Cyclosporine (CsA) is a novel immunosuppressive agent which appears to affect predoninantly T-lymphocyte dependent responses. Despite its increasing clinical usage, the specific mechanism of action of CsA is unknown. The availability of a dansylated derivative of CsA, which retains its native immunosuppressive activity but which is fluorescent, offers new and unique approaches to determine the mechanism of action of CsA. In preliminary studies, using the fluorescent-activated cell sorter (FACS), both peripheral blood T-lymphocytes, and T-cell clones have been shown to have differential staining with dansylated CsA derivatives. T-cell clones will be subjected to functional analysis to determine if diffential staining may represent functionally different cells among the OKT4 and OKT8 subsets of lymphocytes. T-cell clones will also be analyzed for Cyclosporine sensitivity resistance and correlataed with staining characteristics. Competitive inhibition studies will be performed between the dansylated Cyclosporine derivatives, and other both active and inactive derivatives of Cyclosporine. Competetive inhibition studies will also be done between the dansylated probes and reagents with known or purported receptors on T-lymphocytes including the OK monoclonal antibodies, PHA, PMA, ConA, antigen and HLA antibodies. From these competetive inhibition studies binding specificity of Cyclosporine as well as binding coefficients and dissociation constants will be derived. It may indicate if a unique receptor exists on the T-lymphocyte cell membrane for Cyclosporine. The availability of populations of T-cell clones in our laboratory, derived from both the OKT4 and OKT8 pupulations, which show differential staining characteristics with the dansylated derivatives of CsA will allow for examination of a homogeneous pupulation of T-cells. In addition, fluorescent microscopic techniques will be utilized as an adjunct to the above studies as well as a tool to further examine the significance of differential membrane and cytoplasmic binding of CsA among T-lymphocyte subsets.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08CA000958-02
Application #
3079416
Study Section
(SRC)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Colombani, P M; Bright, E C; Wells, M et al. (1989) Drug-drug interaction between cyclosporine and agents affecting calcium-dependent lymphocyte proliferation. Transplant Proc 21:840-1
Scheibel, L W; Colombani, P M; Hess, A D et al. (1989) Calcium/calmodulin functions in P. falciparum in vitro--implications for antiprotozoal drug design. Prog Clin Parasitol 1:21-56
Colombani, P M; Bright, E C; Hess, A D (1988) Comparison of binding to peripheral blood lymphocytes between active and inactive derivatives of cyclosporine. Transplant Proc 20:46-50
Colombani, P M; Bright, E C; Monastyrskyj, O et al. (1987) Subcellular action of cyclosporine: interaction between CsA and calmodulin antagonists. Transplant Proc 19:1171-4
Hess, A D; Colombani, P M (1987) Mechanism of action of cyclosporine: a unifying hypothesis. Adv Exp Med Biol 213:309-30
Scheibel, L W; Colombani, P M; Hess, A D et al. (1987) Calcium and calmodulin antagonists inhibit human malaria parasites (Plasmodium falciparum): implications for drug design. Proc Natl Acad Sci U S A 84:7310-4
Hess, A D; Colombani, P N (1987) Cyclosporin-resistant and -sensitive T-lymphocyte subsets. Ann Inst Pasteur Immunol 138:606-11
Colombani, P M; Robb, A; Hess, A D (1985) Cyclosporin A binding to calmodulin: a possible site of action on T lymphocytes. Science 228:337-9