Browsing by Subject "Erythrocytes"

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    Dependence of red blood cell dynamics in microvessel bifurcations on the endothelial surface layer’s resistance to flow and compression
    (Springer, 2022-06) Triebold, Carlson; Barber, Jared; Mathematical Sciences, School of Science
    Red blood cells (RBCs) make up 40-45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where blood flows from one vessel into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. These studies, however, assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial glycocalyx or endothelial surface layer (ESL) that is on the order of 0.5-1 µm thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional model. In addition, the model is also used to investigate RBC deformation and RBC penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The ESL's resistance to compression is modeled using an osmotic pressure difference. One cell passes through the bifurcation at a time, so there are no cell-cell interactions. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored. Decreasing hydraulic resistivity and/or decreasing osmotic pressure differences (ESL resistance to compression) produced four behaviors: (1) RBC partitioning nonuniformity increased slightly; (2) RBC deformation decreased; (3) RBC velocity decreased relative to blood flow velocity; and (4) RBCs penetrated more deeply into the ESL. Decreasing the ESL's resistance to flow and/or compression to pathological levels could lead to more frequent cell adhesion and clotting as well as impaired vascular regulation due to weaker ATP and nitric oxide release. Potential mechanisms that can contribute to these behaviors are also discussed.
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    Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
    (Wiley, 2013) Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J., Jr.; Cui, Liwang; Pharmacology and Toxicology, School of Medicine
    Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.
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    Extent and Dynamics of Polymorphism in the Malaria Vaccine Candidate Plasmodium falciparum Reticulocyte-Binding Protein Homologue-5 in Kalifabougou, Mali
    (The American Society of Tropical Medicine and Hygiene, 2018-07) Ouattara, Amed; Tran, Tuan M.; Doumbo, Safiatou; Matthew, Adams; Agrawal, Sonia; Niangaly, Amadou; Nelson-Owens, Sara; Doumtabé, Didier; Tolo, Youssouf; Ongoiba, Aissata; Takala-Harrison, Shannon; Traoré, Boubacar; Silva, Joana C.; Crompton, Peter D.; Doumbo, Ogobara K.; Plowe, Christopher V.; Medicine, School of Medicine
    Reticulocyte-binding homologues (RH) are a ligand family that mediates merozoite invasion of erythrocytes in Plasmodium falciparum. Among the five members of this family identified so far, only P. falciparum reticulocyte-binding homologue-5 (PfRH5) has been found to be essential for parasite survival across strains that differ in virulence and route of host-cell invasion. Based on its essential role in invasion and early evidence of sequence conservation, PfRH5 has been prioritized for development as a vaccine candidate. However, little is known about the extent of genetic variability of RH5 in the field and the potential impact of such diversity on clinical outcomes or on vaccine evasion. Samples collected during a prospective cohort study of malaria incidence conducted in Kalifabougou, in southwestern Mali, were used to estimate genetic diversity, measure haplotype prevalence, and assess the within-host dynamics of PfRH5 variants over time and in relation to clinical malaria. A total of 10 nonsynonymous polymorphic sites were identified in the Pfrh5 gene, resulting in 13 haplotypes encoding unique protein variants. Four of these variants have not been previously observed. Plasmodium falciparum reticulocyte-binding homologue-5 had low amino acid haplotype (h = 0.58) and nucleotide (π = 0.00061) diversity. By contrast to other leading blood-stage malaria vaccine candidate antigens, amino acid differences were not associated with changes in the risk of febrile malaria in consecutive infections. Conserved B- and T-cell epitopes were identified. These results support the prioritization of PfRH5 for possible inclusion in a broadly cross-protective vaccine.
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    Malaria induces anemia through CD8+ T cell-dependent parasite clearance and erythrocyte removal in the spleen
    (American Society for Microbiology, 2015-01-20) Safeukui, Innocent; Gomez, Noé D.; Adelani, Aanuoluwa A.; Burte, Florence; Afolabi, Nathaniel K.; Akondy, Rama; Velazquez, Peter; Holder, Anthony; Tewari, Rita; Buffet, Pierre; Brown, Biobele J.; Shokunbi, Wuraola A.; Olaleye, David; Sodeinde, Olugbemiro; Kazura, James; Ahmed, Rafi; Mohandas, Narla; Fernandez-Reyes, Delmiro; Haldar, Kasturi; Microbiology and Immunology, School of Medicine
    Severe malarial anemia (SMA) in semi-immune individuals eliminates both infected and uninfected erythrocytes and is a frequent fatal complication. It is proportional not to circulating parasitemia but total parasite mass (sequestered) in the organs. Thus, immune responses that clear parasites in organs may trigger changes leading to anemia. Here, we use an outbred-rat model where increasing parasite removal in the spleen escalated uninfected-erythrocyte removal. Splenic parasite clearance was associated with activated CD8(+) T cells, immunodepletion of which prevented parasite clearance. CD8(+) T cell repletion and concomitant reduction of the parasite load was associated with exacerbated (40 to 60%) hemoglobin loss and changes in properties of uninfected erythrocytes. Together, these data suggest that CD8(+) T cell-dependent parasite clearance causes erythrocyte removal in the spleen and thus anemia. In children infected with the human malaria parasite Plasmodium falciparum, elevation of parasite biomass (not the number of circulating parasites) increased the odds ratio for SMA by 3.5-fold (95% confidence intervals [CI95%], 1.8- to 7.5-fold). CD8(+) T cell expansion/activation independently increased the odds ratio by 2.4-fold (CI95%, 1.0- to 5.7-fold). Concomitant increases in both conferred a 7-fold (CI95%, 1.9- to 27.4-fold)-greater risk for SMA. Together, these data suggest that CD8(+)-dependent parasite clearance may predispose individuals to uninfected-erythrocyte loss and SMA, thus informing severe disease diagnosis and strategies for vaccine development. IMPORTANCE: Malaria is a major global health problem. Severe malaria anemia (SMA) is a complex disease associated with partial immunity. Rapid hemoglobin reductions of 20 to 50% are commonly observed and must be rescued by transfusion (which can carry a risk of HIV acquisition). The causes and risk factors of SMA remain poorly understood. Recent studies suggest that SMA is linked to parasite biomass sequestered in organs. This led us to investigate whether immune mechanisms that clear parasites in organs trigger anemia. In rats, erythropoiesis is largely restricted to the bone marrow, and critical aspects of the spleen expected to be important in anemia are similar to those in humans. Therefore, using a rat model, we show that severe anemia is caused through CD8(+) T cell-dependent parasite clearance and erythrocyte removal in the spleen. CD8 activation may also be a new risk factor for SMA in African children.
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    Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion
    (Public Library of Science, 2024-09-18) Anaguano, David; Adewale-Fasoro, Opeoluwa; Vick, Grace W.; Yanik, Sean; Blauwkamp, James; Fierro, Manuel A.; Absalon, Sabrina; Srinivasan, Prakash; Muralidharan, Vasant; Pharmacology and Toxicology, School of Medicine
    Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.
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    Public antibodies to malaria antigens generated by two LAIR1 insertion modalities
    (Springer Nature, 2017-08-31) Pieper, Kathrin; Tan, Joshua; Piccoli, Luca; Foglierini, Mathilde; Barbieri, Sonia; Chen, Yiwei; Silacci-Fregn, Chiara; Wolf, Tobias; Jarrossay, David; Anderle, Marica; Abdi, Abdirahman; Ndungu, Francis M.; Doumbo, Ogobara K.; Traore, Boubacar; Tran, Tuan M.; Jongo, Said; Zenklusen, Isabelle; Crompton, Peter D.; Daubenberger, Claudia; Bull, Peter C.; Sallusto, Federica; Lanzavecchia, Antonio; Medicine, School of Medicine
    In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
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    Scalable Preparation and Differential Pharmacologic and Toxicologic Profiles of Primaquine Enantiomers
    (American Society for Microbiology (ASM), 2016-03) Dhammika Nanayakkara, N. P.; Tekwani, Babu L.; Bandara Herath, H. M. T.; Sahu, Rajnish; Gettayacamin, Montip; Tungtaeng, Anchalee; Van Gessel, Yvonne; Baresel, Paul; Wickham, Kristina S.; Bartlett, Marilyn S.; Fronczek, Frank R.; Melendez, Victor; Ohrt, Colin; Reichard, Gregory A.; McChesney, James D.; Rochford, Rosemary; Walker, Larry A.; Department of Pathology & Laboratory Medicine, IU School of Medicine
    Hematotoxicity in individuals genetically deficient in glucose-6-phosphate dehydrogenase (G6PD) activity is the major limitation of primaquine (PQ), the only antimalarial drug in clinical use for treatment of relapsing Plasmodium vivax malaria. PQ is currently clinically used in its racemic form. A scalable procedure was developed to resolve racemic PQ, thus providing pure enantiomers for the first time for detailed preclinical evaluation and potentially for clinical use. These enantiomers were compared for antiparasitic activity using several mouse models and also for general and hematological toxicities in mice and dogs. (+)-(S)-PQ showed better suppressive and causal prophylactic activity than (−)-(R)-PQ in mice infected with Plasmodium berghei. Similarly, (+)-(S)-PQ was a more potent suppressive agent than (−)-(R)-PQ in a mouse model of Pneumocystis carinii pneumonia. However, at higher doses, (+)-(S)-PQ also showed more systemic toxicity for mice. In beagle dogs, (+)-(S)-PQ caused more methemoglobinemia and was toxic at 5 mg/kg of body weight/day given orally for 3 days, while (−)-(R)-PQ was well tolerated. In a novel mouse model of hemolytic anemia associated with human G6PD deficiency, it was also demonstrated that (−)-(R)-PQ was less hemolytic than (+)-(S)-PQ for the G6PD-deficient human red cells engrafted in the NOD-SCID mice. All these data suggest that while (+)-(S)-PQ shows greater potency in terms of antiparasitic efficacy in rodents, it is also more hematotoxic than (−)-(R)-PQ in mice and dogs. Activity and toxicity differences of PQ enantiomers in different species can be attributed to their different pharmacokinetic and metabolic profiles. Taken together, these studies suggest that (−)-(R)-PQ may have a better safety margin than the racemate in human.