|Year : 2019 | Volume
| Issue : 1 | Page : 1
Extracellular vesicles from the aqueous humor of patients with uveitis
Carmen Luz Pessuti1, Deise Fialho Costa1, Kleber S Ribeiro1, Heloisa Nascimento1, Rubens Belfort1, Alessandra G Commodaro1, Ana Claudia Torrecilhas2
1 Department of Ophthalmology, Federal University of São Paulo, Vision Institute, IPEPO, São Paulo, Brazil
2 Department of Pharmaceutical Sciences, Federal University of São Paulo, Diadema, São Paulo, Brazil
|Date of Submission||11-Jul-2019|
|Date of Acceptance||15-Jul-2019|
|Date of Web Publication||08-Aug-2019|
Dr. Carmen Luz Pessuti
Department of Ophthalmology, Federal University of São Paulo, Porto Street, 69, São Paulo, 09426-020
Source of Support: None, Conflict of Interest: None
Context: Extracellular vesicles (EVs) are related to the dissemination of the pathogen and to the regulation of the host immune system in infectious diseases. However, the role of EVs in Ocular toxoplasmosis remains unclear.
Aims: The goal of this study was to identify and characterize the concentration and size of EVs in the aqueous humor (AH) and plasma of patients with ocular toxoplasmosis (OT) compared to other types of uveitis (OTU) and cataract.
Settings and Design: AH and plasma were collected from six patients with active OT, six patients with OTU, and six patients with cataract. All patients were also assessed clinically.
Subjects and Methods: EVs were isolated using the membrane affinity column method. Nanoparticle Tracking Analysis (NTA) was performed to determine the size and concentration of EVs.
Statistical Analysis Used: The ANOVA test was used to determine statistical difference. P < 0.05 was considered statistically significant.
Results: EV was present in the AH of different types of uveitis as well as in patients with cataract. The concentration of EV in AH was significantly lower in OT and OTU compared to cataract (P = 0.03). However, in the plasma, all groups presented a similar concentration of EV. The size of EV was the same in the AH and plasma among the three groups.
Conclusion: This initial study successfully identified and characterized EVs in the AH and plasma from patients with OT as well as OTU and cataract. Further studies are necessary to better understand the role of EVs in different ocular pathologies.
Keywords: Aqueous humor, extracellular vesicles, infectious uveitis, ocular toxoplasmosis, Toxoplasma gondii
|How to cite this article:|
Pessuti CL, Costa DF, Ribeiro KS, Nascimento H, Belfort R, Commodaro AG, Torrecilhas AC. Extracellular vesicles from the aqueous humor of patients with uveitis. Pan Am J Ophthalmol 2019;1:1
|How to cite this URL:|
Pessuti CL, Costa DF, Ribeiro KS, Nascimento H, Belfort R, Commodaro AG, Torrecilhas AC. Extracellular vesicles from the aqueous humor of patients with uveitis. Pan Am J Ophthalmol [serial online] 2019 [cited 2019 Aug 26];1:1. Available from: http://www.thepajo.org/text.asp?2019/1/1/1/264048
| Introduction|| |
Ocular toxoplasmosis (OT) is the main worldwide cause of posterior uveitis. In Brazil, OT affects approximately 50% of the infected patients, leading to visual disability.
Extracellular vesicles (EVs), including exosomes, are small membrane vesicles that may be involved in different diseases, and EV-based therapeutics are being developed.
They can be found in different body fluids, including the aqueous humor (AH). EV can diversify in number, size, and biological functions.
In this preliminary study, we identified and characterized the concentration and size of EV isolated from AH and plasma of the patients with OT compared to other types of uveitis (OTU) and cataract.
| Subjects and Methods|| |
A total of 18 patients (36 samples, 18 from the AH and 18 from the plasma) were recruited from March to July 2018 in the department of ophthalmology. They were divided into three groups: (1) six patients with active OT – 2 males and 4 females; (2) six patients with OTU – 4 males and 2 females; and (3) six patients with cataract – 4 males and 2 females. All patients from OT and OTU groups presented more than 1+ cells in the anterior chamber. All toxoplasmic uveitis patients had positive serological tests for infection and retinochoroiditis, scars, and/or exudative lesions.
The study was approved by the ethics committee investigational review board (CEP Number 2198149) and adhered to the principles of the Declaration of Helsinki and Resolution 196/96 of the Ministry of Health, Brazil. Informed consent was obtained from all participants.
Eighteen peripheral blood and 18 AH samples were collected from the 18 patients. Peripheral blood (10 mL) was collected in EDTA tubes centrifuged for 10 min at 1900 g, and 1 mL of plasma was collected. Approximately 0.1 mL of the AH was collected by anterior chamber paracentesis. All samples were kept at 4°C until the assays.
Extracellular vesicles' isolation and characterization
EV isolation was performed using a commercially available Maxi Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol.
Isolated EV was diluted 100X in phosphate-buffered saline (PBS) and analyzed by nanoparticle tracking analysis (NTA) using an MS300 instrument (Malvern Pananalytical Ltd., Malvern, Worcestershire, UK) coupled to an sCMOS camera and a laser emitting a 50-mW beam at 532-nm wavelength. PBS was used as control. Samples and control were read in triplicates during 30 s at 20 frames per se cond, and other settings were performed according to the instrument protocol. The NanoSight NTA software version 3.2 build 3.2.16 (Malvern Pananalytical Ltd., Malvern, Worcestershire, UK) was used for instrument control, data acquisition and data processing as concentration and size of the particles in the plasma and AH.
Statistical analyses were performed using the GraphPad software (Prism, version 5.00 for Windows; GraphPad, San Diego, CA). The ANOVA test was used to determine statistical difference among the three groups. The results were expressed as mean and standard deviation. A P < 0.05 was considered statistically significant.
| Results and Discussion|| |
The results showed a lower concentration of EV in the AH from OT patients (ranged from 1.0 × 1011 to 8.6 × 1011 particles/mL) and OTU patients (ranged from 8.5 × 1010 to 9.5 × 1011 particles/mL) in comparison with cataract patients (ranged from 2.9 × 1011 to 1.3 × 1012 particles/mL) (P = 0.03) [Figure 1]a. No difference was noticed in the concentration of EV in the plasma from OT, OU, and cataract patients [Figure 1]b.
|Figure 1: Concentration of extracellular vesicles isolated from aqueous humor (a) and plasma (b) and size of extracellular vesicles isolated from aqueous humor (c) and plasma (d) of patients with ocular toxoplasmosis (n = 6), other uveitis (n = 6), and cataract (n = 6). Results are expressed as mean ± standard deviation and *P < 0.05|
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The mean size of EV in AH of OT patients was 248 nm, followed by 243 nm and 229 nm in OU and cataract patients, respectively [Figure 1]c. In relation to plasma, the mean size of EV in OT patients was 227 nm while in OTU patients was 226 nm and in cataract patients was 237 nm [Figure 1]d.
In addition, the size distribution (D10, D50, and D90) analysis of EV in the AH and plasma did not demonstrate any difference among the three groups studied here (data not shown).
Parasites are known to release EV that functions as cell-to-cell effectors in the host–parasite interaction and can exert regulation of the host immune system. EV has been studied in diabetic retinopathy but not in the AH of uveitis patients. However, the relation between EV and OT as other uveitis remains unclear.
In this initial study, EV was successfully isolated from AH and plasma using a membrane affinity column method (Qiagen, Valencia, CA, USA). After isolation, EV was analyzed by NTA to determine the size and concentration in AH and plasma.
It has been demonstrated that exosomes released by the parasite can influence the proliferation and cell cycle of host cells and that various exosomal miRNAs are involved in the regulation of target genes related to cell. In addition, it has been shown that may circulate in the blood of immunocompetent patients with OT. This could probably be related to the lower concentration of EV that we found in OT and OTU patients when compared to cataract patients. However, further studies are necessary to explain the biological meaning of changing the number of EV in AH.
| Conclusion|| |
We successfully isolated and characterized the size and concentration of EV in AH and plasma from patients with different types of uveitis and cataract. The higher number of EV in the AH of cataract patients needs to be further studied to better understand the role of EV.
Financial support and sponsorship
This work was supported by CNPq (AGC–Grant 400040/2014-0), FAPESP, and CAPES. RBJ and ACT are recipients of CNPq Fellowships.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Nussenblatt RB, Belfort R Jr. Ocular toxoplasmosis. An old disease revisited. JAMA 1994;271:304-7.
Costa DF, Nascimento H, Sutili A, Nobrega FAJ, Fowler F, Nobrega MJ, et al.
Frequency of Toxoplasma gondii
in the retina in eye banks in Brazil. Parasitol Res 2017;116:2031-3.
Robbins PD, Morelli AE. Regulation of immune responses by extracellular vesicles. Nat Rev Immunol 2014;14:195-208.
Perkumas KM, Hoffman EA, McKay BS, Allingham RR, Stamer WD. Myocilin-associated exosomes in human ocular samples. Exp Eye Res 2007;84:209-12.
Enderle D, Spiel A, Coticchia CM, Berghoff E, Mueller R, Schlumpberger M, et al.
Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One 2015;10:e0136133.
Trocoli Torrecilhas AC, Tonelli RR, Pavanelli WR, da Silva JS, Schumacher RI, de Souza W, et al.Trypanosoma cruzi
: Parasite shed vesicles increase heart parasitism and generate an intense inflammatory response. Microbes Infect 2009;11:29-39.
Huang C, Fisher KP, Hammer SS, Navitskaya S, Blanchard GJ, Busik JV, et al.
Plasma exosomes contribute to microvascular damage in diabetic retinopathy by activating the classical complement pathway. Diabetes 2018;67:1639-49.
Kim MJ, Jung BK, Cho J, Song H, Pyo KH, Lee JM, et al.
Exosomes secreted by Toxoplasma gondii
infected L6 cells: Their effects on host cell proliferation and cell cycle changes. Korean J Parasitol 2016;54:147-54.
Silveira C, Vallochi AL, Rodrigues da Silva U, Muccioli C, Holland GN, Nussenblatt RB, et al.Toxoplasma gondii
in the peripheral blood of patients with acute and chronic toxoplasmosis. Br J Ophthalmol 2011;95:396-400.