Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

Potential Value of Cellulose Synthesis Inhibitors Combined With PHMB in the Treatment of Acanthamoeba Keratitis.

PURPOSE: The aim of this study was to improve the cytopathic effect (CPE) of antiamebic agents by combining with cellulose synthesis inhibitor as an encystation inhibitor. METHODS: Cellulose synthesis inhibitors, 2,6-dichlorobenzonitrile (DCB) and isoxaben were used to block encystation of Acanthamoeba during cultivation. Cultured human corneal epithelial (HCE) cells and Acanthamoeba were treated with polyhexamethylene biguanide (PHMB) combined with cellulose synthesis inhibitors to evaluate the CPE as an antiamebic agent. RESULTS: 0.02% PHMB showed a 51.9% CPE on HCE cells within 30 minutes but exhibited significant toxic effects on Acanthamoeba. At a level of 0.00125%, PHMB had no significant CPEs on HCE cells, whereas 100 µM DCB and 10 µM isoxaben significantly inhibited the formation of the inner cyst wall of Acanthamoeba during encystation, and Acanthamoeba trophozoites failed to convert into mature cysts. Although a low concentration (0.00125%) of PHMB was used, the novel combinations with 100 µM DCB or 10 µM isoxaben had 23.4% or 18.7% additional amebicidal effects on Acanthamoeba. However, 100 µM DCB and 10 µM isoxaben had no CPEs on HCE cells. CONCLUSIONS: The combination of cellulose synthesis inhibitors with low concentrations of PHMB reduced the CPE on HCE cells and improved the amebicidal effect on Acanthamoeba by inhibition of encystation.

Toll-like receptor 4 signalling pathway activation in a rat model of Acanthamoeba Keratitis.

The pathogenesis of Acanthamoeba keratitis (AK) is complicated. In our previous studies, TLR4 was found involved in the process of infection by Acanthamoeba in human corneal cells. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signalling pathway in Wistar rats challenged with Acanthamoeba. The rat model of AK was established. Corneas were collected and analysed by real-time PCR to assess the mRNA levels of TLR 2, 4, myeloid differentiation protein (MyD)88, nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), interleukin (IL)-8, tumour necrosis factor (TNF)-α and interferon (IFN) -ß. Immunocytochemistry and Western blot were conducted to examine the proteins of TLR2, TLR4, p-Erk1/2 and p-IκB. Specific inhibitors PDTC and U0126 were used to pretreat the animals to determine the exact receptor and signalling pathway involved in pathogenesis. Expressions of TLR4, MyD88, all three cytokines, NF-κB, p-IκB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-ß. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-κB and TLR4-Erk1/2 pathways in corneas of Wistar rats.

Effects of multipurpose contact lens care solutions on the adhesiveness of Acanthamoeba to corneal epithelial cells.

PURPOSE: To study the adhesion of Acanthamoeba castellanii treated with multipurpose contact lens care solutions (MPSs) to human corneal epithelial cells. METHODS: Cell suspensions of A. castellanii (ATCC50514) trophozoites were mixed with six MPSs: MPS-A (polyhexamethylene biguanide [PHMB], macrogolglycerol hydroxystearate, propylene glycol), MPS-B (PHMB, poloxamine, boric acid), MPS-C (polyquad, poloxamine, boric acid), MPS-D (PHMB, poloxamer, propylene glycol), MPS-E (PHMB, poloxamer), or MPS-F (PHMB, poloxamer) for 4 hr. Morphologic changes of A. castellanii after exposure with MPSs were observed with scanning electron microscopy. A. castellanii cells treated with MPS for 4 hr were inoculated onto cultured SV40-immortalized human corneal epithelial cells. After 2-hr incubation, the number of adherent A. castellanii was assessed by 18S-rDNA quantification using real-time polymer chain reaction. RESULTS: After 4-hr incubation, MPS-A- and MPS-B-treated A. castellanii have changed from trophozoite morphology into cyst form; however, MPS-E- and MPS-F-treated A. castellanii maintained trophozoite morphology. In contrast, both cyst and trophozoite forms were observed in MPS-C- and MPS-D-treated A. castellanii. The adherence rate of A. castellanii was approximately two times higher in MPS-E (not significant), and more than three times higher in MPS-F (P<0.05) compared with MPS-A, which produced the lowest adhesiveness as estimated by real-time polymer chain reaction. CONCLUSIONS: Taken together, these results support the possibility that chronic use of MPS with the lowest efficacies on promoting encystment of A. castellanii (MPS-E and MPS-F) by hydrogel contact lens wearers may increase adhesiveness of A. castellanii to corneal epithelial cells.

In vitro amoebacidal activity of photodynamic therapy on Acanthamoeba.

AIM: To evaluate the effectiveness of Hypocrellins B (HB) photodynamic therapy (PDT) on Acanthamoeba trophozoites and cysts, and its cytotoxic potential on the corneal epithelial cells and stromal cells in vitro. METHODS: Acanthamoeba trophozoites and cysts (5 x 10(4)/ml) were incubated with various concentrations of HB, followed by exposure to light (>470 nm) for 30 min at a fluence rate of 50 mW/cm(2). Survival rate was assessed using the live/dead viability/cytotoxicity assay and non-nutrient agar--Escherichia coli culture assay. Rabbit corneal epithelial cells and stromal cells were incubated with various concentrations of HB, followed by exposure to the same light fluence rate as above. The corneal cell survival rate was assessed using the live/dead viability/cytotoxicity assay. RESULTS: HB-PDT showed a dose-dependent inhibition on the trophozoites and cysts. The 100% inhibitory concentration (IC(100)) of HB was 1 microg/ml for the trophozoites and 20 microg/ml for the cysts. The 50% inhibitory concentration (IC(50)) of HB for the cysts was 3.8 microg/ml and for the trophozoites 0.23 microg/ml. The inhibition of the HB-PDT on the rabbit corneal cells and stromal cells was observed, but HB without irradiation had no inhibition on the corneal cells. CONCLUSION: HB-PDT had amoebacidal properties in vitro and showed a better effectiveness on the trophozoites than the cysts. Meanwhile, HB-PDT demonstrated cytotoxicity on the corneal epithelial cells and stromal cells, but HB exhibited no appreciable cytotoxicity in the dark to the corneal cells.

In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis.

OBJECTIVE: This study included in vivo and ex vivo investigations of patients with early-stage Acanthamoeba keratitis by using new-generation laser confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module [HRT 2-RCM]). METHODS: Three patients (2 men and 1 woman; mean age, 22.0 years) with early-stage Acanthamoeba keratitis diagnosed by direct examination (Parker ink-potassium hydroxide stain), culture from corneal epithelial scrapings, or both methods were enrolled in this study. All patients were examined by slit-lamp biomicroscopy. The area of the affected cornea was examined by HRT 2-RCM. Selected images of in vivo corneal layers and ex vivo cultured microorganisms were evaluated qualitatively for shape and degree of light reflection of the corneal structural changes or Acanthamoeba cysts. In addition, cultured Acanthamoeba were examined ex vivo by HRT 2-RCM. RESULTS: In vivo laser confocal microscopy showed highly reflective round-shaped, high-contrast Acanthamoeba cysts (10-20 microm in diameter) in the corneal epithelium in all cases, leading to rapid confirmation of the clinical diagnosis. In all culture samples of Acanthamoeba, ex vivo laser confocal microscopy showed highly reflective round- or stellate-shaped high-contrast particles (10-20 microm in diameter). CONCLUSIONS: In vivo laser confocal microscopy enables rapid and noninvasive diagnosis of early-stage Acanthamoeba keratitis with high resolution. In addition, ex vivo laser confocal images of Acanthamoeba cysts may be helpful when similar structures are identified and have to be interpreted under in vivo conditions.

Acanthamoeba: keratopathogenicity of isolates from domestic tap water in Korea.

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25microg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69microg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.

[Diagnosis of Acanthamoeba corneal infection by impression cytology: case report]. / Citologia de impressão no diagnóstico de infecção corneana por Acanthamoeba: relato de caso.

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.

Intracorneal instillation of latex beads induces macrophage-dependent protection against Acanthamoeba keratitis.

PURPOSE: Instillation of sterile 1.0 microM latex beads into the central corneal epithelium renders Chinese hamsters resistant to corneal infection with Acanthamoeba castellanii. By contrast, activation of the adaptive immune response by subcutaneous immunization with A. castellanii antigens fails to protect against Acanthamoeba keratitis. This study was undertaken to examine the mechanisms that mediate latex bead-induced resistance to Acanthamoeba keratitis. METHODS: In vitro experiments examined the effect of latex bead treatment on the capacity of A. castellanii trophozoites to adhere to and kill corneal epithelial cells. In vivo administration of antineutrophil antiserum was used to evaluate the role of neutrophils in latex-bead-induced protection against Acanthamoeba keratitis. Liposomes containing the macrophagicidal drug clodronate were used to deplete conjunctival macrophages and determine the role of macrophages in the latex-bead-induced resistance. RESULTS: Latex bead treatment did not affect adherence of trophozoites to the corneal epithelium or protect corneal epithelial or stromal cells from trophozoite-mediated cytolysis in vitro. Neutrophil depletion did not abrogate the latex beads' protective effect. Latex bead treatment induced a significant infiltration of macrophages into the corneas that peaked at day 4 of infection. Moreover, depletion of conjunctival macrophages with the macrophagicidal drug clodronate eliminated the latex beads' protective effect. CONCLUSIONS: The results indicate that intracorneal injection of latex beads induces a remarkable resistance to Acanthamoeba keratitis that is largely, if not entirely, mediated by macrophages. These results underscore the importance of the innate immune apparatus in the resistance to Acanthamoeba keratitis.

In vitro pathogenicity of Acanthamoeba is associated with the expression of the mannose-binding protein.

PURPOSE: To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro. METHODS: Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses. RESULTS: Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP. CONCLUSIONS: The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.

Heidelberg retina tomograph II findings of Acanthamoeba keratitis.

PURPOSE: Heidelberg retina tomograph II (HRTII) examination was performed with cornea module in one patient with Acanthamoeba keratitis (AK) to provide images detailing characteristic findings of the disease. METHODS: A 34-year-old woman presented with clinical signs and symptoms of AK. HRTII with cornea module was performed and the patient underwent laboratory investigations. RESULTS: HRTII examination with cornea module revealed numerous 20-26-micro m diameter high-contrast round particles within the corneal epithelium and anterior stroma, resembling Acanthamoeba cysts. Stellate cells as well as ovoid irregular objects, possibly inflammatory cells, trophozoites, altered cysts, or activated keratocytes, were also present in the area of stromal infiltrates. Laboratory investigations confirmed the diagnosis of AK. CONCLUSIONS: HRTII cornea module can be helpful in the diagnosis of AK by identifying acanthamoeba cyst-like structures in the cornea. This technique also has potential uses in monitoring the efficiency of anti-infective treatment.

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells.

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba.

Acanthamoeba are opportunistic protozoan parasites that can cause fatal granulomatous amoebic encephalitis and eye keratitis, however the pathogenic mechanisms of Acanthamoeba remain unclear. In this study, we described the ability of live Acanthamoeba to hydrolyse extracellular ATP. Both clinical and non-clinical isolates belonging to genotypes, T1, T2, T3, T4 and T7 exhibited ecto-ATPase activities in vitro. Using non-denaturing polyacrylamide gel electrophoresis, ecto-ATPases were further characterized. All Acanthamoeba isolates tested, exhibited a single ecto-ATPase band (approximate molecular weight of 272 kDa). However, clinical isolates exhibited additional bands suggesting that ecto-ATPases may play a role in the pathogenesis of Acanthamoeba. This was supported using suramin (ecto-ATPase inhibitor), which inhibited Acanthamoeba-induced host cell cytotoxicity. Previously, we and others have shown that Acanthamoeba binds to host cells using their mannose-binding protein and binding can be blocked using exogenous alpha-mannose. In this study, we observed that alpha-mannose significantly increased ecto-ATPase activities of pathogenic Acanthamoeba belonging to T1, T2, T3 and T4 genotypes but had no effect on non-pathogenic Acanthamoeba (belonging to T7 genotype). Overall, we have shown, for the first time, that Acanthamoeba exhibit ecto-ATPase activities, which may play a role in the pathogenesis of Acanthamoeba as well as their potential role in the differentiation of pathogenic Acanthamoeba.

Acanthamoeba induces cell-cycle arrest in host cells.

Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

Induction of morphological and electrophysiological changes in hamster cornea after in vitro interaction with trophozoites of Acanthamoeba spp.

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.

Impression cytology in the diagnosis of acanthamoeba keratitis with surface involvement.

PURPOSE: To evaluate the use of impression cytology in the diagnosis of Acanthamoeba keratitis with surface involvement. DESIGN: Observational case series. METHODS: Three patients with culture-proven superficial Acanthamoeba keratitis were studied. Patients' medical records were reviewed for clinical information, treatment procedures and disease course. Impression cytology specimens taken from the cornea of those patients were processed and observed by bright field microscopy. The main outcome measure was the cytopathological examination and identification of amoebic organisms. RESULTS: In each of these three patients, impression cytology specimens revealed the amoeba cysts. Some specimens also showed pleomorphic trophozoites with typical amoeba nuclei. CONCLUSION: Corneal impression cytology successfully detected the cysts and trophozoites of Acanthamoeba from patients with superficial amoeba keratitis. This indicates that impression cytology can be a useful technique to facilitate early diagnosis of Acanthamoeba keratitis with surface involvement.

Pathogenic Acanthamoeba induces apoptosis of human corneal epithelial cells.

PURPOSE: 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.

Effects of mannose on Acanthamoeba castellanii proliferation and cytolytic ability to corneal epithelial cells.

PURPOSE: Acanthamoeba trophozoites express a mannose binding receptor that facilitates adhesion of trophozoites to mannosylated proteins on corneal epithelial cells. This study was undertaken to determine the role that mannose stimulation has in the amoeba's growth, secreted products, and ability to desquamate the corneal epithelium. METHODS: Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose (PYG) and PYG with 100 mM methyl alpha-D-mannopyranoside or galactose. The proliferation of trophozoites and cysts was examined by optical density and direct counts. The molecular weight of the mannose-stimulated protein was examined by SDS/PAGE. The cytolytic protein was purified by fast protein liquid chromatography (FPLC) size exclusion and ionic exchange and then tested for cytopathic effect (CPE) and collagenolytic activity in vitro. Collagenolytic activity was examined by zymography. Proteases and protease inhibitors were used to characterize the nature of the cytolytic protein. RESULTS: Methyl alpha-D-mannopyranoside inhibited the growth of A. castellanii by 50% (P < 0.05) and concomitantly induced a threefold increase in the formation of cysts. SDS-PAGE analysis revealed a mannose-induced protein of approximately 133 kDa (MIP-133). The MIP-133 protein was found to be highly cytolytic against corneal epithelial cells, but not human intestinal epithelial cells and also degraded collagen in vitro. Serine protease inhibitors abrogated both CPE and collagenolytic activity of the MIP-133 protein (P < 0.001). CONCLUSIONS: The results suggest that binding of trophozoites to mannosylated proteins on the corneal surface induces A. castellanii to secrete a approximately 133-kDa serine protease that kills both human and hamster corneal epithelium and degrades collagen.