**6. Conclusions and future perspectives**

In an attempt to elucidate the mechanisms responsible for growth of *P. falciparum*, growthpromoting factors were identified and a CDM suitable for complete growth of the parasite was established. The CDM consists of paired NEFA, phospholipids with specific fatty acid moieties, and specific proteins dissolved in basal medium RPMI1640 supplemented with hypoxanthine. The most effective combination of NEFA was C18:1-*cis*-9 and C16:0. The best phospholipid crucial for serum-free culture medium supplemented with NEFA was PCdi18:1 at concentrations of 80–320 μg/ml. A simple protocol for flow cytometry with SYBR Green I was developed and used to analyze the various developmental stages of *P. falciparum*. Different stages of the parasite in RBC and released merozoites were quantified using this flow cytometry protocol. These techniques were applied to investigate the distinct roles of the identified growth-promoting factors in the development of the parasite, demonstrating that different combinations of NEFA and phospholipids exerted distinct roles in the growth of *P. falciparum* by sustaining development at different stages.

These findings can be usefully applied in diverse aspects malaria research, including drug resistance, vaccine development, genetics, parasite biochemistry, and studies of the relationship between the parasites and the host RBC. Culture in CDM produces similar results to those using the original culture method with human serum (Trager & Jensen, 1997), with the added advantage of avoiding the adverse effects caused by human serum. In particular, the methods reported here will allow the components crucial to each developmental stage of the parasite to be established. We have already performed a large-

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**6** 

*1,4USA 2France 3,5India* 

**Development of Humanized Mice** 

*Plasmodium falciparum* **Infection** 

R.K. Tyagi1,2\*, N.K. Garg3**,** T. Sahu4 and P. Prabha5 *1Department of Global Health, College of Public Health,* 

*2Malaria Vaccine Development Laboratory, Institute Pasteur Paris,* 

*3Shantha Biotechnics Ltd., (A Part of Sanofi Aventis Group), Hyderabad, AP,* 

Infectious diseases continue to heavily burden our global society. Endemic and epidemic malaria results in severe disease in an estimated half-a-billion people each year, and causes over 1.5 million deaths annually. Although progress has been made in the prevention and treatment of *falciparum* malaria infections, more effective, tolerable and affordable therapies are urgently needed. This deadly parasite displays unique human tropism, and the development of novel intervention strategies have been hampered by the lack of robust, cost effective, and predictive animal models that accurately reproduce the hallmark of human infections. While rodents and non-human primates have been employed in biomedical research and drug/vaccine development, they often do not yield reliable preclinical results that translate into effective human treatments. "Humanized" mice have recently emerged as powerful tools in the investigation of human diseases (Legrand et al., 2006; Manz, 2007; Shultz et al., 2007). These are amenable animal models transplanted with various kinds of human cells and tissues (and/or equipped with human transgenes) that may be ideally suited for direct investigation of human infectious agents such as malaria. Despite the challenges, humanized mouse technology has made rapid progress over the last few years, and it is now possible to achieve significant levels of human chimerism in various hosts, organ/tissues, particularly the immune systems, liver and muscles. Such humanized mice provide a new opportunity to perform preclinical studies of intractable human malaria

**1. Introduction** 

\*Corresponding Author

**to Study Asexual Blood Stage** 

*4National Institute of Allergy and Infectious Diseases,* 

*5Gene Regulation Laboratory, School Of Biotechnology,* 

*University of South Florida, Tampa, Florida,* 

*National Institutes of Health, Rockville, MD,* 

*Jawaharlal Nehru University, New Delhi,* 

