**5.1 (I.P. Protocol)**

96 Malaria Parasites

the NOD/SCID/IL-2Rc-null mouse (NSG mouse) which, due to the knock out of the γ-chain of the IL-2 receptor, has been shown to better tolerate a variety of transplanted human cells (Ito et al., 2002; King et al., 2007; Watanabe et al., 2007). The resulting new IV model based on NSG mice presents several 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

**4. Strategic planning for the development of humanized mouse model** 

BALB/c, NOD/SCID and NSG mice are purchased from Charles River. Immunodeficient mice were kept in sterile isolators. They are housed in sterilized cages equipped with filter tops during the experimentation, and are provided with autoclaved tap water and a γirradiated pelleted diet ad libitum. They are manipulated under pathogen free conditions

Human whole blood is provided by the blood bank. Blood donors had no history of malaria and all the blood groups are used without observing any difference on parasite survival. Whole blood is washed three times by centrifugation at 900 ×g, 5 minutes at room temperature and buffy coat was separated in order to eliminate white blood cells and platelets. Packed huRBCs are suspended in SAGM (Adenine, Glucose and mannitol solution) and kept at 4°C for a maximum of 2 weeks. Before use huRBCs are washed three times in RPMI-1640 medium (Gibco/BRL, Grand Island, N.Y.) supplemented with 1 mg

*P. falciparum* lines 3D7, UPA, and K1 are employed in the study, along with clinical isolates taken from a patient at Bichat Hospital, Paris (which was used the day after being sampled). The Uganda Palo Alto (UPA) strain employed is the Palo Alto Marburg line, used for all the experiments conducted. Parasite cultures are not synchronized and therefore a mix of various developmental stages was injected to infect mice. Parasites are maintained under *in vitro* conditions at 5% hematocrit at 37oC in a candle jar in complete culture medium (RPMI-1640 medium (Gibco/BRL), 35mM HEPES (Sigma), 24mM NaHCO3, 10% albumax (Gibco/BRL) and 1mg of hypoxanthine (Sigma) per liter. Parasite samples are cryopreserved using the glycerol/sorbitol method (Rowe et al., 1968). The cultures are controlled for mycoplasma contamination by using polymerase chain reaction (PCR) technique. A non-lethal rodent parasite strain *Plasmodium yoelii* XNL1.1 is preserved in 500μl aliquot of cryo-preserving buffer at -80°C at 22% parasitaemia. The strain is thawed at room temperature, diluted twice in RPMI-1640 medium followed by the injection of 50 × 106

hypoxanthine per liter (Sigma, St Louis, MO) and warmed 10 minutes at 37°C.

total erythrocytes infected (Arnold et al., 2010).

**4.1 Materials and methods**

using laminar flux hoods.

**4.1.3 Parasites** 

**4.1.2 Human red blood cells** 

parasite directly into the mice.

**4.1.1 Mice**

## **5.1.1 Immunomodulatory agents to suppress innate immunity**

Numerous attempts have been made to increase the success rate of the grafting of infected RBC. Un-sized dichloromethylene diphosphonate (Cl2-MDP) encapsulated in liposome (clolip) (provided by N. Van Rooijen, Amsterdam, The Netherlands) is injected through intraperitoneal (i.p.) route in order to reduce the number of tissue MP, as described previously (van Rooijen and van Kesteren-Hendrikx, 2003). The anti-PMN monoclonal antibody NIMP-R14 (Lopez et al., 1984) is purified from a hybridoma. Its activity is compared to that of two other anti-PMN monoclonal antibodies: RB6-8C5 (purified from the hybridoma) and 1A8 (BioXcell, Lebanon). The NIMP-R14 monoclonal antibody is used in all the studies, unless specified. Various agents (from Sigma) (Table 1) are used to further reduce innate immunity such as dexamethasone (1-5 mg/kg/day), TGF-β (100 ng - 1 μg/day) (PeproTech, Rocky Hill, NJ), cyclophosphamide (75 mg/kg/day), cisplatinium (1-10 mg/kg/day), and TMβ-1 monoclonal antibody that targets NK cells (1 mg/kg/day).


Table 1. Various immune suppressants to control innate immunity

Asexual Blood Stage Malaria in a Humanized Mouse Model 99

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.

The study blood samples are collected from mice retro-orbital sinus on heparin. Various haematological parameters such as haematocrit, leukocyte number and phenotype (Ly-6C APC, Ly-6G APC (Miltenyi Biotec, Germany), CD115 PE, CD43 FITC, CD62L FITC, CD11b FITC, DX5 FITC, CD122 PE (BD Biosciences, UK) in peripheral blood samples were monitored, as well as the phenotype characterization of monocytes (CD11b+,CD115+), inflammatory monocytes (CD43-, CD62L+, Ly-6C+), PMN (CD11b+, ly6G+), and natural killer cells (DX5+, CD122+). Total leukocyte number (leukocytes/μl blood) is evaluated by lysing 20μl of total blood with BD FACS™ Lysing solution, and counting on Malassez haematocytometer. Since successfully grafted mice have a significant, but variable, percentage of huRBCs in their peripheral blood, parasitaemia in mice is expressed as the overall percentage of *P. falciparum* infected RBCs among total RBCs, i.e. both human and mouse RBCs observed on thin blood smears. In addition, the peritoneal blood parasitaemia

Blood samples drawn from mice are used to determine the percentage of huRBCs in mouse peripheral blood at regular intervals by flow cytometry on a FACScalibur (BD biosciences)

In NOD/SCID mice inflammation is induced by IP injection of 1ml 3% thioglycolate (Sigma) diluted in sterile PBS. 4-5 days after, the peritoneal cavity is washed with HBSS without Ca++ and Mg++. The collected cells are washed twice in RPMI supplemented with Lglutamine, Penicillin (100U/ml), Streptomycin (100ug/ml), and 10% Fetal Calf Serum (FCS) and seeded at 3x105 per well in a 96-well culture plate. Cell suspensions of splenocytes are prepared in cold RPMI 1640 medium supplemented with 10% FCS and filtered on a 100 µm cell strainer to remove debris. Erythrocytes are lysed with ACK lysis buffer, and the splenocytes are washed 2 times with RPMI supplemented with 10% FCS and seeded at 3x105

100 µl blood samples are collected from the retro-orbital plexus with a Pasteur pipette, and sera are stored at -80°C. Conditioned media obtained after 16h stimulation of peritoneal cells and splenocytes with lipopolysaccharide (LPS, 1 µg/ml) (Sigma) are stored at -80°C. Cytokines and chemokines (IL-6, MCP-1, IFNγ, TNFα, IL-12p70 and IL-10) are quantified

using FITC labeled anti-human glycophorin monoclonal antibody (Dako, Denmark).

**5.2.2 Haematological parameters and grafting of** *P. falciparum***-huRBCs** 

is measured on the smears drawn from the peritoneum.

**5.2.4 Cytokines/chemokine/chemiluminesence assay** 

**5.2.3 Mouse cell isolation** 

per well of a 96 well culture plate.

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