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A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

ABSTRACT: The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development.

SUBMITTER: March S

PROVIDER: S-EPMC3780791 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

March Sandra S Ng Shengyong S Velmurugan Soundarapandian S Galstian Ani A Shan Jing J Logan David J DJ Carpenter Anne E AE Thomas David D Sim B Kim Lee BK Mota Maria M MM Hoffman Stephen L SL Bhatia Sangeeta N SN

Cell host & microbe 20130701 1

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platf ...[more]

PMID: 23870318

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Project description:BackgroundMalaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early Plasmodium falciparum and Plasmodium vivax infection.MethodsThis study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated.ResultsThe median peak parasitaemia prior to treatment was 10,277 parasites/ml (IQR 3566-27,815), 71,427 parasites/ml [IQR 33,236-180,213], and 34,840 parasites/ml (IQR 13,302-77,064) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8-13.3), 14.8% (IQR 11.8-15.9) and 11.7% (IQR 8.9-14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5-21), 15 (IQR 7-22), and 8 (IQR 7-15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1-5.3), 7.2% (IQR 5.8-7.8), and 4.9% (IQR 3.7-6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized erythrocytes were estimated to account for 0.015% (IQR 0.006-0.06), 0.128% (IQR 0.068-0.616) and 0.022% (IQR 0.008-0.082) of the malaria-attributable erythrocyte loss in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively.ConclusionEarly experimental P. falciparum and P. vivax infection resulted in a small but significant fall in haemoglobin despite parasitaemia only just at the level of microscopic detection. Loss of parasitized erythrocytes accounted for < 0.2% of the total malaria-attributable haemoglobin loss.

Project description:BackgroundPlasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.ResultsHere, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.ConclusionsThe α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Project description:BackgroundThe mechanisms by which humans regulate pro- and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum.Methodology/principal findingsWe measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-? receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n?=?85), P. falciparum (n?=?30), or both species (n?=?12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-?, IL-10/interferon (IFN)-?, IL-10/IL-6 and sTNFRII/TNF-? ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-? receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species.ConclusionsControlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

Dataset Information

A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

Publications

A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

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