Abstract
Purpose :
Extracellular Vesicles (EVs) are messenger pigeons of the cells which communicate about cellular microenvironment. Our previous in vitro and in vivo studies on profiling of EV proteome in bacterial endophthalmitis revealed the presence of C8α exclusively in the infected eyes while Calpain-2 was upregulated. In this study, we evaluated these proteins in the vitreous of patients with bacterial endophthalmitis.
Methods :
After informed consent, vitreous of patients with presumed (Culture negative/CN, but clinically presumed) and confirmed (Culture positive/CP and clinically confirmed) bacterial endophthalmitis was collected along with controls from patients undergoing vitrectomy for macular hole, retinal detachment and other non-infectious conditions, were included in the study. Following centrifugation to remove debris, 200µL of vitreous was filtered through 0.22µm filter and EVs were isolated by Exosome isolation reagent method. Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS) was performed to confirm the presence of EVs while, ELISA was performed to assess the levels of C8α and calpain-2 directly released in the vitreous and in the EVs.
Results :
SEM and DLS confirmed the presence of EVs in all the samples. The expression level (ng/mL) of C8α in the EVs from CP samples was 144±22 and CN samples was 31.2±9.8 which was significantly higher (<0.0001 and 0.007 respectively) when compared to control 3.7±2.4. However, C8α is not expressed in the vitreous of CN samples and controls. Calpain-2 was significantly downregulated (≤0.0001) in CP (0.94±0.16 ng/mL) and CN (0.70±0.14) samples when compared to control. Additionally, both C8α and Calpain-2 are elevated in fungal endophthalmitis compared to control but their expression levels (41±14 and 5.8±0.6 respectively) were distinct with respect to bacterial endophthalmitis.
Conclusions :
Although both the proteins had significant difference in the expression levels in control, CP and CN, EV C8α is more practical in a diagnostic setting when compared to Calpain-2 due to its absence in control and high expression in infection, making it a potential marker in bacterial endophthalmitis especially in culture negative cases. The presence of these proteins only in the EVs of culture negatives and not in the vitreous promises EVs as the future of diagnostics.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.