Friday, August 27, 2010

Toxoplasma Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

J Vis Exp. 2010 Aug 12;(42). pii: 2091. doi: 10.3791/2091.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Tobin C, Pollard A, Knoll L.

Department of Medical Microbiology and Immunology, University of Wisconsin.

Abstract
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO(2;) starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO(2;) and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO(2;) and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

PMID: 20736916 [PubMed - in process]

1 comment:

jsdaniels said...

There are several great research organizations out there researching this. You can actually participate in the research and get paid for your time if you are diagnosed with diseases like Hepatitis A, Lupus, Rheumatoid Arthritis, Celiac, and many others. The research helps to develop diagnostic tests and cures, and you are paid anywhere from $200 to $1000 every time you participate. The most active options are below.

http://www.idonateplasma.com

https://twitter.com/IDonatePlasma

http://www.myspace.com/seracare

http://www.linkedin.com/pub/i-donate-plasma-for-research/21/b09/726

http://www.facebook.com/pages/PAID-Plasma-Donations-for-Research/101071083280661?ref=ts