18 research outputs found
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Understanding the Determinants of Human Red Blood Cell Tropism of Understudied Plasmodium Species
Malaria control programs in the past decade have been successful in decreasing the global burden of Plasmodium falciparum, the more virulent of the Plasmodium species causing malaria in humans. However, as the public health community gears towards elimination and eradication of malaria, there is need for an attention shift towards research of neglected Plasmodium species.
A central event in malaria pathogenesis is the invasion of host red blood cells (RBCs) mediated by specific interactions between parasite ligands and RBC receptors. These interactions, also called invasion pathways, can be major determinants of host tropism.
Restriction in invasion due to tropism is attributed to limiting disease severity, as it limits the proliferation of the parasite in vivo. In this dissertation, we determine the host cell tropism of Plasmodium knowlesi and Plasmodium vivax, two understudied Plasmodium species. The zoonotic P. knowlesi is now the major cause of malaria in parts of South East Asia, and P. vivax is the most widespread species worldwide causing substantial morbidity. After a review of the current literature on biological and clinical features of these species and their invasion pathways in Chapter 1, we determined the tropism of P. knowlesi in human RBCs in Chapter 2. We used density-based enrichment methods to test the invasion of P. knowlesi into human RBCs of varying age. Incorporating mathematical modeling and experimental adaptation, we demonstrated that an expansion of host cell niche is required for the parasite to reach densities observed clinically. We also obtained parasite lines adapted to efficient proliferation in human RBCs. In Chapter 3, we investigated the molecular basis of increased invasion of human RBCs through whole genome sequencing analysis of newly human-adapted lines, as well as historical strains we adapted to in vitro culture. We showed that different genetic changes in P. knowlesi can lead to the upregulation of PkDBPα-DARC pathway, and have provided evidence of major divergence in invasion ligands in recent field isolates.
Finally, in Chapter 4, we studied the variation in human RBC preference of patient isolates for P. knowlesi and P. vivax. We confirmed that recent human P knowlesi isolates also vary in the niche of susceptible RBCs, with a subset exhibiting a lack of restriction in invading human RBCs. We also evaluated ex vivo P. vivax patient isolates for their host RBC preference. We determined that P. vivax field isolates differ in their level of reticulocyte preference. We further found an association between increased reticulocyte preference and schizont maturation. This body of work aims to contribute to our overall understanding of human RBC tropism of Plasmodium species of public health importance and the implication this has for parasite adaptation to humans.Biological Sciences in Public Healt
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Enrichment of Reticulocytes from Whole Blood using Aqueous Multiphase Systems of Polymers
This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)—immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.Chemistry and Chemical Biolog
Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite
Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.National Institutes of Health (U.S.) (grant AI091787)Centers for Disease Control and Prevention (U.S.) (grant (R36-CK000119-01))National Institutes of Health (U.S.) (Epidemiology of Infectious Disease and Biodefense Training Grant, 2-T32-AI007535-12
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Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite
Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways
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Insights into an Optimization of Plasmodium vivax Sal-1 In Vitro Culture: The Aotus Primate Model
Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous P. vivax in vitro culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that P. vivax may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development
Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates
© 2016 Australian Society for Parasitology Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales
Insights into an Optimization of <i>Plasmodium vivax</i> Sal-1 <i>In Vitro</i> Culture: The <i>Aotus</i> Primate Model
<div><p>Malaria is one of the most significant tropical diseases, and of the <i>Plasmodium</i> species that cause human malaria, <i>P</i>. <i>vivax</i> is the most geographically widespread. However, <i>P</i>. <i>vivax</i> remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous <i>P</i>. <i>vivax in vitro</i> culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term <i>P</i>. <i>vivax</i> culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for <i>in vitro</i> propagation. Reliance on very heterogeneous <i>P</i>. <i>vivax</i> field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined <i>Aotus</i>-adapted <i>P</i>. <i>vivax</i> Sal-1 strain to empirically evaluate different short-term <i>in vitro</i> culture conditions and compare them with previous reported attempts at <i>P</i>. <i>vivax in vitro</i> culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that <i>P</i>. <i>vivax</i> may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of <i>Aotus</i>-adapted Sal-1 for development of <i>P</i>. <i>vivax</i> laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.</p></div
Shaking and static format support cultures similarly.
<p>(<b>A</b>) Glass T flask used for shaking conditions to replicate the conditions used in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref014" target="_blank">14</a>]. (<b>B</b>) Parasitemias of two biological replicates (MN23026b and MN23009b) grown in shaking or static conditions at equal haematocrits. (<b>C</b>) Conversion rates between rings to trophozoites or trophozoites to schizonts in the two biological replicates (<b>D</b>) Parasitized erythrocyte multiplication rate (PEMR) of the two biological replicates in either shaking or static conditions using reticulocytes from hemochromatosis patients enriched by density. Error bars represent the standard error. Results are not statically significant.</p