A) Conditioned medium (CM) of the AGE isolate exhibited HBMEC monolayer disruptions. Acanthamoeba (AGE isolate belonging to T1 genotype) or their CM were added to human brain microvascular endothelial cells (HBMEC) monolayers as described in "Materials and Methods". A) HBMEC monolayers were stained with hematoxylin to observe monolayer disruptions. HBMEC alone represent HBMEC without parasites and/or CM and were used as controls. Both Acanthamoeba as well as CM exhibited HBMEC monolayer disruptions. Note that PMSF blocked CM-mediated HBMEC monolayer disruptions. In contrast, metalloprotease inhibitors (GM 1489 and GM 6001) had no effects on CM-mediated HBMEC monolayer disruptions. B) Micrography of HBMEC cytopathic effects. Micrographs were taken using a phase-contrast microscope, ×200. C) CM of the AGE isolate did not produce HBMEC cytotoxicity. Note that Acanthamoeba (AGE isolate belonging to T1 genotype) exhibited more than 70% HBMEC cytotoxicity, while their CM induced less than 10% HBMEC cytotoxicity. Results are mean of three experiments performed in duplicates. Error bars represent standard error.