| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The objective of this project is establishment of complete cyclic development for a monomorphic stock of Trypanosoma gambiense, the causative agent of African sleeping sickness in man, in vitro. In the beginning of this project, the author established the effective long-term cultivation system for monomorphic Wellcome stock of T. gambiense bloodstream forms in Hepes-buffered RPMI 1640 medium (pH 7.2, 300 milliosmole/kg) supplemented with 20 % fetal calf serum in the presence of rat astroglioma cell line (GA-1) at 37゜C in vitro. Trypanosomes (antigenically O type) cultured in this system for more than 200 days still retained not only their virulence for mice but also their original antigenic type. Secondly, the author described on effective transformation system for a monomorphic T. gambiense bloodstream forms to procyclic forms stimulated by trypsin treatment in the same culture system used for long-term culture of bloodstream forms at 27゜C in vitro. In contrast to typsin treated popul
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ations, transformation of untreated bloodstream forms and forms treated with inhibited trypsin by soybean inhibitor were prolonged. Thirdly, stimulated transformation system for a monomorphic T. gambiense from procyclic forms to epimastigote forms was studied. Transformation of T. gambiense (Wellcome stock) procyclic forms to epimastigote forms could be stimulated by adding 5 mM hemin to Hepes-buffered RPMI 1640 medium supplemented with 20 % fetal calf serum in the presence of GA-1 cell at 27゜C in vitro. In this system, 3-5 % of procyclic forms transformed to epimastigote forms within 10 days, and infective metacyclic forms were detected on day 20 after initiation of the culture. When metacyclic forms could be detected, the culture flasks were incubated at 37゜C for 7-10 days. In this culture system, trypanosomes were propagated as long slender bloodstream fofms in the presence of GA-1 cells. These bloodstream forms were infective to mice. Antigenically, bloodstream forms after cyclic development was not O type. Less
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