After isolation, the P. falciparum or P. cynomolgi sporozoites were suspended in complete medium and added to their respective host's primary hepatocyte cultures. For drug assays, 3×104 sporozoites were added to each well, or 1×105P. cynomolgi sporozoites for cultures initiated in Lab-Tek 8-chambers slides. Infected cultures were fixed with cold 100% methanol on Days 1, 3, 5, 7, 9 and 11 post-infection in order to count and measure the size of the P. cynomolgi or P. falciparum pre-erythrocytic (PE) forms obtained. For the drug assays, the antimalarial drugs were added at different concentrations to complete medium. Infected cells were exposed to the drug for 3 days from Day 5 until fixation at Day 8 post-infection. In a second set of experiments, PE-uni selection was included by treatment of P. cynomolgi hepatic cultures with atovaquone at 67 ng/ml (182 nM) between Day 5 and Day 8. This was followed by a 2 days treatment with either the same concentration of ATO or with PQ at 10 µg/ml (22 nM), until fixation at Day 10 post-infection. In all cases, the medium containing the drug was renewed daily during treatment. Assays for each drug were generally conducted in two or three independent experiments. In an individual experiment, each concentration was tested in duplicate or triplicate wells.
Hepatic parasites were enumerated by immunofluorescence analysis. Briefly, following fixation, the parasites were specifically labelled with a mouse polyclonal serum raised against the P. falciparum heat shock protein 70.1 (PfHSP70.1) obtained after immunization with the recombinant protein. This serum also cross-reacts with P. cynomolgi HSP70. The labelled parasites were visualized with Alexa 488-conjugated goat anti-mouse immunoglobulin (Invitrogen). Parasite and host cell nuclei were stained with 1 µg/ml of diamidino-phenylindole (DAPI; Sigma). Parasites were enumerated by examination of the cultures under a fluorescence microscope at 200× magnification (Leica DMI 4000 B). Photomicrographs were obtained using a confocal Olympus BX61 microscope at 600× magnification on cells cultures in Lab-Tek 8-chambers slides.
The cytotoxicity of the anti-malarial drugs was evaluated on M. fascicularis hepatocytes treated as for the drug activity assays, through the colorimetric methylthiazolyldiphenyl-tetrazolium bromide test (MTT) [27] (link). Briefly, the primary hepatocytes were exposed to various concentrations of drug and three days later, a 100 µl MTT solution (500 µg/mL) was added in each well. After 4 h of incubation at 37°C, the plates were read in a spectrophotometer (540 nm absorbance wavelength) and the results were expressed as percentage of cellular viability compared to the non-treated control.
Hepatic parasites were enumerated by immunofluorescence analysis. Briefly, following fixation, the parasites were specifically labelled with a mouse polyclonal serum raised against the P. falciparum heat shock protein 70.1 (PfHSP70.1) obtained after immunization with the recombinant protein. This serum also cross-reacts with P. cynomolgi HSP70. The labelled parasites were visualized with Alexa 488-conjugated goat anti-mouse immunoglobulin (Invitrogen). Parasite and host cell nuclei were stained with 1 µg/ml of diamidino-phenylindole (DAPI; Sigma). Parasites were enumerated by examination of the cultures under a fluorescence microscope at 200× magnification (Leica DMI 4000 B). Photomicrographs were obtained using a confocal Olympus BX61 microscope at 600× magnification on cells cultures in Lab-Tek 8-chambers slides.
The cytotoxicity of the anti-malarial drugs was evaluated on M. fascicularis hepatocytes treated as for the drug activity assays, through the colorimetric methylthiazolyldiphenyl-tetrazolium bromide test (MTT) [27] (link). Briefly, the primary hepatocytes were exposed to various concentrations of drug and three days later, a 100 µl MTT solution (500 µg/mL) was added in each well. After 4 h of incubation at 37°C, the plates were read in a spectrophotometer (540 nm absorbance wavelength) and the results were expressed as percentage of cellular viability compared to the non-treated control.
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