Malaria is one of the oldest parasitic diseases known to man, and the disease has played a role in shaping civilizations and the success of human populations over many centuries. While the malaria is well studied, it still remains a worldwide killer--claiming about 600,000 lives annually with children under the age of five representing a disproportionate population of those lethally infected. Malaria is caused by the protozoan parasite Plasmodium, which is introduced to the human body through the bite of a female Anopheles mosquito. The most lethal form of the disease is carried by the parasite Plasmodium falciparum, while the most widespread form of malaria is caused by Plasmodium vivax, the latter of which has a specific mode of entry and life cycle that makes it difficult to eradicate. The entry of P. vivax into human reticulocytes is based on the presence of the Duffy antigen chemokine receptor (DARC), which is uniquely absent in two-thirds of the Black population and populations of immediate African descent making it rare in the African region while endemic in Western and Asian countries. Inability to culture the parasite P. vivax in vitro and exhaustible tissue samples makes an accurate model of P. vivax malaria difficult to maintain ex vivo.
The current study focuses on overcoming those limitations by modeling the mode of entry of P. vivax into patient-specific, induced pluripotent stem cell (iPSC)-derived erythrocyte-lineage cells by showing firstly that DARC is a measurable marker of susceptibility in vitro via FACS analysis, and that secondly, P. vivax cell culture limitations can be bypassed by creating a lentivirus designed to specifically infect DARC-expressing cells. To demonstrate the potency of this system, we show that a virus expressing the conserved region of the Duffy binding ligand, Duffy binding protein II (DBPII), can selectively infect peripheral blood mononuclear cells (PBMCs) that express DARC. Moreover, our current study focuses on the development of an iPSC-based disease model using patient samples derived from DARC expressing patients (DARC+) and DARC negative Sickle Cell Disease (SCD) patients (DARC-). We show that DARC+ iPSC-derived erythroid lineage cells express a transient population of DARC-expressing cells via FACS analysis, and we explore different protocols to stabilize this unique population. We hypothesize that DARC is a stage-specific marker for erythrocyte maturation, and we believe that any subset of cells expressing DARC consists of more mature erythrocyte-lineage cells. This study then, provides a novel platform by which to study malaria infection in a patient-specific manner while bypassing the limitations of culturing P. vivax in an in vitro culture system, as well as introducing a new way to measure erythrocyte maturation. Successful establishment of such a disease model has great implications for in-depth drug screenings for novel therapeutics that target the blood stage of the parasitic disease that were previously difficult to validate due to the limitations of currently existing models.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/15082 |
| Date | 22 January 2016 |
| Creators | Nah, Shirley |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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