title: Pathogenic Acanthamoeba induces apoptosis of human corneal epithelial cells.
abstract: OBJECTIVE
  To determine if trophozoites or lysate of pathogenic Acanthamoeba induce apoptosis in cultured human corneal epithelial cells.
  
  
  METHODS
  SV40-immortalized human corneal epithelial (HCE-2) cells were co-incubated with trophozoites or lysate of pathogenic Acanthamoeba castellanii (ATCC 30868) in peptone-yeast-glucose (PYG) medium. Control cells were exposed to PYG only or extracts similarly prepared from fibroblasts. At 6, 12, and 18 h of incubation with trophozoites, HCE-2 cells were examined by light and scanning electron microscopy. Assessment of DNA fragmentation and flow cytometry were performed to detect apoptotic signals in HCE-2 cells treated with Acanthamoeba lysate.
  
  
  RESULTS
  Apoptosis was not detected at 6 h, but became detectable at 12 h and readily evident at 18 h. HCE-2 cells co-incubated with trophozoites displayed morphological features characteristic of apoptosis, including cell shrinkage, membrane blebbing, formation of apoptotic bodies, and nuclear condensation. Characteristic DNA laddering was seen in the DNA of HCE-2 cells exposed to Acanthamoeba lysate. Flow cytometry demonstrated that apoptosis induction was dose- and time-dependent; up to 60% of HCE-2 cells were apoptotic following exposure to parasite lysate for 18 h. In marked contrast, the control cells receiving mouse fibroblast lysate displayed normal morphology at all time points, and only 5% of cells exhibited DNA fragmentation after 18 h of incubation.
  
  
  CONCLUSIONS
  Acanthamoeba trophozoites or lysate can induce apoptosis of cultured human corneal epithelial cells as at least one mechanism of cell death.
authors.affiliation: Department of Ophthalmology, Ehime University School of Medicine, Shigenobu, Japan. xzheng@m.ehime-u.ac.jp
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zheng
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Uno
authors.forename: Tomoko
authors.initials: T
authors.lastname: Goto
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: James M
authors.initials: JM
authors.lastname: Hill
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Ohashi
mesh_terms: D000048:Acanthamoeba / Q000472:pathogenicity*; D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D018920:Coculture Techniques; D004247:DNA / Q000032:analysis; D004847:Epithelial Cells; D019573:Epithelium, Corneal / Q000469:parasitology / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D013997:Time Factors
chemical_list: D004247:DNA