ABSTRACT/ SUMMARY The Immuno-modulatory Biomaterials Laboratory at the University of Florida focuses on the development of novel biomaterial systems that can manipulate the immune system. Our goal is to design the next generation of immunotherapeutics for applications in immune-related diseases. This multidisciplinary work incorporates aspects of biomaterials engineering, drug delivery, immunology, biochemistry, and cell physiology. One of the main research programs in our lab is understanding controlled vomocytosis (non-lytic exocytosis) of fungal cells from phagocytes. Our primary motivation for elucidation of this process is the development of a hybrid fungal- microparticle system that can accomplish phagocytic cell-mediated delivery of drugs, including vaccines to guarded sites such as the brain and lymph nodes. Phagocytic cells (particularly macrophages [MΦ] and dendritic cells [DCs])) have evolved to circulate through peripheral tissue, engulf foreign particulate matter and traffick to specific tissues. Evidently, phagocytic cells can transport particulate materials from peripheral tissues to lymphatic organs. Typically, following phagocytosis, materials that have been engulfed are taken into the phagosome (vacuole in the cytoplasm of a cell containing a phagocytosed particles) where a degradative process occurs due to secretion of reactive oxygen species (ROS), acidic pH and digestive enzymes. This stage would be counterproductive to the integrity of the internalized material. Moreover, for delivery from a phagocyte there has to be expulsion, and there is precedent for such behavior. The fungal cell, Cryptococcus neoformans, elicits vomocytosis or (non-lytic exocytosis) from macrophages. There is still little understanding about the mechanisms governing this phenomena. Here, we seek to demystify vomocytosis - a phenomenon that is understudied. Ultimately, our long-term goal is to develop a universally-deployable, bug-microparticle platform as an effective therapeutic for a plethora of immune and neurological conditions. These goals can only be accomplished through comprehension of the interactions of this remarkable pathogen with innate immune cells. Critically, to address gaps in our knowledge requires (i) the ability to identify vomocytosis in vivo, (ii) cognizance of dissemination and interaction in hosts at the organ, tissue, cellular and subcellular levels and (iii) probing the role of Ca2+ in CN vomocytosis. This proposal seeks to systematically cover these next steps using state-of-the- art engineering tools, including those devised by the PI’s group. Therefore, the proposed program is highly relevant to the mission of the National Institute of General Medical Sciences (NIGMS), which pertains to supporting research that increases understanding of biological processes and lays the foundation for advances in disease diagnosis, treatment and prevention.

PROJECT NARRATIVE The brain is a highly-guarded organ with physiological barriers, in particular, the blood–brain barrier (BBB) and the blood–cerebrospinal fluid barrier, limiting access of most drugs. Interestingly, a bug – Cryptococcus neoformans (CN) has adapted special mechanisms to bypass the BBB. Herein, we seek to understand the underlying mechanisms of innate immune cell vomocytosis of CN through combinatorial engineering and biological tools. Therefore, the proposed program is highly relevant to the mission of the National Institute of General Medical Sciences (NIGMS), which pertains to supporting research that increases understanding of biological processes and lays the foundation for advances in disease diagnosis, treatment and prevention.