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 2018, were enrolled in this study. 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 was collected between 8.00 and 9.00 am for routine biochemical evaluations, including fasting total cholesterol and glucose, 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. Cholesterol analysis was assessed by an enzymatic colorimetric method on a Dimension RXL apparatus (Dade Behring AG, Ziegelbrucke, Switzerland).
ELISA detection of sNox2-dp
Serum Nox2 levels was measured as soluble Nox2-derived peptide (sNox2-dp) with an ELISA method as previously reported. Briefly, we coated reference standards of known concentrations (0–200 pg/ml) of sNox2-dp and platelet supernatant samples (1 µg of protein) into ELISA 96 well plate overnight at 4 °C, after wash away of unbound materials from samples we blocked any free binding site for 120 min at RT, later we washed away of unbound materials from samples and we added in each well anti-sNox2dp-horseradish peroxidase (HRP) monoclonal antibody against the amino acidic sequence of the extra membrane portion of Nox2, finally we quantified immobilized antibody enzyme conjugates by monitoring HRP activity in the presence of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Bethyl Laboratories, TX, USA). The enzyme activity is measured, after acidification of formed products (2 M sulphuric acid), spectrophotometrically by the increased absorbency at 450 nm. Increase in absorbency is directly proportional to the amount of sNox2dp of the unknown sample, then, by interpolation from a reference curve, generated in the same assay with reference standards of known concentrations of sNox2dp, it is possible calculate the concentration of samples. 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 (Abcam and DRG International, Inc).
Serum zonulin levels were measured using a commercially ELISA kit (Elabscience). Antibody specific for zonulin has been pre-coated onto a microplate and 100 μl of standards and patient sera samples were added and incubated 90 min at 37 °C. Then, a biotinylated detection antibody specific for zonulin and Avidin-Horseradish Peroxidase (HRP) conjugate was added to each microplate. The amount of zonulin was measured with a microplate auto-reader at 450 nm. Values were expressed as ng/ml; both intra-assay and inter-assay coefficients of variation were within 10%.
Plasma samples were thawed only once and used to perform specific sandwich enzyme-linked immunosorbent assay (ELISA) to measure LPS (Hycult Biotechnology, Uden, The Netherlands). The kit has a concentration range of 0.04 to 10.0 EU/ml.
Statistical analyses were undertaken using 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 percentages were assessed by the χ2 test. Bivariate analysis was performed by Spearman’s correlation; the variables with evidence of an association P<0.10 were included in a multivariable linear regression using an automated procedure with forward selection. A p value of <0.05 was considered statistically significant.
Sample size determination
We computed the minimum sample size 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.