| Co-Investigator(Kenkyū-buntansha) |
NAKAZAWA Shusuke Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 熱帯医学研究所, 助手 (20180268)
KANBARA Hiroji Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, 熱帯医学研究所, 教授 (20029789)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1985: ¥700,000 (Direct Cost: ¥700,000)
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| Research Abstract |
African trypanosomes evade immunological attack in their mammalian host changing their antigen type or serotype. This antigen can be seen as an electron dense structure of 10-15 nm thickness surrounding outer space of plasma membrane of the parasite. The structure is called surface coat and the variant specific antigen is, substantially, uniform glycoprotein. Trypanosome population of different antigen types appear as an undulating parasitemia in loosely ordered manner in an experimentally infected rabbit. In this project, antigenic variation of Trypanosoma brucei gambiense (Tbg) was studied. In the first place, the variation phenomenon was confirmed analysing antigen types which relapsed in temporarily cured mice and number of antibodies which could benoticed in Tbg infected rabbit serum samples according to days after infection. Meanwhile, monoclonal antibodies specific to four different antigen type, 0, 9-1, 9-2 and 11-1. Each of them could agglutinate trypanosomes of homologous ant
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igen type. In the next stage, it was attempted to analyse the mode of antigenic variation in vivo by treating cloned Tbg infected mice with homologous monoclonal antibody and it was realized that even cloned trypanosome population bore trypanosomes of different antigen type(s) in ratio of 10^<-4>-10^<-5>. In the third, the analysis in vitro was planned introducing monoclonal antibody to Tbg culture System. For that purpose, improvement of Tbg culture was investigated. Feeder layer cells from new born mouse brain and muscle gave a stationary growth of Tbg in Eagle's MEM supplemented with fetal bovine serum (5%) and new born bovine serum (5%). Those feeder cells,nevertheless, had ability of supporting Tbg growth in only primary culture. Another problem was difficulty of collecting Tbg because they mainly increased interstitially in foci. To make the system simple and avoid using different population of feeder cell, the culture method without feeder cells found indispensable. After many trials, a better culture method was established as follows. A basal culture medium consisted of Eagle's MEM and Leibovit's L-15 medium. Tbg was cultured in a final culture medium prepared with the basal medium in addition of 5x10^<-4>M EDTA, 10^<-4> M L-cystein,10^<-3> M sodium pyruvate, 3x10^<-6> M 2-mercaptoethanol and fetal bovine serum, 5%. This culture system could be initiated with even cryo-preserved Tbg, and very useful for various experiments. Less
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