title: Cytopathic effect of Acanthamoeba on human corneal fibroblasts.
abstract: OBJECTIVE
  Acanthamoeba keratitis is associated with keratocyte depletion in humans. We investigated how Acanthamoebae isolated from corneas affected by Acanthamoeba keratitis interacted with human corneal stromal cells in vitro.
  
  
  METHODS
  Acanthamoebae were isolated from 6 patients with Acanthamoeba keratitis and genotyping was done. Whether the isolated Acanthamoebae could invade the corneal stroma was assessed with denuded corneal stroma ex vivo. The cytopathic effect of Acanthamoeba on cultured corneal fibroblasts from donor corneas was quantitatively evaluated by the MTT assay after culture under various conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Annexin V staining were employed to detect apoptotic cells among the corneal fibroblasts co-cultured with Acanthamoebae.
  
  
  RESULTS
  All 6 Acanthamoebae isolated from the patients with Acanthamoeba keratitis were shown to have the T4 genotype by 18S rDNA sequence analysis. Acanthamoebae invaded the denuded corneal stroma in the ex vivo experiments and had a cytopathic effect on human corneal fibroblasts after direct adhesion, but not via chemical mediators. A cytopathic effect was detected with all 6 Acanthamoebae and corneal fibroblasts mainly died by apoptosis, as evidenced by Annexin V staining.
  
  
  CONCLUSIONS
  Acanthamoebae isolated from patients with Acanthamoeba keratitis had a cytopathic effect on human corneal fibroblasts, mainly via induction of apoptosis after direct adhesion. Our findings may provide some clues to the pathophysiology of corneal keratocyte depletion in patients with Acanthamoeba keratitis.
authors.affiliation: Department of Ophthalmology, Tokyo Women's Medical University Medical Center East, Tokyo Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Takaoka-Sugihara
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamagami
authors.forename: Seiichi
authors.initials: S
authors.lastname: Yokoo
authors.forename: Masao
authors.initials: M
authors.lastname: Matsubara
authors.forename: Kenji
authors.initials: K
authors.lastname: Yagita
mesh_terms: D000048:Acanthamoeba / Q000302:isolation & purification / Q000472:pathogenicity* / Q000502:physiology; D015823:Acanthamoeba Keratitis / Q000469:parasitology*; D000293:Adolescent; D000328:Adult; D017304:Annexin A5; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000469:parasitology* / Q000473:pathology; D005260:Female; D005347:Fibroblasts / Q000469:parasitology* / Q000473:pathology; D005838:Genotype; D006790:Host-Parasite Interactions; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D008875:Middle Aged; D058889:Molecular Typing; D016053:RNA, Protozoan / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000032:analysis*
chemical_list: D017304:Annexin A5; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S