Ninety-four subjects were recruited as part of the Childhood Autism Risks from Genetics and Environment (CHARGE) study (Hertz-Picciotto et al., 2006
). For the experiments involving phytohemagglutinin (PHA) stimulation, 34 children with ASD (median age 3.83 (interquartile range 3.17-4.25), 29 males) and 26 typically developing (TD) controls (3.71(3.00-4.50), 21 males) were included. Experiments involving phorbol myristate acetate (PMA) stimulation included 18 ASD children ( 4.25(3.08-4.07), 14 males) and 16 TD controls (3.60(2.08-4.02), 13 males). ASD was diagnosed using gold standard assessments (DSM-IV criteria, Autism Diagnostic Observation Schedules (ADOS) and the Autism Diagnostic Interview-Revised (ADI-R)). Cognitive and adaptive assessments were performed on all participants using the Mullen Scales of Early Learning (MSEL) and the Vineland Adaptive Behavior Scales (VABS). Parents of all participants completed the Aberrant Behavior Checklist (ABC) to assess inappropriate and maladaptive behaviors. All developmental and behavioral testing was conducted by qualified clinicians and methodology is discussed in detail elsewhere (Hertz-Picciotto et al., 2006
). TD controls were included if they did not have a sibling with ASD, had no apparent autism traits based on assessment with the Social Communication Questionnaire (SCQ), and scored within two standard deviations of the mean on MSEL and VABS assessments. No participants had a history of recent infections and/or fever.
Peripheral blood was collected in acid-citrate-dextrose Vacutainers (BD Biosciences, San Jose, CA). PBMC were separated by gradient centrifugation on Histopaque (Sigma), washed twice with Hank’s Balanced Salt Solution (HBSS) Sigma (St. Louis Missouri). The number of viable PBMC was determined by Trypan Blue exclusion (Sigma) and PBMC concentrations were adjusted to 1 × 106 cells/ml in a solution of 0.1% T-Stim (BD Biosciences) in X-Vivo media (Cambrex, Walkersville, MD) in 12-well tissue culture plate (Corning, Corning, NY). PBMC were either cultured in media alone, or stimulated with PHA (10 μg/mL; Sigma) for 24 hours at 37°C in 5% CO2. Following culture, plates were centrifuged before supernatants were harvested and stored at −80°C until the date of assay.
IL-23 and IL-17 concentrations were measured in the media and PHA stimulated cell culture supernatant of each subject by enzyme-linked immunosorbent assay (ELISA). The concentration of IL-23 was measured with the IL-23 ELISA Ready-SET-Go! Kit with pre-coated plates (eBiocience, San Diego, CA). The assay kit allowed for IL-23 detection between 15 pg/ml-2000 pg/ml. IL-17 concentration was measured with the IL-17 ELISA Ready-SET-Go! Kit with pre-coated plates (eBiocience). The kit allowed detection between 4 pg/ml-500 pg/ml. Samples and standards were run in duplicate, and the assay was performed according to the manufacturer’s recommended protocols. Plates were read on a Wallac Victor3 multilabel-plate reader (PerkinElmer, Boston, MA) at 450nm. Sample concentrations were determined by a standard curve correlating a known standard concentration and OD.
Flow cytometric analysis of TH
17 cells in PBMC was determined in cell cultures that were either unstimulated (media alone) or treated with PMA (50 ng/ml) and Ionomycin (1 μM final concentration) for 24 hours, using methods previously described for intracellular staining (Ashwood and Wakefield 2006
; Enstrom et al., 2009a
). PBMC were stained for cell surface markers CD3, CD8 and CD4 (BD Biosciences, CA) before intracellular staining for anti-human IL-17 (eBioscience, San Diego, CA). The cells were analyzed on a LSR II flow cytometer (BD Immunocytometry Systems) and with FlowJo software (BD Immunocytometry Systems).
Statistical analysis to compare the induced cytokine production between groups was conducted with the Mann-Whitney U tests.. Evaluation of induced cytokine levels and clinical assessment and behavioral scores among children with ASD was determined using Spearman correlations. The statistical comparisons were made with SAS software (SAS Institute Inc., Cary, NC). All analyses were two-tailed, and p<0.05 were considered statistically significant.