Caracterização dos mediadores inflamatórios no epitélio conjuntival, no epitélio corneano e no filme lacrimal de pacientes portadores de ceratocone em progressão
Data
2024-08-14
Autores
Silva, Albert Wilson Santos Machado 
Orientadores
Oliveira, Lauro Augusto
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Este estudo teve como objetivo caracterizar mediadores inflamatórios no filme lacrimal de pacientes portadores de ceratocone (KC), investigar a expressão gênica desses mediadores em células epiteliais da córnea e sua imunodetecção em células epiteliais conjuntivais, em comparação com indivíduos sem ceratocone. Métodos: Este estudo transversal envolveu 31 pacientes com KC e 23 indivíduos sem ceratocone. A coleta de lágrima foi obtida por instilação na superfície ocular de 60μl de soro fisiológico estéril (método wash). Os níveis de IL-5, IL-13, IL-2, IL-6, IL-10, IFN-gama, TNF-alfa e IL-4 foram detectados com o kit inflamatório LEGENDplex HU Th1/Th2 e analisados por citometria de fluxo. O epitélio da córnea foi obtido por ceratectomia manual de pacientes com KC candidatos ao crosslinking corneano e de indivíduos sem ceratocone candidatos à ceratectomia fotorrefrativa constituindo o grupo controle. As amostras foram imediatamente armazenadas a -80°C, até posterior extração de mRNA e análise por RT-PCR, para medir a expressão gênica de IL-5 e IL-6. Amostras de epitélio conjuntival foram obtidas por citologia de impressão e analisadas por imuno-histoquímica/microscopia confocal, em busca de imunodetecção de IL-5 e IL-6. Resultados: As concentrações das citocinas IL-13, IL-6, IL-10, INF-γ, TNF-α, IL-4 no filme lacrimal foram maiores nos pacientes com KC do que nos controles, embora sem significância estatística. A expressão gênica de IL-5 e IL-6 no epitélio corneano foi maior em pacientes com KC, quando comparada ao grupo controle (IL-5 foi 50% e IL-6 foi 20% maior, respectivamente). As análises imuno-histoquímicas mostraram maior imunodetecção de IL-5 e IL-6 no epitélio conjuntival de pacientes com KC, em comparação ao grupo controle. Conclusões: Este estudo demonstra que algumas citocinas com maior concentração no filme lacrimal tiveram maior expressão gênica no epitélio corneano e foram detectadas com maior intensidade no epitélio conjuntival de pacientes com KC.
Objective: This study aimed to characterize inflammatory mediators in the tear film of keratoconus patients (KC) and investigate their gene expression on corneal epithelial cells and their immune demarcation on conjunctival epithelial cells compared with individuals without Keratoconus. Methods: This transversal study involved 31 KC patients and 23 individuals without Keratoconus. Tear collection was obtained by ocular surface washing with 60 μl of sterile buffered saline solution. IL-5, IL-13, IL-2, IL-6, IL-10, IFN-γ, TNF-α, and IL-4 levels were detected with the LEGENDplex HU Th1/Th2 panel kit and analyzed by flow cytometer. Corneal epithelia were obtained by manual keratectomy from KC patients’ candidates to corneal cross-linking and from individuals without Keratoconus candidates to PRK (control group). Samples were immediately stored at −80°C until further mRNA extraction and RT-PCR analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were obtained using impression cytology and were analyzed by immunohistochemistry/ confocal microscopy, looking for immunoreaction with IL-5 and IL-6. Results: Tear film concentrations of IL-13, IL-6, IL-10, INF-γ, TNF-α, IL-4 were higher in patients with KC than in controls, although without statistical significance. Gene expression of IL-5 and IL-6 in the corneal epithelium was higher in patients with keratoconus when compared to the control group (IL-5 was 50% and IL-6 was 20% higher, respectively). Immunohistochemical analyses showed more significant immunostaining of IL-5 and IL-6 in the conjunctival epithelium of KC patients compared to the control group. Conclusions: This study demonstrates that some cytokines with higher concentrations in the tear film had higher gene expression in the corneal epithelium and were detected with greater intensity in the conjunctival epithelium of patients with KC.
Objective: This study aimed to characterize inflammatory mediators in the tear film of keratoconus patients (KC) and investigate their gene expression on corneal epithelial cells and their immune demarcation on conjunctival epithelial cells compared with individuals without Keratoconus. Methods: This transversal study involved 31 KC patients and 23 individuals without Keratoconus. Tear collection was obtained by ocular surface washing with 60 μl of sterile buffered saline solution. IL-5, IL-13, IL-2, IL-6, IL-10, IFN-γ, TNF-α, and IL-4 levels were detected with the LEGENDplex HU Th1/Th2 panel kit and analyzed by flow cytometer. Corneal epithelia were obtained by manual keratectomy from KC patients’ candidates to corneal cross-linking and from individuals without Keratoconus candidates to PRK (control group). Samples were immediately stored at −80°C until further mRNA extraction and RT-PCR analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were obtained using impression cytology and were analyzed by immunohistochemistry/ confocal microscopy, looking for immunoreaction with IL-5 and IL-6. Results: Tear film concentrations of IL-13, IL-6, IL-10, INF-γ, TNF-α, IL-4 were higher in patients with KC than in controls, although without statistical significance. Gene expression of IL-5 and IL-6 in the corneal epithelium was higher in patients with keratoconus when compared to the control group (IL-5 was 50% and IL-6 was 20% higher, respectively). Immunohistochemical analyses showed more significant immunostaining of IL-5 and IL-6 in the conjunctival epithelium of KC patients compared to the control group. Conclusions: This study demonstrates that some cytokines with higher concentrations in the tear film had higher gene expression in the corneal epithelium and were detected with greater intensity in the conjunctival epithelium of patients with KC.
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Citação
SILVA, Albert Wilson Santos Machado. Caracterização dos mediadores inflamatórios no epitélio conjuntival, no epitélio corneano e no filme lacrimal de pacientes portadores de ceratocone em progressão. 2024. 99 f. Tese (Doutorado em Oftalmologia) - Escola Paulista de Medicina, Universidade Federal de Sãp Paulo (UNIFESP). São Paulo, 2024.