RESUMO
Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.
Assuntos
Ceratite por Acanthamoeba/metabolismo , Acanthamoeba castellanii/metabolismo , Proteínas de Protozoários/biossíntese , Ceratite por Acanthamoeba/parasitologia , Análise de Variância , Animais , Modelos Animais de Doenças , Humanos , Masculino , Proteômica , Proteínas de Protozoários/genética , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Eletroforese em Gel Diferencial BidimensionalRESUMO
ABSTRACT Purpose: To report our initial experience in the treatment of Acanthamoeba keratitis with accelerated corneal collagen cross-linking. Methods: Retrospective chart review of patients diagnosed with Acanthamoeba keratitis with progressive corneal melting who were treated with accelerated collagen cross-linking. Results: A total of 6 eyes (5 patients) were reviewed. All the patients received adjuvant therapy with moxifloxacin and chlorhexidine. In 4 cases, the ulcer healed with a mean interval to epithelialization of 108.8 days (range 59-217). In 2 eyes, there was a persistent neurotrophic ulcer. The melting was not progressive in any case, nor did any eye required emergency penetrating keratoplasy. Conclusion: This study suggests a beneficial effect of accelerated collagen cross-linking in cases of Acanthamoeba keratitis with corneal melting. Thus, collagen cross-linking may be considered as adjuvant treatment for Acanthamoeba keratitis.
RESUMO Objetivo: Relatar nossa experiência inicial no tra tamento da ceratite por Acanthamoeba com reticulação acelerada de colágeno corneano. Métodos: Revisão retrospectiva de prontuários de pacientes diagnosticados com ceratite por Acanthamoeba, com deformação progressiva da córnea, tratados com reticulação acelerada de colágeno. Resultados: Seis olhos (5 pacientes) foram incluídos. Todos os pacientes receberam terapia adjuvante com moxifloxacina e clorexidina. Em 4 casos, a úlcera cicatrizou com uma média de epitelização de 108,8 dias (amplitude de 59-217 dias). Em dois pacientes, a úlcera apresentou um comportamento neurotrófico. A deformação não foi progressiva em nenhum dos pacientes e nenhum dos olhos exigiu ceratoplastia penetrante de emergência. Conclusão: Este estudo sugeriu um efeito benéfico da reticulação acelerada de colágeno em casos de ceratite por Acanthamoeba infecciosa com deformação corneal. A reticulação de colágeno parece ser uma alternativa coadjuvante possível para casos de ceratite por Acanthamoeba.
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Riboflavina/uso terapêutico , Terapia Ultravioleta/métodos , Ceratite por Acanthamoeba/terapia , Colágeno/metabolismo , Fármacos Fotossensibilizantes/uso terapêutico , Reagentes de Ligações Cruzadas/uso terapêutico , Ceratite por Acanthamoeba/metabolismo , Úlcera da Córnea/metabolismo , Úlcera da Córnea/terapia , Seguimentos , Colágeno/efeitos dos fármacos , Colágeno/efeitos da radiação , Córnea/efeitos dos fármacos , Córnea/efeitos da radiação , Córnea/metabolismoRESUMO
PURPOSE: To report our initial experience in the treatment of Acanthamoeba keratitis with accelerated corneal collagen cross-linking. METHODS: Retrospective chart review of patients diagnosed with Acanthamoeba keratitis with progressive corneal melting who were treated with accelerated collagen cross-linking. RESULTS: A total of 6 eyes (5 patients) were reviewed. All the patients received adjuvant therapy with moxifloxacin and chlorhexidine. In 4 cases, the ulcer healed with a mean interval to epithelialization of 108.8 days (range 59-217). In 2 eyes, there was a persistent neurotrophic ulcer. The melting was not progressive in any case, nor did any eye required emergency penetrating keratoplasy. CONCLUSION: This study suggests a beneficial effect of accelerated collagen cross-linking in cases of Acanthamoeba keratitis with corneal melting. Thus, collagen cross-linking may be considered as adjuvant treatment for Acanthamoeba keratitis.
Assuntos
Ceratite por Acanthamoeba/terapia , Colágeno/metabolismo , Reagentes de Ligações Cruzadas/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Riboflavina/uso terapêutico , Terapia Ultravioleta/métodos , Ceratite por Acanthamoeba/metabolismo , Idoso , Idoso de 80 Anos ou mais , Colágeno/efeitos dos fármacos , Colágeno/efeitos da radiação , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/efeitos da radiação , Úlcera da Córnea/metabolismo , Úlcera da Córnea/terapia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
PURPOSE: To evaluate the efficacy of corneal cross-linking (CXL; riboflavin/ultraviolet A) as a simple therapy for Acanthamoeba keratitis. METHODS: Twenty rabbits were systemically anesthetized and the stroma of their right corneas was inoculated with a suspension of Acanthamoeba. Rabbits were divided into 2 groups: one group was treated with corneal CXL 3 days after infection and the other did not receive any treatment (control). All eyes in both groups were examined before (days 0 and 3) and after (day 7) CXL treatment. On day 7, the eyes were enucleated; 18 corneal buttons (9 of each group) were sent for microbiological examination and 2 (1 of each group) for histopathologic examination. RESULTS: All animals developed Acanthamoeba keratitis. There was no statistically significant difference between groups before treatment (day 0, P = 1, and day 3, P = 0.684). The treated corneas had a higher score (3.48 ± 0.30) at the time of enucleation compared with control corneas (2.60 ± 0.26). This difference was statistically significant (P = 0.008). Microbiological analysis revealed that the treated corneas had a higher protozoal count (2.86 ± 0.09) compared with the control corneas (2.18 ± 0.07); this difference was statistically significant (P = 0.001). CONCLUSIONS: Treatment of Acanthamoeba keratitis by corneal CXL (riboflavin/ultraviolet A) did not prove effective in decreasing the intensity and severity of Acanthamoeba keratitis.