Search

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., Competing Interests: The authors have declared that no competing interests exist.

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.

Purpose: We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A(2α) (cPLA(2α)) pathway., Methods: HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 μg/mL for 6, 12, and 24 hours. The effects of cPLA(2α) inhibitors on cPLA(2α), arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA(2α) involved preincubating HCE cells for 1 hour with cPLA(2α) inhibitors (10 μM methyl-arachidonyl fluorophosphonate [MAFP] or 20 μM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA(2α) mRNA and enzyme was examined by RT-PCR and cPLA(2) activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1β, and IFN-γ was examined by RT-PCR and ELISA., Results: MIP-133 induced significant cPLA(2α) (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA(2α) inhibitors significantly reduced cPLA(2α) (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA(2α) inhibitors significantly inhibited MIP-133-induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA(2α) enzyme activity in HCE cells, which is blocked by preincubation with anti-MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1β, and IFN-γ production, approximately two to three times (P < 0.05)., Conclusions: MIP-133 interacts with phospholipids on plasma membrane of HCE cells and activates cPLA(2α). cPLA(2α) is involved in apoptosis, AA release, and activation of proinflammatory cytokines/chemokines from HCE cells. cPLA(2α) inhibitors may be a therapeutic target in 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) -β. 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-β. 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., (© 2010 Blackwell Publishing Ltd.)

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.

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.

Purpose: Acanthamoebae provoke a vision-threatening corneal infection known as Acanthamoeba keratitis (AK). It is thought that Acanthamoeba-specific IgA antibodies present in mucosal secretions such as human tears, milk, and saliva provide protection against infection by inhibiting the adhesion of parasites to host cells. The goal of the present study was to determine whether human mucosal secretions have the potential to provide protection against the Acanthamoeba-induced cytopathic effect (CPE) by an additional mechanism that is independent of IgA., Methods: Breast milk was used as a model of human mucosal secretions. In vitro CPE assays were used to examine the CPE inhibitory effect of IgA-depleted milk and various milk fractions obtained by gel filtration. The activity of amebic proteinases was examined by zymography., Results: IgA-depleted milk inhibited the Acanthamoeba-induced CPE in a concentration-dependent manner. Milk proteins were separated into four major fractions (F1-F4) by gel filtration. Of these four fractions, CPE inhibitory activity was detected largely in fraction F3. In contrast, fractions F1, F2, and F4 lacked CPE inhibitory activity. Moreover, fraction F3, but not F1, F2, or F4, inhibited amebic proteinases., Conclusions: These data, in conjunction with published findings showing that amebic proteinases are responsible for the induction of Acanthamoeba CPE, led us to propose that human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA and that involves the inhibition of cytotoxic proteinases of amebae.

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.

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.

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.

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 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., (Copyright 2004 SGM)

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.

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.

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.

Acanthamoeba are free-living, harmless organisms, however, given the opportunity and the appropriate conditions, they can cause painful, sight-threatening as well as fatal infections and, thus, are considered opportunistic pathogens. Acanthamoeba infections have become increasingly important in the past few years due to increasing populations of contact lens users and AIDS patients. The mechanisms associated with the pathogenesis of Acanthamoeba tend to be highly complex, depending on parasite, host and the environmental factors. Elucidation of the biochemical, cellular and molecular basis of the pathogenesis of diseases caused by Acanthamoeba may lead to the development of therapeutic interventions.

Acanthamoeba keratitis is sight threatening corneal infection caused by pathogenic Acanthamoeba. Previous studies have shown the genotypic differences between pathogenic and non-pathogenic species/strains of Acanthamoeba. In this study, we examined the morphological differences between pathogenic and non-pathogenic species/strains using scanning electron microscopy. Pathogenic Acanthamoeba exhibited higher number of acanthopodia (structures associated with the binding of amoeba to the target cells) as compared to non-pathogens. In addition, interactions of amoeba with the corneal epithelial cells were studied. Only pathogenic amoeba exhibited adhesion to epithelial cells. Further results indicated that phagocytosis occurs in the pathogenic amoeba by the formation of amoebastome (characteristic of amoeba phagocyte). This study showed that Acanthamoeba phagocytosis may be both an efficient means of obtaining nutrients for the amoeba and a significant factor in the pathogenesis of Acanthamoeba infections.

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.

Acanthamoeba keratitis is a vision-threatening infection caused by pathogenic species of the genus Acanthamoeba. Although not all Acanthamoeba spp. can cause keratitis, it is important to differentiate pathogenic species and isolates from nonpathogens. Since extracellular proteases may play a role in ocular pathology, we used colorimetric, cytopathic, and zymographic assays to assess extracellular protease activity in pathogenic and nonpathogenic Acanthamoeba. Colorimetric assays, using azo-linked protein as a substrate, showed extracellular protease activity in Acanthamoeba-conditioned medium and differentiated pathogenic and nonpathogenic Acanthamoeba. Monolayers of immortalized corneal epithelial cells in four-well plates were used for cytopathic effect (CPE) assays. Pathogenic Acanthamoeba isolates exhibited marked CPE on immortalized corneal epithelial cells, while nonpathogenic isolates did not exhibit CPE. Protease zymography was performed with Acanthamoeba-conditioned medium as well as with Acanthamoeba- plus epithelial-cell-conditioned medium. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. In pathogenic Acanthamoeba isolates, all protease bands were inhibited by phenylmethylsulfonyl fluoride (PMSF), suggesting serine type proteases, while in nonpathogenic strains only partial inhibition was observed by using PMSF. The pathogenic Acanthamoeba strains grown under typical laboratory conditions without epithelial cells exhibited one overexpressed protease band of 107 kDa in common; this protease was not observed in nonpathogenic Acanthamoeba strains. The 107-kDa protease exhibited activity over a pH range of 5 to 9.5.

Acanthamoeba keratitis is a rare, yet sight-threatening corneal infection. Ocular infection does not appear to induce protective immunity as repeated corneal infections occur in both humans and experimental animals. However, we have recently demonstrated that activation of the common mucosal immune system by oral immunization with Acanthamoeba antigens protects both Chinese hamsters and pigs against ocular infection with A. castellanii. Protection correlates closely with the appearance of anti- Acanthamoeba antibodies in the tears. To test the hypothesis that oral immunization induces specific protective IgA antibodies, two monoclonal IgA antibodies specific for Acanthamoeba antigens were generated. Both antibodies detected epitopes on the surface of fixed Acanthamoeba trophozoites. When delivered intraperitoneally, one monoclonal antibody (14E4) was detected in stool and tear samples. This clone also protected naive animals against ocular challenge with Acanthamoeba trophozoites (43% infection rate compared to a 91% infection rate in animals receiving control IgA). In vitro functional studies showed that neither antibody induced encystment or directly killed Acanthamoeba trophozoites. However, both monoclonal anti- Acanthamoeba IgA antibodies produced a three-fold inhibition in the adherence of trophozoites to corneal epithelial cells in vitro. These data show that monoclonal anti- Acanthamoeba IgA antibodies can protect against Acanthamoeba keratitis and suggest that this occurs by inhibiting adhesion of the parasite to the corneal epithelium., (Copyright 1999 Academic Press.)

Purpose: To describe a cytologic technique for the rapid identification of presumed Acanthamoeba organisms from corneal epithelial scrapings., Methods: After administering topical anesthesia, we removed the affected corneal epithelium with a scalpel blade. The tip of the blade, containing the scrapings, was washed off into a cuvette with a solution of an alcohol-based fixative for cytology specimens. The blade was immersed in the cuvette and agitated to ensure that the sample was collected. The specimen was fixed for at least 10 minutes and processed by cytospin centrifugation., Result: Seventy-five patient samples have been studied with this technique, with excellent preservation of the organism., Conclusions: The organism preservation with this technique is superior to that of conventional smears and permits confirmatory organism identification by immunohistochemistry.

Purpose: To determine whether oral immunization mitigates ongoing Acanthamoeba castellanii corneal infections in pigs., Methods: Pigs were orally immunized with aqueous Acanthamoeba antigen mixed with cholera toxin (Ac-CT) or with saline, before or after ocular infection with A. castellanii. Mucosal secretions (i.e., tears and enteric wash) were tested for Acanthamoeba-specific IgA by enzyme-linked immunosorbent assay. Enteric washes were used as a source of IgA in assays measuring the binding of trophozoites to Chinese hamster corneal epithelial (CHCE) cells., Results: Pigs immunized with Ac-CT before ocular challenge with A. castellanii had significant anti-Acanthamoeba IgA antibody titers in their tears and enteric washes and were protected against ocular infection. Enteric washes from orally immunized pigs inhibited trophozoite binding to CHCE cells in vitro by more than 75%. By contrast, pigs immunized after corneal infections had been established displayed keratitis of the same severity and duration as that in control pigs. However, 80% of the orally immunized animals were resistant to rechallenge with Acanthamoeba-laden contact lenses, whereas none of the control animals was resistant., Conclusions: Oral immunization with Ac-CT protects against Acanthamoeba keratitis when administered before corneal challenge. However, delaying oral immunization until after corneal disease is established fails to mitigate keratitis. The appearance of parasite-specific tear IgA correlates with protection and may act by preventing the parasite's binding to the corneal epithelium.

Purpose: Acanthamoeba keratitis is a sight-threatening corneal infection. It is known that: (i) more amoebae bind to the surface of injured corneas than to the normal corneal surface and (ii) mannose-containing glycoproteins (GPs) possess binding sites for Acanthamoeba. The present study was undertaken to determine whether subtle corneal surface injury exposes mannose-GPs and whether more amoebae bind to the mannose-GPs of injured corneas than to those of normal corneas., Methods: Corneal cup assays were developed to determine whether corneal surface injury exposes binding sites for a mannose/glucose-specific lectin, succinylated-concanavalin A (s-ConA). To determine whether injury exposes mannose-GPs, corneal surface proteins were biotinylated, biotin-labeled mannose-GPs were allowed to bind to s-ConA-agarose beads and were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Amoeba binding to mannose-GPs of corneal epithelia was analyzed by PAGE-blot overlay assays., Results: S-ConA binding site density was 2.4 times greater on the injured corneal surface than on the surface of normal corneas. Based on the analysis of the s-ConA-bound, biotin-labeled corneal surface proteins, approximately 5.2 times greater amounts of mannose-GPs were present on the surface of injured corneas than on the normal corneal surface. PAGE-blot overlay assays of s-ConA bound GPs of unlabeled corneal epithelia revealed that, on a per mg total cell protein basis, injured corneal epithelium contained 1.8 times greater amounts of Acanthamoeba-reactive mannose-GPs than normal corneal epithelium., Conclusions: Subtle corneal injury exposes mannose-GPs on the surface of injured corneas. The newly exposed GPs may serve to provide additional attachment sites for the amoebae. This, in turn, could render the cornea susceptible to the infection.

Purpose: Acanthamoeba is an uncommon cause of corneal infection in which the best visual outcome follows prompt diagnosis and a long course of appropriate antimicrobial therapy. Because conventional detection techniques for Acanthamoeba have certain limitations, we investigated the ability of the polymerase chain reaction (PCR) to confirm the clinical diagnosis of Acanthamoeba keratitis, with the ultimate aim of achieving early diagnosis., Methods: Using two different pairs of primers, PCR was performed on representative cultured Acanthamoeba isolates to confirm the assay's ability to amplify Acanthamoeba DNA from a wide range of acanthamoebae. Subsequently, corneal epithelial samples from 19 patients and tear samples from 12 patients with Acanthamoeba keratitis were analyzed by PCR for the presence of Acanthamoeba DNA., Results: Acanthamoeba DNA was amplified by PCR from 16 (84%) of 19 corneal epithelial samples, whereas Acanthamoeba was cultured from 10 samples (53%), all of which were PCR positive. Tear samples from 8 (66%) of 12 patients were positive on PCR testing, and one tear sample was PCR positive, whereas the corresponding epithelial biopsy had yielded a negative PCR result. Samples from culture-positive patients were positive on PCR testing more frequently than those from culture-negative patients (10/10 culture-positive corneal epithelial and 5/7 [71%] culture-positive initial tear samples versus 6/9 [66%] culture-negative corneal epithelial and 2/5 [40%] culture-negative tear samples). All control epithelial (n = 15) and tear (n = 15) samples yielded negative results., Conclusions: PCR was a more sensitive diagnostic test than a culture for Acanthamoeba keratitis, and the use of two different primers achieved better sensitivity than a single set. A PCR of a tear sample also may be a useful complementary test and, in combination with PCR of epithelial samples, would prove particularly helpful in confirming the clinical diagnosis in culture-negative cases.

Acanthamoeba keratitis is a chronic inflammatory disease of the cornea which is highly resistant to many antimicrobial agents. The pathogenic mechanisms of this disease are poorly understood. However, it is believed that the initial phases in the pathogenesis of Acanthamoeba keratitis involve parasite binding and lysis of the corneal epithelium. These processes were examined in vitro, using Acanthamoeba castellanii trophozoites. Parasites readily adhered to Chinese hamster corneal epithelial cells in vitro; however, parasite binding was strongly inhibited by mannose but not by lactose. Although mannose prevented trophozoite binding, it did not affect cytolysis of corneal epithelial cells. Moreover, mannose treatment induced trophozoites to release cytolytic factors that lysed corneal epithelial cells in vitro. These factors were uniquely induced by mannose because supernatants collected from either untreated trophozoites or trophozoites treated with other sugars failed to lyse corneal cells. The soluble factors were size fractionated in centrifugal concentrators and found to be > or = 100 kDa. Treatment of the supernatants with the serine protease inhibitor phenylmethylsulfonyl fluoride inhibited most, but not all, of the cytopathic activity. These data suggest that the binding of Acanthamoeba to mannosylated proteins on the corneal epithelium may exacerbate the pathogenic cascade by initiating the release of cytolytic factors.