KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria 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 of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, 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 for P. vivax malaria prophylaxis and potentially a radical cure

A randomized trial to monitor the efficacy and effectiveness by QT-NASBA of artemether-lumefantrine versus dihydroartemisinin-piperaquine for treatment and transmission control of uncomplicated Plasmodium falciparum malaria in western Kenya

<p>Abstract</p> <p>Background</p> <p>Many countries have implemented artemisinin-based combination therapy (ACT) for the first-line treatment of malaria. Although many studies have been performed on efficacy and tolerability of the combination arthemeter-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP), less is known of the effect of these drugs on gametocyte development, which is an important issue in malaria control.</p> <p>Methods and results</p> <p>In this two-arm randomized controlled trial, 146 children were treated with either AL or DP. Both groups received directly observed therapy and were followed for 28 days after treatment. Blood samples were analysed with microscopy and NASBA. In comparison with microscopy NASBA detected much more gametocyte positive individuals. Moreover, NASBA showed a significant difference in gametocyte clearance in favour of AL compared to DP. The decline of parasitaemia was slower and persistence or development of gametocytes was significantly higher and longer at day 3, 7 and 14 in the DP group but after 28 days no difference could be observed between both treatment arms.</p> <p>Conclusion</p> <p>Although practical considerations could favour the use of one drug over another, the effect on gametocytogenesis should also be taken into account and studied further using molecular tools like NASBA. This also applies when a new drug is introduced.</p> <p>Trial registration</p> <p>Current controlled trials ISRCTN36463274</p

Construction and analysis of fluorescent <i>P. cynomolgi</i> using a novel centromere construct.

<p>(A) Dot matrix analysis of a <i>P. cynomolgi</i> and <i>P. vivax</i> putative centromere (PCEN). Graphical representation of a matrix analysis of a <i>P. cynomolgi</i> PCEN aligned against itself (<i>left</i>), <i>P. cynomolgi</i> PCEN against the <i>P. vivax</i> PCEN (<i>middle</i>) and <i>P. vivax</i> PCEN aligned against itself (<i>right</i>). The analysis was performed using Dotlet <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Junier1" target="_blank">[46]</a> as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Iwanaga1" target="_blank">[22]</a>. The diagonal line within each analysis represents sequence identity, and the diagonal line indicates repetitive regions within each PCEN. Note the absence of the diagonal in the repetitive regions of the <i>P. cynomolgi</i> and <i>P. vivax</i> alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone-0054888-g001" target="_blank">Figure 1</a>, <i>middle</i> panel). (B) Schematic representation of the pPcyC-PAC-GFP_hsp70-mCherry_ef1α plasmid. The plasmid contains the <i>Tgdhfr-ts</i> selectable marker that confers resistance against pyrimethamine and two expression cassettes for constitutive expression of GFP and mCherry. Additionally, to maintain the plasmid throughout the life cycle, a putative <i>P. cynomolgi</i> centromere (PcyCEN) is included. (C) Schematic representation of the procedure used for transfection and analysis of <i>P. cynomolgi</i>. (D) PCR amplification of <i>gfp</i> and <i>mCherry</i> in PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) blood stage parasites. Wild type gDNA of <i>P. cynomolgi</i> M served as negative control. For a control PCR primers for the <i>circumsporozoite protein (csp)</i> were used. For primers used, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888.s001" target="_blank">Table S1</a>. (E) GFP and mCherry expression throughout the life cycle of <i>P. cynomolgi.</i> GFP and mCherry expression in pPcyC-PAC-GFP_hsp70-mCherry_ef1α transfected <i>P. cynomolgi</i> blood stage parasites (a ring and a trophozoite or gametocyte), in oocysts 5 days post mosquito feeding and in salivary gland sporozoites 12 days post feeding. In the Brightfield panel two salivary gland lobes can be distinguished; only one lobe contains sporozoites. In the panel on the right GFP and mCherry expression is shown in Hoechst 33342 stained day 6 liver stages. Note the autofluorescence of hepatocytes in the GFP channel in contrast to the mCherry channel. A small uninucleate (arrow) and a large multinucleate liver stage are visible, confirmed by staining of fixed parasites with anti-HSP70 antibodies (<i>lower right panel</i>). White bars correspond to 10 µm (blood and mosquito stages) and 50 µm (liver stages).</p

Flow cytometry and cell sorting of <i>P. cynomolgi</i> liver stage parasites, including hypnozoite-forms.

<p>(A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP_hsp70-mCherry_ef1α (PcyC-PAC) <i>P. cynomolgi</i> liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP_hsp70-mCherry_ef1α<i>P. cynomolgi</i> liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.</p

Data from: Global methylation in relation to methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia

BACKGROUND Children with acute lymphoblastic leukemia (ALL) often suffer from toxicity of chemotherapeutic drugs such as Methotrexate (MTX). Previously, we reported that 20% of patients receiving high-dose MTX developed oral mucositis. MTX inhibits folate metabolism, which is essential for DNA methylation. We hypothesize that MTX inhibits DNA methylation, which results into adverse effects. We studied DNA methylation markers during high-dose methotrexate treatment in pediatric acute lymphoblastic leukemia (ALL) in relation to developing oral mucositis. MATERIALS &amp; METHODS S-Adenosyl-Methionine (SAM) and S-Adenosyl-Homocysteine (SAH) levels and LINE1 DNA methylation were measured prospectively before and after high-dose methotrexate (HD-MTX 4 x 5g/m2) therapy in 82 children with ALL. Methotrexate-induced oral mucositis was registered prospectively. Oral mucositis (grade ≥ 3 National Cancer Institute Criteria) was used as clinical endpoint. RESULTS SAM levels decreased significantly during methotrexate therapy (-16.1 nmol/L (-144.0 - +46.0),

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

Preventing relapses of Plasmodium vivax malaria 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 of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, 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 [IC(50)] 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 for P. vivax malaria prophylaxis and potentially a radical cure