Detalles del proyecto
Description
The objective of the proposed research is to elucidate the
mechanism of cell killing by cytotoxic T lymphocytes (CTL), an
important phenomenon in cellular immunology. Experiments will
be performed on single cell basis, by using micropipette-
micromanipulation, video recording and computer analysis, to
determine the junction avidity of CTL and target cell (TC)
conjugates, the biophysical properties of these cells individually
and also during their interaction in the killing process, and the
changes in cell nuclei. Studies on junction avidity will be
performed with two micropipettes to hold separately a CTL and a
TC, thus allowing the control of the time of onset for conjugation.
These experiments will be performed by using appropriate TC
with or without antigen expression induced by gamma-interferon,
and non-appropriate target cells resulting from activation (ConA
or PHA) of CTL. The energy of interaction will be estimated
from the forces required to separate the two cells. The role of
cytoskeleton organization in affecting the biophysical properties
(viscosity and elasticity) of CTL and TC will be determined by
using fluorescent labeled monoclonal antibodies (mAbs). The
number and kinds of membrane molecules in the interfacial area
of CTL and TC conjugates will be determined by using fluorescent
and radioactive labeled mAbs. Together with the determination
of the force required to separate the CTL-TC conjugate and using
fluorescent labeling and inhibition of cytotoxicity with mAbs,
these experiments will allow the estimation of the conjugation
force per molecule in the CTL killing process. The membrane
molecules involved in the interdigitation, lethal hit programming,
or secretion in CTL-TC conjugation will be determined by labeling
mAbs with different fluorescent compounds. The effects of
biochemical modification (nonspecific) of TC membrane and
genetic modification of antigen expression on the TC by DNA-
mediated gene transfer (DNA-MGT) or exon shuffling (specific
modification) on the juntional avidity of CTL-TC conjugates and
the biophysical properties of CTL and TC will be assessed during
the killing process. Correlation of the results of these biophysical
studies with the immunochemical events studied by other
investigators will serve to increase our understanding of the
chemico-mechanical transduction process in cell killing by CTL.
Estado | Finalizado |
---|---|
Fecha de inicio/Fecha fin | 6/15/87 → 5/31/91 |
Keywords
- Biofísica
- Biología celular
Huella digital
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