Improved P. falciparum Mouse Model Figure 2. Effect of the aging process on the production of cytokines in NOD/SCID mice. Panel (A): levels of various inflammatory mediators (IL-6, MCP-1, TNFa, IFNc) in supernatants of peritoneal cells (n = 10) and splenocytes (n = 16) from young vs aged mice after stimulation with LPS (1 mg/ml) ; Panel (B) : levels of reactive oxygen intermediates, measured by chemiluminescence, in the presence of PMA (10 mg/ml) produced by peritoneal cells (n = 4) and splenocytes (n = 5) taken from young vs aged mice; Panel (C): in vivo levels of IFNc in aged mice (with long- lasting parasitemia) (n = 4) compared to young mice (with short-lived parasitemia) (n = 5) as measured in serum samples collected 1, 3, and 6 days post infection with P. falciparum. Cytokine levels were measured by using the CBA mouse inflammatory kit by FACS. Results represent the mean 6 SEM from two different experiments. doi:10.1371/journal.pone.0018045.g002 this isolate parasitemia was uniformly composed of young ring forms for the first 4 days, then trophozoites appeared as the parasitemia rose from 0.1 to 1%. Each of the 2 animals infected showed satisfactory growth as did mice sub-inoculated with parasites from the initial animals (not shown). Mice used in the NSG-IV model, when the 550 ml dose is given, demonstrated a life expectancy equivalent to, or slightly better than that seen in NOD/SCID mice used for previously described models (i.e. 2–3 months). The NSG-IV model was reliably reproducible. All mice grafted with huRBC and infected with P. falciparum to date developed a peripheral blood parasitemia (n = 68). The degree of chimerism and parasite growth was consistent and homogenous from one animal to the other (see Figure 4B). This is a critical difference from the SCID, BXN or NOD/SCID models [6,7,8,10]. To confirm the advantage of the IV route, the same NSG mice were used in a protocol using intra-peritoneal (IP) grafting of huRBC, and IP infection with P. falciparum. Despite the advantage of a decreased inflammatory response in this strain of mice, this ‘‘IP-IP’’ protocol provided erratic outcomes, with large individual variations from one animal to the other in terms of presence of parasites in peripheral blood (data not shown). Ultimately, the IP- IP protocol in NSG proved to be as inadequate, and as troublesome as the same route in NOD/SCID. The value of inosine was re-addressed in NSG. In experiments performed using the 400 ml huRBC dose, inosine was administered Figure 3. Effect of inosine and the aging process on production of inflammatory mediators in NOD/SCID mice. Determination of levels of cytokines in supernatants of peritoneal cells (IL-6, MCP1, TNFa) or splenocytes (IFNc) collected from NOD/SCID mice and stimulated in vitro with LPS (1 mg/ml). Results from cells of aged mice (.15 weeks) and young mice (,15 weeks) incubated with or without 1 mM inosine are shown. Results represent the mean 6 SEM from an experiment performed using 3 mice per group. doi:10.1371/journal.pone.0018045.g003 PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e18045