Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Tuberculosis (TB) is a global health problem, which is proving extremely difficult to control in the absence of an effective vaccine. Bacille Calmette-Guérin (BCG), the only vaccine currently licensed against TB, demonstrates variable efficacy in humans and cattle. A greater understanding of what constitutes a protective host immune response is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (T_RM) are a recently-identified subset of T cells, which may represent an important aspect of protective immunity to TB. This thesis aims to characterise the role of lung T_RM in BCG-induced protection against TB. In a mouse model, intravascular staining allowed discrimination between lung-vascular and lung-parenchymal T cells. Experiments demonstrated that BCG vaccination induced a population of antigen-specific lung-parenchymal CD4⁺ T cells, a putative tissue-resident population. This lung-parenchymal population was significantly increased in frequency following mucosal BCG vaccination, compared to systemic BCG vaccination. This correlated with enhanced protection against Mycobacterium tuberculosis (M.tb) infection in the lungs of mice receiving mucosal BCG, compared to those receiving systemic BCG. Mucosal BCG induced lung-parenchymal CD4⁺ T cells with enhanced proliferative capacity and a PD1⁺KLRG1^- cell-surface phenotype, a memory-like phenotype associated with improved protection against M.tb infection. These cells may represent a BCG-induced lung T_RM population responsible for the enhanced protection observed following mucosal BCG. Overall, this thesis highlights the potential of mucosal vaccination to elicit lung T_RM and identifies this as a possible immunological mechanism underlying enhanced protection against M.tb infection. These cells may constitute an important target for future vaccination strategies.

Humans are exposed to high levels of amorphous silica on a daily basis, via the diet and the use of cosmetic and pharmaceutical products. Amorphous silica particles (10-200 nm) have also been developed for use in biomedical applications, including as binding agents in tissue repair, drug and gene therapy delivery agents, coatings for medical contrast agents and as vaccine adjuvants. Numerous studies have already been conducted to evaluate the cellular toxicity of these silica particles but still little is known about their effects both in vitro and in vivo, especially of nanosilica particles under 10 nm in diameter. The aim of this thesis was to investigate the cellular and in vivo activity of < 10 nm diameter nanosilica particles with different properties (e.g., size and dissolution rate in dilute conditions) as it may infer upon safety after exposure via the diet and intravenous administration (biomedical applications). First, the cytotoxicity of sub-10 nm nanosilica particles, fully characterized by size, dissolution rate, zeta-potential and by NMR spectroscopy, on immune cell function was assessed using transformed and cancerous cell lines and primary cells. The particles were toxic to the immune cells in a dose dependent manner and impaired certain cellular functions. Primary cells were most susceptible to nanosilica induced death and, of the primary cells, phagocytes were most susceptible to its cytotoxicity. Further investigations were conducted to assess the effect of nanosilica on T cells, as there was evidence suggesting that nanosilica particles were directly interacting with these cells. Nanosilica particles 3.6 nm in diameter were found to have a significant effect on T cell function. The particles induced numerous markers of T cell activation, including CD25 and CD69 on CD4 T cells, CD8 T cells, gamma-delta T cells and NK/NKT cells, CD95 on CD4 and CD8 T cells, CD40L, FoxP3, LAP, GARP on CD4 T cells, and IFN-gamma production, but it did not induce T cell proliferation. The particles were found to activate T cells regardless of their antigenic specificity. Further investigations showed that nanosilica interacts with the T cell receptor complex, the first documented case of a non MHC-coated nanoparticle directly interacting with this receptor complex. The nanoparticulate induced signalling through Zap70, LAT, and, eventually, through NFAT but not through MAPK. Similar signalling in the literature has been shown to induce a hyporesponsive T cell state (anergy) or activation induced cell death. The induction of the CD25 and CD69 T cell activation markers was limited to nanosilica particles below 10 nm in size, while similarly sized iron hydroxide nanoparticles (3-5 nm) only induced low levels of CD69 expression on T helper cells. Finally, it was shown that nanosilica is capable of inducing T cell activation in whole blood, though the T cell responses were greatly attenuated. Although identification of activation pathway in vivo remains elusive, the nanosilica particles were shown to have therapeutic value, decreasing murine subcutaneous tumour growth rate and significantly reducing the formation of lung metastases. Whether these in vivo responses are related to T cell activation identified in vitro remains unclear.

Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-20Bitstream added on 2014-06-13T21:04:28Z : No. of bitstreams: 1 ramos_op_dr_arafcf.pdf: 948525 bytes, checksum: 0581866624e7c3f57ffffca838856184 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 – Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII – Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... / Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer’s patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 – Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII – Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below)

Interaction between mouse dendritic cells (DCs) and Borrelia afzelii spichochetes was studied on three different levels: phagocytosis of borrelia by DCs, production of cytokines by borrelia-activated DCs and the ablilty of DCs to activate CD4+ T cells. The effect of Ixodes ricinus saliva on each of these levels was examined. Tick saliva was shown to decrease the number of phagocosing DCs. The ability of borrelia-activated DCs to induce both proliferation and IL-2 production in specific CD4+ T cells was significantly reduced by tick saliva. And surprisingly, we have shown an inhibitory effect of I. ricinus saliva on the production of both Th1 (IL-6 and TNF-{$\alpha$}) and Th2 (IL-10) cytokines. Our data reveal a complex inhibitory effect of tick saliva on DC function.

As infecções humanas com a filaria Mansonella ozzardi ocorrem em focos situados em regiões tropicais e subtropicais da América Central e do Sul e frequentemente coexistem com outras doenças endêmicas tropicais. Na Amazônia brasileira, as infecções são geralmente assintomáticas e a maior parte delas, consequentemente, deixam de ser diagnosticada. As filarioses crônicas, geralmente não tratadas, podem criar um ambiente imunorregulador, caracterizado pela expansão de linfócitos T produtores de IL-10, que mediam a supressão de respostas proliferativas de células T frente a antígenos específicos bem como a antígenos não-relacionados. Neste trabalho, utilizamos marcadores de ativação celular (CD69 e HLA-DR) e de atividade reguladora (CD39, CTLA-4, OX40, GITR, LAG3, PD-1, LAP-TGF-&#946; e TNFRII) para caracterizar populações de células mononucleares de sangue periférico (PBMCs) em indivíduos infectados por M. ozzardi bem como em controles saudáveis de uma área endêmica deste parasito na Amazônia Brasileira. A análise de PBMCs, por citometria de fluxo multiparamétrica de 49 pacientes infectados por M. ozzardi, mostrou que pacientes e controles apresentam proporções similares de Treg clássicas circulantes, no entanto, indivíduos infectados apresentam um aumento da proporção de células CD4+ e células T reguladoras (Tregs) que expressam a molécula CD39. Células Treg CD39+ parecem definir uma população distinta entre as Treg, pois ao compararmos os marcadores de regulação e ativação entre Tregs CD39+ e CD39- encontramos proporções aumentas destes marcadores nas Treg CD39+. O bloqueio dessa molécula em condições de reestimulo celular aumenta a produção de citocinas inflamatórias e diminui a produção de IL-10 confirmando seu papel regulador. / Human infections with the filarial parasite Mansonella ozzardi are common in areas of tropical and subtropical Central and South America and often coexist with other endemic tropical diseases, such as malaria. In the Amazonian Basin of Brazil, infections are typically asymptomatic; most of them will remain undiagnosed. These chronic, untreated filarial infections are potentially associated with a regulatory immune environment, dominated by IL-10-producing T-cells, which mediate the suppression of T-cell proliferation in response to filarial and non-related antigens. Here, we used markers of cell activation (CD69 and HLA-DR) and regulatory activity (CD39, CTLA-4, OX40, GITR, and TNFRII) to characterize peripheral-blood mononuclear cell (PBMCs) subpopulations in individuals infected with |M. ozzardi and in healthy controls living in an area of M. ozzardi endemicity in the Brazilian Amazon. Multiparameter flow cytometry analysis of PBMCs from 49 malaria patients showed that patients and controls have similar proportions of classic circulating Tregs, however, the proportion of CD4 + cells and Tregs expressing the CD39 (an ectonucleotidase that regulates the balance of immune responses through Phosphohydrolysis of ATP, an inflammatory molecule in adenosine, an anti-inflammatory molecule), is increased in infected patients. CD39+Treg cells seem to define a distinct population among Tregs, compare activation and regulatory markers between CD39+ and CD39- Tregs - we found increased proportions of these markers in the CD39+ Tregs. Blocking this molecule under cellular restimulation conditions increases production of inflammatory cytokines and decreases IL-10 production, improving its regulatory role.

Os microRNAs são pequenos RNAs, não-codificantes que funcionam como reguladores a nível pós-transcricional da expressão gênica. Nos últimos anos, novas evidências demonstram o papel importante dos microRNAs na regulação e desenvolvimento do sistema imune. Apesar da função de poucos microRNAs ser conhecida, a sua expressão alterada vêm sendo associada a patogênese de diversas doenças autoimunes, incluindo a artrite reumatóide (AR). Recentemente a expressão desregulada de uma série de microRNAs está sendo descrita em pacientes com AR, e o papel patogênico de apenas uma parte deles foi investigada em modelos animais. A artrite reumatóide é uma doença autoimune sistêmica caracterizada por um intenso processo inflamatório na sinóvia, podendo causar destruição óssea e articular. Os linfócitos T apresentam papel importante na indução, manutenção e progressão da doença. A artrite induzida por colágeno é um modelo animal amplamente utilizado por suas características fisiopatológicas muito similares à doença em humanos. A linhagem de camundongos DBA-1/J desenvolve a doença após imunização e booster com colágeno do tipo II, enquanto que a linhagem DBA-2/J se mostra refratária. Isso confere um sistema modelo de susceptibilidade/resistência à artrite, que pode ser estudado em diferentes abordagens. O objetivo do nosso estudo foi identificar o perfil transcricional e as redes de interação entre um grupo de microRNAs e seus respectivos alvos nos timócitos e linfócitos T CD3+ periféricos nos camundongos da linhagem DBA-1/J e DBA-2/J. Para a avaliação da expressão gênica, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos entre as duas linhagens utilizadas. Novos microRNAs foram encontrados nos diferentes estágios de desenvolvimento do linfócito T. Nas redes de interação microRNA-RNAm obtidas, genes importantes associados aos processos de sistema imune, adesão e diferenciação celular, apoptose, recombinação, ativação de linfócitos T e resposta inflamatória, foram encontrados como potenciais alvos. Além disso, em uma perspectiva clínica, baseados nos resultados obtidos em camundongo, nos encontramos a expressão do miR-505 nos linfócitos T de pacientes com AR. Nossos resultados contribuem para a melhor compreensão dos mecanismos molecular envolvidos na resistência/susceptibilidade a CIA. / MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. During the last several years, evidence has emerged to show their critical role for the regulation and development of immune system. Although the function of most mammalian miRNAs has yet to be determined, their aberrant expression has been associated with several autoimmune conditions, including rheumatoid arthritis (RA). Recently, the deregulated expression of a dozen miRNAs has been reported in patients with RA, and the pathogenic role of only a few of these has been investigated in experimental mouse models. RA is a systemic autoimmune disorder mainly characterized by the inflammation of synovial tissue that can lead to destruction of bone and cartilage. The role of effectors T cells in induction, maintenance and progression of the disease is now becoming better understood. Collagen-induced arthritis is an animal model widely studied due to its similarities to human disease. The DBA-1/J mouse strain develops arthritis after immunization process and booster with Type II collagen, and the DBA-2/J strain is refractory to the disease induction. This offers an useful susceptibility/resistance model-system to study RA. The aim of this study was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes in DBA-1/J and DBA-2/J mice strain. For this purpose we used the microarray technology to evaluate the expression of the miRNAs and mRNAs as possible targets involved in this process. The use of bioinformatics software to reconstruct the networks was essential. The results show differential expression of mRNAs and miRNAs in thymocytes and peripheral CD3+ T lymphocytes between both strains. New miRNAs were found during all the stages of T cells development. The microRNA-mRNA interaction networks obtained in this study showed that important genes related to apoptose, immune system, recombination, cell adhesion and differentiation, inflammatory process and T cell activation were found as potential targets. In addition, in a clinical prospects, based on the results obtained in mice, we found the expression of the miRNA miR-505 in T cells of RA patients. Our results contribute to a better understand of the molecular mechanisms evolved in the resistance/susceptibility to CIA.

Acredita-se que a placa dento-bacteriana seja primordial na iniciação do processo de inflamação periodontal. Entretanto, os mecanismos exatos responsáveis pela progressão da doença periodontal ainda não foram completamente elucidados. Além disso, as bases biológicas que ditam a susceptibilidade do hospedeiro ainda permanecem desconhecidas. Portanto, o presente estudo procurou investigar diferenças na expressão gênica de sítios com periodontite. Para tanto, foi utilizada a tecnologia de microarray focado para examinar a expressão de 288 genes relacionados à apoptose, resposta de células T e inflamação. RNA total foi extraído de amostras de tecido gengival e sangue de indivíduos com periodontite crônica (N=10) e de indivíduos periodontalmente saudáveis (N=4). RNA total foi então convertido em cRNA, marcado e hibridizado em lâminas de microarray. Após a exposição, os genes marcados foram quantificados e o nível de expressão genética nas amostras gengivais dos pacientes periodontais foi comparado com o nível de expressão observado no sangue desses mesmos indivíduos, e também com o nível da expressão genética no tecido gengival dos indivíduos sem doença periodontal. Para validação dos resultados dos genes selecionados (p< ou = 0,05 e no mínimo mudança de 2 vezes na expressão) foram submetidos à análise pela técnica de PCR em tempo real. O valor de p das comparações foi calculado por meio do teste t bicaudal. Os dados do microarray, confirmados por PCR em tempo real, demonstraram um total de 8 genes com baixa expressão (log2 < ou= -1 e p<ou= 0,05) e 4 genes com elevada expressão (log2 < +1 e p<ou= 0,05). Entre eles foram detectados 4 genes anti-apoptose (BCL-2, BCL2L1, BAG3 e BAFAR), 2 receptores de citocinas (IL12RB1 e IL17RA), receptor de proteína G (GPR44) e o fator nuclear de células T ativadas (NFATC1). Adicionalmente, os resultados demonstraram 3 genes com expressão elevada, uma citocina (IL-6), uma quimiocina (IL-8) e um membro da família FOS (FOSL-1). As análises da expressão genética por microarray e PCR em tempo real realizadas no presente estudo identificaram uma série de genes candidatos que podem estar relacionados à doença periodontal. A desregulação da expressão desses genes pode contribuir para a severidade desta doença. / It is widely believed that pathogenic bacteria provide the initial trigger in periodontal disease. However, the exact pathogenic mechanisms responsible for the progression of periodontal disease remain unclear. Moreover, the biologic basis dictating the susceptibility to disease has not been elucidated. Therefore, the present study sought to investigate the expression of genes altered in periodontal disease sites. To that aim, a focused microarray system was utilized to examine the expression of 288 genes related to apoptosis, T cell response and inflammation. Total RNA was extracted from gingival tissue and peripheral blood of periodontitis (N=10) and nonperiodontitis (N=4) subjects. The RNA was converted to cRNA, labeled and hybridized to oligonucleotide microarray plates. Following exposure, the positively labeled genes were quantified and the level of expression within periodontitis gingival was compared to the PB of the same subjects, as well as with GT of NP subjects. To validate the results the selected genes (p<or = 0.05 and at least 2 fold change) were subjected to real-time PCR. The p-value of the comparisons was calculated using a 2-tailed T-test. The microarray results and real-time PCR validation showed that 8 genes were downregulated (p<0.05 and log2 <or = -1). Among these, 4 anti-apoptotic genes (BCL-2, BCL2L1, BAG3 and BAFAR), 2 cytokine receptors (IL12RB1 and IL17RA), a G-protein-coupled receptors (GPR44) and a nuclear factor of activated T cells (NFATC1) were detected. In addition, the results showed 3 upregulated genes (p<0.05 and log2 < or = +1), one cytokine (IL-6), one chemokine (IL-8) and one FOS family member (FOSL1). The combined use of oligonucleotide microarrays and realtime PCR identified a number of candidates genes that can be related to the pathogenesis of periodontal disease, and dysregulation in the expression of these genes may contribute to the progression of periodontal disease.

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate similar to 30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4(+)/CD8(+) cells, we hypothesized that Program Death Ligand-1 (PDL1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3K alpha (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (similar to 9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-kappa B expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) similar to 30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI > 90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus. / The protein Themis is a new actor of the T cell receptor (TCR) signaling essential for the positive selection of T cells. The molecular function of Themis has been controversial but recent findings suggest that it acts as positive regulator of TCR signaling. We demonstrated in an initial research that Themis interacts with deubiquitylases and is covalently associated to ubiquitin chains in thymocytes. The aim of my PhD project was to characterize the molecular process that regulates the ubiquitination of Themis and to investigate how these post-translational modifications affect Themis function during T cell development. We demonstrated that Themis mRNA expression is progressively decreased after positive selection whereas Themis protein expression is enhanced at the early stages of positive selection, suggesting that Themis is stabilized by post-translational modifications during positive selection. We demonstrated that USP9X allows the deubiquitination of Themis and its stabilization following TCR engagement. Ours results suggest that USP9X is activated during TCR engagement following its recruitment to proximal signaling complexes through Grb2 and Themis, leading to the deubiquitination and stabilization of Themis expression. We believe that this mechanism is important to sustain Themis expression during positive selection and to promote durable TCR signals required for the efficiency of this process.