Interference with the constitutive activation of ERK1 and ERK2 impairs EWS/FLI-1-dependent transformation.

The chimeric gene EWS/FLI-1, the hallmark of the Ewing's sarcoma and primitive neuroectodermal tumor family, encodes a fusion protein with enhanced transcriptional activation properties and preserved recognition of canonical ETS binding sites. Although EWS/FLI-1 alters the expression of various genes, the precise mechanism by which EWS/FLI-1 acts as an oncogene remains to be defined. In this study we report that members of the mitogen-activated protein kinase (MAPK) signaling pathway, ERK1 and ERK2, are constitutively activated in NIH 3T3 cells expressing EWS/FLI-1. Interference with ERK activation by either highly specific inhibitors of MEK1 or a dominant negative ras mutant profoundly impaired the ability of EWS/FLI-1 to transform NIH3T3 cells to growth in semi-solid medium. An EWS/FLI-1 mutant defective in DNA-binding and transcriptional activation failed to activate ERK and was also defective in 3T3 cell transformation. Constitutive ERK activation was also evident in several human Ewing's sarcoma tumor-derived cell lines. Interestingly, cells expressing the type II EWS/FLI-1 fusion, recently demonstrated more potent in transcriptional activation, showed even greater MAPK activation than cells expressing the more common type I fusion. These results implicate ERK activation in EWS/FLI-1 transformation and suggest that this signaling pathway may be important in the pathogenesis of Ewing's sarcoma. Oncogene (2000) 19, 4523 - 4530.

In vitro amoebicidal activity of propamidine and pentamidine isethionate against Acanthamoeba species and toxicity to corneal tissues.

Effective chemotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of the parasites to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the medical treatment of choice in Europe, we sought to determine whether pentamidine may be equally effective, because the drug is more readily available to ophthalmologists in the United States. In this study, we compared the amoebicidal activity of the Brolene (commercial product), propamidine isethionate and pentamidine isethionate (Pentam) in vitro against three different species of Acanthamoeba, and the drugs' corresponding biocompatibility with rabbit corneal epithelial and endothelial cell cultures. The results indicated that there were significant species differences in drug sensitivity. Propamidine (> 1,000 micrograms/ml) was clearly less effective than pentamidine (> 125 micrograms/ml) against A. castellanii, although equivalent potency (> 250 micrograms/ml) was observed against A. polyphaga. On the other hand, propamidine (> 31.25 micrograms/ml) was slightly more effective than pentamidine (> 62.5 micrograms/ml) against A. hatchetti. Both drugs were also relatively nontoxic after short-term contact with cell cultures, even though the highest concentration of pentamidine caused low-grade injury to the superficial epithelium and reversible membrane damage to the endothelium. Steady-state levels of propamidine at effective amoebicidal concentrations, however, were much more toxic than pentamidine, which indicated that the drug has a much lower therapeutic index. Our data suggest that pentamidine may be an effective therapeutic option because of its potency and low toxicity.

Antibody-dependent neutrophil-mediated killing of Acanthamoeba castellanii.

Neutrophils from naive rats lysed low numbers of Acanthamoeba castellanii in the presence of normal rat serum and significantly higher numbers of the parasite in the presence of serum from immunized rats. With normal rat serum, neutrophils from rats immunized with crude parasite extract or from naive rats killed similar percentages of A. castellanii. However, neutrophils from immunized rats killed a significantly greater percentage of parasites in the presence of serum from immunized rats than was seen with any other combination of serum and neutrophils. The addition of supernatant from cultures of concanavalin A-stimulated rat spleen cells to incubations of the parasite in the presence of neutrophils from naive or immunized rats and immune serum resulted in the highest levels of amoebolysis seen in this study. This study has shown that neutrophils from naive rats or from rats immunized with A. castellanii antigen display a very limited amoebolytic capability which is significantly augmented in the presence of serum from immunized rats and further boosted by the addition of supernatant from Con A-stimulated rat spleen cell cultures.

The role of contact lenses, trauma, and Langerhans cells in a Chinese hamster model of Acanthamoeba keratitis.

PURPOSE: To determine the role of contact lenses, corneal trauma, and Langerhans cells in the development of keratitis caused by Acanthamoeba organisms in Chinese hamsters. METHODS: Various methods were used to induce corneal infections in Chinese hamsters, including application of parasite-laden contact lenses. The role of corneal epithelial defects in promoting parasite binding was examined in vitro in a microscopic binding assay. The role of corneal abrasion in the development of Acanthamoeba keratitis was also examined in Chinese hamsters exposed to parasite-laden contact lenses. Other experiments evaluated the effect of infiltrating Langerhans cells on the incidence and severity of Acanthamoeba keratitis. RESULTS: Corneal epithelial defects promoted extensive parasite binding to abraded corneas compared to intact, nonabraded counterparts. Corneal abrasion was absolutely necessary for the induction of Acanthamoeba keratitis in hamsters infected with contaminated contact lenses. Infection was never detected unless the corneas were abraded before exposure to parasite-laden contact lenses. The presence of Langerhans cells in corneas prevented the development of Acanthamoeba keratitis. CONCLUSIONS: The highest incidence of Acanthamoeba keratitis occurs in corneas expressing epithelial defects and exposed to parasite-laden contact lenses. The presence of Langerhans cells in corneas exposed to parasite-laden contact lenses prevents the development of Acanthamoeba keratitis.

Characterization and pathogenic potential of a soil isolate and an ocular isolate of Acanthamoeba castellanii in relation to Acanthamoeba keratitis.

Acanthamoeba castellanii, one isolate from the eye and one from the soil, were compared on the basis of: (a) pathogenic potential; (b) plasminogen activator activity; (c) chemotactic activity; (d) cytopathic effects; (e) collagenolytic activity; (f) binding ability to contact lenses; and (g) and binding ability to corneal buttons. The ocular isolate of A. castellanii was found to be pathogenic based on its ability to produce corneal infections in Chinese hamsters. By contrast, the soil isolate produced only mild lesions in a single Chinese hamster. Amoebae from the ocular isolate bound to corneal epithelium in greater numbers than the soil isolate counterparts. Moreover, ocular isolate organisms displayed plasminogen activator activity that was not detected in cultures from soil isolates of A. castellanii. Although neither the soil isolate nor the ocular isolate amoebae responded chemotactically to epithelial or stromal components, the ocular isolate displayed a curious and reproducible positive chemotactic response to endothelial extracts. Both A. castellanii isolates produced cytopathic effects on pig corneal epithelium, however the cytotoxicity from the ocular isolate was significantly greater than that of the soil isolate. The results indicate that the pathogenic potential of A. castellanii is correlated with the parasite's capacity to bind to corneal epithelium, respond chemotactically to corneal endothelial extracts, elaborate plasminogen activators, and produce cytopathic effects on corneal epithelium.

Susceptibility of corneas from various animal species to in vitro binding and invasion by Acanthamoeba castellanii [corrected].

A crucial requirement for establishing corneal infection by the extracellular protozoal parasite, Acanthamoeba, is the ability of the parasite to bind to the corneal surface. In a series of in vitro studies, we examined the ability of Acanthamoeba castellanii [corrected] to adhere, invade, and damage normal, intact corneas of 11 mammalian and one avian species. A. castellanii [corrected] (80-90% trophozoites and 10-20% cysts) were incubated with corneas for 24 hours in vitro and examined by scanning electron microscopy (SEM). Results of several independent SEM experiments revealed that parasites not only failed to produce cytopathic effects but did not even bind to the corneal epithelium of mice, rats, cotton rats, horses, guinea pigs, cows, chickens, dogs, and rabbits. However, parasites adhered, invaded, and produced severe damage to human, pig, and Chinese hamster corneas during the 24-hour in vitro incubation period. Additional in vitro experiments quantified the binding of A. castellanii [corrected] to the corneas of selected susceptible and nonsusceptible species. In vitro binding assays revealed scant binding of parasites to mouse, rat, and rabbit (range = 5-20 parasites/7.07 mm2 corneal button). In contrast, extensive binding was observed on Chinese hamster, pig, and human corneas (range = 100-200 parasites/7.07 mm2 button). The results indicate that A. castellanii [corrected] exercises rigid host specificity at the host cell surface.

A pig model of Acanthamoeba keratitis: transmission via contaminated contact lenses.

A model of contact lens-induced Acanthamoeba keratitis was developed in Yucatan micropigs. Pigs fitted with parasite-laden soft contact lenses developed corneal infections that clinically and histopathologically mimicked the human counterpart. Three distinct stages of disease became apparent and were categorized as: acute, condensed infiltrate, and resolution stages. Viable parasites were isolated from corneal scrapings and smears were taken during the acute and condensed infiltrate stages. In addition, cysts could be identified deep within the stroma of histological specimens taken during the resolution stages. The characteristic dense, white ring-like infiltrates, stroma edema, keratic precipitates, and the chronic nature of the infections were similar to those observed in human Acanthamoeba keratitis. Histopathological examination of infected corneas revealed extensive neutrophilic infiltrates, stromal necrosis, and disorganization of the collagen lamellae. The strong correlation between the clinical and histopathologic features of contact lens-induced Acanthamoeba keratitis in the pig as well as the anatomical similarity of the pig eye with the human eye make the porcine model a valuable tool for investigations of the immunology, cell biology, and therapy for Acanthamoeba keratitis.

The role of tetracycline in chronic blepharitis. Inhibition of lipase production in staphylococci.

Tetracycline administered in low doses can be effective in the long-term management of patients with meibomian keratoconjunctivitis (MKC). However, the mechanism of action does not appear to be a reduction of bacteria. Seventy-five percent of the ocular staphylococci in such patients are resistant to tetracycline. An alternative mechanism of action could be the inhibition of production of extracellular enzymes by the ocular flora. Inhibition of lipase production could result in lowered levels of toxic hydrolysis products (free fatty acids), which may exacerbate the disease process. The authors tested this hypothesis by examining the differential effect of tetracycline on growth and lipase production in a tetracycline-resistant and tetracycline-sensitive strain of Staphylococcus epidermidis and S. aureus isolated from patients with MKC and Staphylococcal blepharitis. Tetracycline caused significant decreases in the production of lipase in the sensitive and resistant strains of S. epidermidis without concominant decreases in growth. In contrast, S. aureus strains showed parallel decreases in both lipase production and inhibition of growth. The authors propose that the sensitivity of lipase production to tetracycline, in tetracycline-resistant S. epidermidis, may partially explain the clinical improvement observed in MKC patients.

Effect of contact lens preservatives on Acanthamoeba.

Single preservatives used in contact lens solutions were evaluated for their effectiveness in killing Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites and cysts. Preservatives were tested against amoebae at intervals varying from 30 minutes to 24 hours. The preservatives were tested with axenically and nonaxenically grown organisms. Chlorhexidine (0.001% and 0.005%), polyaminopropyl biguanide (0.0015%), benzalkonium chloride (0.001% and 0.004%), and hydrogen peroxide (3%) were very effective preservatives. Lower concentrations of these same preservatives were less effective. Thimerosal (0.001% and 0.004%), sorbic acid (0.1%), potassium sorbate (0.13%), EDTA (0.1%), polyaminopropyl biguanide (0.00005%), and polyquaternium-1 (0.001%) were not effective as tested. However, thimerosal 0.004% when combined in solution with EDTA was effective. Preservatives were more effective when tested against axenically prepared organisms than when tested against co-cultured organisms.

The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga.

Contact lens disinfection systems were evaluated for their effectiveness in killing Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites and cysts. Amoebae were inoculated into commercially available contact lens cleaning and soaking solutions. At intervals varying from 30 minutes to 24 hours, solutions were filtered. The filters were removed and cultured for Acanthamoeba organisms. Striking differences were observed in the abilities of the different disinfecting solutions to kill the organisms. Solutions containing chlorhexidine were effective at very short exposure times. Solutions containing benzalkonium chloride required slightly longer exposure times but were faster than solutions containing only thimerosal. Solutions containing sorbate, polyaminopropyl biguanide, or polyquaternium-1 were not effective at killing Acanthamoeba organisms in the time allotted for the experiment. Solutions containing hydrogen peroxide were quite effective if the agent was not prematurely catalyzed. A. polyphaga generally required longer exposure to disinfectants than did A. castellanii for complete inhibition to occur.

The rapid detection of Acanthamoeba in paraffin-embedded sections of corneal tissue with calcofluor white.

Acanthamoeba keratitis is a difficult diagnosis to make with routine stains and cultures. Gram's, Giemsa, and hematoxylin-eosin stains do not differentially stain Acanthamoeba, making the detection of organisms difficult. Trophozoite and cyst forms in paraffin-embedded corneal tissue sections can be rapidly and differentially stained with calcofluor white. Under the fluorescence microscope, the trophozoites are bright red-orange, and cyst cell walls fluoresce bright apple-green with red-orange cytoplasm. Retrospective identification can be made by destaining hematoxylin-eosin-stained sections. Digesting background corneal tissue with trypsin or collagenase and hyaluronidase solutions helps to more readily identify trophozoites.