**5.1.2 Chemical immunomodulation protocol and mouse infection**

A previously described immunomodulation protocol (Badell et al., 2000), modified as described in (Moreno et al., 2006) is employed. On day -13, each mouse receives a dose of 10 mg/kg of mAb NIMP-R14 by i.p. injection. On day -12, each mouse receives 0.2 ml of the suspension of clo-lip by the same route. On days -9, -6, -3 each mouse receives 0.5 ml of huRBCs i.p. mixed with a dose of 10 mg/kg of mAb NIMP-R14 and 0.2 ml of clo-lip. On day 0 mice are infected with 50µl huRBCs parasitized by *P. falciparum* at a parasitaemia of 1% (all the developmental forms, i.e. trophozoite, schizont and rings, were present) mixed with a dose of 10 mg/kg of NIMP-R14 antibody. Afterwards, a dose of 10 mg/kg of antibody NIMP-R14, 0.2 ml of clo-lip and 0.5 ml of huRBCs is injected i.p. at 3 day intervals, until the end of the study. The infection is followed-up by daily Giemsa stained thin blood films drawn from the tail vein.

Fig. 2. Schematics of intraperitoneal immunomodulation protocol

### **5.2 Protocol for intravenous injection**

#### **5.2.1 Mouse infection and immunomodulation protocol**

NOD/SCID mice are retro-orbitally injected with 400 µl huRBCs every 3 days to ensure a satisfactory proportion of huRBCs (i.e. chimerism), at the time of infection (≈ 60%). Simultaneously, 0.1 ml of un-sized dichloromethylene diphosphonate (Cl2MDP) encapsulated in liposome (clo-lip) (provided by Nico Van Rooijen) diluted in 0.4 ml RPMI is intraperitoneally injected. Four injections at 2-3 day intervals are given before parasite infection. At the time of the fifth injection, mice are retro-orbitally injected with 300 µl of a *P. falciparum* infected huRBCs suspension in RPMI at a parasitaemia of 1% (all the developmental forms, i.e. rings, trophozoites and schizonts were present). After infection huRBCs and clo-lip are supplied every 3 days as described for the pre-infection step. In

The effect of splenectomy and of irradiation (100 - 300 cGy) is also tested. Other experiments that evaluates the addition of metabolic agents such as pABA (400 mg/kg/day), and folinic acid (400 mg/kg/day), or of antioxidants, such as vitamin E (20 mg/kg/day; Nepalm, Cenexi, Fontenay-sous, France), N-acetyl cysteine (100 mg/kg/day), trolox (4-100 mg/kg

A previously described immunomodulation protocol (Badell et al., 2000), modified as described in (Moreno et al., 2006) is employed. On day -13, each mouse receives a dose of 10 mg/kg of mAb NIMP-R14 by i.p. injection. On day -12, each mouse receives 0.2 ml of the suspension of clo-lip by the same route. On days -9, -6, -3 each mouse receives 0.5 ml of huRBCs i.p. mixed with a dose of 10 mg/kg of mAb NIMP-R14 and 0.2 ml of clo-lip. On day 0 mice are infected with 50µl huRBCs parasitized by *P. falciparum* at a parasitaemia of 1% (all the developmental forms, i.e. trophozoite, schizont and rings, were present) mixed with a dose of 10 mg/kg of NIMP-R14 antibody. Afterwards, a dose of 10 mg/kg of antibody NIMP-R14, 0.2 ml of clo-lip and 0.5 ml of huRBCs is injected i.p. at 3 day intervals, until the end of the study. The infection is followed-up by daily Giemsa stained thin blood films

**Means to control Innate Immunity**

**Infection**


NOD/SCID mice are retro-orbitally injected with 400 µl huRBCs every 3 days to ensure a satisfactory proportion of huRBCs (i.e. chimerism), at the time of infection (≈ 60%). Simultaneously, 0.1 ml of un-sized dichloromethylene diphosphonate (Cl2MDP) encapsulated in liposome (clo-lip) (provided by Nico Van Rooijen) diluted in 0.4 ml RPMI is intraperitoneally injected. Four injections at 2-3 day intervals are given before parasite infection. At the time of the fifth injection, mice are retro-orbitally injected with 300 µl of a *P. falciparum* infected huRBCs suspension in RPMI at a parasitaemia of 1% (all the developmental forms, i.e. rings, trophozoites and schizonts were present). After infection huRBCs and clo-lip are supplied every 3 days as described for the pre-infection step. In

**Blood 0.6 mL (60 % hematocrit) + clo-Iip + NIMP-R14**

/day), 8-aminoguanidine (100 mg/kg/day).

drawn from the tail vein.

**Clo-α PMN lip 250 μg 0.2 mm** 

**5.1.2 Chemical immunomodulation protocol and mouse infection** 

**Blood 0.6 mL (60 % hematocrit) + clo-Iip + NIMP-R14**

Fig. 2. Schematics of intraperitoneal immunomodulation protocol

**5.2.1 Mouse infection and immunomodulation protocol** 

**5.2 Protocol for intravenous injection** 

some experiments, 250 mg/kg of inosine (Sigma) is injected intraperitoneally every day as the half-life of inosine is very short (Mabley et al., 2009). In experiments using NSG mice the protocol has been adopted in order to achieve varying levels of adequate huRBCs chimerism and to avoid overloading the mice. As such, different amounts of blood are employed, and either 200, 400, 550 or 750 µl huRBCs are injected 3 times per week (i.e. Monday, Wednesday and Friday) mixed with 250 µl human AB serum, as it has previously been described that human serum improves huRBCs survival in immunocompromised mice (Angulo-Barturen et al., 2008); 4 injections are done prior to infection, and clo-lip is injected as described above. The infection is followed-up by daily Giemsa stained thin blood films drawn from the tail vein.
