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 Mechanisms of fungal pathogenesis: In the paragraphs that follow you will see that we study several different aspects of C. neoformans pathogenesis. In their own unique way, each project advances our knowledge of how C. neoformans causes disease and how innate and humoral immunity influences the outcome of infection.
 Capsule function: The capsule of C. neoformans is one of the main virulence factors of this pathogenic fungus. A main feature of the capsule is that it can dramatically change its size depending on the environment. We are interested in the process of capsule growth, and the influence that such growth has on the interaction with the host. We have found that capsule growth inhibits complement-mediated phagocytosis by macrophages. These results show that capsule enlargement is a dynamic process and such a process can affect the host response.
 Melanin structure and function: Melanin production in C. neoformans, is associated with virulence. Melanin is a pigment with an undefined chemical structure and tremendous physical stability. This pigment accumulates in the cell wall of C. neoformans. We are interested in understanding the fundamental biological process of how melanin in the cell wall is remodeled to allow growth and budding to occur. In addition, we are also studying how bacterial-fungal interactions, which may occur in the environment, cause melanization of C. neoformans, ultimately allowing resistance against amoeboid predators and mammalian hosts.
 Mathematical modeling of antibody-mediated phagocytosis: We are investigating how protective and non-protective antibodies to C. neoformans affect the phagocytosis process of macrophages. Using monoclonal antibodies we have developed to the capsular polysaccharide, we can experimentally evaluate this host-pathogen interaction and use the data to formulate a mathematical model of the kinetics of phagocytosis and determine how different antibodies change the model.
 Relationship between antibody structure and function: This area of research focuses on understanding the relationship of antibody structure and its biological function. To answer such questions the generation, characterization and molecular analysis of monoclonal antibodies against antigens of C. neoformans and Bacillus anthracis are necessary. In addition, we are also investigating epitope specificity and idiotypic reactivity of antibodies.
 Effects of antibody on innate immunity: We are also investigating the host-pathogen interaction between macrophages and C. neoformans. Currently a comparative study of three C. neoformans strains with respect to their interaction with host macrophages is being conducted. We are studying the intracellular replication of the yeasts with live imaging and analyzing the consequences of such events on the host macrophage. In addition, we are examining the more general question of how the cell cycle of the macrophage protects the host against microbial infections. We have found that macrophages were driven into replication after they phagocytosed C. neoformans by either Fc- or complement-mediated phagocytosis. This novel observation suggests a potential mechanism for increasing the number of effector cells after microbial ingestion, but can also have the negative consequence of promoting the spread of infection.
 Structures of capsular polysaccharides: C. neoformans is an encapsulated yeast that uses its polysaccharide capsule to evade the immune system. We are currently studying the composition and physical properties of the major and minor components of the capsule GXM and GalXM. We are characterizing these polysaccharides in order to better understand the macromolecular structure of the capsule and to resolve key issues such as the identity of the polysaccharide epitopes recognized by protective antibodies and the contribution of capsular components to virulence.
 Biofilm formation: Biofilms are communities of microorganisms that attach to surfaces and allow them to survive in new and sometimes hostile environments. The ability of microbes to generate biofilms on indwelling and prosthetic medical devices has become a troublesome medical problem. C. neoformans biofilms allow for increased antifungal resistance. We are currently developing an in vivo model for C. neoformans biofilm formation. In addition, we are also investigating how antibodies may protect the host by preventing C. neoformans biofilm formation.
 Origin of virulence in pathogenic fungi: The mechanism of acquiring and maintaining virulence by C. neoformans is unknown. The evolution of virulence traits is being studied in three different systems. The interactions of C. neoformans with the free-living soil amoeba Acanthamoebae castellanii and the slime mold Dictyostelium discoideum are being characterized. These interactions help us to understand the environmental survival strategy of C. neoformans and relate to the emergence of fungal virulence for humans. In addition, mouse-passaged C. neoformans strains are also being used to understand how this pathogenic yeast evolves virulence in mammals.
 Development of antibody therapy: We are generating and characterizing monoclonal antibodies (mAbs) to the poly (g-D-glutamic acid) capsule of Bacillus anthracis. These mAbs will be used to gain a better understanding of the potential of humoral immunity to protect by targeting the capsule, for passive antibody therapy and to gain greater insight into the relationship between antibody structure and function for an unusual antigen composed of a polymerized D-amino acids. These studies will produce new information on antibody structures and gene usage that is of fundamental importance for vaccine design and the treatment of anthrax.
 Validating the Damage-Response Framework: Several years ago, we proposed a new way to view pathogenesis. We called it the Damage-Response framework (see review: Casadevall and Pirofski (2003) Nat Rev Microbiol 1:17-24) to incorporate the concept that both the host and the microorganism effect the outcome of infection. There are three tenets to this model. First, that microbial pathogenesis is the outcome of an interaction between a HOST and a microorganism, and is attributable to neither the microorganism nor the host alone. Second, that the pathological outcome of the host. Microorganism interaction is determined by the amount of DAMAGE to the host. Third, that damage to the host can result from microbial factors and/or the host response. Current efforts to experimentally test the Damage-Response framework include mathematical modeling of the phagocytosis of C. neoformans by macrophages.
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