This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Candidiasis is a life-threatening infection that occurs in patients whose immune systems are not working properly. Candida albicans is the leading cause of Candida infections originating from the blood stream. Despite antifungal therapy, mortality resulting from these infections due to C. albicans remains unacceptably high and continues to increase. In light of these concerns, it is critical that mechanisms by which C. albicans initiate the infection in the blood stream be identified. C. albicans must bind to and traverse the vascular endothelium (cells that line the blood vessels)to escape from the blood vessel and infect a target organ such as kidneys. Therefore, a critical step in crossing the endothelial barrier involves adherence to and invasion of vascular endothelial cells by C. albicans. Additionally, during these processes C. albicans must evade killing by circulating white blood cells. Glycosyl-phosphatidylinositol-anchored proteins (GPI-proteins)are cell surface proteins of C. albicans and are known to encode key elements that promote infection. Amongst these GPI-proteins are those that regulate adherence to and invasion of endothelial cells, and resistance to white blood cells-mediated killing. In the studies proposed herein, we will use a new, genetic approach that depends on over producing these proteins under specific conditions to investigate the contribution of these proteins to the infectious process. After overproduction of these proteins in C. albicans, the generated organisms will be used to compare their ability to the parent strain (containing regular amount of the protein under study)in adhering adhering to and damaging endothelial cells as well as resistance to white blood cell killing in the test tube. To perform these experiments, it is necessary to grow in the test tube the same type of human cells that the fungus normally interacts with while in the blood stream (i.e. human endothelial cells and white blood cells). The most available, richest source of endothelial cells is the umbilical cord veins. The umbilical cords are anonymously removed from placentas that are to be discarded after delivery. There is no link between the patient and the umbilical cord that would allow the patient?s identity to be known. Given the absence of any link between the umbilical cord, the fact that the umbilical cords are anonymously removed from the placenta after delivery of the child, the fact that the umbilical cords would be destroyed if they were not used for this research, the risk to the patient is minimal. White blood cells (neutrophils)will be harvested from healthy volunteers after signing an informed consent. The volunteers will be from our division which is will diversified in ethnicity and gender, including African Americans, Asians, Hispanics and Caucasians. Approximately 10 ml blood will be harvested per draw. Phlebotomy will not be performed on any given volunteer more frequently than once per month. Phlebotomy is a routine procedure with minimal adverse effects that might include discomfort and some swelling in the arm due to needle insertion. Given the potential for discoveries to be made that could lead to a vaccine to prevent common and life-threatening Candida albicans, the potential benefit of this research is high. Therefore, the risk:benefit ratio is highly favorable. It is improtant to mention that all procedures involving harvesting endothelial cells from umbilical cords and obtaining blood samples are required only for performing the research outlined in the attached grant application.
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