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

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 is measured on the smears drawn from the peritoneum.

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

## **5.2.3 Mouse cell isolation**

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 per well of a 96 well culture plate.

#### **5.2.4 Cytokines/chemokine/chemiluminesence assay**

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

Asexual Blood Stage Malaria in a Humanized Mouse Model 101

The present protocols illustrate an improvement of the mice with genetic deficiencies in adaptive immunity for successful grafting of human cells or pathogens to study human biological processes *in vivo.* Since non-adaptive immunity received little attention, we have deployed our efforts to study innate defences, responsible for the substantial control of *P. falciparum* and highlighted some of the remaining limitations on the development of the optimal humanized mouse. In addition, numerous attempts were made to enhance the success rate of infected huRBC by employing various immunomodulatory agents intraperitoneally to further suppress the residual innate immunity. This study came up with some conclusions such as, 1) stable parasitaemia with only a subset of mice 2) parasite clearance and *P. falciparum* induced inflammation are correlated 3) *P. yoelii* induces less inflammation than *P. falciparum* 4) huRBC, and the anti-inflammatory agents, induce low grade inflammation. 5) repeated administrations of huRBC, clo-lip and anti-PMN reduce inflammation and improve HuRBC grafting 6) MO/MPs are critical in controlling *P. falciparum* and huRBC grafting in NOD/SCID. A strong pro-inflammatory effect of the parasite itself and poor understanding of migration of infected and uninfected huRBC into blood stream across the peritoneum and subsequently, uneven distribution of huRBC are two barriers to achieving an improved, workable humanized mouse model*.* The results obtained by the IP protocols indicate that the SCID mice to study human biological processes *in vivo* need to be carefully explored and that further attempts are required to address the remaining limitations such as residual innate immunity and route of delivery of

The complex biological processes often require *in vivo* analysis of human cells: humanized mice or mouse-human chimeras have been developed to meet this requirement, however, with low percentage of infectivity and reproducibility supporting a long standing parasitaemia only in a proportion of animals. We have obtained improved parasitaemias based on intravenous delivery of huRBC and *P. falciparum* instead of the intraperitoneal

In essence the genetic background has also played an important role to optimize the humanized mouse model. The intravenous mouse model (NSG-IV) shows the role of ageing and inosine in controlling *P. falciparum* induced inflammation. The success of humanized mouse (NSG mice) model, with IV delivery of huRBC and *P. falciparum* in clo-lip treated (immunosuppression of macrophages) mice, was 100% in terms of infectivity and reproducibility. Synchronization, partial sequestration and receptivity to various strains of *P. falciparum* without preliminary adaptation are some of the lucrative features of the developed mouse model which is reliable and more relevant, and better meet the needs of

In conclusion, the chapter demonstrates that the careful selection of mice with combined deficiencies of adaptive and innate immunity, and the better control of innate immune defenses by improving immunomodulatory protocols and using anti-inflammatory substances, could provide a reproducible, long lasting and straightforward mouse model of

**6. Anticipated results** 

huRBC to create an optimized humanized mouse model.

route (IP) by testing various immunosuppressive drugs.

biomedical translational research.

**7. Conclusion** 

immunomodulatory protocol

Since production of reactive oxygen intermediates (ROI) closely mirrors the state of activation of macrophages and polymorphonuclear cells, luminol dependent photometric assay is used to measure Reactive Oxygen Intermediates (ROI). Blood samples are collected from NOD/SCID mice, washed with HBSS with freshly added Ca++ and Mg++. Washed blood is diluted 1/10 in HBSS and 90 µl blood were added to each well in a 96 well plate (Nunc, Denmark) and incubated for 30 minutes at 370C after adding 10 µl PMA (final concentration 1 µg/ml) to stimulate the cells. 50µl of luminol (final concentration 200 µg/ml) solution are added immediately before measuring emissions.

#### **5.2.5 Analysis of deep-seated organs for parasite differential count**

NSG mice are used for the comparison of parasite differential counts in the peripheral blood, with that in deep-seated organs. Four mice are infected with UPA strain, and when a parasitaemia of >10% is reached a thin smear from peripheral blood is drawn before killing the mouse, and harvesting its organs. Kidney, Liver, Spleen, Lung, and Brain are removed from each mouse. Parasite content is assessed from blots made by repeatedly spotting sections from each organ. These slides are then stained with Giemsa. The last blots taken, are considered to be the most representative of the parasite content in the organ's vascular bed, and are examined at 1000x magnification to perform differential counts of each stage (> 200 parasites from each organ counted).
