Patients
For this study, both patients and controls were recruited from the Ophthalmology Department of Egas Moniz Hospital, West Lisbon Hospital Center, between October 2014 and October 2016.
Patients presenting with active uveitis from a presumed viral/herpetic etiology were included in the uveitis group. The diagnosis of active uveitis followed the clinical criteria based on inflammatory cell reaction in the anterior chamber or vitreous as per standardization of uveitis nomenclature (SUN) and National Eye Institute (NEI) grading systems [4].
At the time of sampling, all patients had active disease and both blood and AqH samples were collected at presentation. Intraocular samples were examined for the presence of cytomegalovirus (CMV), herpes simplex virus (HSV)-1 and 2, and varicella zoster virus (VZV) by real-time polymerase chain reaction (PCR) analysis as previously described [5].
Controls were selected among healthy subjects undergoing cataract or refractive surgery, with no known history of intraocular inflammation.
The study protocol was approved by the Ethics Committee of Egas Moniz Hospital, West Lisbon Hospital Center, and informed consent was obtained from each patient.
Sample collection
The AqH samples were collected with a 30-gauge needle under topical anesthesia and sterile conditions by slit lamp with the aid of one drop of povidone iodine before and after puncturing the anterior chamber. The AqH samples of control subjects were collected with a 30-gauge needle before starting surgery. Undiluted aqueous samples of at least 0.1 mL were collected from each subject and immediately sent to the laboratory for analysis.
Peripheral blood samples were also collected in order to obtain serum.
Quantification of serum cytokine expression by multiplexed flow cytometry
A multiplex bead-based immunoassay (BD CBA Flex Set, BD Biosciences, San Jose, CA, USA) was used to determine serum and AqH levels of TNF-α, IFN-ɣ, IL-17A and IL-10. A similar single-plex bead-based immunoassay was used for TGF-β.
The protocol was performed following the instructions of the manufacturer. In brief, standards and serum samples were incubated with specific capture beads for 1 h at room temperature. After adding the detection reagent, the mixtures were incubated for 2 h at room temperature in the dark. After a final wash, beads were acquired in a BD FACS Canto II, previously set up for the BD CBA Flex Set. For each cytokine, at least 300 beads were acquired per sample. The FCAP Array Software (BD Biosciences) was used for data analysis. Standard curves covered a 0–2500 pg/mL concentration range and the minimum detection levels were: 0.13 pg/mL for IL10; 0.3 pg/mL for IL17A; 1.8 pg/mL for IFN-γ and 0.7 pg/mL for TNF-α.
For TGF-β, analyzed separately, samples were previously activated with the Sample Activation Kit 1 (R&D, Minneapolis, MN, USA) according to the recommended procedure. After activation, samples were incubated with capture beads for 2 h, washed and incubated with detection reagent. Acquisition and analysis were performed as described above. For TGF-β, standard curves covered a 0–10,000 pg/mL concentration range, and minimum detection level was 14.9 pg/mL.
Statistical analysis
The Mann-Whitney U test was used to compare each 2 independent groups. A P value of < 0.05 was considered for statistical significance. Data were analyzed using GraphPad Prism, version 8 for Windows (GraphPad Software, La Jolla, California).
