Malaria

A hallmark trait of P. falciparum malaria is sequestration, in which parasite infected erythrocytes (IEs) adhere to the vasculature, causing organ failure and death. Current antimalarials only kill the parasites, necessitating development of anti-adhesion drugs. Using our two-step approach, we can efficiently screen for anti-adhesion small molecules. Screenings of 75libraries using Bio-Plex 200 identified the most active TPI libraries, which were deconvoluted to single compounds. Screenings library TPI 1319 yielded 3 inhibiting non-optimized compounds, each of which inhibits binding between two receptors, CSA and ICAM1, and their binding PfEMP1 domains. Two compounds deconvoluted from TPI 2103 prevent binding between PfEMP1 and ICAM1. Cytoadhesion assays with live IEs support the results seen with Bio-Plex, with best hits showing inhibition below 200 nM. Cytotoxicity testing of active compounds showed minimaltoxicity. Identified hits appear to be amenable to Structure Activity Relationship studies to develop powerful anti-adhesion drugs to treat severe malaria.

Placental malaria infection, during which infected red blood cells sequester in the placenta, is a substantial cause of pregnancy-related complications in areas where malaria is endemic. Accumulation of infected red blood cells creates an inflammatory environment and induces an immune response that can be deleterious to the placenta. This response can cause complications that include low birth weight, which is a major risk factor for neonatal and infant death. A decrease in the megalin transport and signaling system has been demonstrated to be linked with placental malaria infection and to be connected with low birth weight pathology. In this study we analyze the abundance of a protein related to megalin, LRP1 (LDL receptor related protein 1) in pregnancy malaria. Protein expression was analyzed in placental tissue samples by immunofluorescence staining. A statistically significant decrease was observed in the expression of LRP1 in placental samples of patients stratified by presence of placental malaria infection and infants born with low birth weight. Findings were supported using an in vitro cell model of placental syncytial trophoblast during malarial infection. In this model BeWo cell line was incubated with erythrocytes infected with malaria parasite CS2 line that is known for binding to malaria placental receptor. LRP1 expression in BeWo cells was analyzed by immunostaining and Western Blot, and a reduction was found by both methods. Analysis of LRP1 mRNA levels by RT-qPCR revealed no difference compared to control samples, indicating that changes happen at the protein level.

Electrical impedance of cells is a sensitive indicator of changes in cellular structure and biophysical characteristics. Integration of electrical impedance sensing in microfluidics can be a useful tool for characterization of blood cells for their disease state, such as sickle cell disease and malaria. The first part of this dissertation presents application of a microfluidics-based electrical impedance sensor for the study of sickle cell disease. Dynamic cell sickling-unsickling process of blood cells in response to cyclic hypoxia was measured. Strong correlation was found between the electrical impedance data and patients’ hematological parameters such as levels of sickle hemoglobin and fetal hemoglobin. In addition, application of electrical impedance spectroscopy in narrow microfluidic channel was used for label-free flow cytometry and non-invasive assay of single sickle cells under controlled oxygen level. We demonstrate the capability of this new technique in differentiating normal red blood cells from sickle cells, as well as sickled cells from unsickled cells, using normoxic and hypoxic conditions. The second part of this dissertation reports an application of electrical impedance sensing for the study of placental malaria. Testing conditions were optimized so that electrical impedance can be used for real time monitoring of different cellular and molecular level variations in this in vitro model of placental malaria. Impedance characteristics of cell proliferation, syncytial fusion and long-term response of BeWo cells to adhesion of infected erythrocytes were obtained and related to the immunostaining results and inflammatory cytokines measurements. Comparing to the conventional optical microscope-based methods, electrical impedance sensing technique can provide a label-free, real-time monitoring tool to study erythrocytes and cytoadhesion, and can further be extended to other disease models and cell types.

Malaria is a severe global health problem that causes approximately 435,000 deaths per year. Any non-immune individual traveling to malaria endemic regions can be affected too, including humanitarian volunteers, travelers, and US troops.
Under physiological conditions, damaged or malaria-infected RBCs would be removed within the spleen, but Plasmodium falciparum infected RBCs (iRBCs) sequester to microvascular endothelial cells to avoid entering the spleen. Adhesion interactions and parasite sequestration to endothelial cells are mediated by Plasmodium falciparum erythrocyte membrane protein 1 family (PfEMP1) proteins expressed on the iRBC’s surface. The PfEMP1 proteins bind to existing endothelial cell surface receptors that already serve primary functions, including ICAM-1, integrin αVβ3, and CD36.
Traditionally, these receptors are explored in the context of endothelial cell sequestration, but this project examines the consequence of receptor::PfEMP1 interaction on immune cells, namely monocyte-like THP-1 cells.