Neurosciences Unit, Unit of Child Health and Great Ormond Street Hospital, and Neuroinflammation Unit, Institute of Neurology, London
Neurosciences Unit, Unit of Child Health and Great Ormond Street Hospital, and Department of Child and Adolescent Psychiatry, Institute of Psychiatry, London
Neuroinflammation Unit, Institute of Neurology, London, UK
Correspondence: Dr Isobel Heyman, Department of Child and Adolescent Psychiatry, PO Box 085, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK. E-mail: I.Heyman{at}iop.kcl.ac.uk
Declaration of interest None. Funding detailed in Acknowledgements.
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ABSTRACT |
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Aims We tested the hypothesis that post-streptococcal autoimmunity may have a role inidiopathicobsessivecompulsive disorder (OCD).
Method We examined 50 children with OCD for ABGA using enzyme-linked immunosorbent assay (ELISA) and western immunoblotting. The findings were compared with paediatric autoimmune (n=50), neurological (n=100) and streptococcal (n=40) controls.
Results The mean ABGA binding on ABGA binding on ELISA was elevated in the patient cohort compared with all control groups (P<0.005 in all comparisons). Western immunoblotting revealed positive antibody binding (as seen in Sydenhams chorea) in 42% of the patient cohort compared with 210% of control groups (P<0.001 in all comparisons).
Conclusions Our findings support the hypothesis that central nervous system autoimmunity may have a role in a significant subgroup of cases of OCD. Further studyis required to examine whether the antibodies concerned are pathogenic.
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INTRODUCTION |
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METHOD |
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Controls
For comparison in the serological study, we recruited paediatric control
groups: a neurological control group with stroke, metabolic movement disorders
and encephalitis (n=100, mean age 8.2 years, range 116 years,
50 males); a group with uncomplicated streptococcal infections, defined as
laboratory-confirmed streptococcal pharyngitis without autoimmune or invasive
complications (n=40, mean age 9.8 years, range 215 years, 25
males); and a third group of children with autoimmune disorders without
neurological involvement, including rheumatic carditis and post-streptococcal
glomerulonephritis (n=50, mean age 9.2 years, range 216 years,
25 males). Some of these control groups had been previously reported
(Church et al,
2003b). The control groups were recruited during the same
period as the obsessivecompulsive disorder cohort (between August 2001
and August 2002).
Serology
All serum samples were coded and stored at 80 °C prior to
streptococcal serological investigation, in which anti-streptolysin O titres
were measured using the Dade Behring BN II nephelometer
(http://www.dadebehring.com).
All control group levels were within acceptable parameters. Titres greater
than 200 IU/ml were considered significant according to World Health
Organization guidelines (Spaun et
al, 1961). Streptococcal serological measurements took place
in a different laboratory from the other tests, with investigators masked to
the anti-basal ganglia antibody results.
The methods for assaying anti-basal ganglia antibodies have been described by Church et al (2002). All participants in the patient and control groups had their antibody levels measured using both enzyme-linked immunosorbent assay (ELISA) and western blotting (all assays were performed by A.J.C.). The former technique gives a semi-quantitative measurement of antibody binding to antigens, whereas western blotting can define specific antibodyantigen interactions. For both assays, the antigen was homogenised delipidated human basal ganglia (caudate and putamen). Human immunoglobulin G (IgG) was first removed from the homogenised antigen using Protein A. Samples from patients and controls were coded and assayed at the same time on the same ELISA plates and western blots. A 96-well ELISA plate was incubated with basal ganglia homogenate overnight. The plate was then blocked with 2% bovine specific albumin, and washed with normal saline (0.9% sodium chloride) with 0.2% milk proteins and 0.025% Tween. Duplicate serum samples were diluted 1:300 and incubated for 1 h. After washing, 1:1000 rabbit anti-human IgG conjugated with horseradish peroxidase (Dako, Glostrup, Denmark) was incubated for a further hour. After washing, the plate was developed with the reagent o-phenylenediamine for 15 min and stopped with 1 mol/l hydrochloric acid. A known positive and negative control was used on all ELISA plates. For western blotting, the basal ganglia homogenate was mixed with lithium dodecyl sulphate and 0.05 mol/l dithiothreitol and heated at 65 °C for 15 min; 30 µg of protein was loaded onto a 412% BisTris gel and subjected to electrophoresis. The gel was transferred to nitrocellulose (BioRad, Hemel Hempstead, UK) and the proteins blocked with 2% milk proteins. The blot was loaded onto a manifold and serum samples diluted 1:300 were incubated overnight. Samples were washed with normal saline with 0.2% milk proteins and 0.025% Tween. The secondary antibody (rabbit anti-human IgG conjugated with horse-radish peroxidase) was diluted 1:1000 and incubated for 2 h. The blot was then washed again and developed with substrate 4-chloro-1-naphthol for 15 min. A positive western blot result was reported when there was discrete antibodyantigen binding demonstrated on colorimetric detection. Positive antibody binding was compared with the previously reported findings in Sydenhams chorea (Church et al, 2002).
Statistics
All statistical tests were performed using the Statistical Package for the
Social Sciences version 12. The distribution of the ELISA data was positively
skewed, therefore the planned comparisons between the
obsessivecompulsive disorder patient cohort and the control cohorts
were made with MannWhitney tests. Western blotting antibody positivity
was compared using the chi-squared test. Clinical characteristics of patients
testing positive and negative for anti-basal ganglia antibodies were compared
using chi-squared tests.
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RESULTS |
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DISCUSSION |
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Obsessivecompulsive disorder after infectious disease
Further evidence for specific brain mechanisms in
obsessivecompulsive disorder is the long-recognised association between
this disorder and infection or immunemediated central nervous system disease.
For example, obsessivecompulsive behaviours were reported in patients
with encephalitis lethargica during the 19161927 epidemic, although
parkinsonism was the most striking clinical feature
(Von Economo, 1931). The
pathological abnormalities of encephalitis lethargica were inflammatory
infiltrates that were relatively localised to the basal ganglia, leading to
early speculation that obsessivecompulsive disorder could occur
secondary to basal ganglia dysfunction
(Von Economo, 1931).
Another infection-mediated cause of secondary obsessivecompulsive disorder is Sydenhams chorea, a latent manifestation of group A streptococcal infection. Psychiatric studies using operationalised criteria have revealed that obsessivecompulsive symptoms are common in Sydenhams chorea (Mercadante et al, 2000) and are even more evident in chronic or relapsing disease (Asbahr et al, 1999). Like encephalitis lethargica, Sydenhams chorea is characterised by an inflammatory infiltrate, predominantly of the basal ganglia (Von Economo, 1931). Furthermore, recent magnetic resonance volumetric measurement of brain regions demonstrated specific volumetric enlargement of the basal ganglia in patients with Sydenhams chorea, although conventional magnetic resonance images are normal (Giedd et al, 1995).
In the late 1980s an apparently new post-streptococcal neuropsychiatric phenotype was recognised: motor tics and/or obsessivecompulsive disorder (Swedo et al, 1998). The syndrome was named paediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), and was characterised by an abrupt onset of symptoms after streptococcal infection, with a relapsing course associated with further infections (Swedo et al, 1998). The recognition of the PANDAS phenotype has led to speculation that post-streptococcal autoimmunity might play a contributory part in a subgroup of idiopathic tic disorders and obsessivecompulsive disorder. The PANDAS concept and the role of autoimmunity in movement and psychiatric disorders (including Tourette syndrome) remain controversial (Kurlan & Kaplan, 2004; Swedo et al, 2004).
Immunopathogenesis of post-streptococcal brain disease
The favoured hypothesis regarding Sydenhams chorea and PANDAS
pathogenesis states that an immune response raised against group A
streptococcus cross-reacts with brain proteins owing to similarity in the
antigens, a concept called molecular mimicry. To date, most investigators have
focused on cross-reactive anti-neuronal antibodies as possible mediators of
disease, although T cells may also be involved
(Husby et al, 1976;
Kiessling et al,
1993; Church et al,
2002). Antibodies against human basal ganglia and brain antigens
are more common in Sydenhams chorea
(Husby et al, 1976;
Church et al, 2002,
2003a) and other
post-streptococcal movement disorders compared with controls
(Dale et al, 2004).
Using western immunoblotting in two cohorts with Sydenhams chorea and
post-streptococcal parkin-sonism respectively, we have previously proposed
that a conserved group of neuronal antigens are involved in antibody binding,
of molecular weights 40, 45 and 60 (Church et al,
2002,
2003a;
Dale et al, 2004). We
have demonstrated that the 45 kDa protein exists as a doublet. The 40 kDa, 60
kDa and one of the 45 kDa bands are brain-specific
(Dale et al, 2004).
Limitations of western blotting and ELISA include the fact that both
techniques alter the conformation of the antigens and could therefore affect
antibodyantigen interaction. It would be preferable to use the specific
autoantigens in their physiological state. We are currently attempting to
identify these antigens. A separate group have found similar antibody findings
(using immunofluorescence rather than western immunoblotting) in individuals
with motor tics in association with streptococcal infection
(Kiessling et al,
1993). Pathogenic effect of the antibodies in Sydenhams
chorea and PANDAS has been inferred by the induction of disease in animals
after PANDAS antibody (IgG) infusion
(Hallett et al, 2000)
and symptom reduction after immunotherapies that remove IgG
(Perlmutter et al,
1999).
An interesting recent development is the demonstration of an antibody in people with Sydenhams chorea that cross-reacts with lysoganglioside (a neuronal cell surface molecule), resulting in altered neuronal cell signalling (Kirvan et al, 2003). Identifying the 40 kDa, 45 kDa and 60 kDa autoantigens to see whether they are related to lysoganglioside or represent other neuronal markers will further our understanding of the possible role of autoimmunity in movement and neuropsychiatric disorders. Possible mechanisms of antibody action could include upregulation (Kirvan et al, 2003) or downregulation of neuronal metabolism or cell signalling.
Role of post-streptococcal autoimmunity in obsessivecompulsive disorder
We report here that 42% of patients with obsessivecompulsive
disorder (a consecutive cohort of children and adolescents attending a
specialist clinic) had circulating anti-basal antibodies. This is a highly
significant finding, as these antibodies are uncommonly found in the control
groups studied. In contrast, patients with the neurological disorder most
robustly established as a post-streptococcal autoimmune disorder
Sydenhams chorea almost always test positive for these
antibodies using the same assays (Church
et al, 2002). These findings demonstrate that a subgroup
of people with obsessivecompulsive disorder have antibody findings
similar to those seen in Sydenhams chorea, suggesting that autoimmunity
many have a role in the genesis and/or maintenance of the former disorder.
Only a few other studies have looked for anti-neuronal antibodies in
obsessivecompulsive disorder. An indirect immunofluorescence method has
been used in one small study of idiopathic disease, and did demonstrate
increased brain antibody binding in the group with the disorder
(Kiessling et al,
1994). Two other studies found no evidence of anti-neuronal
autoantibodies in people with the disorder
(Murphy et al, 1997;
Hoekstra et al, 2003).
These discre-pancies in autoantibody findings in different study cohorts could
be due to different methods of antigen preparation (e.g. delipidation, used in
methods described here) or antibody detection (colorimetric v.
enhanced chemiluminescence). The detection method is particularly important,
as sensitive techniques such as enhanced chemiluminescence can pick up low
titres of clinically insignificant antibodies in healthy people. We use
colorimetric detection, which we believe has reduced sensitivity but improved
specificity. Although the presence of anti-neuronal antibodies is important in
establishing a possible autoimmune aetiology in obsessivecompulsive
disorder, it is also essential to demonstrate that immune factors are
pathogenic (rather than simply markers). It could be argued that antibodies
are produced as a result of neuronal damage or are a non-specific response to
recent streptococcal infection, therefore representing an epiphenomenon.
However, the low prevalence of these antibodies in the neurological and
streptococcal control groups makes this less likely.
The presence of anti-basal ganglia antibodies in some of the patients with obsessivecompulsive disorder raises the possibility that these cases may differ from those in which these antibodies are absent. We therefore compared the two groups to define any possible clinical differences. In support of the role of streptococcal infection, antibody-positive patients were more likely to have positive results on streptococcal serological testing. However, titres of anti-streptolysin O were elevated in 31% of antibody-negative patients. Streptococcal infection is common in children and therefore cannot be used as a marker of post-streptococcal neuropsychiatric disorder alone. The antibody-positive patients were also more likely to have tics or Tourette syndrome, further supporting a link between post-streptococcal autoimmunity and movement disorders. Other than these findings, there was no clear difference between the antibody-positive and antibody-negative patients in terms of obsessivecompulsive symptoms, comorbidity and family history. Importantly, the mean CYBOCS score in the antibody-negative patients was higher than in the antibody-positive patients. This suggests that the absence of these antibodies was not due to remission of disease: rather it suggests that there are discrete subgroups of obsessivecompulsive disorder with a differing pathogenesis.
The patients recruited to this study attended a specialist clinic for children with obsessivecompulsive disorder, so it is conceivable that our cohort contained children whose disorder was complicated, atypical or therapy-resistant. Whether the autoimmune subgroup in our study had disorders with a different natural history (perhaps more spontaneous remission, or better long-term outcome) or differential treatment responsiveness requires longitudinal studies. However, all 50 patients had obsessivecompulsive disorder using internationally defined criteria (DSMIV) and exhibited the range of obsessions, compulsions (and comorbidities) previously described in the idiopathic condition. It would be important to compare these findings with epidemiological samples of childhood- and adult-onset disorder, and also with other psychiatric disorders. Further examination of this autoimmune subgroup might provide insight into the neurobiology of obsessivecompulsive disorder and offer alternative therapies to patients with refractory illness in the future.
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Clinical Implications and Limitations |
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LIMITATIONS
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ACKNOWLEDGMENTS |
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Received for publication July 29, 2004. Revision received January 7, 2005. Accepted for publication January 18, 2005.
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