RESUMO
The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.
Assuntos
Acanthamoeba , Amebíase , Epitélio Corneano , Peptídeo Hidrolases/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/enzimologia , Amebíase/patologia , Animais , Cricetinae , Cães , Epitélio Corneano/metabolismo , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Junções Intercelulares/metabolismo , Junções Intercelulares/parasitologia , Junções Intercelulares/ultraestrutura , Células Madin Darby de Rim Canino , Masculino , MesocricetusRESUMO
The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman s membrane in 3h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested.
Assuntos
Acanthamoeba castellanii/fisiologia , Córnea/parasitologia , Acanthamoeba castellanii/patogenicidade , Acanthamoeba castellanii/ultraestrutura , Técnicas de Cocultura , Lentes de Contato/parasitologia , Córnea/ultraestrutura , Meios de Cultivo Condicionados , Epitélio Corneano/parasitologia , Epitélio Corneano/ultraestrutura , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de VarreduraRESUMO
To describe three cases of corneal infection due to Acanthamoeba sp in which was possible to detect Acanthamoeba sp cysts by the corneal impression cytology technique. Three patients referred to the External Eye Disease Laboratory in 2004 with superficial corneal alterations were submitted to corneal specimen collection by impression cytology filter paper to investigate the presence of Acanthamoeba sp cysts. Two impression cytology samples were obtained from each patient and were stained by PAS, hematoxylin and Papanicolaou. Routine microbiological investigation and culture were also performed using corneal scraping. Positive culture and impression cytology for Acanthamoeba sp was observed in all patients while smears with Giemsa stain were positive in two. Impression cytology Acanthamoeba sp cysts were observed among sheets of corneal epithelial cells and as isolated cells. Cysts were also found in the superficial epithelium in one of these patients after treatment while corneal scraping did not reveal any cyst. Histopathology revealed cysts in the epithelium and stroma in a transplanted cornea in one of these patients. The first description of impression cytology as a diagnostic method for Acanthamoeba keratitis occurred recently. In this study corneal impression cytology detected Acanthamoeba sp cysts successfully in these patients with only superficial involvement. Impression cytology as a non invasive technique can be used to facilitate early recognition of Acanthamoeba infection playing a useful role in the follow-up of the disease.
Assuntos
Ceratite por Acanthamoeba/patologia , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/parasitologia , Animais , Lentes de Contato Hidrofílicas/efeitos adversos , Citodiagnóstico , Epitélio Corneano/parasitologia , Epitélio Corneano/patologia , Humanos , Coloração e RotulagemRESUMO
Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.