Acanthamoeba pathogenicity for corneal cells
Introduction
Acanthamoeba keratitis is painful and potentially sight threatening. The condition is also noted for its wide variety of clinical presentations.1., 2., 3., 4., 5. Indeed a recent confocal scanning study, confirmed by histology on epithelial scrapes, has suggested that amoebic keratitis may occur more commonly than currently recognised.6 This would imply that certain cases either resolve on non-specific therapy or are self-resolving. One possible explanation is that in some instances, perhaps due to less pathogenic organisms or in less favourable conditions, the organism fails to penetrate through the epithelium into the corneal stroma.
The association between Acanthamoeba keratitis and contact lens wear is well recognised. Microtrauma to the corneal epithelium is assumed to be an important factor in facilitating invasion of this small free-living amoeba. Indeed it has been demonstrated in vitro that injury increases exposure of mannose-glycoproteins which are known to be involved in binding of Acanthamoeba.7 It is also of note that no animal model has been developed which adequately mimics this process. Those that are available either involve direct inoculation of the organism into the stroma,8., 9., 10., 11., 12., 13. or prior abrasion of the epithelium.14., 15. Taken in conjunction, these factors suggest that the intact epithelium plays a very important role in preventing amoebic invasion.
It has been demonstrated that, for invasion to occur, the trophozoites must first bind to the corneal epithelial cells16 and then gaps appear in the cell junctions allowing the organism to penetrate, burrowing between cells.17 Initially the amoebae are presumably confined to this tissue but progressive stromal invasion occurs as the infection advances.18 Late disease is also associated with keratocyte depletion.18 It is therefore assumed that the amoebae are consuming these cells. Indeed it has been demonstrated that they are capable of utilising either keratocyte or corneal epithelial cells as a food source.19 The pathogenesis of this condition is, however, only incompletely understood.
In this paper we compare the relative susceptibility of keratocyte and corneal epithelial cells to the cytopathic effect produced by Acanthamoeba castellanii. Selected results are formally assessed by image analysis. The findings are discussed with regard to our understanding of the role of the epithelium with respect to the pathogenesis of the condition in vivo.
Section snippets
Cells
Human corneal epithelial cells and keratocytes were obtained by primary culture from corneal buttons removed at grafting from patients with keratoconus and from corneal buttons recovered from eyes enucleated for treatment of choroidal melanoma. Over a dozen different attempts were required to amass sufficient numbers of cells. This was mainly due to difficulties encountered with primary isolation of epithelial cells: they had more demanding growth requirements than keratocytes and were readily
Results
Results of the preliminary experiments measuring the effect of different concentrations of Acanthamoeba castellanii on epithelial cells and keratocytes are summarised in Table 1. A ‘cut-off point’ could be demonstrated for each of the mammalian cell types: for epithelial cells this was 105/ml/well while for keratocytes it was 104/ml/well, although the damage produced at these concentrations was slight. Concentrations of amoebae below these respective levels had no demonstrable effect. The
Discussion
Co-incubation of Acanthamoeba castellanii at a concentration of 106/ml or 105/ml with keratocytes (50 000/ml/well) led to progressive destruction of the majority of the ‘monolayer’ within 24 h. In both cases, however, destruction of the remaining cells occurred at a comparatively lesser rate. This may at least be partly due to the fact that the cells that survive longer tend to be relatively protected (i.e. physically more difficult to attack because of clumping). Interestingly, the reaction
Acknowledgements
This work was supported by grants from the Tennent Institute of Ophthalmology, the Ross Foundation, the Wallace Brown Memorial Trust and the Pocklington Trust. The author would like to thank J. Hay and G. Reid for instruction in amoebal and cell culture techniques respectively. The author is also grateful to W. Sehu for assistance with image analysis and illustrations and W.R. Lee for advice.
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Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis
2011, Clinical Microbiology and InfectionCitation Excerpt :Eventually, the pathophysiology of AK can result in corneal transplantation, blindness and eye enucleation [4,17]. The role of the corneal epithelium as an essential physical barrier in the prevention of amoebic infection has been described previously [18]. Most of the cytopathic factors involved in these infectious processes have been described as resulting from the activity of proteolytic enzymes secreted by trophozoites in the surface of host corneal tissue [4,5,7,19,20].
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