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Sammy H. Liu, John D. Gottsch; Apoptosis Induced by a Corneal-Endothelium–Derived Cytokine. Invest. Ophthalmol. Vis. Sci. 1999;40(13):3152-3159.
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purpose. The purpose of this study was to isolate and characterize cDNA clones
encoding target proteins for autoantibodies in patients at high risk
for transplant rejection.
methods. A pool of 10 sera from patients at high risk for rejection who had
undergone corneal transplantation was used for immunoscreening of an
endothelial cDNA library, and the cDNA fragments were subcloned into
prokaryotic expression vectors to generate recombinant fusion proteins.
Cytotoxicity of recombinant protein was determined by a modified 51Cr-release assay. Apoptosis induced by recombinant
protein was determined by fluorescent dye–chromatin fragmentation
assay and by TdT-dUTP terminal nick-end labeling (TUNEL) assay. An
enzyme-linked immunosorbent assay was used to detect the presence of
antibodies to recombinant protein in the sera of high-risk patients
undergoing corneal transplantation and of control subjects.
results. Screening of 500,000 plaques identified six positive clones, one of
which demonstrated extensive homology with a novel tumor-derived
cytokine termed endothelial monocyte–activating polypeptide (EMAP).
EMAP was synthesized as a 39-kDa precursor that was proteolytically
cleaved to generate an active 22-kDa cytokine. The mature peptide of
EMAP alone was capable of inducing the death of cultured endothelial
cells, whereas the propeptide was inactive. The protein synthesis
inhibitor cycloheximide potentiated EMAP-induced apoptosis in
endothelial cells. Cell death by apoptosis was evidenced by DNA
fragmentation, extensive surface bleb formation, and chromatin
condensation. A statistically significant difference was found in the
level of antibodies specific to EMAP between patients at high risk for
corneal transplant rejection and control subjects
(P < 0.001). The antibody levels were elevated in
patients with severe graft reaction when compared with patients with no
graft reaction (P < 0.001).
conclusions. These studies demonstrated that EMAP is a novel protein in corneal
endothelial cells that is capable of inducing programmed cell death.
Overexpression of this cytokine could initiate endothelial cell damage
leading to stromal edema and corneal
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