Introdução: Doenças pulmonares ou infecções que ocorrem no início da vida podem ter permanente impacto na vida adulta. Pouco se sabe sobre o perfil de células do sistema imunológico em pulmões de crianças lactentes. Objetivo: Descrever o perfil de células do sistema imunológico no pulmão de lactentes sem doença pulmonar. Métodos: Amostras de pulmões histologicamente normais, obtidas através de autópsia de dez crianças que morreram de causas acidentais ou de doenças não pulmonares, foram marcadas por anticorpos contra linfócitos B e T, macrófagos, células NK (natural Killer), células citotóxicas, células dendríticas e mastócitos. As células foram quantificadas no epitélio, na camada interna, na camada externa das vias aéreas e nos septos alveolares. Membrana basal e septos alveolares foram medidos através de análise de imagem. Resultados expressos em células/mm de membrana basal epitelial brônquica ou alveolar. Resultados: A mediana das idades foi 2,5 meses (1-730 dias). Os resultados mostraram que a camada interna apresentou pequena densidade celular. No epitélio da via aérea e no parênquima houve predominância de células que estão relacionadas com a imunidade inata, tais como: CD56+, Granzyme + e CD68+. A camada externa e o parênquima alveolar apresentaram a maior densidade celular. Poucas células T CD4+ e células dendríticas foram encontradas na maioria dos compartimentos do pulmão. Conclusão: Há uma compartimentalização de células relacionadas com o sistema imunológico ao longo da via aérea e parênquima dos pulmões das crianças estudadas. Esta configuração pode estar relacionada com o desenvolvimento dos mecanismos de defesa da imunidade inata e da imunidade adquirida. Este conhecimento é importante para entender os mecanismos da imunocompetência pós-natal dos pulmões / Introduction: Pulmonary diseases or infections occurring early in life may have a permanent impact in adulthood. Little is known about the normal immune cell profile in the lungs of infants. Objective: To describe the immune cell profile of infants without lung disease. Methods: Histologically normal lung samples obtained at autopsy of ten infants that died either due to incidental or inflicted causes or non-pulmonary diseases were stained for antibodies against B and T lymphocytes, macrophages, NK cells, cytotoxic cells, dendritic cells and mast cells. Cells were quantified in the airway epithelial layer, inner layer, outer layer and alveolar septa. Basement membrane or alveolar septa lengths were assessed by image analysis. Results are expressed as cells/mm. Results: The median age of patients was 2.5 months and ranged from 1- 730 days. The inner layer of the airways was the region with the smallest density of cells. There was a predominance of cells related to the innate immunity such as CD56+, Granzyme B+ and CD68+ cells in the epithelial layer and alveolar parenchyma. The outer layer and the lung parenchyma presented the highest cellular density. There were very few CD4+ T cells or dendritic cells in most of the lung compartments. Conclusions: There was a compartmentalization of immune cells along airways and parenchyma in infants, which may be related to the development of innate and acquired lung defense mechanisms. This knowledge is important to understand mechanisms of postnatal immune competence of the lungs

Despite Human Leukocyte Antigen (HLA) matching and use of immunosuppressive drugs, graft-versus-host disease (GVHD) following hematopoietic stem cell transplant (HSCT) is prevalent and often fatal. Additionally, older HSCT recipients experience increased morbidity and mortality. Prophylactic treatment with age-matched syngeneic (recipient strain-derived) cultured regulatory DC (DCreg) has been shown to decrease GVHD-associated mortality in young bone marrow transplanted (BMT) mice. The purpose of this study was to investigate: 1) the potential to generate DCreg from older mice and their subsequent ability to ameliorate GVHD in older BMT mice, 2) the mechanism(s) by which DCreg mitigate GVHD in vivo, 3) the ability of DCreg-treated BMT mice to respond to infectious pathogens, and 4) whether DCreg can be generated under clinically relevant conditions from healthy donor and HSCT recipient PBMCs. To evaluate the efficacy of DCreg treatment in older mice, complete MHC-mismatched BMT mice were treated with DCreg (hereafter referred to as DCreg-treated BMT mice). Although DCreg treatment ameliorated GVHD in older BMT mice, these mice had increased morbidity and decreased survival compared to their young counterparts. Following transfer into BMT mice, older DCreg failed to increase inhibitory molecule (PD-L1 and PIR B) expression while significantly upregulating co-stimulatory molecule (CD40 and CD80) expression, conversely young DCreg upregulated inhibitory molecules as well as co-stimulatory molecules. These phenotypic differences between young and older DCreg in vivo provide a potential mechanism for modestly increased morbidity and mortality in older DCreg-treated BMT mice relative to their young counterparts. Indeed, BMT mice treated with DCreg deficient in PD-L1 or PIR B had significantly reduced overall survival, thus both molecules are required for optimal GVHD mitigation. A murine H1N1 influenza (IAV) infection model was used to assess the donor immune system's capacity to respond to relevant antigens other than those responsible for GVHD. Surprisingly, sub-lethally IAV-infected DCreg-treated BMT mice began to die after d. +21 and all were deceased by d. +25. Virus-specific CD8+ T cell and antibody (Ab) responses were undetectable following primary infection. Interestingly, following a prime-boost infection strategy, DCreg-treated BMT mice survived lethal IAV challenge with no signs of morbidity and had demonstrable IAV-specific Ab and CD8+ T cell responses. Thus a prime-boost IAV infection strategy establishes a protective immune response in the DCreg-treated BMT mice and underscores the potential role vaccination may play in establishing immune competence in DCreg-treated BMT mice. We investigated whether human DCreg can be generated under clinically relevant conditions: 1) following peripheral blood mononuclear cell (PBMC) cryopreservation, 2) in bovine serum-free media, and 3) from older individuals and HSCT recipients. DCreg were generated from healthy donor and HSCT patient PBMCs isolated from young (old) and older (> 50 years old) individuals by culturing cells in X-vivo serum-free. Human DCreg generated from both young and older healthy donor PBMCs had comparable numbers, surface molecule phenotype, cytokine production, and able to induce Treg. Cryopreserved and fresh PBMCs generated DCreg with similar phenotypes and cytokine production. DCreg generated from HSCT recipients maintained low co-stimulatory molecule and high inhibitory molecule expression as well as immunosuppressive cytokine production. These studies confirm DCreg can be generated under clinically relevant conditions.

The 21st century has been called the age of genomic medicine, yet gene therapy for medicinal use remains a theory. One reason that there are no safe and effective treatments for human disease is the lack of a vehicle capable of delivering genetic material to a specific target. In nature we observe gene pathology by viral vectors, which deliver their own genetic material to specific host cells efficient at spreading the viral blueprint throughout the organism. The aim of my research into gene therapy has been to develop a synthetic vector with the delivery capability of viral vectors found in nature. This includes the ability to protect genetic cargo from modification and degradation in vivo, target to a desired cell type within a specific tissue, facilitating absorption into the cell, and delivery to the nucleus, where expression of genetic material occurs. The goal of this thesis project was to synthesize a novel vector which would selectively target the dendritic cell SIGN receptor, mirroring the method of pathogens such as HIV, which target this receptor and subsequently the immune system, resulting in chronic infection. The vector we designed contains two major components, the high mannose N-glycan Man9GlcNAc2Asn, and a peptide composed of nine amino acids: four lysine spacing residues, four lysines derivatized with acridine on the epsilon amine of their side chains, and a cysteine for conjugation to the glycan. This compound, the Man9-AcrLys Glycopeptide, was engineered to intercalate into plasmid DNA via the acridine functional groups and to bind the DC-SIGN receptor through the glycan's mannose residues. The vehicle was tested in vitro in CHO cells bearing a recombinant DC-SIGN receptor in the context of luciferase reporter gene delivery. We found that under equal treatment conditions, DC-SIGN (+) CHO cells expressed more luciferase and were 100-fold more luminescent than control DC-SIGN (-) CHO cells. My delivery method was further analyzed in a cell-sorting FACS experiment. I covalently labeled pGL3 reporter plasmid with a fluorophore, and transfected the CHO cells under typical transfection conditions. The experimental results confirmed preferential DC-SIGN mediated gene delivery.

The discovery that cationic lipids can independently stimulate the immune system has generated interest in their potential as vaccine adjuvants. Here, we show that the cationic lipid R-DOTAP can independently stimulate type 1 interferon production in dendritic cells in both primary culture and immortalized cell culture. Levels of type 1 interferon production are cell line-dependent and limited in vitro by lipid-induced cell death. We show that cationic lipids can independently activate TLR-7 and TLR-9, suggesting a mechanism for type 1 interferon induction. This TLR-stimulatory activity is not restricted to R-DOTAP and can be extended to other similar cationic lipids in a lipid-specific and TLR-specific manner.

Measurements are carried out for dendrite tip growth of succinonitrile-acetone alloys solidifying freely in an undercooled melt. The current experimental investigation is conducted using the equiaxed dendritic solidification experiment (EDSE). This setup allows for precise measurements of the dendrite tip velocity, radius and shape for a range of undercoolings and solute concentrations. The collected data are compared to available theories of free dendritic growth, such as the Lipton-Glicksman-Kurz and Li-Beckermann models. It is found that for dilute succinonitrile-acetone alloys, the measured dendrite tip Péclet numbers agree well with previous theories of free dendritic growth, if the effects of melt convection are taken into account. The tip selection parameter deviates significantly from the pure succinonitrile value and is inversely related to the applied undercooling. Besides, the selection parameter shows no dependence on the solute concentration. These results are consistent with phase-field simulations and preceding experimental investigations. In addition, scaling relationships for the sidebranching shape were obtained in terms of the dendritic envelope, projection area and contour length. These new scaling relations agree well with previous measurements in pure succinonitrile dendrites by Li and Beckermann.

The severe disease associated with seasonal epidemics of influenza A virus (IAV), as well as pandemic outbreaks, have highlighted the necessity for novel, broadly cross-reactive vaccination and therapeutic strategies against IAV. Our studies have focused on the contribution of IAV-specific CD8 T cells to mediating protection following IAV vaccination and infection as IAV-specific CD8 T cells are required for clearance of IAV. Further, IAV-specific CD8 T cells are typically cross-protective as they are generally directed at highly conserved areas of IAV. Recently, influenza virus-like particles (VLPs) have been developed from recombinant baculoviruses containing influenza proteins hemagglutinin (HA) and/or neuraminidase (NA) on the surface and matrix (M1) in the VLP core. Influenza VLPs induce potent antibody responses and have been shown to provide protection from morbidity and mortality during lethal homo- and hetero-subtypic IAV challenge. This suggests that conserved, VLP-induced CD8 T cell responses may also contribute to the overall protective ability of VLPs. However, whether influenza VLPs can induce influenza-specific CD8 T cell responses and if these T cells are protective during IAV challenge remains unknown. Here, I demonstrate that a single, intranasal vaccination with VLPs containing HA and M1 leads to a significant increase in HA533-specific CD8 T cells in the lungs and lung-draining lymph nodes. Our results also indicate that HA533-specific CD8 T cells primed by influenza VLP vaccination are significantly increased in the lungs following lethal IAV challenge. These VLP-induced memory CD8 T cells are critical in providing protection from lethality following subsequent challenge infections, as depletion of CD8 T cells leads to increased mortality, even when total, but not VLP-induced memory, CD8 T cell numbers have been allowed to recover prior to lethal dose IAV challenge. In addition, my studies also importantly demonstrate that these VLP-induced, HA533-specific CD8 T cells aid in protection from high-dose, heterosubtypic IAV infections where CD8 T cell epitopes are conserved, but the targets of neutralizing antibodies have been destroyed. This dissertation further elucidates the requirements for the regulation of the IAV-specific CD8 T cell response in the periphery (i.e. lung) by pDC and CD8α+ DC. Our studies have previously demonstrated that pDC or CD8α+ DC must present viral antigen in the context of MHC class I along with trans-presentation of IL-15 to effector, IAV-specific CD8 T cells in the lungs to protect the T cells from apoptosis and allow generation of the full magnitude CD8 T cell response needed to clear IAV infection. Herein, I demonstrate that in addition to antigen presentation and IL-15, costimulatory molecules on the surface of pDC and CD8α+ DC are also required. However, the specific costimulatory molecules required depends upon both the mouse strain utilized for IAV infection as well as DC subset. In addition to costimulatory molecules, I also demonstrate that the requirement for pDC and CD8α+ DC to be infected differs in order for them to participate in this pulmonary rescue of the IAV-specific CD8 T cell response. While CD8α+ DC are able to efficiently cross-present exogenous antigen, pDC must be directly infected and utilize the endogenous, direct antigen presentation pathway to present viral antigen to IAV-specific CD8 T cells in the lungs during IAV infection. These data suggest there are distinct differences between pDC and CD8α+ DC in their mechanism of regulating the pulmonary IAV-specific CD8 T cell response, which had not been previously appreciated. Together, the results presented herein further detail the mechanism of regulation of effector IAV-specific CD8 T cells by DC as well as the contribution of IAV-specific CD8 T cells to a novel, IAV VLP vaccination strategy. These findings highlight the importance of IAV-specific CD8 T cells in mediating protection following IAV vaccination and infection.

Sphingoid bases (sphingosine, dihydrosphingosine and phytosphingosine) have been recently found in the oral cavity where they may serve to fortify innate immunity against commensals and periodontal pathogens. In fact, sphingoid bases have potent antimicrobial activity against Gram- positive and Gram- negative bacteria including oral pathogens like Porphyromonas gingivalis. It is not known whether these lipids are cytotoxic or alter the chemokine and cytokine responses of human dendritic cells, a finding important to their future potential as a therapeutic for treatment of periodontal disease. Objectives: The objective of this study was to determine the effects of sphingoid bases on the cytotoxicity and cytokine responses of human myeloid dendritic cells. Methods: Dendritic cells were treated with sphingoid bases (0.2-80.0 μM) for 16 hours in the presence or absence of 0.02 μM hemagglutinin B, a nonfimbrial adhesin of P. gingivalis used as a pro-inflammatory stimulus. The cytotoxicity of the inocula and its ability to induce the production of chemokines and pro-inflammatory cytokines was determined after 16 hours. Results: Higher concentrations of sphingoid bases were cytotoxic (e.g., 40.0-80.0 μM), but physiologic concentrations of sphingoid bases (e.g., 0.2-20.0 μM) were not. At 5, 10, or 20 μM, sphingosine did not enhance or attenuate any HagB-induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. At 5 or 10 μM, neither phytosphingosine nor dihydrosphingosine enhanced or attenuated any HagB- induced IL-8, GM-CSF, MIP-1α, MIP-1β, or TNFα response of human myeloid dendritic cells. Conclusion: Sphingoid bases exhibit dose-dependent cytotoxicity and cytokine responses against human myeloid dendritic cells. But at physiologic concentrations sphingoid bases appear to be safe and efficacious at the doses needed to prevent or treat microbial infections in the oral cavity.

L'athérosclérose est une maladie cardio-vasculaire immuno-inflammatoire se développant sur un terrain de dyslipidémie. De nombreuses composantes de la réponse immunitaire sont capables de moduler le développement des plaques d'athérome. Notamment, les lymphocytes T (LTs) CD4+ dont le rôle dans le processus athérogène dépend de la voie de polarisation. Le mécanisme de polarisation des LTs CD4+ est sous le contrôle des cellules dendritiques conventionnelles (cDCs) CD11b+. Ainsi, moduler ces cDCs et orienter la réponse adaptative vers une polarisation anti-athérogène pourraient représenter une potentielle cible thérapeutique dans la pathologie. Dans cette perspective, nous avons évalué le rôle des cDCs CD11b+ dans le développement de l'athérosclérose qui à ce jour reste totalement inexploré. Nous avons montré que la baisse du nombre des cDCs CD11b+ n'a pas d'impact sur le développement des lésions d'athérome. Cependant, nous montrons que l'absence du facteur de transcription IRF4 nécessaire au développement des cDCs CD11b+ altère le rôle anti-athérogène reconnu de l'adjuvant vaccinal à base d'aluminium (Alum). Nos données suggèrent que les cDCs CD11b+ n'ont pas d'impact direct sur le développement de l'athérosclérose, cependant, elles contrôlent l'effet athéroprotecteur de l'Alum. / Atherosclerosis is a disease characterized by arterial blood vessel thickening due to the accumulation of inflammatory cells in the arterial intima in response to cholesterol deposition. Several components of the immune response are able to modulate the development of atheromatous plaques. In particular, the role of conventional of CD4+ lymphocytes in the atherogenic process depends on their polarization pathway. The polarization mechanism of CD4+ T cells is under the control of conventional CD11b+ dendritic cells (cDCs). Thus, modulating these cDCs and orienting the adaptive response towards anti-atherogenic polarization could represent a potential therapeutic target in pathology. In this context, we evaluated the role of CD11b+ cDCs in the development of atherosclerosis which still remains totally unexplored. We have demonstrated that the decrease of the number of CD11b+ cDCs has no impact on the development of atheroma lesions. However, we show that the deletion of IRF4, the transcription factor necessary for the development of CD11b+ cDCs alters the recognized anti-atherogenic role of the aluminum-based vaccine adjuvant (Alum). However our data suggest that CD11b+ cDCs have no direct impact on the development of atherosclerosis but can control the atheroprotective effect of Alum adjuvant.

A infecção por HIV-1 leva a uma séria imunodeficiência causada principalmente pela depleção de linfócitos T auxiliadores, a principal célula-alvo do vírus. Além dos linfócitos T CD4, o HIV-1 também pode interagir e infectar macrófagos e células dendríticas (DCs). As DCs são resistentes à infecção pelo HIV-1, mas podem internalizar vírions em compartimentos e transferi-los para linfócitos T CD4&#43, em um processo chamado trans-infecção. Para promover sua infecção, o HIV-1 subverte o citoesqueleto da actina da célula hospedeira em várias etapas de seu ciclo. Em DCs o citoesqueleto também é essencial para internalização do HIV-1 e formação dos compartimentos. Miosinas são proteínas motoras que interagem com filamentos de actina e estão envolvidas em diversos processos celulares, incluindo migração, transporte de moléculas, endocitose e reciclagem de componentes de lipid rafts. Apesar de existirem mais de 40 tipos de miosinas em humanos, apenas a miosina 2a foi estudada no contexto da trans-infecção. Por isso, nosso objetivo nesse trabalho foi estudar o papel das miosinas 1c e 1e na maturação de células dendríticas derivadas de monócitos (MDDCs) e na internalização de HIV-1 por estas células. Confirmamos por Real Time PCR a expressão de 10 miosinas em MDDCs de doadores saudáveis, depois verificamos que há regulação negativa da expressão do gene da miosina (myo1c) em MDDCs de pacientes HIV&#43. Analisamos a ativação das células em resposta ao lipopolissacarídeo (LPS) por meio da expressão de CD86 e HLA-DR em MDDCs silenciadas para myo1c e 1e. Não houve diferença na expressão dos marcadores de ativação em células silenciadas para miosina 1e (myo1e) em relação ao controle. No entanto, na maioria dos doadores testados, o silenciamento da myo1c interferiu com o aumento de expressão desses marcadores, indicando que a myo1c possa ter um papel na ativação celular por LPS. Ademais, a localização subcelular do HIV-1 em MDDCs silenciadas para myo1c e ativadas com LPS ficou mais próxima ao fenótipo de células imaturas. Contudo, não houve diferença na quantidade HIV-1 internalizado por MDDCs silenciadas para as miosinas 1c e 1e ou tratadas com um inibidor específico de miosinas do tipo 1. Estes resultados sugerem que a myo1c pode estar envolvida na ativação de células dendríticas e consequentemente alterar o mecanismo de internalização do HIV-1 por MDDCs. / Infection by human immunodeficiency virus (HIV) leads to severe immunodeficiency caused by depletion of T helper cells, the main targets of the virus. Besides T CD4&#43 cells, HIV-1 can infect and interact with other immune cells, including dendritic cells and macrophages. Dendritic cells are resistant to HIV infection, however, they can bind and internalize HIV in compartments and then transfer the virus to CD4&#43 T cells in a process called trans-infection. To promote infection, HIV-1 subverts actin cytoskeleton of host cell at several points of its cycle. In DCs, cytoskeleton is also essential to HIV-1 internalization and compartment assembly. Myosins are motor proteins that can interact with actin and take part in several cellular processes, including migration, molecular trafficking, endocytosis and lipid raft recycling. Even though there are about 40 myosin types, only myosin 2a has been investigated in trans-infection. Thus, our aim was to evaluate the role of myosins 1c and 1e in monocyte derived dendritic cell (MDDC) activation and HIV-1 internalization. We have validated the expression of 10 myosins in MDDCs by real-time PCR, and observed a down regulation of myosin 1c gene in HIV&#43 patients. We have evaluated cell activation in response to lipopolysaccharide (LPS) through CD86 and HLA-DR expression in myosin 1c and 1e knocked down MDDCs. There was no change in expression of activation markers in myosin 1e knocked down MDDCs compared with control cells. However, in most donors, myosin 1c knock down impaired the increase of activation markers following LPS treatment, suggesting that myosin 1c may play a role in cell activation by LPS. In addition, subcellular location of HIV-1 in MDDCs knocked down for myosin 1c and activated with LPS, was similar to immature cell phenotype. Nevertheless, we have not observed changes in the amount of HIV-1 internalized by myosin 1c or 1e knocked down MDDCs or in MDDCs treated with myosin I inhibitor. These data suggest that myosin 1c may play a role in MDDC activation and therefore alter the mechanism of HIV-1 internalization by MDDCs.

Airway hyperresponsiveness (AHR) is one of the primary features of allergic airways disease. Despite continuous allergen exposure atopic asthmatics do not develop progressively worsening AHR. The mechanism(s) that limit AHR are unknown. Two valid candidates are regulatory T cells (Treg) and antigen presenting cells (APC). Dendritic cells (DC) are the main APC within the airways. Presentation of allergens to T cells can result in the differentiation and expansion of different subsets of T cells including effector Treg cells. The precise role of Treg and DC in the attenuation of allergen-induced AHR remains unknown. The general aim of this thesis is to investigate mechanisms to limit AHR in a murine model of atopic asthma. Specific aims are to: 1. develop a murine model of allergen-induced attenuation of AHR, 2. determine the potential role of regulatory T cells (Treg) in allergen-induced AHR attenuation, and 3. determine the potential role of airway dendritic cells (DC) in allergen-induced AHR attenuation. Balb/c mice were sensitised with intraperitoneal Ovalbumin (OVA) in aluminium hydroxide and challenged with a single, 3-weeks or 6-weeks of OVA aerosols. Aerosols were 1% OVA in sterile saline delivered for 30 minutes for three days per week. Animals were sacrificed 24 hours after the final aerosol for measurements of lung function and Methacholine (MCh) responsiveness (low-frequency forced oscillation technique), collection of bronchoalveolar lavage fluid (BALF) and serum. '...' In contrast, 6-weeks of OVA challenges decreased Treg numbers back to control levels. Adoptive transfer of 1x106 Treg taken from DLN of 3-week challenged mice attenuated AHR in single-OVA recipients (p<0.05). Furthermore, in vivo depletion of Treg in 3-week OVA challenged mice restored AHR (p<0.05 compared with control). Similar proportions of CD4+ T cells became activated following both aerosol regimes, however total numbers of airway CD4+ T cells were decreased (p<0.05), and OVA-specific CD4+ T cell proliferation in DLN was reduced (p<0.05) after 3-weeks versus one OVA aerosol. Analysis of antigen handling by airway APC populations showed antigen uptake (OVA-647) and processing (DQ-OVA) by macrophages and airway DC subsets to be down-regulated (p<0.05) after 3-weeks of OVA aerosols. In addition, adoptive transfer of Treg into single-OVA recipients did not affect antigen handling by airway APC populations. These data suggest that Treg are responsible for allergen-induced attenuation of AHR in vivo in established airways disease. AHR attenuation was associated with an altered function of airway DC, resulting in reduced antigen capture and processing, leading to limited clonal expansion of antigen-specific CD4+ T cells with limited production of Th2 cytokines. Furthermore, Treg were not directly responsible for the down-regulation of allergen capture in the airways. In conclusion, knowledge of the role of Treg and DC in attenuation of AHR could potentially result in improved and more directed therapies for the attenuation of AHR in atopic asthmatics.