cytokines and lymphocyte subsets in childhood Tourette syndrome with positive streptococcal markers

Cytokine 96 (2017) 49–53

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Case-control, exploratory study of cerebrospinal fluid chemokines/cytokines and lymphocyte subsets in childhood Tourette syndrome with positive streptococcal markers Michael R. Pranzatelli ⇑, Elizabeth D. Tate, Tyler J. Allison 1 National Pediatric Myoclonus Center and National Pediatric Neuroinflammation Organization, Inc., Orlando, FL, USA

a r t i c l e

i n f o

Article history: Received 19 December 2016 Received in revised form 28 February 2017 Accepted 2 March 2017

Keywords: CSF B cells CSF immunophenotyping CSF chemokine profiling GABHS OCD Pediatric neuroinflammation

a b s t r a c t A longstanding question is whether neuroinflammation is present in children symptomatic for Tourette syndrome (TS) with positive streptococcal serology and throat cultures. The objective was to directly test for it using modern hypothesis-driven approaches. Profiling studies for 14 immune cell types (flow cytometry), 7 chemokines/cytokines (ELISA), oligoclonal bands, and other immunoglobulins were performed in this IRB-approved study of 5 children with TS and streptococcal markers compared to data from 26 non-inflammatory pediatric neurological controls. Subjects were well-characterized clinically and with standardized scales for tics and obsessions/compulsions. Three subjects with TS (60%) had positive throat cultures for Group A beta-hemolytic strep, five had elevated anti-deoxyribonuclease-B titers (mean = 444), and 4 (80%) had elevated anti-streptolysin O titers (981). There were no significant differences between groups in the frequency of CSF B and T cell subsets or NK cells; the proportion of intracellularly-stained T helper type 1 (IFN-c) or type 2 (IL-4) cells; the concentrations of B cell chemoattractants CXCL13, CXCL10; the B cell proliferation/survival cytokines BAFF and APRIL, or other chemokines (CCL19, CCL21, CCL22). None of the patients had positive CSF oligoclonal bands or an abnormal IgG index/synthesis rate. Parallel blood studies were negative. This novel study found no group CSF lymphocyte phenotypic abnormalities or elevated inflammatory mediators in childhood TS despite positive serology and throat cultures for Group A beta-hemolytic streptococci. It demonstrates feasibility of the methodology, and should serve as the basis for a larger study of putative streptococcal-associated neuroimmunological disorders. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Group A beta-hemolytic streptococci (GABHS), or S. Pyogenes, is a pathogen of global import. Controversy exists as to whether it causes a diversity of purported autoimmune pediatric neuroinflammatory disorders beyond the well-substantiated immunogenicity of GABHS in Sydenham Chorea, as recently reviewed [1]. One such disorder caught in the crosshairs is Tourette syndrome Abbreviations: ASO, anti-streptolysin O; ADN-B, anti-deoxyribonuclease-B; CSF, cerebrospinal fluid; GABHS, Group A beta-hemolytic streptococci; OCD, obsessivecompulsive disorder; OCB, oligoclonal immunoglobulin G; TS, Tourette syndrome. ⇑ Corresponding author at: National Pediatric Neuroinflammation Organization, Inc., 12001 Research Parkway, Suite 236, Orlando, FL 32826, USA. E-mail addresses: [email protected] (M.R. Pranzatelli), etate@omsusa. org (E.D. Tate), [email protected] (T.J. Allison). 1 Present address: Division of Child Neurology, Children’s Mercy Hospital, 2401 Gillham Road, Kansas City, MO 64108, USA. http://dx.doi.org/10.1016/j.cyto.2017.03.003 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.

(TS), a childhood-onset movement disorder diagnosed by the combination of motor and vocal tics for a minimum of one year, usually with neuropsychiatric comorbidities. Tic exacerbations may or may not occur in the setting of positive serology for GABHS, noted to be more prevalent in TS than in pediatric controls [2]. Other than antibiotic treatment, a clinical management directive is lacking due to a gap in knowledge about whether the GABHS-associated subgroup has neuroinflammation. The focus of immunological studies has been on disputed antibasal ganglia antibodies [2–4], and most other markers have been studied in blood [5–8] rather than cerebrospinal fluid (CSF). However, CSF is in greater proximity to the central nervous system and more likely to be informative [9]. Also, routine CSF testing for inflammation has a much lower yield for diagnosing neuroinflammation than revealing the immunophenotype through flow cytometry [9,10] and measuring concentrations of inflammatory mediators [11–15].

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Drawing from established neuroinflammatory disorders, we hypothesized that if neuroinflammation giving rise to autoantibodies was present, evidence of recruitment and proliferation of B cells in the CNS with possible dysregulation and skewing of T cell subsets would be found. This approach represents a paradigm shift in TS from the antibody to the B lymphocyte, which can act as an antigen-presenting cell, looking for direct evidence of B cell expansion. The aims of this study were to (1) determine the CSF and blood immunophenotype of well-characterized TS compared to non-inflammatory neurological disorders without TS [9,10]; (2) measure chemokines and other cytokines involved in recruiting B cells (CXCL13, CXCL10) [11,13] and maintaining an abnormal CNS niche (BAFF, APRIL) [16]; and (3) identify markers of intrathecal humoral involvement, such as oligoclonal immunoglobulin G (OCB) and IgG synthesis. Because some B cell mechanisms are T cell-dependent, we surveyed T cell subsets [9,10], including T helper type 1 (Th1) and Th2 cells as identified by intracellular staining of cytokines. Other chemokines included were CCL22, which preferentially attracts Th2 lymphocytes, and CCL19 and CCL21, both critically involved in antigen-engaged B cells, dendritic cells, and central memory T cells [14]. CSF B cell expansion and marked elevations of CSF BAFF, CXCL13, and CXCL10 have been found in anti-N-methyl-D-aspartate receptor encephalitis, multiple sclerosis, neuromyelitis optica, neuropsychiatric lupus, and paraneoplastic neurological disorders [9–15]. The present panel of markers has not been previously evaluated in TS.

2.3. Clinical assessments The principal investigator conducted a diagnostic interview in the company of parents (as informants) or separately (when teenagers) concerning tic disorder symptoms, associated symptoms, and psychosocial issues. Patients were examined off all medications for TS for one week: clonidine (patients 1–3), olanzapine (patient 3), methylphenidate and sertraline (patient 4), and risperidone (patient 5). The Tic Inventory of the Yale Global Tic Severity Scale (YGTSS) was administered by the co-investigator (E.D.T) [16]. Additional information concerning each of the YGTSS anchor points was obtained. The Children’s Yale-Brown Obsessive Compulsive Scale (CY-BOCS) was used to rate obsessions and compulsions [17]. 2.4. Controls Controls were age- and gender-matched from a cohort of 26 children with a variety of non-inflammatory neurological disorders, comprising ataxia, developmental delay, headaches, seizures, and non-TS movement disorders, who were already immunophenotyped in the same manner as part of diagnostic evaluations [9,15]. Control samples were collected on a regular basis throughout the study period. 2.5. Sample collection (lumbar puncture and blood drawing)

2. Materials and methods 2.1. Study design This was a cross-sectional, case-control study in a convenient sample. Five school-aged children with TS and waxing and waning of tics and neuropsychiatric symptoms in the context of GABHS were recruited through Dr. Pranzatelli’s Pediatric Movement Disorders Clinic at SIU School of Medicine (Springfield, IL). None had explosive onset of obsessive-compulsive disorder. Exclusion criteria were prior immunotherapy, other autoimmune disorders, non-streptococcal febrile illness or immunization within 1 month, contraindications to lumbar puncture, and other symptomatic causes of TS. Parents of subjects meeting inclusion/exclusion criteria signed informed consent and children also gave assent for this Institutional Review Board-approved protocol entitled ‘‘Immunophenotyping of Cerebrospinal Fluid Lymphocytes in Tourette Syndrome.” The clinical aspects of the study were conducted from 2004 to 2008, but chemokine/cytokine assays, included to strengthen the study, were performed on the banked samples from 2008 to 2013 as they became available in the laboratory of the principal investigator.

Lumbar puncture usually was performed the day following clinical assessments. Extra precautions to prevent traumatic lumbar puncture were taken [9]. The first 3 ml of CSF went for quantitative immunoglobulins, IgG synthesis rate, oligoclonal bands, cell count, and chemistries. An additional 8–10 ml was collected on ice for lymphocyte subset analysis and cytokine measurements. Blood for parallel studies also was drawn. TS and non-TS samples were handled in the same manner. 2.6. Flow cytometry/immunophenotyping Fresh CSF and blood were brought promptly to the flow cytometry lab, and lymphocyte subset analysis, using anti-CD45 (pan leukocyte) and anti-CD-14 (monocyte) for gating, was performed according to published methods [9]. To determine if the Th1 subset predominated and cytokine production was increased, we also intracellularly stained CSF CD4(+) T cells for IFN-c and IL-4 as prototypic Th1 and Th2 cytokines, respectively. Th1 cells were CD4(+) IFN-c(+); Th2 cells, CD4(+)IL-4(+). 2.7. Chemokine/cytokine assays and other tests

2.2. Screening Reference range values for serum streptococcal markers were used: anti-streptolysin O (ASO) < 200 and antideoxyribonuclease-B (ADN-B) < 1:170. Only throat cultures positive specifically for GABHS were included. Subjects were required to have at least 2 positive streptococcal markers. Patient 1 had pharyngitis with 2 negative throat cultures despite tic reemergence and labile mood. Patient 2 had a prior GABHS infection, elevated C-reactive protein of 6.4, ASO of 929, and required antibiotic retreatment before slow, partial, self-reported resolution of tics. Patient 3 had a previous ADN-B of 680, but normal ASO. Screening of 16 patients with TS revealed 44% positive for ASO, 71% for ADN-B, 15% for GABHS throat culture, and 21% reporting tic exacerbation with pharyngitis (see Supplementary table). ASO is elevated in 15% of pediatric controls [2].

All cytokine assays were performed in duplicate on batched CSF/ serum samples stored at 80 °C [15], with controls and TS on the same assay plates. BAFF, CXCL10, CXCL13, CCL19, CCL21, and CCL22 were measured using Quantikine human-specific enzymelinked immunosorbent assay kits (R & D Systems, Inc., Minneapolis, MN) as per the manufacturer. The assay sensitivity was 3–4 pg/ml for BAFF, 0.41–4.46 pg/ml for CXCL10, 2 pg/ml for CXCL13, 15.6 pg/ml for CCL19, 9.9 pg/ml for CCL21, and <62.5 pg/ml for CCL22. The kit for APRIL, purchased from Bender MedSystems, Inc. (Burlingame, CA), had an assay sensitivity of 0.4 ng/ml. For CSF studies, the intra-assay CV ranged from 3 to 7%. The inter-assay CV values were also acceptable (3–8.4%). Tests for OCB, which were measured by isoelectric focusing and immunofixation, and CSF and serum immunoglobulins were performed in the clinical lab.

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2.8. Data analysis Statistical comparisons of means were made between controls and TS by t tests using the GraphPad Prism statistical software program. The significance level was a = 0.05. 3. Results 3.1. Patient characteristics The clinical features are displayed in Table 1. Evaluation was performed an average of 4 years after tic onset age. The family history was positive for acute rheumatic fever in one, tics in two, and OCD in four. All subjects had elevated ADN-B titers and ASO, and 4 had both. Three (60%) had a positive throat culture for GABHS, each with positive streptococcal serology. Three subjects had all 3 positive streptococcal markers. On the YGTSC, the global score, which is the sum of scores for motor tics, phonic tics, and overall impairment, was 50. On the CY-BOCS, the rating for obsessions and compulsions were summated to a total score of 22, which fell within the test’s moderate range (16–23). 3.2. CSF and blood immunophenotype No statistically significant group differences in the mean (Table 2) or median frequency of CSF or blood B cells was found. The mean CSF/blood ratio of 0.04 for CD19(+) B cells was identical

in TS and controls. Individual CSF CD19(+) B cell frequencies for patients 1–5 were 1.5%, 0.52%, 0%, 0.27%, and 1.0%, respectively. CD20(+) B cell frequencies also were normal at 0.72%, 0%, 0%, 1.2%, and 0.57%. There were no significant group abnormalities in the percentages of CSF total conventional T cells, T helper/inducer or cytotoxic/suppressor T cells, unconventional T cells, NKT cells, or NK cells. The mean CSF/blood ratio and the frequency of helper/ inducer T cells stained for Th1 or Th2 intracellular cytokines did not differ significantly between groups. The relative and absolute pool of blood lymphocyte subsets were not significantly different between TS and controls. 3.3. Chemokines and other cytokines CSF CXCL10, CXCL13, CCL19, BAFF, and APRIL concentrations were within the control range, as were serum BAFF, APRIL, CCL21, and CCL22 concentrations. 3.4. Quantitative immunoglobulins and other tests No CSF or serum Ig concentrations fell outside the reference range, and there were no significant group differences. Patient 3 had one CSF oligoclonal band, which was reported as negative by the reference lab. IgG synthesis rate, IgG index, albumin index, CSF leukocyte count, protein, and glucose were uniformly normal, without evidence of a traumatic tap. Serum quantitative

Table 1 Clinical data. Subject

Gender Age (y) Tic onset age (y) Recent tic exacerbation Comorbid features ADD/ADHD Rage Sleep disturbance Mood problems Obsessions/compulsions Learning problems Family history Tics Strep. pharyngitis Acute rheumatic fever Obsessions/compulsions

Group total

1

2

3

4

5

F 6 3.5 +

M 12 5.5 +

M 12 10 +

M 10 8.5 +

F 11 7 +

+

+

+ +

+

+ + +

3:2 M/F 10 ± 2 6.9 ± 2 5 (100%)

+ + +

+ + +

+ + + + + +

+ +

+

+

+

+

2 5 1 4

+ +

4 3 2 4 4 5

(80%) (60%) (40%) (80%) (80%) (100%) (40%) (100%) (20%) (80%)

+

+ + + +

YGTSC Motor tics Phonic tics Overall impairment Global score

13 9 10 32

15 13 18 20

18 16 50 84

15 17 50 83

18 15 63 33

16 ± 2 14 ± 3 38 ± 23 50 ± 31

CY-BOCS Obsessions Compulsions Total scores

10 9 19

12 9 21

15 15 30

11 14 25

6 9 15

11 ± 3 11 ± 3 22 ± 6

Serology ASO (IU/mL) ADN-B titer

556a 480a

780a 340a

131 680a

238a 240a

3200a 480a

981 ± 1267a 444 ± 166a

+

+ +

Throat culture Positive for GABHS On antibiotics

+

ADD/ADHD = attention deficit disorder with or without hyperactivity. YGTSC = Yale Global Tic Severity Scale. CY-BOCS = Children’s Yale-Brown Obsessive Compulsive Scale. Symbols: += yes, = no. a Positive.

+

3 (60%) 1 (20%)

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M.R. Pranzatelli et al. / Cytokine 96 (2017) 49–53 Table 2 Immunological data. Markers

TS

Controls

CSF B cells (%) Pan-B Mature B Naïve Memory Activated ‘Fetal’/B1

CD3(-)CD19(+) CD19(+)CD20(+) CD19(+)CD27( )IgD(+) CD19(+)CD27(+) CD19(+)CD38(+) CD19(+)CD5(+)

0.67 ± 0.6 0.67 ± 0.5 0.33 ± 0.3 0.48 ± 0.6 1.3 ± 1.4 0

0.5 ± 0.8 0.4 ± 0.6 0.3 ± 0.7 2.0 ± 2.2 5.3 ± 6.3 0.2 ± 0.7

T cells (%) TCR ab Helper/inducer Suppressor/cytotoxic Natural killer-like (NKT) Gamma/delta Th1 Th2 Natural killer (NK) cells (%)

CD3(+)CD45(+) CD3(+)CD4(+) CD3(+)CD8(+) CD3(+)CD16/56(+) CD3(+)TCR-cd(+) CD4(+)IFN-c(+) CD4(+)IL-4(+) CD3(-)CD16/56(+)

89 ± 4 65 ± 4 24 ± 5 5.2 ± 3 4.4 ± 2 14 ± 8 19 ± 8 5.5 ± 2

90 ± 5 57 ± 17 23 ± 7 3.0 ± 2 5.3 ± 4 13 ± 12 12 ± 12 3.4 ± 3

90 ± 15 0.04 ± 0.07 19 ± 16

69 ± 26 0.01 ± 0.04 29 ± 28

162 ± 24 2.9 ± 5

150 ± 62 2.0 ± 3

2.1 ± 1 0.04 ± 0.02 0.19 ± 0.2

1.2 ± 0.04 0.03 ± 0.01 0.19 ± 0.2

IgG synthesis rate [0–8 mg/24 h] IgG index [0.26–0.66] Albumin index [0–9] Oligoclonal bands [<4] Total leukocytes (mm3)

<0.1 0.47 ± 0.2 3.6 ± 2 <4 0.9 ± 0.2

<0.1 0.63 ± 0.1 2.9 ± 2 <4 1.3 ± 1.3

CSF/blood ratios Total B cells Total T cells T helper/inducer T suppressor/cytotoxic NK cells

0.04 ± 0.04 1.3 ± 0.1 1.6 ± 0.3 1.0 ± 0.3 0.61 ± 0.4

0.04 ± 0.06 1.2 ± 0.6 1.1 ± 0.5 1.1 ± 0.6 0.4 ± 0.4

Blood/serum Blood cell frequencies Pan-B cells%

17 ± 6

20 ± 6

T cells% TCR-ab Helper/inducer Suppressor/cytotoxic Activated CD4/CD8 ratio NK cells%

69 ± 3 40 ± 6 24 ± 4 5.6 ± 5 1.8 ± 0.5 11 ± 7

61 ± 12 37 ± 7 20 ± 5 2.5 ± 1 2.0 ± 0.6 10 ± 6

Blood cell counts Total B cells Total T cells T helper/inducer cells T suppressor/cytotoxic cells NK cells T activated cells

491 ± 250 1860 ± 315 1100 ± 287 600 ± 118 330 ± 176 117 ± 79

664 ± 541 1798 ± 726 1093 ± 432 587 ± 261 304 ± 180 89 ± 89

Chemokines CCL21 (pg/mL) CCL22 (pg/mL)

454 ± 92 1223 ± 431

424 ± 143 1206 ± 475

Cytokines BAFF APRIL

1043 ± 151 2029 ± 998

1292 ± 212 3763 ± 2247

Chemokines CXCL10 (pg/mL) CXCL13 (pg/mL) CCL19 (pg/mL) Cytokines BAFF (pg/mL) APRIL (lg/mL) Quantitative Ig IgG [0.5–6.1 mg/dL] IgM [<0.10 mg/dL] IgA [0.15–0.60 mg/dL]

CD3(+)HLA-DR(+)

Data are means ± SD. Reference ranges are given in brackets. There were no statistically significant inter-group differences.

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immunoglobulins IgG, IgM, and IgA, and complete blood count with differential were within normal limits and without significant group differences (data not shown).

4. Discussion In this novel but exploratory study of TS with positive streptococcal markers, no consistent CSF lymphocyte subset, chemokine/ cytokine, or immunoglobulin abnormalities were found. None of the children had CSF pleocytosis, abnormal albumin index, elevated IgG synthesis rate, or an imbalance in proportions of intracellularly stained Th1 and Th2 T helper cells. The results demonstrate proof-of-concept, however, more general conclusions cannot be drawn from this sample size. This CSF neuroinflammation biomarker approach holds promise for larger studies of TS and related disorders. We found no pediatric CSF chemokine, immunophenotyping, or OCB studies in TS for comparison. In blood, regulatory CD4(+)CD25 (+)CD69( ) T cells were decreased in one study [6], and CD3(+), CD4(+) T cells, and the CD4/CD8 ratio were decreased in another [8]. In adults, the percentage of blood CD69(+)CD22(+) B cells and CD95(+)CD4(+) T cells was increased, but multiple other B and T subsets were not [7]. Elevations in plasma interleukin-2 (IL-2) and IL-12 were reported [5]. Positive CSF OCB were found in 29% of adult TS, but did not correlate with clinical data [18]. The strengths of our study include the unique application of lymphocyte immunophenotyping and chemokine/cytokine profiling to CSF in TS, and broadening the research scope beyond a focus on autoantibodies. A variety of methodologies were applied and performed at the same site by the same researchers. Multiple lymphocyte subsets were evaluated and cytokines were analyzed both by intracellular staining and paired CSF/serum analysis. Clinical phenotypes were well-characterized and control data were ample. To our knowledge, the CSF marker panel we used was not previously measured in TS. A limitation of the study was the small TS sample, hence, these results bear replication in a larger cohort. Subject accrual was hampered by the test invasiveness without clinical benefit to the individual. A different time of sampling, other immunological tests, and comparison of seronegative and seropositive TS may have yielded different results. Our chemokine/cytokine panel was not exhaustive, rather, hypothesis-driven. Multiplex cytokines panels could not be run because a much larger sample would be needed given their variability and low detectability in CSF. Finally, determination of immune cell function cannot be made exclusively from immune phenotypic studies. In conclusion, this proof-of-concept study presents new neuroimmunological data in children with TS and positive streptococcal markers. Neither positive throat cultures nor serology for GABHS was associated with evidence of neuroinflammation as assessed by the panel of CSF inflammatory mediators and immunophenotyping markers utilized. Such a direct approach should be applied to the study of TS and other putative autoimmune neurological disorders associated with streptococcal infections.

5. Study funding This study was funded by a research grant from the Tourette Syndrome Association, Inc. (Bayside, NY) to M.R.P., the principal investigator. The funding source had no role in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; or in the decision to submit the article for publication.

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Acknowledgements The authors thank the former Tourette Syndrome Association Scientific Advisory Board (Chair, Neal R. Swerdlow, MD, PhD) for funding the grant application; Sue Levi-Pearl and Heather Cowley, Ph.D., at the Tourette Syndrome Association for project extension and advertising for recruitment; Anna L. Travelstead, BS, MT (ASCP), for flow cytometry; Nathan R. McGee, BS, for chemokine/ cytokine assays, and all participating patients and families. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2017.03.003. References [1] A. Macerollo, D. Martino, Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS): an evolving concept, Tremor Other Hyperkinet Mov. (N Y) 3 (2013), http://dx.doi.org/10.7916/ D8ZC81M1. 2013 Sep 25, Pii: tre-03-167-4158-7. [2] A.J. Church, R.C. Dale, A.J. Lees, G. Giovannoni, M.M. Roberston, Tourette’s syndrome: a cross sectional study to examine the PANDAS hypothesis, J. Neurol. Neurosurg. Psychiatry 74 (5) (2003) 602–607. [3] J.J. Hallett, C.J. Harling-Berg, P.M. Knopf, E.G. Stopa, L.S. Kiessling, Anti-striatal antibodies in Tourette syndrome cause neuronal dysfunction, J. Neuroimmunol. 111 (2000) 195–202. [4] C.M. Morris, C. Pardo-Villamizar, C.D. Gause, H.S. Singer, Serum autoantibodies measured by immunofluorescence confirm a failure to differentiate PANDAS and Tourette syndrome from controls, J. Neurol. Sci. 276 (2009) 45–48. [5] V. Gabbay, B.J. Coffey, L.E. Guttman, L. Gottlieb, Y. Katz, J.S. Babb, M.M. Hamamoto, C.J. Gonzales, A cytokine study in children and adolescents with Tourette’s disorder, Prog. Neuropsychopharmacol. Biol. Psychiatry 33 (6) (2009) 967–971. [6] I. Kawikova, J.F. Leckman, H. Kronig, L. Katsovich, D.E. Bessen, M. Ghebremichael, A.L. Bothwell, Decreased numbers of regulatory T cells suggest impaired immune tolerance in children with Tourette syndrome: a preliminary study, Biol. Psychiatry 61 (2007) 273–278. [7] J.C. Möller, B. Tackenberg, M. Heinzel-Gutenbrunner, R. Burmester, W.H. Oertel, O. Bandmann, K.R. Müller-Vahl, Immunophenotyping in Tourette syndrome – a pilot study, Eur. J. Neurol. 15 (2008) 749–753. [8] E. Li, Y. Ruan, Q. Chen, X. Cui, L. Ly, P. Zheng, L. Wang, Streptococcal infection and immune response in children with Tourette’s syndrome, Childs Nerv. Syst. 31 (7) (2015) 1157–1163. [9] M.R. Pranzatelli, A.L. Travelstead, E.D. Tate, T.J. Allison, E.J. Moticka, D.N. Franz, M.A. Nigro, J.T. Parke, D.A. Stumpf, S.J. Verhulst, B- and T-cell markers in opsoclonus-myoclonus syndrome: immunophenotyping of CSF lymphocytes, Neurology 62 (2004) 1526–1532. [10] S. Alvermann, C. Hennig, O. Stüve, H. Wiendl, M. Stangel, Immunophenotyping of cerebrospinal fluid cells in multiple sclerosis: in search of biomarkers, JAMA Neurol. 71 (2014) 905–912. [11] M.R. Pranzatelli, E.D. Tate, Trends and tenets in relapsing and progressive pediatric opsoclonus-myoclonus, Brain Dev. 38 (5) (2016) 439–448. [12] Z. Liba, J. Kayserova, M. Elisak, P. Marusic, H. Nohejlova, J. Hanzalova, V. Komarek, A. Sedina, Anti-N-methyl-D-aspartate receptor encephalitis: the clinical course in light of the chemokine and cytokine levels in cerebrospinal fluid, J. Neuronflamm. 13 (1) (2016) 55, http://dx.doi.org/10.1186/s12974016-0507. [13] E. Alvarez, L. Piccio, R.J. Mikesell, E.C. Klawiter, B.J. Parks, R.T. Naismith, A.H. Cross, CXCL13 is a biomarker of inflammation in multiple sclerosis, neuromyelitis optica, and other neurological conditions, Mult. Scler. 19 (9) (2013) 1204–1208. [14] M.R. Pranzatelli, N.R. McGee, Z.Y. Wang, B.K. Agrawal, Characteristics and pharmacodynamics of severe neuroinflammation in a child with neurolupus, Neurol.: Neuroimmunol. Neuroinflamm. 4 (2017) e316, http://dx.doi.org/ 10.1212/NXI.0000000000000316. [15] M.R. Pranzatelli, E.D. Tate, N.R. McGee, A.L. Travelstead, J.A. Colliver, J.M. Ness, R.M. Ransohoff, BAFF/APRIL system in pediatric OMS: relation to severity, neuroinflammation, and immunotherapy, J. Neuroinflamm. 10 (2013) 10, http://dx.doi.org/10.1186/1742-2094-10-10. [16] J.F. Leckman, M.A. Riddle, M.T. Hardin, S.I. Ort, K.L. Swartz, J. Stevenson, D.J. Cohen, The Yale global tic severity scale: initial testing of a clinician-rated scale of tic severity, J. Am. Acad. Child Adolesc. Psychiatry 28 (1989) 566–573. [17] L. Scahill, M.A. Riddfle, M. McSwiggin-Hardin, S.I. Ort, R.A. King, W.K. Goodman, D. Cicchetti, J.F. Leckman, Children’s Yale-Brown Obsessive Compulsive Scale: reliability and validity, J. Am. Acad. Child Adolesc. Psychiatry 36 (1997) 844–852. [18] C. Wenzel, U. Wurster, K.R. Müller-Vahl, Oligoclonal bands in cerebrospinal fluid in patients with Tourette’s syndrome, Mov. Disord. 26 (2) (2011) 343– 346.