species in the majority of experimental malaria studies - for reasons of convenience rather than scientific merit. A main barrier to achieving an improved, workable P. falciparum mouse model is the strong pro-inflammatory effect of the parasite itself. In humans the asexual erythrocytic stages of P. falciparum are known to result in a systemic inflammatory process that is responsible for many of the symptoms of the disease [13]. Although the two situations differ in several respects, a similar inflammatory response was observed in immunodeficient mice, lacking T and B cells. In this case, as recently described [6], P. falciparum triggers strong pro-inflammatory reactions in monocytes and macrophages, contributing to partial control of the parasite and the human erythrocyte (huRBC) graft. A second significant practical problem with all existing models developed to date is that huRBC are injected by the intra-peritoneal (IP) route, which relies on the successful migration of huRBC into the blood stream across the peritoneum. This is a process that is not properly understood, and, in turn, this prevents any rational analysis and improvement of the model. Existing models have been useful in proving the concept that P.falciparum can survive in a small rodent model, however they have also shown that unless inordinate volumes of blood are injected IP daily [11,14], trans-peritoneal passage to the peripheral blood is uneven, particularly in the long term. This leads to important variations from one animal to the other, most likely related to the inflammatory reaction triggered by high parasite loads, as shown previously [6]. With these limitations of current models in mind, we thought it was essential to attempt to improve the P. falciparum humanised mouse model, particularly in terms of control of inflammatory reactions, and reproducibility of parasitemia. We decided to address these issues by using the IV route for huRBC and parasite administration, and by investigating other means to increase control over the mouse innate immune response. The use of this IV model led us to identify, among several factors investigated, the effect of aging and that of inosine as significant in reducing inflammatory reactions, and therefore improving P. falciparum growth. Moreover, after using various strains of immunodeficient mice, we investigated, as others [14], the value of the NOD/ SCID/IL-2Rc-null mouse (NSG mouse) which, due to the knock out of the c-chain of the IL-2 receptor, has been shown to better tolerate a variety of transplanted human cells [15,16,17,18]. The resulting new IV model using NSG mice presents several significant advantages over previously available models. It offers greater reproducibility, with 100% of mice successfully grafted without the need for mouse-adapted parasites, consistent curves of parasitemia, and high levels of infection with up to 40–50% of total erythrocytes infected. Materials and Methods 1. Mice All procedures were carried out in line with the European Community Council Directive, 24th November 1986 (86/609/ EEC), and the European Union guidelines. All procedures were reviewed and approved by the Pasteur Institut Animal ethical committee (approval number A 75 15–27). Every effort were made to minimize suffering. BALB/c, NOD/SCID and NSG mice were purchased from Charles River. Immunodeficient mice were kept in sterile isolators. They were housed in sterilized cages equipped with filter tops during the experimentation, and they were provided with autoclaved tap water and a c-irradiated pelleted diet ad libitum. They were manipulated under pathogen free conditions using laminar flux hoods. Improved P. falciparum Mouse Model 2. Human Red Blood Cells (huRBC) Human whole blood was provided by the French blood bank (Etablissement Franc¸ais du Sang, Paris, France) and used in accordance with French legislation. Blood donors had no history of malaria and all blood-groups were used without observing any difference on parasite survival. Whole blood was centrifuged thrice at 9006g, for 5 minutes at room temperature and the buffy coat was separated in order to eliminate white blood cells and platelets. Packed RBC were suspended in SAGM (Adenine, Glucose and mannitol solution) and kept at 4uC for a maximum of 2 weeks. Before use, huRBC were washed three times with RPMI-1640 medium (Gibco/BRL, Grand Island, N.Y.), supplemented with 1 mg of hypoxanthine per liter (Sigma, St Louis, MO) and warmed at 37uC. Blood samples drawn from mice were used to determine the percentage of huRBC in mouse peripheral blood at regular intervals by flow cytometry on a FACScalibur (BD biosciences) using FITC labeled anti-human glycophorin monoclonal antibody (Dako, Denmark). 3. Parasites P. falciparum lines 3D7, UPA, and K1 were employed in this study, along with one clinical isolate taken from a patient at Bichat Hospital, Paris (used the day after sampling). The Uganda Palo Alto (UPA) strain employed was the Palo Alto Marburg line, this was used for all experiments conducted unless otherwise specified. Parasite cultures were not synchronized and therefore a mix of various developmental stages was injected to infect mice. Parasites were maintained under in vitro conditions at 5% hematocrit at 37uC in a candle jar in complete culture medium (RPMI-1640 medium (Gibco/BRL), 35 mM HEPES (Sigma), 24 mM NaHCO3, 10% albumax (Gibco/BRL) and 1 mg of hypoxanthine (Sigma) per liter. Parasite samples were cryopreserved using the glycerol/sorbitol method [19]. The cultures were controlled for Mycoplasma contamination by using PCR testing. 4. Mouse infection and immunomodulation protocol NOD/SCID mice were retro-orbitally injected with 400 ml huRBC every 3 days to ensure a satisfactory proportion of huRBC (i.e. chimerism), at the time of infection (<60%). Simultaneously, 0.1 ml of unsized dichloromethylene diphosphonate (Cl2MDP) encapsulated in liposome (clo-lip) (provided by Nico Van Rooijen) diluted in 0.4 ml RPMI was intraperitoneally injected. Four injections at 2–3 day intervals were given before parasite infection. At the time of the fifth injection, mice were retro-orbitally injected with 300 ml of a P. falciparum infected huRBC suspension in RPMI at a parasitemia of 1% (all the developmental forms, i.e. rings, trophozoites and schizonts were present). After infection huRBC and clo-lip were supplied every 3 days as described for the pre- infection step. In some experiments, 250 mg/kg of inosine (Sigma) was injected intraperitoneally every day as the half-life of inosine is very short [20]. In experiments using NSG mice the protocol has been adapted in order to achieve varying levels of adequate huRBC chimerism and to avoid overloading the mice. As such, different amounts of blood were employed, and either 200, 400, 550 or 750 ml huRBC was injected 3 times per week (i.e. Monday, Wednesday and Friday) mixed with 250 ml human AB serum, as it has previously been described that human serum improves huRBC survival in immunocompromised mice [11]; 4 injections were done pre- infection, and clo-lip was injected as described above. Follow-up of the infection was performed by daily Giemsa stained thin blood films drawn from the tail vein. PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e18045