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 December 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 clinic 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, renal disease, cancer, treatment with immuno-suppressive drugs or antioxidants, liver disease, 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 samples were collected, between 8.00 and 9.00 am, 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 serum samples were prepared and the wells of a microtiter plate were coated overnight at 4 °C. After washing and blocking the remaining protein-binding sites, a specific 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, after the addition of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Bethyl Laboratories, TX, USA) and the stop solution, the absorbance of each well was read spectrophotometrically at 450 nm with a plate reader. Values were expressed as pg/ml; intra-assay and inter-assay coefficients of variation were 8.95% and 9.01%, respectively.
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 with a commercially ELISA kit (Elabscience). Briefly, standards and samples were added to a pre-coated microplate with a specific antibody for zonulin 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 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 into 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 analysis 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. Simple linear regression 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.