Published online June 2, 2008
PEDIATRICS Vol. 121 No. 6 June 2008, pp. 1198-1205 (doi:10.1542/peds.2007-2658)
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My File Cabinet
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singer, H. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singer, H. S.
Related Collections
Right arrow Infectious Disease & Immunity
Right arrowRelated AAP Red Book topics:
Non-Group A or B Streptococcal and...
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

ARTICLE

Serial Immune Markers Do Not Correlate With Clinical Exacerbations in Pediatric Autoimmune Neuropsychiatric Disorders Associated With Streptococcal Infections

Harvey S. Singer, MDa, Colin Gause, BSa, Christina Morris, MSa, Pablo Lopez, PhDb and the Tourette Syndrome Study Group

Departments of a Neurology
b Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVE. Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections is hypothesized to be a poststreptococcal autoimmune disorder. If clinical exacerbations are triggered by a streptococcal infection that activates cross-reacting antibodies against neuronal tissue or alters the production of cytokines, then a longitudinal analysis would be expected to identify a correlation between clinical symptoms and a change in autoimmune markers.

PATIENTS AND METHODS. Serial serum samples were available on 12 children with pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections participating in a prospective blinded study: 2 samples before an exacerbation point, 1 during the clinical exacerbation, and 2 after the exacerbation. Six subjects had a well-defined clinical exacerbation in association with a documented streptococcal infection, and 6 had a clinical exacerbation without an associated streptococcal infection. All of the serum samples were assayed for antibodies against human postmortem caudate, putamen, and prefrontal cortex; commercially prepared antigens; and complex sugars. Cytokines were measured by 2 different methodologies.

RESULTS. No correlation was identified between clinical exacerbations and autoimmune markers, including: enzyme-linked immunosorbent assay measures of antineuronal antibodies; Western immunoblotting with emphasis on brain region proteins located at 40, 45, and 60 kDa or their corresponding identified antigens; competitive inhibition enzyme-linked immunosorbent assay to evaluate lysoganglioside GM1 antibodies; and measures of inflammatory cytokines. No differences were identified between individuals with pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections with or without exacerbations triggered by streptococcal infections.

CONCLUSIONS. The failure of immune markers to correlate with clinical exacerbations in children with pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections raises serious concerns about the viability of autoimmunity as a pathophysiological mechanism in this disorder.

Key Words: PANDAS • longitudinal analysis • antineuronal antibodies • cytokines • lysoganglioside GM1

Abbreviations: PANDAS—pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections • GABHS—group A β-hemolytic streptococci • ELISA—enzyme-linked immunosorbent assay • ExWS—exacerbation with streptococcal infection • ExWOS—exacerbation without streptococcal infection • BA10—Brodmann's area 10 • PBS—phosphate-buffered saline • Ig—immunoglobulin • TBS-T—Tris-buffered saline containing 0.1% Tween 20 • BSA—bovine serum albumin • GlcNAc—N-acetyl-β-D-glucosamine • Th—T-helper • IL—interleukin • TNF—tumor necrosis factor • MCP—macrophage chemoattractant protein • RANTES—regulated upon activation, normal T cell expressed and secreted • AUC—area under the curve

Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) is characterized by recurrent, acute fulminant tics and/or obsessive-compulsive behaviors that are temporally associated with a streptococcal infection.1,2 The existence of this disorder has generated considerable clinical and scientific interest, as well as controversy.1–7 Pathophysiologically, PANDAS is proposed to be a poststreptococcal autoimmune disorder similar to that of the hallmark entity Sydenham chorea. More specifically, it is hypothesized that tics and obsessive compulsive symptomatology result from group A β-hemolytic streptococci (GABHS) activation of the adaptive immune system, either by the induction of antibodies that cross-react against neuronal tissue (molecular mimicry) or by the production of secreted proteins that mediate and regulate immunity and inflammation (cytokines and chemokines).8 Thus, to confirm an autoimmune etiology in PANDAS, investigators have been actively seeking to identify abnormalities of immune markers.

Several studies have measured serum antineuronal antibodies in children with PANDAS, usually on serum samples unrelated to the presence of a GABHS infection or the timing of clinical exacerbations.9–12 Results in these studies have been conflicting. Several investigators have suggested, based on enzyme-linked immunosorbent assay (ELISA) or immunofluorescent histochemical staining, that this cohort is readily differentiated from a variety of disease controls.9,10 In addition, Western immunoblot analysis of serum antibodies in poststreptococcal subjects has identified significantly more reactive bands at 60, 45, and 40 kDa.9 In contrast, other researchers, using ELISA and immunoblotting against a variety of brain epitopes, were unable to distinguish PANDAS subjects from children with Tourette syndrome or control subjects.11,12 In these studies, immunoreactivity suggested no diagnostic specificity at previously reported molecular weights nor to their putative antigenic proteins: pyruvate kinase M1, {alpha}- and {gamma}-enolase, and aldolase C. Furthermore, the failure to identify differences in immune testing in PANDAS subjects after preabsorption of sera with streptococci has suggested that there is no evidence for molecular mimicry.12

The availability of serial serum samples from subjects with PANDAS, prospectively evaluated by experienced experts, using standardized evaluations plus periodic laboratory testing for markers of GABHS over a 2-year period, provides a unique opportunity to evaluate longitudinal markers of autoimmunity. Identification that antibody reactivity or cytokine levels correlate with documented clinical exacerbations, occurring within 5 weeks of a GABHS infection, would provide definitive evidence in support of an autoimmune pathophysiologic hypothesis. In a desire to be comprehensive, the presence of autoantibodies in PANDAS sera was sought by several methodologies, including ELISA and Western immunoblotting with supernatant fractions from homogenized human postmortem caudate, putamen, and prefrontal cortex, as well as the use of commercially available antigens for the putative antigens pyruvate kinase M1, {alpha}- and {gamma}-enolase, and aldolase C.13 Furthermore, recognizing that recent reports have suggested that immune epitopes could involve complex sugars rather than brain proteins 14–16 or the excess release of cytokines,17,18 investigations were included to evaluate these possibilities. We hypothesized that, if the clinical exacerbation in PANDAS is associated with an autoimmune factor, with or without a streptococcal trigger, then the appropriate marker would increase during a pre-exacerbation period, peak at the time of clinical exacerbation, and then gradually decline.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Subjects
Serial serum samples were available from 12 children with the diagnosis of PANDAS (mean age: 11.4 years; 8 boys and 4 girls). All of the disease subjects met the diagnostic clinical criteria for PANDAS from Swedo et al2 and were participants in a longitudinal Tourette Syndrome Study Group multicenter study described in detail in an accompanying article. The 12 participants in this immune analysis included 6 children with an association between a streptococcal infection and clinical exacerbation (exacerbation with streptococcal infection [ExWS]): 4 "definite" and 2 "probable." A definite GABHS infection required the presence of 3 criteria including a new M/emm streptococcal typing, a 0.2 log rise in antistreptolysin O and/or antideoxyribonuclease B titer, and clinical symptoms (fever, sore throat, pharyngeal erythema, or exudates). A probable GABHS infection had 2 positive criteria. The comparison group consisted of 6 children with PANDAS who had a clinical exacerbation having no association with a GABHS infection (ExWOS). An exacerbation was declared when the site clinical expert determined that the subject experienced a significant worsening of tics or obsessive-compulsive disorder that lasted for ≥5 days and was not related to an alteration of prescribed medications. Demographic information on each cohort is provided in Table 1.


View this table:
[in this window]
[in a new window]

  		TABLE 1 Subject Data

 
Five serial serum samples were available on each subject, 2 before the exacerbation point (samples 1 and 2), 1 during the clinical exacerbation (sample 3), and 2 after the exacerbation (samples 4 and 5). In all of the analyses for each subject, samples 1 through 5 were assayed simultaneously to reduce the possibility of interassay variability. Laboratory personnel were unaware of the subject grouping, and all of the analyses were completed before diagnostic codes were made available.

Tissue Preparation
Fresh, nonfrozen, human caudate, putamen, and prefrontal Brodmann's area 10 (BA10) were obtained at autopsy from a 76-year-old man who died of a cardiac problem and had no evidence of neurologic disease. The postmortem interval was unknown. A supernatant (S1) fraction for each region was obtained by homogenizing the tissue in 0.9% normal saline (2.5 g of tissue per 10 mL of saline) containing protease inhibitors (1 µg/mL of aprotinin, 10 µg/mL of leupeptin, 10 µg/mL of pepstatin, and 1 mM of phenylmethylsulfonyl fluoride) in a Teflon-glass homogenizer on ice. Homogenized tissue was centrifuged for 30 minutes at 12000 x g. The supernatant fraction was collected, and aliquots were stored at –80°C. Protein concentrations were measured by the bicinchoninic acid method (Pierce, Rockford, IL).

ELISA
ELISA assays were performed on all of the serum samples with use of tissue protein from caudate, putamen, and BA10 using a previously described methodology.12 Assays were performed in sterile 96-well microtiter ELISA plates to which 150 µL of protein (2.5 µg/mL) were added. The plates were incubated overnight at 4°C and then rinsed with washing buffer (0.05% Triton X-100 in phosphate-buffered saline [PBS]). To block the plates, 5% nonfat Carnation dry milk in PBS was added to each well, and the plates were then maintained for 90 minutes at room temperature. Plates were rinsed in washing buffer and then exposed to serum (diluted 1:250) for 90 minutes at room temperature with agitation. Goat anti-human immunoglobulin G (IgG) conjugated with horseradish peroxidase (Amersham International, Arlington Heights, IL) diluted 1:3000 in PBS was used as the secondary antibody. The amount of antibody-antigen interaction was detected using a tetramethylbenzidine kit purchased from Vector Laboratories (Burlingame, CA). Freshly prepared tetramethylbenzidine solution (tetramethylbenzidine with buffer and hydrogen peroxide) was incubated on the ELISA plate for 5 minutes, followed by the addition of H3PO4. Immediately after tetramethylbenzidine exposure, each plate was read at optical density 450 nm on an automated Microplate Reader (Bio-Rad, Hercules, CA). All of the samples were assayed in triplicate.

Western Immunoblotting
Immunoblotting using caudate, putamen, and BA10 tissue was performed on all of the longitudinal samples from each subject. All of the serial samples from a specific individual were assayed on the same gel following methodology reported previously.12 Thirty µg of brain protein per sample were denatured at 100°C for 5 minutes, electrophoresed in 10% acrylamide ready gels (Bio-Rad), and transferred to 0.45 µm of nitrocellulose at 100 V (Schleicher and Schuell Protran, Dassel, Germany) for 80 minutes. The nitrocellulose was incubated overnight at 4°C in blocking solution containing 5% Carnation nonfat milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). The nitrocellulose was washed 3 times in TBS-T for 10 minutes per wash and then exposed to serum (diluted 1:500) for 90 minutes at room temperature with agitation. The nitrocellulose was washed and then exposed to secondary antibody, horseradish peroxidase-conjugated sheep anti-human IgG (Amersham Biosciences, Piscataway, NJ) diluted 1:3000, and agitated for 60 minutes. After washing 3 times with TBS-T for 10 minutes per wash, the membranes were developed with Amersham electrochemiluminescence reagents according to the vendor protocol. The blots were exposed to Denville Blue Bio Films (Denville Scientific, Metuchen, NJ) for a timed 60-second period. Molecular weights were estimated for each band on the basis of the distance migrated for 8 known molecular weight standards (Bio-Rad). After the primary analysis, the nitrocellulose blot was then stripped of antibodies and reassayed for actin content as described by Yoon et al.19 Digital image analysis and evaluation of Western blots were performed using Quantity One software (Bio-Rad), which created densitometric data of the blots showing the gray-intensity values (8-bit gray values) versus retardation factor values. For all of the bands on each blot, Quantity One generated a peak for each band, assigned each peak a molecular weight, and determined the peak height and area under the curve.

For immunoblotting using specific epitopes, in the place of brain protein, 1 µg of pyruvate kinase M1 (United States Biochemical, Cleveland, OH), {alpha}-enolase (US Biological, Swampscott, MA), {gamma}-enolase (Polysciences, Inc, Warrington, PA), and aldolase C (United States Biochemical) were electrophoresed along with a constant volume of actin (US Biological), transferred to nitrocellulose, blocked overnight, and then exposed to serum (diluted 1:500) for 90 minutes at room temperature, with agitation.12 After washing with TBS-T, the membranes were developed with Amersham electrochemiluminescence reagents as described above, stripped, and reassayed for actin content. Serial samples from each individual subject were assayed on the same gel.

ELISA Based Anti-Ganglioside Assays
IgG and IgM binding to lysoganglioside GM1 and the GM1 ganglioside were performed on all of the samples using published methodology.20 Stock specific ganglioside (Sigma-Aldrich, St Louis, MO) was evaporated under nitrogen and resuspended in a combination of ethanol, phosphatidylcholine-cholesterol solution (100 µM phosphatidylcholine and 400 µM cholesterol), and distilled water. Each well, in prepared microplates, received 200 pmol of ganglioside. Blanks received no ganglioside. Microplates were stored overnight at 4°C, and decanted. Blocking solution (1 mg/mL of bovine serum albumin [BSA] in Dulbecco's PBS), 200 µL per well, was added and incubated at 37°C for 30 minutes. After washing, 50 µL per well of a 1:500 dilution of serum in PBS-BSA were added, incubated for 90 minutes at room temperature, and washed 3 times in PBS. Secondary antibody, alkaline phosphatase-conjugated rabbit anti-human IgG or goat anti-human IgM (Jackson ImmunoResearch, West Grove, PA) diluted 1:500 in PBS-BSA, was added to each microplate. After incubation for 45 minutes at room temperature and washing with distilled water, plates were developed with a 2-mg/mL solution of p-nitrophenylphosphate in ELISA developing buffer (pH 9.5; 100 mM Tris, 100 mM NaCl, and 5 mM MgCl2). Optical density values were measured at 405 nm after 30 minutes using a model 680 microplate reader (Bio-Rad). All of the assays were run simultaneously with a known positive control sample.

N-Acetyl-β-D-Glucosamine-Lysoganglioside GM1 Competitive-Inhibition Assay
The competitive-inhibition ELISA was performed in triplicate using sera from time points 1 to 5 from all of the subjects, following the methodology of Galvin.21 N-acetyl-β-D-glucosamine (GlcNAc)-BSA, purchased from Dextra Laboratories (Reading, United Kingdom), was immobilized on the ELISA plate, and lysoganglioside GM1 (Sigma Chemical, St Louis, MO) was used to inhibit the serum IgG reactivity with GlcNAc. Lysoganglioside GM1 inhibitor solution was prepared in PBS and mixed with an equal volume of patient serum, then incubated at 37°C for 1 hour and overnight at 4°C. Fifty µL of serum-inhibitor mixture were added to wells coated with GlcNAc-BSA (10 µg/mL). Plates were developed with alkaline phosphatase-labeled goat anti-human IgG (1:500; Sigma Chemical) and p-nitrophenyl phosphate (Sigma 104 phosphatase substrate), prepared in diethanolamine buffer. Optical density was measured at 405 nm using a Microplate Reader 680 (Bio-Rad). The percentage of inhibition was calculated as follows: 100 x [1 – (A405 inhibitor + serum/A405 PBS + serum)]. Maximal (100%) reactivity was determined by incubating serum with PBS without inhibitor.

Cytokines
Assays were initially performed in duplicate using a Beadlyte multicytokine detection kit (Upstate Biological, Charlottesville, VA) according to vendor protocol on all of the subjects using sera from time points 2 to 4. Analyses included T-helper (Th) 1 cytokines (interferon-{gamma}) and interleukin (IL)-12, CD8+ releasing cytokines interferon-{gamma} and tumor necrosis factor (TNF)-{alpha}, Th2 cytokines (IL-4, IL-5, IL-6, IL-10, and IL-13), the immunomodulatory cytokine IL-1β, and chemokines (macrophage chemoattractant protein [MCP]-1 and RANTES (CCL5). After initial assays were performed, samples 1–5 were sent to Pierce Biotechnology, Inc (Woburn, MA) for Searchlight testing, a commercial analysis of IL-4, IL-10, IL-12, and TNF{alpha} using an ELISA-based protocol.

Statistical Analyses
Statistical analyses were performed using SPSS 12.0 software (SPSS Inc, Chicago, IL), Microsoft Excel (Redmond, WA) statistical tools, and the Mann-Whitney rank-sum test to test the null hypothesis that antineuronal antibody profiles, antiganglioside profiles, and measurements of cytokines would be similar across the 2 diagnostic groups: ExWS and ExWOS. Optical density readings for caudate, putamen, BA10, and gangliosides were analyzed as continuous variables and compared by both a Mann-Whitney rank-sum test and a 2-tailed t test. Specific ELISA optical density values (total reading minus tissue blank) were determined for each sample before statistical analysis. Western blot analyses provided information regarding the total number of peaks, peak height, and the area under each peak (area under the curve [AUC]). These data were treated as continuous. In addition to the aforementioned statistics, comparisons between groups for antineuronal and antiganglioside antibodies, antibodies against specific antigens, and cytokine measurements were also analyzed for differences in slope. Slope is the measurement of incline as defined by the change in the y coordinate divided by the corresponding change in the x coordinate, representing values at time points 1-2-3 and 3-4-5.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Serum Measurement Time Points
The exacerbation sample (3) was typically drawn within 2 weeks of clinical exacerbation and is herein designated "time 0." The mean timing of the pre-exacerbation (1 and 2) and postexacerbation samples (4 and 5) are shown in Fig 1. There was no significant difference in the timing of blood draws between the ExWS and ExWOS clinical groups.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 1 Number of days (mean ± SD) from the exacerbation time point for the ExWS and ExWOS groups.

 
ELISA
Median (interquartile range) optical density readings for serum antibodies directed against postmortem epitopes from tissue supernatant preparations from caudate, putamen, and BA10 are presented in Table 2. In no instance was there a significant difference among the groups at any specific time point or when analyzed as slopes between time points 1 to 3 and 3 to 5.


View this table:
[in this window]
[in a new window]

  		TABLE 2 Median ELISA Values at Exacerbation (Sample 3) and Surrounding Time Points

 
Immunoblots With Brain Proteins
Western blot analyses on postmortem tissues from the caudate, putamen, and frontal cortex showed no molecular weight reactivity exclusive to either the ExWS or ExWOS group. The number of individuals with bands identified at 60, 45, and 40 kDa at the exacerbation time point is shown in Table 3. There was no clear trend in either group among the number of bands at pre-exacerbation, exacerbation, and postexacerbation times. Mean analysis of reactive band peak heights and AUC at these molecular weights, evaluated both as measures of density and slope, showed no significant trends (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 3 Number of Individuals in Each Group With Reactive Bands Identified at the Exacerbation Point Against Brain Region Proteins Located at 40, 45, and 60 kDa and Against Corresponding Antigens

 
Immunoblots Against Specific Epitopes
The numbers of subjects with reactive bands identified against pyruvate kinase M1, nonneuronal {alpha}-enolase, and aldolase C, are shown in Table 3. Greater reactivity was observed at sites of interest using specific antigens as compared with that detected by homogenized brain protein. No PANDAS sera had antibodies against neuronal ({gamma}) enolase. At each single time point, average peak height and AUC showed no difference between the ExWS and ExWOS groups. Comparisons of peak height slopes for time points 1 to 3 and 3 to 5, presented in Fig 2, failed to show rise to or fall from the exacerbation point.


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 2 Slopes of peak height measurements for specific antigens pyruvate kinase, nonneuronal ({alpha}) enolase, and aldolase C in PANDAS subgroups. No group showed an increase in antibody levels (density) from pre-exacerbation to the exacerbation point or a decline from the exacerbation point to postexacerbation times.

 
Gangliosides
Serum IgG and IgM antibody reactivity against ganglioside GM1 and lysoganglioside GM1 were evaluated in all of the subjects at the 5 time points. No optical density value attained the requirement for positivity, that is, twice blank. For comparison, a positive control sample attained values of 2.8 times blank for lysoganglioside GM1 and 8.4 times blank for ganglioside GM1.

GlcNAc Competitive Inhibition
A competitive-inhibition ELISA was used to determine whether PANDAS serum IgG reacted with lysoganglioside GM1 and GlcNAc at time points 1 to 5. Seven of 12 PANDAS subjects had serum binding to GlcNAc-BSA inhibited by soluble lysoganglioside GM1 at a level <500 µg/mL15 at any point in time. Only 1 subject, a member of the ExWS group, had a reduced requirement of lysoganglioside GM1 to produce 50% inhibition of anti-GlcNAc IgG reactivity at the exacerbation point as compared with pre-exacerbation and postexacerbation levels (Table 4).


View this table:
[in this window]
[in a new window]

  		TABLE 4 Competitive Inhibition ELISA in PANDAS Serum

 
Cytokines
Analysis of serum cytokines using the Beadlyte methodology showed that most values fell below the level of detection except for MCP-1 and RANTES (Table 5). In rare instances where individual values were detectable, no longitudinal trends were appreciated (data not shown). Serial measures of IL-4, IL-10, IL-12, and TNF{alpha} as determined commercially by Searchlight are presented in Table 5. The latter were within the provided reference ranges and showed no consistent correlation with clinical exacerbation.


View this table:
[in this window]
[in a new window]

  		TABLE 5 Median Serum Concentration of Inflammatory Cytokines

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
PANDAS is proposed as an autoimmune disorder in which the host's natural defense mechanisms, triggered by GABHS, are directed against normal host neurons. If bacteria are not immediately eliminated by the innate immune response, antigenic fragments are presented to cognate CD4+ T cells triggering their differentiation into either Th1 or Th2 cells, thus initiating the adaptive immune response. Activated Th1 cells secrete cytokines IL-2 and IFN-{gamma}, which work to activate macrophages and cytotoxic T cells. The cellular immune system is further enhanced by factors that stimulate acute phase reactions (TNF-{alpha}) and serve as chemotactic agents for T cells (RANTES) and macrophages (MCP-1). Activated helper Th2 cells secrete cytokines IL-4, IL-5, IL-6, and IL-10 and also trigger the proliferation and differentiation of B cells and plasma cells that secrete immunoglobulins (humoral system). If 2 antigens share an identical epitope or if 2 different epitopes have similar shapes and charges, an antibody produced against a GABHS epitope could cross-react with neuronal tissue through the process of molecular mimicry.

Whether PANDAS is an autoimmune disorder associated with antineuronal antibodies is controversial. ELISA and immunoblotting studies in single-point-in-time studies have been inconsistent in their ability to identify the presence of specific antibodies.9–12 Hence, longitudinal studies provide a unique opportunity to evaluate the association between immune factors and changes in clinical symptomatology. In this report, serial serum samples were available from a prospective, multicenter, blinded, controlled, intensive clinical and laboratory cohort study that identified children with PANDAS who had clinical exacerbations associated with GABHS and others with an exacerbation unassociated with streptococcal infection. Our results do confirm the presence of autoantibodies in children with PANDAS, but autoantibodies have been identified in healthy individuals12,22 and have been speculated to provide important regulatory functions, such as tissue repair and neuroprotection.23,24 More important, however, is the inability to identify an association between clinical worsening or a GABHS infection (culture, antistreptococcal titers) and quantitative measures of autoantibodies obtained by the use of 3 separate methodologies: ELISA, immunoblotting against 3 brain regions as well as putative specific antigens, and competitive inhibition with lysoganglioside. This study is, therefore, consistent with the findings of others who have shown that antineuronal antibodies at 40, 45, and 60 kDa detected against basal ganglia do not predict a specific tic phenotype, phenomenology, severity, or duration.25 Discrepancies from studies defining specific differences in autoantibodies in PANDAS could, in part, be related to variations in methodologies.26

Although most investigators have focused on antibodies against brain proteins, in Sydenham's chorea, a monoclonal antibody has been shown to have specificity for GlcNAc, a streptococcal surface antigen, and mammalian lysoganglioside GM1 and GM1 ganglioside.14 In our study, 58% of subjects with PANDAS had a positive competitive-inhibition ELISA assay (serum IgG reacted with lysoganglioside GM1 and GlcNAc) in ≥1 serum sample, consistent with a previous report.15 Only 1 subject, however, showed an alteration of binding (lower concentration of lysoganglioside GM1 required to produce 50% inhibition of anti-GlcNAc reactivity) that correlated with a clinical exacerbation. Our analyses did not include measures of calcium-calmodulin-dependent protein kinase II activity, hypothesized to be activated by antibodies in Sydenham chorea and, to a lesser extent, with PANDAS sera.15

In addition to antibodies against brain proteins or gangliosides, pathophysiological mechanisms in PANDAS might also involve immune abnormalities associated with cytokines18 or lymphocyte dysfunction.27 Cytokines IL-4 and IL-10 are elevated in the cerebrospinal fluid of ~30% of patients with acute Sydenham chorea,17 and IL-12 and TNF-{alpha} are elevated in the sera of patients with tics at baseline as compared with control subjects.18 Serial analyses of cytokines in our study showed no meaningful differences between PANDAS groups, with or without exacerbation associated with streptococci, and no definite increases during exacerbations. These results differ from a study that has suggested increased levels of IL-12 and TNF-{alpha} during clinical exacerbations in patients with Tourette syndrome, primarily in those individuals with exacerbations unrelated to the presence of infection.18 Concerns about the use of serum cytokine measurements as predictors of activity in the central nervous system are raised by their inconsistent and highly variable levels, as well as discrepancies between cytokine measurements in cerebrospinal fluid and serum.28 A prospective longitudinal study using flow cytometry to quantify B lymphocytes expressing a known marker for GABHS infection (D8/17) found no clear relationship between new GABHS infections or amplification of B lymphocyte populations relevant to GABHS infections and tic symptom exacerbations.29

This study has attempted to confirm the validity of proposed biological mechanisms for PANDAS using serial serum samples from well-characterized patients. Our findings do not support the role of autoantibody or cytokine abnormalities in PANDAS, irrespective of the issue of a GABHS trigger. We emphasize that substantiation of an autoimmune disorder requires experimental confirmation in 5 areas: the identification of autoantibodies, the presence of immunoglobulins at the pathologic site, a positive response to immunomodulatory therapy, the induction of symptoms with autoantigens, and an ability to passively transfer the disorder to animal models.24

This study has several limitations including a small number of subjects, the use of tissue antigens from only selected brain regions located within a proposed pathologic fronto-striatal-thalamo-cortical circuit,30–32 the lack of a healthy control or well-established autoimmune group, and the failure to include other specific epitopes, such as tubulin32 or measures of regulatory T cells or immunohistochemical studies. The small number of available samples is explained in an accompanying article, which showed that only 5 of 64 exacerbations in 40 subjects with PANDAS were temporally associated (within 4 weeks) with GABHS. Immunofluorescent histochemical studies on frozen tissue were excluded based on a single-point-in-time study that failed to identify statistical differences between control and PANDAS subjects (Morris et al, unpublished data). Nevertheless, we believe that the use of serial serum samples from patients with PANDAS with rigorously characterized clinical symptomatology and carefully documented streptococcal infections makes this a valuable report.


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In summary, taken in conjunction with reports demonstrating a lack of specificity of antibodies in single-point-in-time studies,11,12 methodologic concerns about the single immunomodulatory treatment study in PANDAS,33,34 and the failure of microinfused PANDAS sera into ventral and ventrolateral rodent striatum to produce changes in animal behavior,35 our serial antibody results raise serious concerns about the role of autoimmunity in PANDAS and the use of immunomodulatory therapies.


   ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health grants MH61940 and NS42240. We thank the faculty and staff of the Tourette Syndrome Study Group under the leadership of Roger Kurlan, MD, for providing the samples used in this study and Edward Kaplan, MD, and Dwight Johnson for their contributions.


   FOOTNOTES
 
Accepted Nov 12, 2007.

Address correspondence to Harvey S. Singer, MD, Division of Pediatric Neurology, Johns Hopkins Hospital, Rubenstein Child Health Building, Suite 2158, 200 N Wolfe St, Baltimore, MD 21287. E-mail: hsinger{at}jhmi.edu

The authors have indicated they have no financial relationships relevant to this article to disclose.


What's Known on This Subject

PANDAS, an acronym for pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections, is proposed to be an autoimmune disorder. Antibody reactivity in PANDAS assessed by single-point-in-time studies has been controversial.

 

What This Study Adds

This study provides a longitudinal assessment of antineuronal and lysoganglioside antibodies and cytokines in patients with PANDAS. Results compare changes in patients with exacerbations with and without temporally associated streptococcal infections.

 


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Snider LA, Swedo SE. Post-streptococcal autoimmune disorders of the central nervous system. Curr Opin Neurol. 2003;16 (3):359 –365[CrossRef][Web of Science][Medline]
  2. Swedo SE, Leonard HL, Garvey M, et al. Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections: clinical description of the first 50 cases. Am J Psychiatry. 1998;155 (2):264 –271[Abstract/Free Full Text]
  3. Kurlan R. Tourette's syndrome and ‘PANDAS’: will the relation bear out? Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection. Neurology. 1998;50 (6):1530 –1534[Abstract/Free Full Text]
  4. Kurlan R, Kaplan EL. The pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS) etiology for tics and obsessive-compulsive symptoms: hypothesis or entity? Practical considerations for the clinician. Pediatrics. 2004;113 (4):883 –886[Abstract/Free Full Text]
  5. Kurlan R. The PANDAS hypothesis: losing its bite? Mov Disord. 2004;19 (4):371 –374[CrossRef][Web of Science][Medline]
  6. Singer HS, Loiselle C. PANDAS: a commentary. J Psychosom Res. 2003;55 (1):31 –39[CrossRef][Web of Science][Medline]
  7. Pavone P, Parano E, Rizzo R, Trifiletti RR. Autoimmune neuropsychiatric disorders associated with streptococcal infection: Sydenham chorea, PANDAS, and PANDAS variants. J Child Neurol. 2006;21 (9):727 –736[Abstract/Free Full Text]
  8. Harris K, Singer HS. Tic disorders: neural circuits, neurochemistry, and neuroimmunology. J Child Neurol. 2006;21 (8):678 –689[Abstract/Free Full Text]
  9. Church AJ, Dale RC, Giovannoni G. Anti-basal ganglia antibodies: a possible diagnostic utility in idiopathic movement disorders? Arch Dis Child. 2004;89 (7):611 –614[Abstract/Free Full Text]
  10. Pavone P, Bianchini R, Parano E, et al. Anti-brain antibodies in PANDAS versus uncomplicated streptococcal infection. Pediatr Neurol. 2004;30 (2):107 –110[CrossRef][Web of Science][Medline]
  11. Singer HS, Loiselle CR, Lee O, Minzer K, Swedo S, Grus FH. Anti-basal ganglia antibodies in PANDAS. Mov Disord. 2004;19 (4):406 –415[CrossRef][Web of Science][Medline]
  12. Singer HS, Hong JJ, Yoon DY, Williams PN. Serum autoantibodies do not differentiate PANDAS and Tourette syndrome from controls. Neurology. 2005;65 (11):1701 –1707[Abstract/Free Full Text]
  13. Dale R, Candler P, Church AR, Pocock JG. Glycolytic enzymes on neuronal membranes are candidate auto-antigens in post-streptococcal neuropsychiatric disorders. Mov Disord. 2004;19 (suppl 9):S33 .
  14. Kirvan CA, Swedo SE, Heuser JS, Cunningham MW. Mimicry and autoantibody-mediated neuronal cell signaling in Sydenham chorea. Nat Med. 2003;9 (7):914 –920[CrossRef][Web of Science][Medline]
  15. Kirvan CA, Swedo SE, Snider LA, Cunningham MW. Antibody-mediated neuronal cell signaling in behavior and movement disorders. J Neuroimmunol. 2006;179 (1–2):173 –179[CrossRef][Web of Science][Medline]
  16. Kirvan CA, Swedo SE, Kurahara D, Cunningham MW. Streptococcal mimicry and antibody-mediated cell signaling in the pathogenesis of Sydenham's chorea. Autoimmunity. 2006;39 (1):21 –29[CrossRef][Web of Science][Medline]
  17. Church AJ, Dale RC, Cardoso F, et al. CSF and serum immune parameters in Sydenham's chorea: evidence of an autoimmune syndrome? J Neuroimmunol. 2003;136 (1–2):149 –153[CrossRef][Web of Science][Medline]
  18. Leckman JF, Katsovich L, Kawikova I, et al. Increased serum levels of interleukin-12 and tumor necrosis factor-alpha in Tourette's syndrome. Biol Psychiatry. 2005;57 (6):667 –673[CrossRef][Web of Science][Medline]
  19. Yoon DY, Gause CD, Leckman JF, Singer HS. Abnormal dopaminergic neurotransmission in Tourette syndrome: a postmortem analysis. J Neurol Sci. 2007;255 (1–2):50 –56[CrossRef][Web of Science][Medline]
  20. Lopez PH, Schnaar RL. Determination of glycolipid-protein interaction specificity. Methods Enzymol. 2006;417 :205 –220[CrossRef][Web of Science][Medline]
  21. Galvin JE, Hemric ME, Ward K, Cunningham MW. Cytotoxic mAb from rheumatic carditis recognizes heart valves and laminin. J Clin Invest. 2000;106 (2):217 –224[Web of Science][Medline]
  22. Singer HS, Giuliano JD, Hansen BH, et al. Antibodies against human putamen in children with Tourette syndrome. Neurology. 1998;50 (6):1618 –1624[Abstract/Free Full Text]
  23. Lacroix-Desmazes S, Kaveri SV, Mouthon L, et al. Self-reactive antibodies (natural autoantibodies) in healthy individuals. J Immunol Methods. 1998;216 (1–2):117 –137[CrossRef][Web of Science][Medline]
  24. Archelos JJ, Hartung HP. Pathogenetic role of autoantibodies in neurological diseases. Trends Neurosci. 2000;23 (7):317 –327[CrossRef][Web of Science][Medline]
  25. Martino D, Defazio G, Church AJ, et al. Antineuronal antibody status and phenotype analysis in Tourette's syndrome. Mov Disord. 2007;22 (10):1424 –1429[CrossRef][Web of Science][Medline]
  26. Dale RC, Church AJ, Candler PM. Serum autoantibodies do not differentiate PANDAS and Tourette syndrome from controls. Neurology. 2006;66 (10):1612; author reply 1612[Free Full Text]
  27. Kawikova I, Leckman JF, Kronig H, et al. Decreased number of regulatory T cells suggests impaired immune tolerance in children with Tourette's syndrome. Biol Psychiatry. 2007;61 (3):273 –278[CrossRef][Web of Science][Medline]
  28. Zimmerman AW, Jyonouchi H, Comi AM, et al. Cerebrospinal fluid and serum markers of inflammation in autism. Pediatr Neurol. 2005;33 (3):195 –201[CrossRef][Web of Science][Medline]
  29. Luo F, Leckman JF, Katsovich L, et al. Prospective longitudinal study of children with tic disorders and/or obsessive-compulsive disorder: relationship of symptom exacerbations to newly acquired streptococcal infections. Pediatrics. 2004;113 (6). Available at: www.pediatrics.org/cgi/content/full/113/6/e578
  30. Singer HS, Minzer K. Neurobiology of Tourette syndrome: concepts of neuroanatomical localization and neurochemical abnormalities. Brain Dev. 2003;25 (suppl):S70 –S84[CrossRef][Web of Science][Medline]
  31. Berardelli A, Curra A, Fabbrini G, Gilio F, Manfredi M. Pathophysiology of tics and Tourette syndrome. J Neurol. 2003;250 (7):781 –787[CrossRef][Web of Science][Medline]
  32. Hoekstra PJ, Anderson GM, Limburg PC, Korf J, Kallenberg CG, Minderaa RB. Neurobiology and neuroimmunology of Tourette's syndrome: an update. Cell Mol Life Sci. 2004;61 (7–8):886 –898[CrossRef][Web of Science][Medline]
  33. Singer HS. PANDAS and immunomodulatory therapy. Lancet. 1999;354 (9185):1137 –1138[CrossRef][Web of Science][Medline]
  34. Perlmutter SJ, Leitman SF, Garvey MA, et al. Therapeutic plasma exchange and intravenous immunoglobulin for obsessive-compulsive disorder and tic disorders in childhood. Lancet. 1999;354 (9185):1153 –1158[CrossRef][Web of Science][Medline]
  35. Loiselle CR, Lee O, Moran TH, Singer HS. Striatal microinfusion of Tourette syndrome and PANDAS sera: failure to induce behavioral changes. Mov Disord. 2004;19 (4):390 –396[CrossRef][Web of Science][Medline]
PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:


Home page
 JWatch PediatricsHome page
PANDAS: Two Studies of Clinical Exacerbations
Journal Watch Pediatrics and Adolescent Medicine, July 23, 2008; 2008(723): 6 - 6.
[Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My File Cabinet
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Singer, H. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Singer, H. S.
Related Collections
Right arrow Infectious Disease & Immunity
Right arrowRelated AAP Red Book topics:
Non-Group A or B Streptococcal and...
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?