Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references.

Hyaluronic acid (HA) is one of the primary chemical building blocks of the extracellular matrix and thus is an attractive material for biomedical applications. FDA approved HA-based materials are available as dermal fillers, joint viscosupplements, vitreous substitutes, and abdominal adhesion barriers. The engineering of new HA-based materials and applications is an active area of research. Here we develop several new types of HA-based hydrogels with unique and useful properties. To address the challenge of delivering hydrophobic drugs from hydrophilic hydrogel matrices we have grafted HA hydrogels with [Beta]-cyclodextrin to create hydrogels capable of binding poorly water soluble drugs. To create HA hydrogels with unique anisotropic swelling behavior we have developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked domains. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. To address the challenge of creating hydrogel scaffolds with biomimetic branched porosity we have invented a "crystal templating" technique. This technique grows dendritic crystals throughout a biopolymer solution, crosslinks the biopolymer around the crystals, and washes the crystals away to yield a hydrogel with a dendritic macroporous network. Lastly, we invented a method for patterning a substrate with a microarray of hydrogel compartments. A microarray of living cells is obtained when cells are seeded on the hydrogel patterned substrate. This method addresses the need for an inexpensive, simple method for obtaining living cell microarrays that does not require clean room labs and lithographic expertise. Each of these new materials were based on hyaluronic acid hydrogels but the methods are generalizable to hydrogels of other polymers too. In conclusion, the novel methods in this dissertation are a significant contribution to the engineering of HA-based materials. / text

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Formins, also known as formin homology (FH) proteins, are involved in a wide range of actin-mediated processes. The Diaphanous-related formin Daam1 (Dishevelled-associated activator of morphogenesis) interacts with the PDZ domain protein Dishevelled, and is required to establish planar cell polarity in Xenopus. Through a yeast two-hybrid screen, I characterized a PDZ-mediated interaction between the C-terminus of Daam1 and the PDZ domains 456 of GRIP1. In dissociated rat hippocampal cultures, Daam1 expression was seen throughout the soma and dendrites in a punctate pattern. Furthermore, co-staining with a synaptic marker suggests that Daam1 could be associated with post-synaptic specializations. Dendritic spines are enriched with actin filaments, and based on the subcellular localization of Daam1 and the evidence that formins are involved in regulating actin polymerization, I hypothesized that Daam1 might play a role in dendritic spine morphology. In order to investigate the functional roles for Daam1, viral vectors were developed using the Semliki-Forest defective viral vector to over-express the full-length Daam1 protein and a Daam1 lacking the PDZ-binding motif. The over-expression of the full-length Daam1 in organotypic hippocampal slices showed a punctate distribution throughout the dendritic shaft, with the occasional appearance in spines, resulting in an overall increase in dendritic spine length. This suggests that formins, such as Daam1, could potentially regulate spine morphology.

I examined the relationship between landscape attributes and population differentiation among lake trout (Salvelinus namaycush) populations inhabiting a hierarchically structured dendritic freshwater system in northern Labrador, the Kogaluk River system. Samples were collected from a total of 10 lakes which differed in size, elevation, level of connectivity, and position within the system. STRUCTURE analysis provided evidence of significant population structure within the system likely attributed to a varying degree of asymmetric gene flow. Gene flow estimates were generally low and appear to be influenced by the presence of waterfalls as well as geographic distance. Isolation by distance tests coupled with decomposed pairwise regression analysis suggest a significant influence of geographic distance on population differentiation. Mantel testing also showed that population differentiation is significantly correlated with the position of waterfalls. Estimates of effective population size reveal significantly smaller population sizes in headwater lakes, a pattern not attributed to lake size.

Les anomalies phénotypiques et fonctionnelles des lymphocytes B (LB) sont typiques d'une infection au VIH et se traduisent principalement par une activation polyclonale, une perte de la mémoire immunitaire ainsi qu'une réponse humorale déficiente et des phénomènes auto-immunitaires souvent précurseurs de lymphomes B. Ces anomalies se retrouvent principalement chez les patients lors de la phase chronique de la maladie et semblent être reliées en partie au niveau de la charge virale ainsi qu'à un compartiment de lymphocytes T CD4+ altéré. Cependant, quoique controversé, des éléments d’activation polyclonale ont également été observés chez les non-progresseurs à long terme (LTNPs) qui présentent une charge virale faible et un compartiment T CD4+ semblable aux individus séronégatifs. Ainsi, les objectifs principaux de cette étude sont 1) d’établir une chronologie des anomalies du compartiment des cellules B chez des individus infectés par le VIH qui ont une progression différente de la maladie (PHI normaux, rapides, sains et LTNP). 2) corréler les niveaux sériques du stimulateur de lymphocytes B (BLyS), un facteur de croissance des cellules B, avec les phénotypes observés chez ces mêmes patients. L’hyperglobulinémie, les niveaux sériques de BLyS et d’auto-anticorps ont été mesuré longitudinalement chez une cohorte d’individus en primo-infection (PHI) avec des progressions différentes de la maladie (rapides et normaux), LTNP et sujets sains. Nos résultats démontrent que l’activation polyclonale des LB survient indépendamment de la vitesse de progression et persiste chez les LTNP ou malgré une thérapie antirétrovirale efficace chez les progresseurs rapides. Des niveaux élevés de BLyS dans le sérum des progresseurs rapides corrèlent avec des fréquences altérées de monocytes et cellules dendritiques, suggérant un rôle de celles-ci dans l’atteinte du compartiment des cellules B. / B lymphocyte abnormalities are an important consequence of HIV infection, where both polyclonal activation and loss of B cell memory and humoral immunity have been described, and often evolve towards rheumatic-like autoimmunity and lymphoma. Although these abnormalities are prevalent in chronically infected patients, polyclonal B cell activation is also reported in patients with primary HIV-infection (PHI), who already present signs of defective humoral immunity. Although controversial, elements of B cell dysregulation have been reported in long term non progressor (LTNP) patients, even though they bear low viral loads and present a relatively "normal" CD4+ T cell compartment, suggesting that other factors are involved. Therefore, the main objectives of this study are to 1) establish a timeline for specific B cell abnormalities in HIV-infected patients with different rates of disease progression (PHI normal and fast progressors, LTNP), and controls 2) to correlate serum levels of the B lymphocyte stimulator (BLyS) a B cell growth factor, among these patients and controls. Thus we have longitudinally assessed hyperglobulinemia, auto-antibody and soluble BLyS levels in the serum of subjects undergoing primary HIV infection (PHI) with different rates of disease progression; rapid and normal progressors, long term non-progressors (LTNPs), and healthy donors. Here, we report that B cell polyclonal activation occurs independently of the rate of disease progression, with hypergammaglobulinemia persisting beyond successful therapy in rapid progressors and despite non-progressing clinical disease in LTNPs. High levels of BLyS in the serum of PHI rapid progressors correlate with altered blood monocyte and dendritic cell frequencies suggesting their contribution in triggering B cell dysregulations.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production, which can eventually lead to immune complex (IC)-mediated organ damage. Due to the stimulation of plasmacytoid dendritic cells (pDC) by nucleic acid-containing ICs (DNA- or RNA-IC), patients with SLE have an ongoing interferon (IFN)-α production. IFN-α induces a general activation of the immune system that may initiate or propagate an autoimmune process if not properly regulated. Previous studies have shown that natural killer (NK) cells potently enhance the IFN-α production by pDCs. In study I, the mechanisms behind the NK cell-mediated increased IFN-α production by RNA-IC-stimulated pDCs were investigated. ICs triggered CD56dim NK cells via FcγRIIIA to the secretion of cytokines (e.g. MIP-1β) that promoted IFN-α production. Additionally, an LFA-1-dependent cell-cell interaction between pDCs and NK cells strongly contributed to the increased production of IFN-α. In study II, the RNA-IC-induced regulation of surface molecules on pDCs and NK cells was investigated. The expression of CD319 and CD229, which are two SLAM family receptors genetically associated with SLE, was induced on pDCs and NK cells by RNA-IC. IFN-α-producing pDCs displayed an increased expression of CD319 and CD229, whereas pDCs from patients with SLE had a decreased expression of CD319. In study III, we serendipitously identified an SLE patient harboring autoantibodies to the NK cell receptor CD94/NKG2A. In study IV, sera from 203 patients with SLE were analyzed for autoantibodies to the CD94/NKG2A, CD94/NKG2C and NKG2D receptors. Seven patients harbored anti-CD94/NKG2A autoantibodies, and two of these patient’s autoantibodies also reacted with CD94/NKG2C. Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with the HLA-E-mediated regulation of NK cell cytotoxicity, and facilitated the elimination of target cells expressing these receptors. Furthermore, these autoantibodies were found in a group of severely diseased SLE patients and their titers closely followed disease activity. In conclusion, this thesis provides insights to molecular mechanisms whereby NK cells regulate the IFN-α production, it further links the SLAM receptors to SLE, and it describes novel autoantibodies to receptors regulating NK cell cytotoxicity. Together these findings strengthen the assumption that NK cells are involved in the pathogenesis of SLE.

Les travaux antérieurs du laboratoire ont démontré le rôle du CD47 dans la fonction des cellules dendritiques ainsi que dans l’induction des lymphocytes T régulateurs (Tregs) chez l’humain in vitro. Notre premier objectif était de déterminer le rôle de CD47 sur la fonction des DCs in vivo. Nos travaux démontrent que le CD47 contrôle sélectivement la migration des DCs au travers des vaisseaux lymphatiques et des barrières cellulaires endothéliales in vivo sans interférer avec celle des lymphocytes T et B. Des expériences de migration compétitive et d’ immunisation active avec des DCs myéloïdes démontrent que la migration des DCs est dépendante de l’expression du CD47 sur les DCs et non sur les cellules endothéliales. Ce défaut de migration est corrélé avec l’absence de DCs spléniques dans la zone marginale chez nos souris CD47-/-. Notre second objectif était de déterminer le rôle de CD47 dans l’homéostasie et la fonction des Tregs. Nous démontrons que l’expression du CD47 contrôle sélectivement l’homéostasie d’une sous-population de Tregs CD103+ à l’état de base. La proportion de cellules activées/mémoires (CD44hi CD62Llo ) Foxp3+ CD103+ augmente rapidement au cours du vieillissement chez nos souris CD47-/- comparée aux souris CD47+/+ du même âge, tandis que le pourcentage de cellules (CD44loCD62Lhi) Foxp3+ CD103- reste comparable entre les deux souches de souris. En conclusion, le CD47 inhibe la prolifération excessive des Tregs CD103+ empêchant ainsi l’accumulation de ces cellules en absence d’inflammation. Les DCs et les Tregs sont étroitement régulées de manière reciproque. Cette régulation croisée contribue au maintien d’un équilibre entre l’immunité protectrice et la tolérance. La perspective de nos travaux est d’approfondir nos connaissances sur le rôle du CD47 et de ses ligands dans la régulation des DCs par les Tregs et vice et versa. Les DCs et les Tregs étant impliqués dans la pathogenèse de multiples maladies telles que le cancer, les maladies infectieuses et les maladies auto-immunes. Par conséquent, nos études pourraient ouvrir des portes à de nouvelles stratégies thérapeutiques. / Previous work in the laboratory have demonstrated the role of CD47 in the function of dendritic cells and in the induction of regulatory T cells in humans. Here, we show that the ubiquitous self-marker CD47 selectively regulates DC, but not T and B cell trafficking across lymphatic vessels and endothelial barriers in vivo. Competitive DC migration assays and active immunization with myeloid DCs demonstrate that CD47 expression is required on DCs but not on the endothelium and not vice and versa for efficient DC trafficking and T-cell responses. This migratory defect correlates with the quasi-disappearance of splenic marginal zone DCs in non manipulated CD47-deficient mice. Our data reveal that CD47 on DCs is a critical factor in controlling migration and efficient initiation of the immune response. Mutual or reciprocal regulation exists between DCs and Tregs. For instance, DCs efficiently induce Tregs in vivo. We here examine how CD47 deficiency that selectively decrease myeloid DCs impact on Treg homeostasis. We here show that CD47 expression, selectively regulated CD103+Foxp3+ Treg homeostasis. The proportion of effector/memory-like (CD44highCD62Llow) CD103+ Foxp3+ Tregs rapidly augmented with age in CD47-deficient mice (CD47-/-) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44lowCD62Lhigh) CD103-Foxp3+ Tregs remained stable. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103+ Tregs that may overwhelmingly inhibit Ag specific T cell responses. DCs and Tregs are closely regulated to maintain the balance between protective immunity and tolerance. When that balance is broken, several diseases such as cancer, infectious diseases and autoimmune diseases may develop. Our ultimate goal is to understand how CD47 regulates DCs and Tregs function. Manipulation of the two cells types may open to the door to unexplored therapeutic avenues.

L’asthme est une maladie multifactorielle hétérogène qui engendre une inflammation pulmonaire associée à une variété de manifestations cliniques, dont des difficultés respiratoires graves. Globalement, l’asthme touche environ une personne sur 6 et présente actuellement un sérieux problème de santé publique. Bien que de nombreux traitements soient disponibles pour soulager les symptômes de la maladie, aucun traitement curatif n’est actuellement disponible. La compréhension des mécanismes qui régissent l’état inflammatoire au cours de la maladie est primordiale à la découverte de nouvelles cibles thérapeutiques efficaces. Les cellules dendritiques captent les allergènes dans les poumons et migrent vers les ganglions drainants pour les présenter aux cellules T et engendrer la réponse inflammatoire pathogénique chez les asthmatiques. Nous avons contribué à l’avancement des connaissances mécanistiques de l’asthme en identifiant chez la souris la sous-population de cellules dendritiques responsable de l’initiation et du maintien de la réponse inflammatoire locale et systémique associée à l’asthme. En effet, nous avons démontré que le SIRPα, récepteur extracellulaire impliqué dans la régulation de la réponse immune, est sélectivement exprimé à la surface des cellules dendritiques immunogéniques. L’interruption de la liaison entre le SIRPα et son ligand, le CD47, interfère avec la migration des cellules dendritiques SIRPα+ et renverse la réponse inflammatoire allergique. Ce mécanisme constitue une avenue thérapeutique prometteuse. D’ailleurs, les molécules de fusion CD47-Fc et SIRPα-Fc se sont avérées efficaces pour inhiber l’asthme allergique dans le modèle murin. Nous avons également démontré l’implication des cellules dendritiques SIRPα dans un modèle d’inflammation pulmonaire sévère. L’administration répétée de ces cellules, localement par la voie intra-trachéale et systémiquement par la voie intra-veineuse, mène au développement d’une réponse inflammatoire mixte, de type Th2-Th17, similaire à celle observée chez les patients atteints d’asthme sévère. La présence de cellules T exprimant à la fois l’IL-17, l’IL-4, l’IL-13 et le GATA3 a été mise en évidence pour la première fois in vitro et in vivo dans les poumons et les ganglions médiastinaux grâce à ce modèle. Nos expériences suggèrent que ces cellules Th2-Th17 exploitent la plasticité des cellules T et sont générées à partir de la conversion de cellules Th17 qui acquièrent un phénotype Th2, et non l’inverse. Ces résultats approfondissent la compréhension des mécanismes impliqués dans l’initiation et le maintien de l’asthme allergique et non allergique, en plus d’ouvrir la voie à l’élaboration d’un traitement spécifique pour les patients asthmatiques, particulièrement ceux pour qui aucun traitement efficace n’est actuellement disponible. / Asthma is a heterogeneous multifactorial disease resulting in airway inflammation associated with a variety of clinical manifestations, which include severe breathing difficulties. Asthma affects approximately one out of six people and is currently a serious public health problem. As of now, many treatments are available to relieve the symptoms of the disease, but no definitive cure is available. Understanding the mechanisms that regulate the inflammatory condition during the disease is essential to the discovery of effective new therapeutic targets. Dendritic cells capture allergens in the lungs, migrate to the draining lymph nodes where they activate cognate T cells, which cause the pathogenic inflammatory response. My work help defined and deepened the mechanistic understanding of asthma by identifying the subpopulation of dendritic cells responsible for the initiation and maintenance of local and systemic inflammatory response. We demonstrated that SIRPα is selectively expressed on the surface of immunogenic dendritic cells. Indeed, the interruption of the ligation between SIRPα and its ligand, CD47, interferes with the migration of SIRPα+ dendritic cells and reverses the allergic inflammatory response. This mechanism is a promising new therapeutic avenue. Moreover, we showed that the soluble fusion molecules CD47-Fc and SIRPα-Fc are potent inhibitors of the allergic asthma in a mouse model. In addition, we demonstrated the involvement of SIRPα+ dendritic cells in a model of severe airway inflammation induced upon local and systemic repeated administration of those cells. Either treatment led to the development of a mixed Th2-Th17 inflammation, a phenotype recently described in patients with severe asthma. This model allowed us to show the presence of T cells expressing at once IL-17, IL-4, IL-13 and GATA3 in vitro and in vivo in the lungs and in the mediastinal lymph nodes. Our results suggest that these Th2-Th17 cells are generated from the conversion of Th17 cells acquiring a Th2 phenotype, and not the other way around, a hallmark of Th17 cells plasticity. These results deepen the understanding of the mechanisms involved in the initiation and maintenance of allergic and non-allergic asthma. Besides, we open a way to the development of a specific treatment for asthmatic patients, particularly those for whom no effective treatment is currently available.