large numbers of peptides, simultaneously and reproducible .. In order to assess the ability to discriminate infections caused by serotypes of dengue those negative for this disease, we performed a test cross-reactivity of peptides identified with pooled sera from patients with DENV-1, DENV-2 and DENV-3, followed by evaluation using pool negative sera to Dengue, pool of serum from subjects previously vaccinated for yellow fever and pool of sera from patients with rubella, measles, leptospirosis, malaria, smallpox and indicated the existence of a total of 195 epitopes common to the group 3 and dengue-specific epitopes for DENV-1, 9 for DENV-2 and 11 for DENV-3. Molecular modeling of the proteins of DENV allowed confirmation of the location of epitopes on the surface of the molecule. These results provide the first complete description of continuous epitopes of DENV and may contribute to the understanding of the interaction of antibody-epitope at the molecular level and, consequently, the pathogenicity of dengue, as well as serve as a basis for the rational construction of preventive vaccines and to development of serological diagnostic tests virus-specific.

Feed costs are approximately 70% of total production cost for poultry producers. Poultry diets in the United States generally consist of 2 grains: corn and soybean meal. In recent years, the cost of these grains has dramatically increased. Due to these price increases, producers seek alternative feeds that provide adequate nutrition, and are also more affordable than "traditional" grains. Cottonseed meal is one alternative that is both affordable and an excellent source of crude protein. However, cottonseed meal contains gossypol, a pigment toxic to chickens. This study had two main objectives. The first objective was to determine if silymarin, an extract from milk thistle, could offset or prevent gossypol toxicosis. The second objective was to determine if divergent selection for humoral immune response would have an impact on the ability of the chicken to cope with gossypol toxicosis. Two preliminary studies were conducted. One determined basal activities of liver detoxification enzymes at various ages. The other determined concentrations of gossypol and silymarin that should be added to the diet to elicit a response. The information gathered from the second preliminary study was used to conduct the final experiment. In the final experiment, chickens from each of 2 lines selected for humoral immunity were exposed to diets containing gossypol, silymarin, gossypol and silymarin, and a control. Humoral immunity had no impact on the ability of the chicken to cope with gossypol toxicosis. Silymarin did not alleviate gossypol toxicosis. Future studies will focus on using a lower gossypol concentration in the diet. / Master of Science

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