Effect of age on inflammation We define young mice as those less than 15 weeks old and old mice as greater than 15 weeks old. While 16.6% of the aged mice were able to support a parasitemia lasting more than 12 days, none of the young mice supported a parasitemia lasting this long, and average length of parasitemia for old vs young was 7.1 vs 3.9 days respectively. The gender of the mice made no difference. To explain this phenomenon, we analysed the inflammatory responses of healthy non-infected ‘‘young’’ and ‘‘aged’’ NOD/ SCID mice. The levels of inflammatory mediator production were investigated in peritoneal cells and splenocytes from these mice in vitro after stimulation with LPS for 16 hours. The levels of IL-6, MCP-1, TNFa and IFNc were 1.7, 2.8, 1.8, and 6.4 times lower in the supernatant of peritoneal cells from aged mice as compared to that from young mice, respectively. Despite these important differences, probably due to group size, they were not statistically significant. The same investigation was performed using spleno- cytes, and the findings were more modest. The levels of IL-6, MCP-1, TNFa and IFNc were 1.18, 1.16, 2.14 and 1.54 times lower in supernatants of splenocytes from aged mice as compared to young mice, respectively (Figure 2A). Interestingly, cells from young mice produce very strong inflammatory responses when incubated in serum from young mice, whereas these responses are decreased when they are incubated in serum from old mice. This would imply that these age-related differences are mediated by humoral factors. Furthermore, a study of O2- free radical emission from PMA- stimulated peritoneal cells detected by chemiluminescence showed major differences for young vs aged mice cells, i.e.: 11046354 vs 3086152 CU respectively, and in splenocytes 21006941 vs 13986528 CU respectively. These findings were not statistically significant either, however (Figure 2B). The same was addressed in P. falciparum infected NOD/SCID mice using serum collected 1, 3 and 6 days post-inoculation, either from young mice with short-lived parasitemia or aged mice with long-lasting parasitemia. Among the cytokines/chemokines mea- sured only IFNc differed, particularly on days 1 and 3 (102638 vs 4546168 at day 1, and 2468 vs 3696196 at day 3, in aged vs young mice, respectively) (Figure 2C). Effect of immunosupressants on NOD/SCID mice In order to improve sustainability of parasitemia in the mouse model further, we investigated immunosuppressive agents that might help to minimize the innate immune response. Inosine, a purine nucleoside, proved to be the most effective agent at improving peripheral blood parasitemia from a group of agents tested, including TGFb1, dexamethasone, and IL-10. In vitro experiments found that at a concentration of 1 mM inosine decreased IL-6, MCP-1, and TNFa secretion from young NOD/SCID LPS stimulated peritoneal cells by a ratio of 1.72, 3.55, and 2.56 respectively as compared to similar inosine- untreated cells. This effect was less pronounced on cells from aged mice, with respective decreases of 1.23, 1.49, and 2.24, although age related differences were not significant (Figure 3). The other cytokines/chemokines tested (IL-12p70, IFNc and IL-10) re- mained unchanged. In the case of NOD/SCID mouse spleno- cytes, only IFNc release was decreased by treatment with inosine in vitro. The reduction of IFNc production in young and aged mice was 2.3 and 1.7 times respectively. In vivo inoculation of inosine confirmed the above data in both young and aged mice. In young NOD/SCID mice daily intra- peritoneal injection of inosine at a dose of 250 mg/kg led to 10% of mice sustaining parasitemia for .12 days, as compared to none in the inosine-free group. Using the combination of aged mice and inosine Improved P. falciparum Mouse Model administration resulted in 37% of mice sustaining parasitemia for .12 days, and an average length of parasitemia of 9.8 days. Development of an improved IV model using NSG mice Experiments were performed in NSG mice using various amounts of huRBC (200, 400, 550 or 750 ml of RBC pellets) injected IV 3 times a week, and also using the IV route to introduce the parasite infection. Clo-lip was delivered IP on the same day as huRBC administration. This is called the NSG-IV model below. Injecting 750 ml of huRBC resulted in a high proportion of huRBC in mouse peripheral blood (chimerism), ranging from 83% to 92% of total erythrocytes (Figure 4A). This high level of chimerism was stable over a number of months and supported rapid, optimal growth of P. falciparum (Figure 4B) with abundant and very healthy parasites (Figure 5). Indeed peripheral blood parasitemia levels reached 30–35% within 3 weeks (Figure 4B) and went on to reach a maximum of 56.2% (which corresponds to a parasitemia of 67% in the huRBC subset of mouse peripheral blood). The total haematocrit in mouse peripheral blood was on average 70% using this protocol (data not shown). Use of a lower dose of huRBC (400 ml) resulted in an initial chimerism of about 60% (Figure 6A), and parasitemia of up to 7.2% (ca. 12% of huRBC parasitized). Use of this lower huRBC volume however, was followed by a decrease in chimerism most likely due to inflammation induced by the parasite, producing a parallel reduction in parasitemia, which fluctuated at an intermediate level, i.e. without complete parasite clearance (Figure 6B). An analysis of inflammatory markers was performed in mice from the latter protocol to investigate the observed decrease seen in chimerism. The results revealed substantial differences from NOD/SCID mice. The decreased huRBC chimerism and parasitemia seen in NSG mice correlated with the release of TNFa and IL-12p70. In contrast IL-6 and IFNc remained almost unchanged (Figure 6B), whereas in NOD/SCID mice IL-6 and IFNc levels increased at the time of parasite decrease [6]. Hence the inflammation produced by NSG mice is reduced in comparison to that seen in NOD/SCID, and this is likely to be one of the reasons for improved P. falciparum survival. We observed from the variety of protocols experimented with, that provided the chimerism is maintained above 10–20%, parasitemia can be supported for a number of months (i.e. the mouse life-span). So, to avoid volume overload in the mice, intermediary protocols were designed requiring lower volumes of IV administered huRBC: i) satisfactory results were achieved in all mice where 550 ml of huRBC was injected three times a week (n = 47), with a mean 6 SD chimerism of 74%67% over the first month and half, decreasing to 56% over the second and third month, and long- lasting parasitemia reaching up to 20–40% (25.6% to 51.3% of huRBC parasitized); ii) when 550 mL of huRBC was injected only twice a week, a chimerism of 40–60% was achieved and this supported a moderate 5%–12% parasitemia during the first month. In this manner, adjusting the quantity of huRBC injected leads to various levels of prolonged, stable parasitemia. The level can be tailored in this manner to the needs of each experiment. In this model there was no need for preliminary adaptation of the parasites, contrary to what has been reported for another model based upon the NSG mouse [11]. All strains tested were supported (UPA, 3D7, K1), and parasite growth was readily apparent from the first days post-inoculation. Naturally, there were some variations in the maximum parasitemia reached with each strain, particularly for 3D7, which produced the lowest. Mice were also infected with a fresh clinical isolate of P. falciparum. With PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e18045