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Comparing biguanide and diamidine associated with enzymatic inhibitors on cell viability of corneal cells and trophozoites of Acanthamoeba spp

Grant number: 15/06664-3
Support Opportunities:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2015
Effective date (End): May 31, 2016
Field of knowledge:Health Sciences - Collective Health - Public Health
Principal Investigator:Fábio Ramos de Souza Carvalho
Grantee:Mariana Corinti Son
Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
Associated research grant:11/51626-1 - Acanthamoeba spp. toxins as virulence factors in infections of the ocular surface, AP.JP


Free-living amoebae of Acanthamoeba genus are directly related to the corneal epithelium infections. In general, infection of the corneal surface by the protozoan, called amoebic keratitis, affects young and healthy individuals and the exponential increase in the number of cases of Acanthamoeba keratitis has attracted the world's scientific curiosity and attention of the competent health authorities. Prior trauma to the corneal surface associated with poor hygiene and/ or inappropriate contact lens use are the main risk factors of amoebic keratitis. The initial adhesion of Acanthamoeba spp to the corneal epithelium is preceded by the secretion of different proteolytic enzymes by the protozoan, which induce the migration of macrophages and neutrophils to the site of infection, resulting in the establishment of an intense inflammatory process. In this sense, the treatment is mainly based on the use of topical chemical compounds (to inactivate cysts and/ or trophozoites of Acanthamoeba spp) as biguanides and diamidines, associated with corticosteroids (metalloproteinase enzyme blockers derived neutrophil degranulation). However, the use of these drugs at high concentrations for long periods of time (~ 4-6 months) may induce the development of cataracts and progressive iris atrophy. In addition, the possibility of using corticosteroids helps not only in immunosuppression of the patient but also excystment and proliferation of trophozoites of Acanthamoeba spp, which is another important aspect to be considered in increased clinical severity. For these reasons, the absence of standardized protocol as the antimicrobial agent, concentration and time of usage have been considered as the main limiting factors for therapeutic success. Thus, this research project aims to achieve the following objectives: to evaluate the lowest concentration of biguanide and diamidine which is effective for inactivation of cysts and / or trophozoites of Acanthamoeba spp and toxic effects on corneal cells; to study the association of antimicrobial agents with different enzyme inhibitors of toxins secreted by the protozoan in the inactivation of cysts and / or trophozoites of Acanthamoeba spp and the possible toxic effects on corneal cells; to correlate parasite interaction studies and corneal cells in the presence of different enzyme inhibitors and antimicrobial agents compounds. From the concentration of use in clinical practice, will be studied by serial dilution, the action of these compounds on cell viability of Acanthamoeba spp and its toxic effect on corneal cells: polihexametil biguanide 0.02%, isethionate propamidine 0.1 hexamidine and diisotionato% to 0.1%. The antimicrobial agents are associated with different enzyme inhibitors such as PMSF, aprotinin and EDTA. SIRC (rabbit corneal fibroblast) and HCECs (human corneal epithelial cells) strains will be used as model mammalian cell. Qualitative and quantitative analysis will be performed in triplicate for each experiment and the results will be compared and statistically analyzed. Based on the implementation of the study results are expected to aid (1) standard in particular therapeutic setting for amoebic keratitis, in order to minimize potential cytotoxic effects of biguanide and diamidines on healthy epithelium of the host cornea; (2) in the understanding of the effect of enzyme inhibitors on the viability of the corneal cells; (3) to propose, based on the obtained results, the analysis of experiments for the animal model and (4) provide perspectives of qualitative improvements to patients with amoebic keratitis, minimizing the pain associated with infection and possible post-treatment complications.

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