Abstract
BackgroundEradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.MethodsA novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.ResultsimHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.ConclusionsimHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.
Highlights
Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients
After induction of differentiation, resulting immortalized hepatocyte-like cell line (imHC) were examined for hepatocyte phenotype and metabolic function maintenance, and for continuous cell line property using population doubling level (PDL) [38]. imHCs exhibited a typical homogenous hepatocyte morphology, polygonal shape, and cord-like structure (Fig. 1a), and expressed basic hepatocyte-specific markers, including ALB, AFP, HNF-4ɑ, low-density lipoprotein receptor (LDLR), and NTCP, as demonstrated by immunofluorescence staining (Fig. 1b–f, respectively)
This result was consistent with the previous study, which showed that the expression levels of the hepatic marker in imHCs were comparable to those in primary hepatocytes [38]
Summary
Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Of the five Plasmodium species that infect humans, Plasmodium falciparum is thought to cause the majority deaths, of which most cases (70%) involve children aged < 5 years in sub-Saharan Africa [1]. Plasmodium vivax is the most widespread Plasmodium species outside Africa This organism is recognized as a major obstacle in malaria eradication campaigns because it can hide in a form called a hypnozoite [2, 3] in the human liver, and can reactivate weeks, months or years after the primary infection to cause a relapse [4,5,6,7,8]
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