Improved P. falciparum Mouse Model Figure 6. Residual inflammation present in the IV-NSG model. Panel A: using the sub-optimal dose of 400 mL huRBC grafting results in a reduction of chimerism (red line) and parasitemia (blue line); Panel B: inflammatory cytokine responses were measured in parallel using the CBA kit. Note the initial rise of IL6, followed by a major increase of IL-12p70, and a modest increase of TNFa and MCP-1 that are temporally associated with anemia, and might play a role in its etiology. Results represent the means 6 SEM from 2 experiments (n = 7). doi:10.1371/journal.pone.0018045.g006 ination of slides is very impressive with over 95% of parasites seen being at the same stage. This synchronisation results in a very characteristic step-wise increase in parasite density at 48 hour intervals - reminiscent of tertian Malaria - at least during the initial rising phase. The various stages seen in the peripheral blood do not differ morphologically from those that are observed in human peripheral blood smears, although very high densities of mature trophozoites can be seen in NSG which, obviously, is seldom seen in humans. Partial sequestration takes place with an overwhelming dominance of rings or mature trophozoites (i.e. parasites at one nucleus stage) in peripheral blood, and conversely rare schizonts. However the presence of schizonts seems to increase at high parasitemia. Examination of blood from deep-seated capillary beds from various organs shows that the most mature segmenters are far more frequent, ca. 4–5 fold, in blood from the brain and the kidney, than in peripheral blood. This partial sequestration could result from sub-optimal affinity of the P. falciparum infected huRBC for the heterologous murine endothelium. Conversely mature forms are also occasionally seen in human peripheral blood particularly in severe cases at high parasite loads [26]. Thus, it is also possible that at high parasite loads, phagocytosis of numerous parasites by neutrophils results in neutrophil necrosis, causing release of neutrophil proteases, which together with parasite proteases, may in turn contribute to desequestrate mature forms from the endothelium [27]. Whatever the case, the picture that emerges is one of intermediate, rather than absolute sequestration. Further work will be required to determine if the model can help understand the sequestration phenomenon which is characteristic of P. falciparum, and to see if it has relevance to the human situation. The model also proved to support the development of the P. falciparum sexual blood cycle. Stage III and IV gametocytes could be seen in the peripheral blood; however stage V gametocytes were rarely seen, most likely as a result of the high turnover rate of huRBC caused by the residual inflammatory response, and the long duration needed for the sexual blood cycle. From a practical point of view the described NSG-IV model is very flexible, and levels of parasitemia can be adjusted, by modulating the amount of huRBC injected, to anywhere from 1– 2% to 30–40% in order to meet the needs of the experiment, and of the investigator. The model remains demanding however, with 2–3 IV inoculations required per week. Results also show, that innate defences are still very active and hence that the model remains suboptimal. Detailed analysis better defined the role of each of the cytokines/chemokines produced in this process, and helps to identify which ones need to be controlled to improve success. Our findings contribute in this way to the understanding of the delicate balance between inflammation control and P. falciparum survival. The relationship between aging and the mouse’s ability to both tolerate the graft and produce less inflammatory mediators is an interesting finding that emerged from the analysis. Aging is well known to be associated with increased susceptibility to infections PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e18045