Persistence of Plasmodium cynomolgi hypnozoites in cynomolgus monkey iPS-derived hepatocytes

Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.

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Safety, pharmacokinetics, and liver-stage Plasmodium cynomolgi effect of high-dose ivermectin and chloroquine in Rhesus Macaques

10.1101/2020.04.27.065409 ◽

2020 ◽

Author(s):

Pattaraporn Vanachayangkul ◽

Rawiwan Im-erbsin ◽

Anchalee Tungtaeng ◽

Chanikarn Kodchakorn ◽

Alison Roth ◽

...

Keyword(s):

Plasmodium Berghei ◽

Rhesus Macaques ◽

High Dose ◽

Radical Cure ◽

The Novel ◽

Liver Stage ◽

Plasmodium Cynomolgi ◽

Stage Development ◽

Pharmacokinetic Drug

AbstractPreviously, ivermectin (1–10 mg/kg) was shown to inhibit liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (IC50 = 10.42 μM) and hypnozoites (IC50 = 29.24 μM) in primary macaque hepatocytes when administered in high-dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for seven consecutive days was evaluated for prophylaxis or radical cure of Plasmodium cynomolgi liver-stages in Rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for seven days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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Functional immunoassays using an in-vitro malaria liver-stage infection model: where do we go from here?

Trends in Parasitology ◽

10.1016/j.pt.2009.08.004 ◽

2009 ◽

Vol 25 (11) ◽

pp. 525-533 ◽

Cited By ~ 8

Author(s):

Brent L. House ◽

Michael R. Hollingdale ◽

John B. Sacci ◽

Thomas L. Richie

Keyword(s):

Infection Model ◽

Liver Stage ◽

Stage Infection

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KAI407, a Potent Non-8-Aminoquinoline Compound That Kills Plasmodium cynomolgi Early Dormant Liver Stage ParasitesIn Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01927-13 ◽

2013 ◽

Vol 58 (3) ◽

pp. 1586-1595 ◽

Cited By ~ 45

Author(s):

Anne-Marie Zeeman ◽

Sandra M. van Amsterdam ◽

Case W. McNamara ◽

Annemarie Voorberg-van der Wel ◽

Els J. Klooster ◽

...

Keyword(s):

Plasmodium Vivax ◽

Inhibitory Concentration ◽

Radical Cure ◽

Malaria Prophylaxis ◽

Activity Profile ◽

Liver Stage ◽

Content Type ◽

Plasmodium Cynomolgi ◽

Malaria Eradication

ABSTRACTPreventing relapses ofPlasmodium vivaxmalaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay ofin vitro-cultured hypnozoite forms ofPlasmodium cynomolgi(an excellent and accessible model forPlasmodium vivax). In this assay, primary rhesus hepatocytes are infected withP. cynomolgisporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class forP. vivaxmalaria prophylaxis and potentially a radical cure.

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A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation

Communications Biology ◽

10.1038/s42003-019-0737-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 4

Author(s):

Annemarie M. Voorberg-van der Wel ◽

Anne-Marie Zeeman ◽

Ivonne G. Nieuwenhuis ◽

Nicole M. van der Werff ◽

Els J. Klooster ◽

...

Keyword(s):

Cell Infection ◽

Liver Stage ◽

Plasmodium Cynomolgi ◽

Prolonged Culture ◽

Blood Stage Infection ◽

Transgenic Parasites ◽

Stage Development ◽

Green Fluorescent ◽

Stage Infection

AbstractPlasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.

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Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00741-20 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Pattaraporn Vanachayangkul ◽

Rawiwan Im-erbsin ◽

Anchalee Tungtaeng ◽

Chanikarn Kodchakorn ◽

Alison Roth ◽

...

Keyword(s):

Rhesus Macaques ◽

High Dose ◽

Radical Cure ◽

The Novel ◽

Liver Stage ◽

Content Type ◽

Plasmodium Cynomolgi ◽

Stage Development ◽

Pharmacokinetic Drug

ABSTRACT Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 μM) and hypnozoites (IC50, 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

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In Vitro Differentiation of T Cell: From Human iPS Cells in Feeder-Free Condition

Methods in Molecular Biology - In Vitro Differentiation of T-Cells ◽

10.1007/978-1-4939-9728-2_8 ◽

2019 ◽

pp. 77-80

Author(s):

Yutaka Yasui ◽

Yasumichi Hitoshi ◽

Shin Kaneko

Keyword(s):

T Cell ◽

Ips Cells ◽

In Vitro Differentiation ◽

Free Condition ◽

Human Ips Cells

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328 NONVIRAL REPROGRAMMING OF mCHERRY-EXPRESSING PORCINE FIBROBLASTS INTO INDUCED PLURIPOTENT STEM CELLS BY piggyBac TRANSPOSONS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab328 ◽

2015 ◽

Vol 27 (1) ◽

pp. 253

Author(s):

D. Kumar ◽

T. R. Talluri ◽

W. A. Kues

Keyword(s):

Stem Cells ◽

Ips Cells ◽

Large Animal ◽

Suitable Model ◽

Differentiation Potential ◽

Piggybac Transposon ◽

Increased Risk ◽

Fetal Fibroblasts ◽

Induced Pluripotent

The generation of induced pluripotent stem (iPS) cells is a promising approach for innovative cell therapies, as well as for animal biotechnology. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity due to the preferential integration into active genes. The domestic pig is an attractive large animal model for preclinical testing of safety and efficacy of cell-based therapies. Porcine organs are similar in size and physiology to their human counterparts, and a suitable model for cardiovascular disease, muscular dystrophies, atherosclerosis, wound repair, diabetes, and ophthalmological diseases. Therefore, the present study was carried out to derive porcine iPS cells from transgenic fetuses systemically expressing mCherry (Garrels et al. 2011 PLOS ONE 6) through a nonviral piggyBac transposon. The piggyBac transposon system has several advantages: (i) piggyBac has no bias to integrate in expressed gene-like lenti- or retroviral vectors, (ii) the cargo capacity is >100 kb, (iii) seamless removal is possible, and (iv) the production of transposon plasmid is cost-efficient and does not require S2 safety cabinets. Porcine fetal fibroblasts isolated from CAGGS-mCherry founder porcine line fetuses (passage 2), were co-electroporated with a PB transposon carrying a multigene cassette consisting of human cDNA for OCT4, SOX2, KLF4, c-MYc, NANOG, and LIN28 separated by self-cleaving 2A peptide sequences, driven by a CAGGS promoter and a helper plasmid expressing the pCMV-PB transposase. On Day 6 postelectroporation, morphology of fibroblasts started change to round structure, and on Day 9 loose aggregates of cells developed. Putative iPS cell colonies were cultured, propagated, and characterised through morphology and expression of pluripotency markers, such as AP, OCT4, SSEA-1, and SSEA-4, through immunostaining. Further, various stemness genes, including OCT4, SOX2, NANOG, and UTF, were detected by porcine-specific primers through endpoint RT-PCR. In vitro differentiation potential was assessed by embryoid body (EB) formation. The formed EB exhibited the expression of mCherry in their cells and expressed differentiation markers, such as NESTIN, TUJI, GATA4 and AFP. To test their tumorigenic potential, 1 × 106 iPS cells were injected under the skin of nude mice. An mCherry-positive tumour was recovered 6 weeks later. Presently the tumour is being prepared for histological analysis. This study indicates that piggyBac transposon containing 6 transcription factors is able to reprogram porcine fetal fibroblasts into iPS cells. These cells could be cultured and maintained in vitro for a prolonged period, exhibit characteristics of stem cells, and offer a potential source for future blastocyst complementation experiments.

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The Filter Loop Technique as a Method of Measuring Platelet Aggregation in the Flowing Blood of the Rat; the Inhibitory Activity of 5-oxo-l-cyclopentene-l-heptanoic Acid (AY-16,804) on Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649110 ◽

1973 ◽

Vol 30 (01) ◽

pp. 138-147 ◽

Cited By ~ 1

Author(s):

Christopher R. Muirhead

Keyword(s):

Platelet Aggregation ◽

Prostaglandin E1 ◽

Suitable Model ◽

Heptanoic Acid ◽

Simple Method ◽

Loop Technique ◽

Flowing Blood ◽

Filter Loop

SummaryThe filter loop technique which measures platelet aggregation in vivo in the flowing-blood of the rat was compared to the optical density technique of Born which is carried out in vitro with platelet rich plasma. Using these two experimental models the effect on platelet aggregation of three known inhibitors sulfinpyrazone, dipyridamole and prostaglandin E1, and a novel compound 5-oxo-l-cyclopentene-l-heptanoic acid (AY-16, 804) was determined.The effects on platelet aggregation of the known inhibitors were consistent with information in the literature. Prostaglandin E1 was the most potent inhibitor in both techniques; sulfinpyrazone inhibited aggregation in both models but was less potent than prostaglandin E1. AY-16, 804 exhibited activity in vitro and in vivo similar to that of sulfinpyrazone. Dipyridamole did not inhibit platelet aggregation in vivo and did not inhibit aggregation in vitro in concentrations at which it remained soluble.The filter loop technique is a suitable model for measuring platelet aggregation in the flowing blood of the rat. It is a relatively simple method of determining aggregation and easily adapted to other species.

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