Are these hypnozoites?

<p>HepG2-A16 cells were infected, maintained for 9 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#pone-0014275-g004" target="_blank">Fig 4</a> and stained with PvCSP mAb. Approximately 10% of the Pv liver stage parasites that expressed PvCSP were similar in size to 3-day trophozoites, and much smaller than the 9-day schizonts. Are these hypnozoites?</p

Multiple Pv liver stage parasites were seen in single hepatocyte in a 3 Day culture.

<p>HepG2-A16 cells were infected with Pv spz and the liver stage trophozoites were stained with the anti-PvCSP mAb, NVS3. Some HepG2-A16 cells were seen with multiple liver stage parasites (400X magnification). <b>N</b>: Nucleus of hepatocyte, C: Cytoplasm of hepatocyte, White Arrows: Individual 3 Day hepatocyte stage Pv.</p

<i>In vitro</i> development of hepatocyte stage parasites.

<p>25,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 3 days and 50,000 Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 9 days, and the numbers of early liver stage trophozoites (3 day assay) or late liver stage schizonts (9 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3 (20 µg/mL).</p

<i>In-vitro</i> development of late liver stage schizonts expressing PvMSP1.

<p>20,000 HepG2-A16 cells were infected with 50,000 Pv spz. Three hrs later uninfected Pv spz were washed off and the culture was maintained for 9 days with daily media changes. Mature Pv liver stage schizonts (400 X magnification) in HepG2-A16 cells were stained with (A) the mAb to the PvCSP, NVS3 (20 µg/ml) or (B) with a mAb against Pv merozoite surface protein 1(PvMSP1), 3F8.A2 (1∶50 dilution). As a negative control, uninfected HepG2-A16 cells were incubated with the individual mAbs and labeled secondary antibodies. There was no evidence of staining in these negative control cultures (data not shown).</p

No change in infectivity after cryopreservation of Pv spz.

<p>25,000 Pv spz (Fresh or thawed after cryopreserved in LNVP for one year) were added to wells containing 20,000 hepatoma cells and cultured for 3 days. The numbers of early liver stage trophozoites (3 day assay) were counted by immunofluorescence microscopy after staining with the anti-PvCSP mAb, NVS3, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>.</p

<i>In-vitro</i> development of liver stage parasites.

<p>25,000 Pv spz was used to infect 20,000 HepG2-A16 cells. Uninfected spz were washed off after three hrs and cells were maintained for 3 days with daily media change. Cells were fixed and Pv liver stage trophozoites (400 X magnification) were stained with mAb NVS3 against the PvCSP (20 µg/ml). <b>N</b>: Nucleus of HepG2-A16 cells, <b>C</b>: Cytoplasm of HepG2-A16 cells, <b>P</b>: Developing 3 day old Pv hepatocyte stage parasite (trophozoite).</p

Dose dependent effect of primaquine on the development of 9 day hepatocyte stage parasites.

<p>50,000 fresh Pv spz were added to wells containing 20,000 hepatoma cells and were cultured for 9 days in medium alone or in the presence of different concentrations of primaquine (PQ). The numbers of late liver stage schizonts (9 day assay) were counted by immunoflourescence microscopy after staining with the anti-PvCSP mAb, NVS3 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>.</p

Hepatotoxic changes in HepG2-A16 cells after treatment with high dose primaquine for 9 days.

<p>Culturing HepG2-A16 cells with high concentrations of primaquine (10 µg/mL) induced hepatotoxic changes. <b>P</b>: Parasite; <b>White arrows</b>: Dense bodies in cytoplasm; <b>E</b>: Empty spaces due to focal cell loss in the cell culture well.</p

Immunofluorescence Assay.

<p>Air dried Pv spz were immunostained with monoclonal antibody against PvCSP (NVS3) at 0.98 ng/mL as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014275#s2" target="_blank">Materials and Methods</a>. The end point titer is 1∶1,024,000.</p