Two subject groups were established for this study: subjects with TD and healthy comparison subjects. The TD sample consisted of 32 children (27 males), ages 7–18 years (11.2 ± 3.2), who met DSM-IV-TR criteria for TD [mean total tic Yale Global Tic Severity Scale (YGTSS) score = 22.0 ± 6.11]. The TD sample was further divided into two subgroups: one consisting of 17 subjects (14 males) with TD plus comorbid OCD (TD+OCD), and a second consisting of 15 subjects (13 males) with TD without comorbid OCD (TD−OCD). Demographics, diagnoses, and treatment profiles are compiled in . Patients were recruited from the Institute for Tourette and Tic Disorders at the New York University (NYU) Child Study Center and from the Tourette Syndrome Association. Sixteen healthy comparisons (7 males), ages 9–19 (15.1 ± 3.3), were recruited from families of NYU staff. Data on 15 of the controls were reported elsewhere (Gabbay et al., 2009
Demographic and clinical characteristics of children and adolescents with Tourette's disorder (TD) and healthy control subjects.
Participants ages 18 years and over signed informed consent; those under age 18 provided assent, and a parent or guardian provided signed consent as approved by the NYU School of Medicine IRB.
A board-certified child and adolescent psychiatrist with expertise in diagnosis and treatment of TD, OCD, and PANDAS (VG or BC) interviewed all subjects (patients with TD and controls) and parents using the Schedule for Affective Disorders and Schizophrenia-Present and Lifetime Version for Children (K-SADS-PL) (Kaufman et al., 1997
). Full DSM-IV-TR criteria had to be fulfilled to meet diagnostic criteria for TD and OCD. Tic severity was rated on the YGTSS (Leckman et al., 1989
), and OCD severity on the Children's Yale-Brown Obsessive Compulsive Scale (CY-BOCS) (Scahill et al., 1997
). PANDAS was diagnosed using Swedo et al.'s five research diagnostic criteria (Swedo et al., 1998
). Baseline medical evaluation incorporated medical history and laboratory studies, including complete blood count, metabolic panel, liver functions, throat culture, and a urine toxicology test.
Exclusion criteria for all subjects were: immune-affecting medications taken in the past 6 months (other than psychotropic medications), any immunological or hematological disorder, Sydenham's chorea, any infectious disease in the month prior to enrollment (including a common cold), and significant medical or neurological disorders (other than a tic disorder). Exclusionary diagnoses for subjects with TD were bipolar disorder, major depressive disorder, pervasive developmental disorder, psychotic disorder, and a substance-related disorder in the past 12 months (based on history and urine toxicology test). Control subjects could not meet criteria for any current or past DSM-IV-TR psychiatric disorder. In addition, control subjects who endorsed taking allergy medication(s) were excluded.
2.2. Cytokine measurements
Venous blood samples of 10 ml were drawn directly into plastic tubes containing EDTA-K3. All blood samples (10 ml) were drawn between 08:00 and 09:00 AM after an overnight fast; the EDTA blood was processed within 20 min of collection. Aliquots of the plasma samples were stored at −80 °C for appropriate serial immunoassay analysis.
The quantitative determinations of the plasma levels of TNF-α, IL-1β, IL-12, IL-6, and IL-2 were performed in duplicate for each of the serial aliquots by commercial enzyme-linked immunosorbent assays (ELISA) in accordance with the manufacturers’ instructions. The TNF-α, also known as cachectin and TNFSF1A, the Quantikine Human TNF-α/TNFSF1A assay (R&D Systems, Minneapolis, MN) was used; for IL-1β, also known as IL-1F2, the Quantikine HS Human IL-1β/ILF2 immunoassay (R&D Systems, Minneapolis, MN) was used. For IL-12, the High Sensitivity Human Quantikine IL-12 (R&D Systems, Minneapolis, MN) was used; IL-6 was assayed with High Sensitivity Human Quantikine IL-6 (R&D Systems, Minneapolis, MN); and for IL-2, the QuantiGlo Human IL-2 immunoassay (R&D Systems, Minneapolis, MN) was used.
The lower limits of detection of the assays were: 0.038 pg/ml, 0.057 pg/ml, 0.18 pg/ml, 0.039 pg/ml, and 0.1 pg/ml, respectively. Intra-assay variability was less than 10%. The mean of the duplicate sample values was used. All assays were performed by CG, who was blind to the subjects’ clinical status.
2.3. Statistical analysis
As data were not normally distributed, the non-parametric Mann–Whitney test was used to compare subjects with TD and controls. A separate analysis was conducted for each cytokine. When cytokine levels were too low for detection, cytokines were given the lowest detectable value. Additionally, when cytokines were not detectable for more than 20% of either controls or subjects with TD, a binary logistic regression analysis was conducted to compare the groups in terms of the percentage of subjects with detectable levels. Further, ANCOVA based on ranks was used to assess the interaction of subject group with age and gender in terms of their impact on each measure.
Significance levels were set at p ≤ 0.05. Spearman rank correlation coefficients were used to characterize the association of cytokine levels with severity of TD symptoms on the YGTSS and severity of OCD symptoms on the CY-BOCS, as well as with age and gender. SAS version 9.0 (SAS Institute, Cary, NC) was used for all statistical computations.