We propose to develop more sensitive and specific methods for diagnosis of acute toxoplasmosis in pregnant women, congenitally infected newborns, acute ocular toxoplasmosis and immunocompromised patients. To achieve this, we will attempt to identify antigens/epitopes recognized by Ig subclasses during the acute infection in adults and by those produced in the congenitally infected fetus/newborn. Recombinant antigens and selected synthetic peptides derived from surface and secreted antigens of T.gondii will be studies. To identify genes that encode antigens recognized by antibodies early during infection, tachyzoite cDNA expression libraries will be screened with purified IgM, IgA and IgE antibodies from sera of acutely infected patients and with antibodies raised against the unique acute phase AC antigen. To identify linear and conformational epitopes recognized by acute phase antibodies, we will attempt to identify peptides that mimic epitopes from T.gondii acute phase antigens using a phage-display random peptide library. Our observation of unique antigens recognized by the fetus but not by their mothers will be further examined to improve diagnosis of congenital toxoplasmosis. A new model of toxoplasmosis in mice genetically resistant to development of toxoplasmic encephalitis (TE) that more nearly resembles the natural resistance of humans to T. gondii (in humans, life threatening TE only develops when the immune system is severely impaired), will be used to study the role of alpha beta, gamma/delta, CD4+ and CD8+T cells (using adoptive transfer), cytokines and the major surface antigen SAG1 in resistance against and the histopathology of TE. Previous models were in mice genetically susceptible to development of TE> Cytokine production by and cytotoxic activity of gamma/delta and alpha/beta cells from brains of mice will be examined following infection. We will attempt to quantify parasites in the brain using transgenetic tachyzoites that express beta-galactosidase. We recently demonstrated for the first time that a clinical manifestation of toxoplasmosis, (TE in AIDS), may be under genetic regulation. We propose to determine whether toxoplasmic lymphadenopathy is also under genetic regulation since only approximately 10% of acutely infected, immunocompetent individuals develop this clinical manifestation. We propose to continue seeking more active and less toxic therapies for toxoplasmosis. We will identify the most satisfactory in vitro method for this purpose by comparing 3 methods now in use. Drugs will be tested alone or in combination with other drugs and/or cytokines in vitro and in mouse models of acute toxoplasmosis or TE. Drug activity against the cyst form will also be studied.
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