Figure 4. Results obtained using the NSG-IV P. falciparum model. Panel (A): proportion of huRBC (% of total erythrocytes), or ‘‘chimerism’’ seen in peripheral mouse blood, as determined by FACS analysis using an anti-human glycophorin A antibody (average of 18 mice). HuRBC grafting started 10 days before infection with P. falciparum; Panel (B): parasitemia patterns obtained after inoculation with either UPA (red; n = 10), 3D7 (blue; n = 4), or K1 (green; n = 4) P. falciparum strains. Results represent the means 6 SEM from 2 different experiments. Panel (C): detailed pattern of P. falciparum growth over time in the NSG-IV model. Arrows indicate points at which .95% of parasites were at ring stage. Parasitemia is expressed as a percentage of total erythrocytes found in mouse peripheral blood. Mice received 750 ml of huRBC delivered IV 3 times a week, along with IP administration of liposomal clodronate (100 ml). doi:10.1371/journal.pone.0018045.g004 IP on a daily basis in addition to the standard use of clo-lip. While the benefits of inosine on parasite survival were initially not as obvious as in NOD/SCID mice, statistically significant improve- ment of parasitemia in NSG mice receiving inosine (n = 3), as compared to NSG without inosine (n = 4), became apparent (Figure S1) after about 2 weeks of treatment, i.e. when strong inflammatory responses are induced by high parasitemia (non parametric Wilcoxon test on 55 paired values: Z = 3.65, p value = 0.0003). Additional characteristics of P. falciparum in the NSG-IV model Remarkably, parasite growth occurred in a synchronous fashion (Figure 4C and Figure S2), with the vast majority of parasites in Improved P. falciparum Mouse Model peripheral blood being at the same stage at a given time (Figure 5). As shown in Figure 5 A, B, C, D, over 95% of parasites were simultaneously either at young ring stage or at trophozoite stage. This resulted in a stepwise increase in parasitemia. As demon- strated in Figure 4C, over the first 24 hours the parasite matures without an increase in parasitemia. This is then followed the next day by the reinvasion of new huRBC, leading to a steep increase in the proportion of infected erythrocytes (Figure 4C and Figure S2). As asynchronous parasite cultures were used to infect animals, this suggests a synchronizing effect of the mouse innate defences. This is the first-ever animal model for P. falciparum that exhibits a synchronised asexual blood stage cycle as occurs in human beings. However this characteristic was not constant, in particular when there was long-standing high parasitemia; this synchronism eventually disappeared in some of the mice as illustrated in Figure S3. Fully mature schizonts, i.e. segmenters, were seen very rarely in peripheral blood and this occurred mostly at high levels of parasitemia. However, the high rate of re-invasion implied that they were developing and releasing merozoites in a synchronised manner elsewhere. To investigate this further we analyzed the parasite stages in peripheral blood, as compared to those in deep-seated capillary beds from various organs. In peripheral blood segmenters were only seen rarely when parasitemia was low, particularly during the initial increase following infection of mice, i.e. ,1% of all parasites detected. When the parasitemia increased, their proportion slightly increased, i.e. reaching 3–5% of those huRBC parasitized. Among all parasites seen, the majority were rings or very mature pigmented trophozoites (parasites at a single nucleus stage). The very mature trophozoites seen did not differ in morphology from similar stages that can be appreciated in peripheral blood from a malaria patient. The simultaneous presence of 3–4 mature trophozoites in the same RBC (Figure 5C and D) sometimes can be mistaken for a schizont. Schizonts were present however, but were only seen rarely. The parasite load in blood from various organs investigated was similar to that seen in peripheral blood at the time of sampling. However capillary blood from the brain, kidney, spleen, and liver revealed a 4–5 fold higher proportion of segmenters (detailed counts of brain and kidney showed that 14–16% of parasites were fully mature schizonts, at a time when the peripheral blood contained only 3% of them). Tissue sections examined by IFAT using an anti-HSP antibody also revealed numerous pigmented mature forms in the organs; however it was not possible to perform a differential count as rings were hardly identified at all by this means (not shown). These findings are suggestive of an intermediate level of sequestration in the NSG-IV model, with high numbers of very mature trophozoites and occasional presence of schizonts in peripheral blood, but with markedly higher prevalence of mature schizonts in organ vascular-beds. Gametocytes were also able to develop in the NSG-IV model. These sexual forms (Figure 5G, H) were occasionally seen up to stage III or IV, and developed rarely to stage V. The observation of stage V gametocytes implies that infected huRBC are able to survive in the mouse circulation for, at least, 9 days without suffering any significant immune-mediated insults when the NSG- IV protocol is used. Discussion This study is part of our longstanding effort aimed at creating a satisfactory experimental mouse model for P. falciparum malaria. By employing the IV route for both parasite and huRBC delivery, PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e18045