Engraftment of hE in NOD<i>^{scid/β2m−/−}</i>.

<p>(A) Long term kinetics of engraftment of NOD<i>^{scid β2m−/−}</i> mice with hE. Data are the mean percentage±SE of hE (TER-119⁻ or human glycophorin-A⁺ mouse) erythrocytes pooled from 142 mice. The inset plot shows the effect of human serum deprivation from daily-inoculated hE on engraftment of hE in peripheral blood of NOD<i>^{scid β2m−/−}</i> (n = 3 mice·group⁻¹). Data shown are from a representative experiment out of three. (B) Histological analysis of brain, kidney, liver and spleen of NOD<i>^{scid/β2m−/−}</i> control, conditioned with hE and conditioned mice infected with <i>P. falciparum</i> (day 8 after infection). Non-infected mice conditioned with hE showed a marked vascular congestion compared to control mice. Infected mice, showed decreased vascular congestion, increased numbers of myelomonocytic cells and enhanced phagocytic activity in the spleen (×600 magnification). (C) Concentration of erythrocytes, hE and mE in peripheral blood of NOD<i>^{scid β2m−/−}</i> during conditioning before i.v. infection with <i>P. falciparum</i>. Data are the mean±SE of n = 21 mice.</p

Optimized selection and characterization of competent <i>P. falciparum</i> isolates.

<p>(A) A cohort C1 of mice is conditioned for 7–10 days with daily i.p. injections of hE. Then, the mice are infected by i.p. or i.v. route with 25·10⁶ or 50·10⁶ parasitized erythrocytes from <i>in vitro</i> cultures. Parasitemia in peripheral blood is followed up to 1 month after infection or until about 1% of parasitemia is achieved (see point B). Mice having productive infections are used as donors to infect by i.v. route a cohort C2 of conditioned mice. After 1 week of infection, the mice with the highest parasitemia is selected as donor for next <i>in vivo</i> passage. After ten passages showing a stable kinetics of growth (see point C), a final expansion step is performed to establish a standard <i>P. falciparum</i> strain, which is frozen and used in experiments. (B). Course of parasitemia in peripheral blood from the mice that originated the competent isolates <i>Pf</i>3D7^0087/N9 (i.p. infection), <i>Pf</i>V1/S^0176/N7 (i.p. infection) and <i>Pf</i>V1/S^0176/N10 (i.v. infection). (C) Dot plot analysis of the kinetic stability of the isolate <i>Pf</i>3D7^0087/N9 growth during sequential i.v. infections with 20·10⁶<i>P. falciparum</i>-infected erythrocytes. Stability in 10 sequential i.v. infections was used as the criterion for establishing the reference standard <i>Pf</i>3D7^0087/N9 strain. Each data point is the mean of three mice per <i>in vivo</i> passage for ten consecutive passages. (D) Microsatellite <i>Pf</i>RRM of the <i>P. falciparum</i> 3D7 and <i>Pf</i>3D7^0087/N9 strains. (E) Growth of <i>Pf</i>3D7^0087/N9 in different strains of HM after i.v. infection with 20·10⁶<i>Pf</i>3D7^0087/N9 parasites. Data are the mean±SE of four mice·group⁻¹.</p

Validation of the <i>P. falciparum</i> 4-day test.

<p>(A) Therapeutic efficacy of chloroquine, artesunate, and pyrimethamine using the standard <i>P. falciparum</i> 4-day test. Data are the mean parasitemia±SE of n = 3 mice·group⁻¹ from a single experiment using 45 mice. (B, C, D, E) Representative blood smears 48 h after starting treatment with saline, chloroquine (20 mg·Kg⁻¹), artesunate (25 mg·Kg⁻¹) or pyrimethamine (20 mg·Kg⁻¹), respectively. The remaining parasites in peripheral blood after treatment with chloroquine or artesunate were pycnotic cells and disrupted trophozoites. Pyrimethamine led to swollen late trophozoites with prominent granules of hemozoin (×1000 magnification). (F) Logarithmic growth of <i>Pf</i>3D7^0087/N9 during recrudescence after treatment with chloroquine (10 and 20 mg·Kg⁻¹, p.o., u.i.d.) in a standard 4-day test. Data are the mean parasitemia±SE of three mice/group. (G) Exposure-therapeutic efficacy relationships of chloroquine in the <i>P. yoelii</i> or the <i>P. falciparum</i> murine models of malaria. Data are the mean concentration of chloroquine in blood of n = 3 mice·group⁻¹.</p

Comparison of the activity of selected antimalarials against <i>P. falciparum</i> 3D7 or <i>Pf</i>3D7^0087/N9¹.

1<p>The activity of antimalarials is expressed as the concentration in ng·ml⁻¹ that inhibits by 50% the incorporation of [³H]-hypoxanthine (IC₅₀).</p>2<p>Differences between IC₅₀ were not statistically significant in any case.</p

Infection dynamics in HM infected with <i>Pf</i>3D7^0087/N9.

<p>Concentration of hE, ihE and mE in peripheral blood of infected mice. Data are the mean±SE of three mice per data point. The results are from a representative experiment out of three. (B) Abrogation of infection-induced elimination of hE by treatment with chloroquine (10 mg·Kg⁻¹) and exponential growth of <i>Pf</i>3D7^0087/N9 during recrudescence. Data are the mean concentration±SE of three mice per data point. The results are from a representative experiment out of two. (C) The effect of splenectomy on the percentage of hE and parasites in peripheral blood. The data are the means±SE of n = 6 mice·group⁻¹ pooled from two independent experiments.</p

Therapeutic efficacy of diamidine derivatives against <i>P. berghei</i>, <i>P. vinckei</i> and <i>P. falciparum</i>.

<p>(A, B, C) Therapeutic efficacy of pentamidine (40 mg·Kg⁻¹, u.i.d., s.c.), DB75 (10 mg·Kg⁻¹, u.i.d., s.c.) or DB289 (100 mg·Kg^-1, u.i.d., p.o.) against <i>P. berghei</i>, <i>P. vinckei</i> and <i>P. falciparum</i>, respectively. We started treatment when parasitemias where comparable and administered compounds for 4 days. (D, E) Giemsa-stained blood smears from mice infected with <i>P. berghei</i> obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Neither relevant cellular damage nor significant inhibition of parasitemia were observed at the time of sampling. (F, G) Giemsa-stained blood smears from mice infected with <i>P. vinckei</i> obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Parasites from treated mice were mostly abnormal late trophozoites. (H, I) Giemsa-stained blood smears from mice infected with <i>P. falciparum</i> obtained 48 h after starting treatment with vehicle or DB75 at 10 mg·Kg⁻¹, respectively (×1000 magnification). Parasites from treated mice were mostly abnormal late trophozoites.</p

Effect of repeated blood injections on clinical parameters of mice¹.

1<p>Each experimental group was n = 5.</p>2<p>Analysis of organ weight was performed using two factor-ANOVA test followed by Bonferroni post test. Differences were considered significant if P<0,05. Values reported are the probability of the difference in weight with respect to day 0. In italics groups that differed significantly from control at day 0.</p>3<p>ALT: Alanine aminotransferase; BUN: Blood Urea Nitrogen; AST: Aspartate aminotransferase; TBIL: Total bilirrubine; TP: Total protein. Analysis of biochemical markers in serum was conducted using a general linear model multivariate contrast of means followed by a bilateral Dunnett's t test. In italics groups that differed significantly from control at day 0.</p>4<p>NS: Not significant.</p

Dynamics of infection in HM¹.

1<p>Data are the mean±SEM of n = 3 mice infected at day 0 with 20·10⁶<i>Pf</i>3D7^0087/N9-parasitized erythrocytes/mouse obtained from peripheral blood of a donor mouse.</p>2<p>Total parasitemia.</p

Characterization of the <i>P. falciparum</i>-malaria model for antimalarial drug testing.

<p>(A) Kinetics of parasitemia in peripheral blood of HM infected i.v. with 20·10⁶<i>Pf</i>3D7^0087/N9 parasites. Data are the mean±SE from 88 mice. The inset plot shows the regression line to fit an exponential growth model of the means of parasitemia of the pooled data up to day 7 after infection. (B) Giemsa-stained smears from peripheral blood of HM infected i.v. with <i>Pf</i>3D7^0087/N9 showing the different stages of <i>P. falciparum</i>: ring (upper row on the left) to mature schizont (lower row on the right) (×1000 magnification). (C) Replication of <i>Pf</i>3D7^0087/N9 in hE. Flow cytometry panels depict erythrocyte populations in control (left panel), uninfected HM (middle panel) and infected HM (right panel). The percentage of mE TER-119⁺ cells is indicated. <i>Pf</i>3D7^0087/N9 (parasitemia 6%) are in TER-119⁻SYTO-16⁺ events. Pycnotic forms of <i>P. falciparum</i> were found in Giemsa stained-cytospin preparations of mE TER-119⁺ purified immunomagnetically. Viable parasites were found in purified hE TER-119⁻. Data are representative of three independent experiments. (D) Q–Q plot of normality for the variable log₁₀ (parasitemia at day 7) showing observed values vs expected normal values (n = 327 mice).</p

Selection of immunodeficient mice.

<p>The percentage of hE (TER-119⁻ or human glycophorin A⁺ cells) in peripheral blood of NIH-III<i>^{beige/xid/nude}</i>, CB17<i>^scid</i>, CB17<i>^scid/beige</i>, NOD<i>^scid</i> and NOD<i>^{scid/β2m−/−}</i> murine strains upon daily intraperitoneal injection of hE is shown. The regression curves for fitting to an exponential association equation are shown for CB17<i>^scid</i>, CB17<i>^scid/beige</i>, NOD<i>^scid</i> and NOD<i>^{scid/β2m−/−}</i>. The strain NIH-III<i>^{beige/xid/nude}</i> was discarded from the study at the point indicated (★). Data are the mean±SE of n = 4 mice per data point.</p