Thirty consecutive subjects (24 males and 6 females, mean age 9±3), who were referred to the Allergology and Pediatric Neurology clinic of “Sapienza” University of Rome from January 2018 to November 2019, were enrolled. Thirty control subjects (24 males and 6 females, mean age 9±3), matched for aged and gender, were enrolled at the same pediatric department at the same period. Controls were recruited through a screening program in childhood.
Inclusion criteria were represented by: subjects aged between 3-16 years affected by PANDAS.
PANDAS was defined according to the criteria elaborated by Dr. Swedo and collaborators[2, 3]:
1) presence of OCD (diagnosed according to DSM IV criteria) and/or tic disorders
2) onset of symptoms between 3 years and puberty
3) episodic course of the disease
4) symptoms and exacerbations temporally associated with GAS infections
5) association with neurological anomalies (choreiform movements and motor hyperactivity during symptoms exacerbations).
Exclusion criteria were represented by: PANS not related to GAS, Sydenham corea, Tourette syndrome, Autoimmune encephalitis, Systemic autoimmune diseases, Wilson's disease, congenital heart disease, existence of renal disease, malignancy, treatment with immuno-suppressive drugs or antioxidants, liver failure, acute disease.
The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Sapienza University of Rome Ethics Committee (n. 5377).
Blood Sampling
Blood sampling was collected between 8.00 and 9.00 am for routine biochemical evaluations and for oxidative stress analysis. Blood samples were collected in Vacutainers (Vacutainer Systems, Belliver Industrial Estate, Plymouth, UK) after an overnight fast (12 hours). Samples were centrifuged at 300g for 10 minutes, and the supernatant was collected and stored at -80°C until dosage.
ELISA detection of sNOX2-dp
Serum NOX2 levels were measured as soluble NOX2-derived peptide (sNOX2-dp) with an ELISA method as previously reported[20]. Briefly, reference standards of sNOX2-dp and samples (1 µg of protein) were coated into ELISA 96 well plate overnight at 4 °C. After washing unbound materials and blocking any free binding site for 120 min at RT, anti-sNOX2dp-horseradish peroxidase (HRP) monoclonal antibody against the amino acidic sequence of the extra membrane portion of NOX2 was added in each well. Finally, immobilized antibody enzyme conjugates was quantified by monitoring HRP activity in the presence of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Bethyl Laboratories, TX, USA). After acidification, the enzyme activity is measured spectrophotometrically at 450 nm. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were 8.95% and 9.01%, respectively.
Serum 8-iso-prostaglandin F2α (8-iso-PGF2α)
8-iso-PGF2α levels were measured in serum by using a colorimetric assay kit (DRG International, Inc). Values were expressed as pmol/L. Intra-assay and inter-assay coefficients of variation were 5.8 % and 5.0 %, respectively.
Serum zonulin
Serum zonulin levels were measured using a commercially ELISA kit (Elabscience). Briefly, the microplate has been pre-coated with a specific antibody for zonulin and 100 μl of standards and samples were added and incubated 90 min at 37 °C. Then, a biotinylated detection antibody and Avidin-Horseradish Peroxidase (HRP) conjugate were added to each well. The amount of zonulin was measured spectrophotometrically at a wavelength of 450 nm with a microplate auto-reader. Values were expressed as ng/ml; both intra-assay and inter-assay coefficients of variation were within 10%.
LPS
Samples were thawed only once and used to perform specific sandwich enzyme-linked immunosorbent assay (ELISA) to measure LPS (Cusabio, Wuhan, China). The standards and samples were plated for 2 h onto a micro-plate pre-coated with the antibody specific for LPS. After incubation, samples were read at 450 nm. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were <10%.
Statistical analysis
Statistical analyses was performed with SPSS 18.0 software for Windows (SPSS, Chicago, IL, USA). The Kolmogorov-Smirnov test was used to determine whether variables were normally distributed. Normally distributed data are described as means±standard deviations (SDs). Group differences were analyzed by Kruskal-Wallis tests (for non-normally distributed data) or analysis of variance (ANOVA). Differences between categorical variables were assessed by the χ2 test. Bivariate analysis was performed by Spearman’s rank correlation test; the variables with evidence of an association p<0.10 were included in a multivariable linear regression using an automated procedure. A p value <0.05 was considered as statistically significant.
Sample size determination
The minimum sample size was computed with respect to a two-tailed, one-sample Student t test considering, on the basis of data from a previous pilot study (data not shown): a difference of 4 pg/ml for sNOX2dp levels between children affected by PANDAS and controls, 4.7 as SD, 0.05 (α) as type I error probability and 0.95 as power 1−β. The sample size was n=30 patients/group.