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3R-Project 107-07
Evaluation of an in vitro model to identify host parameters associated with virulence of Toxoplasma gondii strains
Sushila D’Souza and Vijay Morampudi
Pasteur Institute of Brussels-ISP,1180-Uccle, Belgium
sdsouza@pasteur.be
Keywords: human; parasites, ectoparasites: toxoplasma gondii; epithelia; epithelia; intestine; infectious diseases; replacement; infectiosity
Duration: 2 years End of the Project: 2009
Background and Aim
The global prevalence of T. gondii, the causative agent of toxoplasmosis is estimated to be 30%. This intracellular protozoan parasite is one of the three common causes of congenital disease which can lead to severe malformation, mental retardation and spontaneous abortion. T. gondii is also the second most common cause of encephalitis in immunocompromised individuals.
Epidemiological studies have classified T. gondii populations into three clonal lineages which display only discrete genetic polymorphisms. Virulence in mice to T. gondii clonotypes however differs markedly. We aim to develop an in vitro model to test the presence and virulence of T. gondii in cell culture. Human intestinal epithelial cells, known to be an early site of T. gondii replication are being tested as the prototype host cell.
Method and Results
in progress (present status)
Human ileocecal epithelial carcinoma cells (HCT-8) are being tested as an in vitro model. We have studied the replication of Type I (RH) and Type II (76K) strains of T. gondii within these cells, using a parasite specific quantitative real time PCR. These host cells clearly permit the infection and the intracellular replication of both the Type I and Type II parasites even at a very low multiplicity of infection. Comparison of the two clonotypes of parasites in the same experiment demonstrated that Type I parasites had a significantly higher rate of replication than Type II parasites. Further, the foci of cell destruction in the monolayer were rapidly formed and more abundant with Type I compared with Type II strains. These in vitro findings seem to correspond so far with the knowledge in mice that Type I strains are virulent.
Studies on the expression of early host genes involved in pathogenesis are being conducted after infection of HCT-8 cells with Type I or Type II T. gondii strains, in order to pin down gene expression that can be associated with virulence.
Conclusions and Relevance for 3R
Since the use of mice is the only source of biological test to detect virulence of T. gondii strains, there is an urgent need for an ethically acceptable in vitro model. At the toxoplasmosis reference laboratory of our institute alone, diagnosis of T. gondii entails the use of 5000 mice for the analysis of 500 clinical samples. Hence the use of intestinal epithelial cell lines would significantly reduce the number of animals required for identifying the parameters associated with the virulence of T. gondii. By using this in vitro model we could correlate the rate of replication, production of inflammatory factors by the host cell and cellular mortality with the virulence of T. gondii. The data accumulated from this study could finally serve as the experimental basis to design and develop in vitro tests for the diagnosis of T. gondii infections, as well as to determine the virulence of the different type of T. gondii strains.
References
1. Bout. D., Moretta M., Dimier-Poisson I., Gatel DB. 1997. Interaction between Toxoplasma gondii and enterocyte. Immunology. 201:225-228.
2. Liesenfeld, O. 1999. Immune responses to Toxoplasma gondii in the gut. Immunobiology. 201:229-239.
3. Abreu, MT., Fukata, M. and Arditi, M. 2005. TLR Signaling in the Gut in Health and Disease. J. Immunol. 174:4453-4460.
4. Butcher, BA, Kim, L., Panopoulos AD., Watowich, SS., Murray, PJ and Denkers EY. 2005. Cutting Edge: IL-10-Independent STAT3 Activation by Toxoplasma gondii mediates suppression of IL-12 and TNFalpha in host macrophages. J. Immunol. 174:3148-3152.
5. Mun HS, Aosai F, Norose K, Chen M, Piao LX, Takeuchi O., Akira S, Ishikura H. and Yano A. 2003. TLR2 is an essential molecule for protective immunity against Toxoplasma gondii infection. Int. Immunol. 15:1081-1087.
6. Furuta T., Kikuchi T., Akira S., Watanabe N. and Yoshikawa Y. 2006. Role of the small intestine for induction of toll-like receptor 4 mediated innate resistance in naturally acquired murine toxoplasmosis. Int. Immun. 10:1093-1099.
7. Molestina RE., Payne TM, Coppens I., and Sinai AP. 2003. Activation of NF-kappa B by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated I kappaB to the parasitophorous vacuole membrane. J. Cell Science 113:4359-4371.
Figures
Figure 1: HCT-8 cell cultures under native conditions.
Figure 2: Plaque formation of HCT-8 cells infected with T. gondii (MOI=20)48hrs after infection.
Figure 3: Increased number of plaques in HCT-8 cells infected with T. gondii (MOI=20) 72hrs after infection.
| Dernières modifications: 29.08.2010 |  |