Pneumocystosis, a serious and often fatal disease caused by Pneumocystis carinii, has become a serious health problem. Patients with AIDS and those undergoing immunosuppressive therapy (especially cancer patients and transplantation recipients) have a high morbidity and mortality due to pneumocystosis. Early diagnosis and subsequent treatment could reduce both the morbidity and mortality of this disease. Unfortunately, current diagnostic methods lack sensitivity and involve invasive procedures which place the patient at risk. The reason why current diagnostic methods are so inadequate and why so little is known about the hosts immune response to P. carinii has been the inability to purify the organism from host tissues. During the past three years we have used enzyme digestion and density gradient centrifugation to obtain partially purified organisms from lungs of rats with pneumocystosis. Analysis of these organisms revealed greatly reduced lung tissue contamination and several molecules unique to P. carinii. We are using this material to produce mouse monoclonal antibodies, which, when used in an ELISA, react with sera from patients with pneumocystosis. We propose to further develop and refine the ELISA for P. carinii using rat and mouse monoclonal antibodies. Using these antibodies, we will study antigenemia and antibody response in rats during induction and the manifestation of pneumocystosis. P. carinii will be further purified and separated into cysts and trophozoite forms using gradient centrifugation, gel permeation and cell affinity chromatography. The biochemical and immunological nature of the purified macromolecules of P. carinii will be studied using PAGE, Western blotting techniques and monoclonal antibodies. Pneumocystis antigens associated with antigenemia will be identified. We will evaluate the ability of the ELISA to detect subclinical and clinical pneumocystosis in patients with immunodeficiencies (AIDS, leukemias, cancer, transplants) and in patients with lung diseases using monoclonal antibodies with known antigenic reactivity. Our data will provide valuable information on the nature of antigens and antibodies, as well as the role of antigenemia in P. carinii infections. This information, along with a sensitive ELISA, will allow the early detection of human pneumocystosis and thus help to decrease human morbidity and mortality due to this disease.
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