Both freshly prepared and frozen-stored amniotic membrane cells express the complement inhibitor CD59.

Amniotic membrane proved to be very effective tool in the treatment of a number of ocular surface diseases. The amniotic membrane, however, has to be stored before its transplantation onto the ocular surface followed by mandatory serologic control in order to exclude the transmission of certain viruses. Therefore it is most important to study if cryopreservation of the membrane affects cell surface expression of the molecules. We measured cell surface expression of CD59, a membrane-bound complement inhibitor on the cells of freshly prepared and cryopreserved amniotic membrane. Cells of amniotic membrane were separated mechanically. Epithelial and mesenchymal cells were identified by the intracellular expression of nanog and the cell surface ICAM1 positivity, respectively. Multicolor flow cytometric immunophenotyping was used for determination of the CD59 expression. CellQuest-Pro software program (Becton Dickinson) was used both for measurements and analysis. CD59-positive cells could be detected in all investigated samples and in all investigated cell types, although the expression level of CD59 differed. CD59 was expressed both on freshly prepared and frozen-stored samples. Higher level of CD59 was detected on ICAM1+ mesenchymal cells than on nanog+ epithelial cells. Our findings indicate that amniotic membranes maintain their complement inhibiting capacity after cryopreservation.

P21, p27, bax, cathepsin and survivin pathways in macular dystrophy corneas.

The purpose of our study was to elucidate pathways of genetically programmed cell death (apoptosis) in corneas with macular dystrophy. 10 corneal buttons (10 patients) with macular dystrophy and 8 buttons (8 patients) from enucleated eyes with chorioideal melanoma (controls) were analysed histologically. Immunohistochemical analysis was performed to investigate the presence of p21, p27, bax, cathepsin and survivin proteins. The number of positive cells was determined by analysis of 100 cells and given in percentages. The bax protein was present in 25.6% of epithelial cells in macular dystrophy corneas but was absent in controls. P21 and p27 were found in 35.7 and 87.5% of epithelial cells of macular dystrophy corneas, respectively, but again not in control tissue. In contrast, a lower percentage of cathepsin-positive (30.7% vs 58.8%) and survivin-positive cells (37.6% vs 52.1%) were present in epithelial cells of macular dystrophy corneas than in control epithelial cells. The difference reached statistical significance in the expression of p21 and p27 genes (p<0.05 in both). P21 was positive in 3% of keratocytes, p27 in 1% of endothelial cells of macular dystrophy corneas but negative in controls (0%). Bax, cathepsin and survivin immunopositivity was not detected in keratocytes or endothelial cells of either group. We conclude that the down-regulation of p21, p27 and cathepsin in epithelial cells of macular dystrophy corneas may be related to defense mechanisms against apoptotic cell death.