METHODS
From April 2019 to June 2019, children affected by VKC, between 6 and 14 years of age, were enrolled at the Department of Pediatrics, Division of Allergy and Immunology, ‘Sapienza’ University of Rome.
Healthy children, cross-matched to the enrolled VKC children for gender and age, were recruited as controls.
Exclusion criteria were the diagnosis of ocular pathologies and the use of antihistamines and/or corticosteroids in the four weeks before the enrollment.
Diagnosis of VKC was performed by an ophthalmologist investigating ocular signs (conjunctival hyperemia, tarsal and/or limbal papillae, giant papillae) and subjective ocular symptoms (itching, photophobia, tearing, foreign body sensation, and burning sensation), according to the two disease severity scales, graded as follows: 0 = absent; 1 = mild; 2 = moderate; 3 =severe.
Children were classified as having severe VKC if the score was >3 points for one eye for each scale.17
Half of them were treated with CsA 1% eye drops suspension (Sandimmun galenical collyrium Pharmacy Umberto I hospital, Rome, Italy) 1 drop/eye twice daily, prepared by the Chemistry Service Institute at the University of Rome, following a formulation which included one part of commercially available CsA solution (Sandimmun Novartis Farma S.p.A.S.S., Varese, Italy), diluted in an aqueous vehicle (Vismed Light_TRB Chemedica, Haar/ Munich, Germany).
Parents were asked to keep the vial with the eye drops shielded from light and to use it within 15 days from the opening. CsA treatment was started for at least four weeks; antihistamines and/or corticosteroids had been suspended at least eight weeks before the beginning of CsA treatment.
The remaining VCK children were enrolled at the onset of disease and tested before starting CsA therapy. Atopic status was assessed by skin prick tests (SPTs) to aeroallergens and food allergens (Lofarma, Milan, Italy) and /or elevated specific (>0.35 kU/l) and total IgE (>100kU/l). SPT panels included: Dermatophagoides pteronissinus (DPT), Dermatophagoides farinae, dog/cat dander, Olea Europea, Lolium perenne, Alternaria tenuis, Parietaria officinalis, lactalbumin, ß-lactoglobulin, casein, egg white and yolk, soy, codfish. Histamine dihydrochloride 10 mg/mL and glycerol saline solution were used as positive and negative controls, respectively. Wheal reactions, read after 15 minutes, ≥3 mm was regarded as positive.18
Serum was obtained from peripheral blood samples to evaluate routine blood count, total, and specific IgE levels (sIgE) and oxidative stress. Serum total IgE and sIgE levels were detected for the same allergens tested in the SPTs, using a fluorescence enzyme immunoassay (FEIA) with capsulated cellulose polymer solid‐ phase (Immuno CAP®) coupled allergens (Thermo Fisher Scientific Inc, Phadia AB, Uppsala, Sweden). Results were expressed in kU/L: a cutoff point of 0.35 kU/L has been used as positivity for the specific IgE and > 100 kU/L for the total IgE.
Tear samples were obtained as follows: 20–50 μl of open eye tears were gently collected from the external canthus of the most affected eye using a microcapillary tube and avoiding the tear reflex as much as possible. The samples were placed in Eppendorf Tubes, centrifuged at 160 ×g for 8 min, and stored at −80°C.19
The concentration of H2O2 was analyzed in serum and tear samples using a commercial colorimetric assay (Sigma, Saint Louis, Missouri).
The sensitivity, determined by subtracting two standard deviations from the mean absorbance value of sixteen zero standard replicates and calculating the corresponding concentration, was around 50ng/ml.
The inter-assay Precision coefficient was 1,7 %, and the inter-assay Precision coefficient was 3,0%
Values are expressed in terms of micro molarity, where 1,0 m corresponds to 8,46ng/ml. This study was approved by the International Review Board of ‘Sapienza’ University of Rome and performed with the written informed consent of the parents of all children.