[28,29]. There is currently a renewed interest in the effect of age on the immune system, both adaptive and innate. Several recent studies have described modifications of immune function in aged subjects, leading either to increased, or more frequently decreased inflammatory processes with a reduction in LPS-induced IL-6, and IFNc levels, similar to what we observed [30,31,32,33]. However we are not aware of any previous data pertaining specifically to NOD/SCID mice. The present findings of improved graft survival associated with a decreased release of inflammatory cytokines by macrophages in ‘‘aged’’ mice complements previous findings in other mouse strains [34], and indicates that this process reduces defences against Plasmodium. The effect of aging was not evident in the NSG mice however. This is likely to be due to a blurring of the effect by interposition with the IL-2Rc mutation, along with a sample size too small to show a marginal benefit of aging in these mice. Among a large number of immunomodulating substances investigated, inosine emerged as one of the few drugs beneficial in this scenario. It is well known that adenosine possesses anti- inflammatory properties [35], and that its derivative analogues such as inosine, can provide protection to mice from a variety of inflammatory diseases including endotoxic and septic shock [35,36], acute lung injury [20,37], pleural inflammation [38], and colitis [39] by inhibiting the release of free radicals and pro- inflammatory cytokines [40]. Inosine, a naturally occurring purine, and breakdown product of adenosine by adenosine deaminase [41], has been shown to have both pro and anti- inflammatory properties, probably mediated by its ability to bind to more than one type of adenosine receptor. The results of the present study showing that inosine reduces the release of IL-6, MCP-1 and TNFa by macrophages from NOD/SCID mice, is consistent with previous reports using cells from immunocompe- tent mice [42]. These effects are supported by the significant improvement that was seen in parasitemia for mice that received inosine, compared to those that did not. Inosine may also act through its ability to suppress the function of neutrophils. It has been reported to inhibit neutrophil recruitment in vivo, and inhibit neutrophil superoxide production in vitro [40]. While the effect of increasing age on an NSG background was not evident, the benefit of administering inosine was significant at high parasitemia, i.e. after 15 days of treatment. The relationship observed in this study, as in our previous study [6], between the peak of inflammation and a reduction in huRBC count should be stressed. It suggests that the anemia seen correlates with increased inflammation, and that it could potentially be the inflammation itself that causes the anemia, independent of the parasite, through destruction of non-infected huRBC. This might be mediated by free oxygen radicals damaging the huRBC membrane [43]. In this respect the model may provide novel research hypotheses that could lead to better characterize the poorly understood pathophysiological process of severe anemia in humans, which is one of the two major causes of mortality [44], although admittedly the human situation differs substantially from that of P.falciparum in the NSG mice. Our results confirm that the NSG mouse strain has a significantly attenuated inflammatory response, leading to an improvement in parasitemia. These mice have proven to be excellent hosts for the grafting of various human cells, and notably they are better recipients than NOD/SCID for CD34+ hemato- poietic stem cells and human leukocytes [15,18,45]. This improved receptivity results from the reduced function of macrophage and dendritic cells, in addition to the lack of T, B, and NK cells [18,45]. The improved survival of P. falciparum in NSG was associated with an absence of significant production of the two Improved P. falciparum Mouse Model major inflammatory cytokines IL-6 and IFNc, whereas both were elevated in the NOD/SCID model [6]. This is not to say that the inflammatory response was quashed completely in the NSG strain. The need for further efforts to control the deleterious responses of the murine immune system in this model remains, likely by including additional genetic modifications, for example, by knocking out CCR2 on an NSG genetic background to reduce the egress, the recruitment and the number of inflammatory monocytes [46,47,48]. The improved survival of P. falciparum in NSG mice is in agreement with results recently reported by Jime´nez-D´ıaz et al., where massive loads of huRBC were administered intra-peritone- ally [14]. At the expense of injections on a daily basis of as much as 2 ml of blood (1 ml huRBC pellet) IP in 25 gram mice, and using a mouse adapted strain of P. falciparum, improved results as compared to NOD/SCID/b2m mice [11] were obtained. The fact that the mice were able to survive such severe treatment implies that there must have been massive intraperitoneal destruction, and intravascular hemolysis of grafted erythrocytes. Inflammatory mediator levels were not reported for this IP model however, and it is unclear how the huge huRBC loads operate. It has been reported in patients receiving blood transfusions, that large amounts of RBC can exert an immuno-suppressor effect upon macrophages [49]. It is also possible that macrophages overloaded by phagocytosis of an inordinate number of huRBC become unable to cope with additional parasitized cells. Similarly, by saturating the mouse with huRBC, a subset of them sufficient to permit P. falciparum development is more likely to remain intact, despite the massive amount of erythrocyte destruction. Whichever way, we repeated the protocol described by Jime´nez-D´ıaz et al [14] and confirm their results (not shown). The use of liposomes containing clodronate (clo-lip), as described in the macrophage ‘‘suicide’’ approach developed by Nico van Rooijen [50,51] brings several advantages with respect to both parasite survival and the quantity of huRBC that need to be administered. The reduction in macrophage numbers induced by clo-lip, and its consequence on parasite survival has been described in previous work on NIH-III and NOD/SCID mice [6,7]. In our NSG-IV murine model, clo-lip complemented the IL- 2Rc mutation very nicely in controlling inflammation, and thereby reducing erythrophagocytosis. For example, using this drug allowed us to reduce the total number of huRBC injected into mice by nearly 5 times as compared to the IP-IV model [14], i.e. a total of 1.6 ml vs 7 ml per week, per mouse (or nearly 10 times, i.e. only 0.8 ml vs 7 ml when a moderate parasitemia is desired), avoiding overload of the peritoneal cavity with [13] unreasonable volumes of fluid, proteins, membranes and the local pathological consequences. In total, there are now three possible protocols that can be used in NSG mice, each with different outcomes. They differ in the routes of delivery used for huRBC and parasites - in the ‘‘IP-IP model’’, both components are delivered intraperitoneally; in the model employed by Jime´nez-D´ıaz et al. the former are delivered intraperitoneally while the infection is delivered intravenously leading to the ‘‘IP-IV model’’; and in this study we demonstrate the ‘‘IV-IV model’’ where both components are delivered intravenously. The IP-IP model presents the same limitations as were described when using this protocol in NOD/SCID mice, with poor reproducibility of peripheral parasitemia. The IP-IV model remains effective at the expense of administering large amounts of huRBC to the mouse, daily interventions, and pre- adaptation of the parasite. Therefore, the IV-IV model seems to be more physiological, requiring lower amounts of huRBC and only 2–3 interventions per week. The requirement for 3 injections PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e18045