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• W3087629065 abstract "Standard in vitro culture environments differ dramatically from in vivo conditions in nutrient levels, hematocrit, and rheology and have lower variability in gas levels and temperature.Nutritional and physical differences lead to pronounced, and often rapid, changes in phenomenon, important for understanding virulence in Plasmodium.Parasite drug sensitivity may be altered due to culture adaptation selection, supraphysiological metabolite concentrations, and in vitro media formulations.Parasites propagated in vitro, versus in vivo, show altered transcriptomic and genomic patterns related to virulence factors, metabolism, gametocytogenesis, and more.Direct-from-host methodologies avoid the impacts of in vitro culture adaptation but limit the types of assessments that can be performed as many experiments either require equipment not readily available in endemic settings or necessitate long-term manipulation. Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes. Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes. In humans, five species of Plasmodium lead to malaria infections, with Plasmodium falciparum being responsible for the majority of malaria morbidity and mortality [1.WHO World Malaria Report 2019. World Health Organization, 2019Google Scholar]. Malaria has burdened humanity for millennia. Even as recently as the 20th century, malaria infections accounted for an estimated 2–5% of all deaths [2.Carter R. Mendis K.N. Evolutionary and historical aspects of the burden of malaria.Clin. Microbiol. Rev. 2002; 15: 564-594Crossref PubMed Scopus (416) Google Scholar]. Yet, successful vector control, infection prevention, and antimalarial drug treatments have put malaria elimination in reach for progressively more countries [1.WHO World Malaria Report 2019. World Health Organization, 2019Google Scholar]. The advent of a standardized P. falciparum continuous laboratory 'in vitro' culture system in the 1970s has been essential to these successes by accelerating understanding of parasite biology and drug sensitivity. In vitro culture allows for the continuous cultivation of the intraerythrocytic developmental cycle (IDC) (see Glossary) of not only P. falciparum but, more recently, of Plasmodium knowlesi as well [3.Lim C. et al.Expansion of host cellular niche can drive adaptation of a zoonotic malaria parasite to humans.Nat. Commun. 2013; 4: 1638Crossref PubMed Scopus (75) Google Scholar,4.Moon R.W. et al.Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 531Crossref PubMed Scopus (161) Google Scholar]. Notably, conditions suitable for robust long-term in vitro propagation of Plasmodium vivax have yet to be deciphered, and therefore samples of P. vivax and other human malaria species (Plasmodium ovale, Plasmodium malariae) must come directly from human infections. Within the human body, known as 'in vivo', the IDC is responsible for all symptoms of malaria, including cyclical fevers and hypoglycemia. Asexual stages that occur late during the IDC, as well as immature gametocytes, sequester in the microvasculature and bone marrow [5.Cao P. et al.Modeling the dynamics of Plasmodium falciparum gametocytes in humans during malaria infection.eLife. 2019; 8e49058Crossref PubMed Scopus (22) Google Scholar, 6.Neveu G. et al.Plasmodium falciparum gametocyte-infected erythrocytes do not adhere to human primary erythroblasts.Sci. Rep. 2018; 8: 17886Crossref PubMed Scopus (11) Google Scholar, 7.Obaldia N. et al.Bone marrow is a major parasite reservoir in Plasmodium vivax infection.mBio. 2018; 9e00625-18Crossref PubMed Scopus (107) Google Scholar], respectively, to avoid removal by the host’s spleen [8.Blasco B. et al.Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic.Nat. Med. 2017; 23: 917-928Crossref PubMed Scopus (286) Google Scholar,9.David P.H. et al.Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes.Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5075-5079Crossref PubMed Scopus (276) Google Scholar]. Thus, a blood sample taken directly from an infected human will contain primarily early-stage asexual parasites and mature gametocytes, whereas a sample from in vitro culture can contain an asynchronous mix of all stages. Similar to samples directly from in vivo, ex vivo samples contain parasites within the first asexual developmental cycle outside of the host and, like in vivo samples, will contain a synchronous population of parasites but at a stage dependent on the time elapsed since phlebotomy. While modern in vitro culture systems have been wholly necessary for many of the biological breakthroughs in Plasmodium biology, no in vitro system can perfectly recapitulate an in vivo infection. Parasites in vivo are subjected to a myriad of physical, nutritional, and immunological factors differing from those found in vitro. Pressure exerted by these factors in vivo induces dynamic reactions in parasites that are difficult to appreciate in vitro but result in meaningful changes affecting virulence, transmissibility, and drug sensitivity. Many of the differences between in vitro and in vivo Plasmodium environments and their implications were reviewed by LeRoux et al. over a decade ago [10.LeRoux M. et al.Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions.Trends Parasitol. 2009; 25: 474-481Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. Here, we review knowledge that has been gained in the intervening decade on how the differences in nutrient levels and physical forces between in vivo/ex vivo and in vitro culture environments lead to changes in Plasmodium biology. Further, we present various methodologies for studying Plasmodium biology directly from the human host, which avoids the impact of the artificial culture environment discussed within this review. Finally, we advocate for methodological improvements that will drive research progress of physiologically relevant parasite phenotypes. In vitro P. falciparum and P. knowlesi culture systems are almost universally maintained in RPMI 1640-based media, supplemented either with (i) 10% human serum, (ii) 0.5% AlbuMAX Lipid-Rich bovine serum albumin (BSA) (ALB) plus hypoxanthine, or (iii) a serum–ALB combination. Reducing human serum usage through ALB-supplemented RPMI media is a popular choice due to lower cost, reduced batch-to-batch variability, and elimination of the logistical challenge of obtaining non-immune serum in endemic localities. Parasites cultured in ALB-supplemented media have generally demonstrated good growth compared with those grown with human serum supplementation, yet evidence suggests large underlying effects of different media formulations. Through a comparison of human serum-supplemented versus ALB-supplemented P. falciparum cultures, close to 1000 genes were significantly differentially expressed between cultivation medium, with the large majority exhibiting increased expression in ALB [11.Tilly A.K. et al.Type of in vitro cultivation influences cytoadhesion, knob structure, protein localization and transcriptome profile of Plasmodium falciparum.Sci. Rep. 2015; 5: 16766Crossref PubMed Scopus (11) Google Scholar]. Gene ontology terms most enriched in ALB upregulated genes indicate significant metabolic changes (e.g., metal ion transport, DNA catabolic processes, divalent inorganic transmembrane transport activity, and oxidoreductase activity), which highlights the impacts of differing nutrient environments. Whether supplemented with ALB or serum, all RPMI 1640-based media formulations provide a source of critical lipids and purines in addition to the abundant glucose, amino acids, cofactors, and salts. The nutrient-rich base media contain many metabolites at levels far higher than those present in the host in vivo environment (i.e., tenfold higher hydroxyproline and 100-fold higher folic acid [10.LeRoux M. et al.Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions.Trends Parasitol. 2009; 25: 474-481Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]). This nutritional strategy serves to promote growth in diverse cell types where specific metabolic needs may be poorly defined; however, divergence in metabolite levels in vivo and in vitro likely contributes to discordant results discussed throughout this review. For example, reduced cholesterol abundance in vitro compared with in vivo proved to be impactful for a recently developed method that enriches infected red blood cells (RBCs) by selectively lysing uninfected RBCs using a cholesterol-dependent lytic agent (Figure 1A , Key Figure) [12.Brown A.C. et al.Cholesterol-dependent enrichment of understudied erythrocytic stages of human Plasmodium parasites.Sci. Rep. 2020; 10: 4591Crossref PubMed Scopus (12) Google Scholar]. The considerably lower cholesterol levels of in vitro media formulations maximize the cellular discrimination required for this technique but these levels remain adequate to support normal parasite invasion in vitro [13.Dluzewski A.R. et al.Relation of red cell membrane properties to invasion by Plasmodium falciparum.Parasitology. 1985; 91: 273-280Crossref PubMed Scopus (22) Google Scholar]. In vivo, cytoadherence can lead to sequestration and rosetting in P. falciparum infections, which serve to avoid parasite removal by splenic filtration and increase immune evasion, respectively [14.Henry B. et al.The human spleen in malaria: filter or shelter?.Trends Parasitol. 2020; 36: 435-446Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,15.Niang M. et al.STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting.Cell Host Microbe. 2014; 16: 81-93Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar]. Therefore, accurate knowledge about these phenomena is critical for understanding Plasmodium virulence. Factors present in human serum, including lipoproteins and components of the immune system, are necessary in vitro for high levels of erythrocyte membrane protein 1 (PfEMP1) presentation on the P. falciparum-infected RBC surface as well as the formation of rosettes (mediated by stevor gene products) [16.Frankland S. et al.Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface.Eukaryot. Cell. 2007; 6: 1584-1594Crossref PubMed Scopus (35) Google Scholar, 17.Luginbühl A. et al.Complement factor D, albumin, and immunoglobulin G anti-band 3 protein antibodies mimic serum in promoting rosetting of malaria-infected red blood cells.Infect. Immun. 2007; 75: 1771-1777Crossref PubMed Scopus (22) Google Scholar, 18.Ribacke U. et al.Improved in vitro culture of Plasmodium falciparum permits establishment of clinical isolates with preserved multiplication, invasion and rosetting phenotypes.PLoS One. 2013; 8e69781Crossref PubMed Scopus (30) Google Scholar]. However, the presence of serum factors may not be the whole story; transcription of PfEMP1-encoding var genes is decreased even in parasites transitioned into human serum-supplemented cultures [19.Peters J.M. et al.Differential changes in Plasmodium falciparum var transcription during adaptation to culture.J. Infect. Dis. 2007; 195: 748-755Crossref PubMed Scopus (42) Google Scholar,20.Zhang Q. et al.From in vivo to in vitro: dynamic analysis of Plasmodium falciparum var gene expression patterns of patient isolates during adaptation to culture.PLoS One. 2011; 6e20591Crossref PubMed Scopus (17) Google Scholar]. Thus, it remains unclear to what extent expression of key virulence genes in vivo are dependent on human serum factors compared with other host–parasite interactions, such as rheology, interaction with endothelial cells, and febrile temperature (see the 'Physical Environmental Factors' section, later; Box 1).Box 1Mechanistic Insight into the Effects of Physical Factors on CytoadherenceThe Contribution of Cellular InteractionsExpression of var genes in P. falciparum grown in serum-supplemented media was shown to be further increased by panning selection for endothelial cell binding by Claessens et al. [84.Claessens A. et al.A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells.Proc. Natl. Acad. Sci. U. S. A. 2012; 109E1772Crossref PubMed Scopus (157) Google Scholar]. Physical interaction of infected RBCs with endothelial cells represents a key difference between in vitro and in vivo environments.The Contribution of RheologyA transcriptomics study by Nyarko et al., utilizing parasites grown statically or in suspension, revealed that over 200 genes were differentially expressed between conditions, largely representing increases in erythrocyte-binding, rigidity, and invasion-related genes in suspended cultures [32.Nyarko P.B. et al.Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level.Sci. Rep. 2020; 10: 245Crossref PubMed Scopus (6) Google Scholar]. These properties enhance P. falciparum cytoadherence; thus, studying these phenomena in a static environment plausibly leads to an underappreciation of genes relevant to successful cytoadherence and immune evasion under the shear stress of the vascular system in vivo.The Contribution of TemperatureIncreased PfEMP1 presentation on the infected RBC surface has been observed in response to heat shock by Udomsangpetch et al. but not in more recent studies by Oakley et al. nor Zhang et al. These discordant results may be time-dependent as levels of PfEMP1 presentation were measured 2–4 h following the onset of febrile temperatures by Zhang et al. and Oakley et al. compared with a ≥4 h incubation by Udomsangpetch et al. [35.Udomsangpetch R. et al.Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11825-11829Crossref PubMed Scopus (77) Google Scholar,36.Zhang R. et al.Febrile temperature elevates the expression of phosphatidylserine on Plasmodium falciparum (FCR3CSA) infected red blood cell surface leading to increased cytoadhesion.Sci. Rep. 2018; 8: 15022Crossref PubMed Scopus (9) Google Scholar,85.Oakley M.S.M. et al.Molecular factors and biochemical pathways induced by febrile temperature in intraerythrocytic Plasmodium falciparum parasites.Infect. Immun. 2007; 75: 2012-2025Crossref PubMed Scopus (112) Google Scholar]. Other studies have also implicated higher levels of phosphatidylserine (PS) in the outer leaflet of the infected RBC membrane in increased cytoadherence following a 2 h pulse of febrile heat shock [86.Engelbrecht D. Coetzer T.L. Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro.Cell Death Dis. 2013; 4e971-e971Crossref Scopus (20) Google Scholar], which is consistent with the observation by Zhang et al. that the adhesion of infected RBCs to mammalian cells was abrogated by the PS-binding protein, Annexin V [36.Zhang R. et al.Febrile temperature elevates the expression of phosphatidylserine on Plasmodium falciparum (FCR3CSA) infected red blood cell surface leading to increased cytoadhesion.Sci. Rep. 2018; 8: 15022Crossref PubMed Scopus (9) Google Scholar].While evidence for heat shock-induced infected RBC adhesion to endothelial cells mounts, others have found that a different dynamic occurs between infected RBCs and the surrounding uninfected RBCs. Singhaboot et al. detected a significant decrease in rosette formation in all stages of the IDC under febrile temperatures [87.Singhaboot Y. et al.Temperature dependence of Plasmodium falciparum erythrocytic stage development.Am. J. Trop. Med. Hyg. 2019; 100: 1191-1195Crossref PubMed Scopus (3) Google Scholar]. However, a caveat of this in vitro study design is that parasites were subjected to constant elevated temperatures for 48 h, contrary to the cyclical tertian fever seen in vivo with P. falciparum infection [34.Oakley M.S. et al.Clinical and molecular aspects of malaria fever.Trends Parasitol. 2011; 27: 442-449Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. The Contribution of Cellular Interactions Expression of var genes in P. falciparum grown in serum-supplemented media was shown to be further increased by panning selection for endothelial cell binding by Claessens et al. [84.Claessens A. et al.A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells.Proc. Natl. Acad. Sci. U. S. A. 2012; 109E1772Crossref PubMed Scopus (157) Google Scholar]. Physical interaction of infected RBCs with endothelial cells represents a key difference between in vitro and in vivo environments. The Contribution of Rheology A transcriptomics study by Nyarko et al., utilizing parasites grown statically or in suspension, revealed that over 200 genes were differentially expressed between conditions, largely representing increases in erythrocyte-binding, rigidity, and invasion-related genes in suspended cultures [32.Nyarko P.B. et al.Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level.Sci. Rep. 2020; 10: 245Crossref PubMed Scopus (6) Google Scholar]. These properties enhance P. falciparum cytoadherence; thus, studying these phenomena in a static environment plausibly leads to an underappreciation of genes relevant to successful cytoadherence and immune evasion under the shear stress of the vascular system in vivo. The Contribution of Temperature Increased PfEMP1 presentation on the infected RBC surface has been observed in response to heat shock by Udomsangpetch et al. but not in more recent studies by Oakley et al. nor Zhang et al. These discordant results may be time-dependent as levels of PfEMP1 presentation were measured 2–4 h following the onset of febrile temperatures by Zhang et al. and Oakley et al. compared with a ≥4 h incubation by Udomsangpetch et al. [35.Udomsangpetch R. et al.Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11825-11829Crossref PubMed Scopus (77) Google Scholar,36.Zhang R. et al.Febrile temperature elevates the expression of phosphatidylserine on Plasmodium falciparum (FCR3CSA) infected red blood cell surface leading to increased cytoadhesion.Sci. Rep. 2018; 8: 15022Crossref PubMed Scopus (9) Google Scholar,85.Oakley M.S.M. et al.Molecular factors and biochemical pathways induced by febrile temperature in intraerythrocytic Plasmodium falciparum parasites.Infect. Immun. 2007; 75: 2012-2025Crossref PubMed Scopus (112) Google Scholar]. Other studies have also implicated higher levels of phosphatidylserine (PS) in the outer leaflet of the infected RBC membrane in increased cytoadherence following a 2 h pulse of febrile heat shock [86.Engelbrecht D. Coetzer T.L. Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro.Cell Death Dis. 2013; 4e971-e971Crossref Scopus (20) Google Scholar], which is consistent with the observation by Zhang et al. that the adhesion of infected RBCs to mammalian cells was abrogated by the PS-binding protein, Annexin V [36.Zhang R. et al.Febrile temperature elevates the expression of phosphatidylserine on Plasmodium falciparum (FCR3CSA) infected red blood cell surface leading to increased cytoadhesion.Sci. Rep. 2018; 8: 15022Crossref PubMed Scopus (9) Google Scholar]. While evidence for heat shock-induced infected RBC adhesion to endothelial cells mounts, others have found that a different dynamic occurs between infected RBCs and the surrounding uninfected RBCs. Singhaboot et al. detected a significant decrease in rosette formation in all stages of the IDC under febrile temperatures [87.Singhaboot Y. et al.Temperature dependence of Plasmodium falciparum erythrocytic stage development.Am. J. Trop. Med. Hyg. 2019; 100: 1191-1195Crossref PubMed Scopus (3) Google Scholar]. However, a caveat of this in vitro study design is that parasites were subjected to constant elevated temperatures for 48 h, contrary to the cyclical tertian fever seen in vivo with P. falciparum infection [34.Oakley M.S. et al.Clinical and molecular aspects of malaria fever.Trends Parasitol. 2011; 27: 442-449Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. In addition to nutrient levels and serum factors, the fundamental design of in vitro culture alters parasite access to nutrients. RBC cell density is traditionally limited in vitro because parasite replication suffers at high hematocrit, with most experiments performed between 1% and 5% hematocrit [10.LeRoux M. et al.Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions.Trends Parasitol. 2009; 25: 474-481Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. This is compared with >30% hematocrit in malaria-infected patients [21.Chotivanich K. et al.The mechanisms of parasite clearance after antimalarial treatment of Plasmodium falciparum malaria.J. Infect. Dis. 2000; 182: 629-633Crossref PubMed Scopus (97) Google Scholar,22.Price R.N. et al.Factors contributing to anemia after uncomplicated falciparum malaria.Am. J. Trop. Med. Hyg. 2001; 65: 614-622Crossref PubMed Scopus (274) Google Scholar]. This limitation in vitro is likely due to the local depletion of nutrients and buildup of waste products in this static environment (i.e., a flask sitting in an incubator) [23.Ferrer J. et al.Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures.Malar. J. 2008; 7: 203Crossref PubMed Scopus (7) Google Scholar]. Parasite density also limits growth, presumably for a similar reason. Using typical in vitro static culture methods, parasite growth suffers above 10% parasitemia, even considering the relatively low hematocrit in vitro compared with in vivo [23.Ferrer J. et al.Effect of the haematocrit layer geometry on Plasmodium falciparum static thin-layer in vitro cultures.Malar. J. 2008; 7: 203Crossref PubMed Scopus (7) Google Scholar]. However, patients with severe malaria have been reported with parasitemia values over 40% [24.Branco A. Melo-Cristino J. Extreme parasitemia in P. falciparum malaria.Blood. 2018; 132: 868Crossref PubMed Scopus (3) Google Scholar]. Gentle shaking of Plasmodium cultures can partially overcome the challenges associated with high hematocrit and parasitemia, while also increasing chances for non-motile merozoites to contact and invade uninfected RBCs. Utilization of non-traditional culture systems that allow for continual flow of RBCs through an in vivo-like environment, such as hollow-fiber capillary bioreactors (HFBRs), further overcomes this problem; HFBRs allow for high parasite replication at physiological hematocrit levels by not only increasing surface area for nutrient exchange but also by allowing continual addition of fresh medium [25.Bakshi R.P. et al.Model system to define pharmacokinetic requirements for antimalarial drug efficacy.Sci. Transl. Med. 2013; 5205ra135Crossref PubMed Scopus (26) Google Scholar, 26.Caton E. et al.Kinetic driver of antibacterial drugs against Plasmodium falciparum and implications for clinical dosing.Antimicrob. Agents Chemother. 2019; 63: e00416-e00419Crossref PubMed Scopus (2) Google Scholar, 27.Li T. et al.A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities.J. Infect. Dis. 2003; 187: 159-162Crossref PubMed Scopus (13) Google Scholar, 28.Meyer K.J. et al.Model system identifies kinetic driver of Hsp90 inhibitor activity against African trypanosomes and Plasmodium falciparum.Antimicrob. Agents Chemother. 2018; 62e00056-18Crossref PubMed Scopus (8) Google Scholar, 29.Preechapornkul P. et al.Optimizing the culture of Plasmodium falciparum in hollow fiber bioreactors.Southeast Asian J. Trop. Med. Pub. Health. 2010; 41: 761-769PubMed Google Scholar]. The pumped fluid flow design of HFBRs has specifically facilitated precise in vitro modulation of drug concentrations over time, generating conditions that more closely mimic the pharmacokinetics of in vivo drug treatment [25.Bakshi R.P. et al.Model system to define pharmacokinetic requirements for antimalarial drug efficacy.Sci. Transl. Med. 2013; 5205ra135Crossref PubMed Scopus (26) Google Scholar,26.Caton E. et al.Kinetic driver of antibacterial drugs against Plasmodium falciparum and implications for clinical dosing.Antimicrob. Agents Chemother. 2019; 63: e00416-e00419Crossref PubMed Scopus (2) Google Scholar,28.Meyer K.J. et al.Model system identifies kinetic driver of Hsp90 inhibitor activity against African trypanosomes and Plasmodium falciparum.Antimicrob. Agents Chemother. 2018; 62e00056-18Crossref PubMed Scopus (8) Google Scholar]. HFBR-based studies can also facilitate the collection of parasite antigens within cartridges for assessments of immune evasion or vaccine development [27.Li T. et al.A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities.J. Infect. Dis. 2003; 187: 159-162Crossref PubMed Scopus (13) Google Scholar]. However, the HFBR system requires a considerably more complex setup than flask-based culturing, and the polymer used in commercially available cartridges is toxic for P. falciparum, necessitating custom-made glass cartridges [25.Bakshi R.P. et al.Model system to define pharmacokinetic requirements for antimalarial drug efficacy.Sci. Transl. Med. 2013; 5205ra135Crossref PubMed Scopus (26) Google Scholar]. Physical factors apart from nutrient availability play important roles in shaping which parasites survive and proliferate in vivo; this makes it prudent to understand the effect of varying these factors. In vivo P. falciparum parasites are subjected to the constant shear flow within the vascular system, either while circulating as rings or during sequestration as trophozoites and schizonts. The implications of removing fluid flow as part of in vitro culture limits nutrient access, as mentioned above, but it also alters parasite physiology. For example, maintaining cultures in suspension by shaking, along with other serum factors mentioned previously, contributes to the formation of rosettes in vitro [18.Ribacke U. et al.Improved in vitro culture of Plasmodium falciparum permits establishment of clinical isolates with preserved multiplication, invasion and rosetting phenotypes.PLoS One. 2013; 8e69781Crossref PubMed Scopus (30) Google Scholar]. Furthermore, parasites grown in static in vitro culture systems were not able to switch between glycan moiety-dependent and -independent invasion pathways, whereas counterparts suspended with gentle shaking were able to successfully switch [30.Awandare G.A. et al.Plasmodium falciparum strains spontaneously switch invasion phenotype in suspension culture.Sci. Rep. 2018; 8: 5782Crossref PubMed Scopus (17) Google Scholar]. While cytoadherence is beneficial in vivo [14.Henry B. et al.The human spleen in malaria: filter or shelter?.Trends Parasitol. 2020; 36: 435-446Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar], the parasite’s effort to avoid host clearance may reflect a fitness cost in the absence of splenic filtration in vitro. The density of PfEMP1 knobs on the RBC surface (Figure 1B), which mediate sequestration in P. falciparum to avoid physical filtration by the spleen, was reduced fivefold upon adaptation of ex vivo isolates to in vitro culture [31.Quadt K.A. et al.The density of knobs on Plasmodium falciparum-infected erythrocytes depends on developmental age and varies among isolates.PLoS One. 2012; 7e45658Crossref PubMed Scopus (35) Google Scholar]. Transcription of many P. vivax cytoadherence genes was reduced in vivo in splenectomized Aotus monkeys relative to spleen-intact monkeys, demonstrating that Plasmodium cytoadherence is likely regulated by many factors, including serum lipoproteins [16.Frankland S. et al.Serum lipoproteins promote efficient presentation of the malaria virulence protein PfEMP1 at the erythrocyte surface.Eukaryot. Cell. 2007; 6" @default.
• W3087629065 created "2020-09-25" @default.
• W3087629065 creator A5021815731 @default.
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• W3087629065 date "2020-11-01" @default.
• W3087629065 modified "2023-10-02" @default.
• W3087629065 title "From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro" @default.
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