A JOURNAL OF NEUROLOGY
N-methyl-D-aspartate antibody encephalitis:
temporal progression of clinical and
paraclinical observations in a predominantly
non-paraneoplastic disorder of both sexes
Sarosh R. Irani,1Katarzyna Bera,1Patrick Waters,1Luigi Zuliani,1Susan Maxwell,1
Michael S. Zandi,2Manuel A. Friese,1,3Ian Galea,4Dimitri M. Kullmann,5David Beeson,1
Bethan Lang,1Christian G. Bien6and Angela Vincent1
1 Department of Clinical Neurology, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
2 Department of Neurology, Box 165, Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK
3 Institut fu ¨r Neuroimmunologie und Klinische Multiple Sklerose-Forschung, Zentrum fu ¨r Molekulare Neurobiologie, Universita ¨tsklinikum
Hamburg-Eppendorf, D-20251 Hamburg, Germany
4 Division of Clinical Neurosciences, School of Medicine, University of Southampton, Southampton SO17 1BJ, UK
5 Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK
6 Department of Epileptology, University of Bonn Medical Centre, Sigmund-Freud Str. 25, 53105, Bonn, Germany
Correspondence to: Prof. Angela Vincent,
Department of Clinical Neurology,
L6 West Wing,
John Radcliffe Hospital,
University of Oxford,
Oxford OX3 9DU, UK
Antibodies to the N-methyl-D-aspartate subtype of glutamate receptor have been associated with a newly-described encephal-
opathy that has been mainly identified in young females with ovarian tumours. However, the full clinical spectrum and treat-
ment responses are not yet clear. We established a sensitive cell-based assay for detection of N-methyl-D-aspartate receptor
antibodies in serum or cerebrospinal fluid, and a quantitative fluorescent immunoprecipitation assay for serial studies. Although
there was marked intrathecal synthesis of N-methyl-D-aspartate receptor antibodies, the absolute levels of N-methyl-D-aspartate
receptor antibodies were higher in serum than in cerebrospinal fluid. N-methyl-D-aspartate receptor antibodies were of the
immunoglobulin G1 subclass and were able to activate complement on N-methyl D-aspartate receptor-expressing human em-
bryonic kidney cells. From questionnaires returned on 44N-methyl-D-aspartate receptor antibody-positive patients, we identified
a high proportion without a detected tumour (35/44, 80%: follow-up 3.6–121months, median 16months). Among the latter
were 15 adult females (43%), 10 adult males (29%) and 10 children (29%), with four in the first decade of life. Overall, there
was a high proportion (29%) of non-Caucasians. Good clinical outcomes, as defined by reductions in modified Rankin scores,
correlated with decreased N-methyl-D-aspartate receptor antibody levels and were associated with early (540 days) adminis-
tration of immunotherapies in non-paraneoplastic patients (P50.0001) and earlier tumour removal in paraneoplastic patients
(P=0.02). Ten patients (23%) who were first diagnosed during relapses had no evidence of tumours but had received minimal
or no immunotherapy during earlier episodes. Temporal analysis of the onset of the neurological features suggested progression
through two main stages. The time of onset of the early features, characterized by neuropsychiatric symptoms and seizures
doi:10.1093/brain/awq113 Brain 2010: 133; 1655–1667 |
Received August 14, 2009. Revised April 7, 2010. Accepted April 8, 2010
? The Author(s) 2010. Published by Oxford University Press on behalf of Brain.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5),
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
preceded by a median of 10–20days, the onset of movement disorders, reduction in consciousness and dysautonomia. This
temporal dichotomy was also seen in the timing of cerebrospinal fluid, electroencephalographic and in the rather infrequent
cerebral imaging changes. Overall, our data support a model in which the early features are associated with cerebrospinal fluid
lymphocytosis, and the later features with appearance of oligoclonal bands. The immunological events and neuronal mechan-
isms underlying these observations need to be explored further, but one possibility is that the early stage represents diffusion of
serum antibodies into the cortical grey matter, whereas the later stage results from secondary expansion of the immunological
repertoire within the intrathecal compartment acting on subcortical neurons. Four patients, who only had temporal lobe epilepsy
without oligoclonal bands, may represent restriction to the first stage.
Keywords: N-methyl-D-aspartate receptor-antibody encephalitis; autoimmunity; non-paraneoplastic; paraneoplastic;
Abbreviations: cDNA = complementary DNA; EGFP = enhanced green fluorescent protein; IgG = immunoglobulin G;
NMDAR = N-methyl-D-aspartate receptor
Human auto-antibodies directed against neuronal proteins are of
neurological diseases. Auto-antibodies with specificity towards
intracellular proteins act as diagnostic markers of diseases that
are usually associated with an underlying cancer and are rarely
immunotherapy-responsive. By contrast, antibodies that target
the extracellular domain of surface-expressed neuronal proteins
are likely to be pathogenic and their presence usually indicates
the possibility of successful immunotherapy. In the peripheral ner-
vous system, it is well established that such antibodies are patho-
genic (Vincent et al., 2006).
It is increasingly recognized that some CNS disorders can also be
antibody-mediated. Antibodies to voltage-gated potassium chan-
nels are often associated with limbic encephalitis, presenting with
seizures, amnesia and medial temporal lobe inflammation. These
patients do not usually have an underlying neoplasm, and do well
following immunotherapies, with a substantial and sometimes
complete recovery (Buckley et al., 2001; Thieben et al., 2004;
Vincent et al., 2004). Moreover, with the expansion of the
voltage-gated potassium channels-antibody-associated phenotype
to patients whose diagnosis was drug-resistant epilepsy, new
immunotherapy-responsive seizure phenotypes are beginning to
be recognized (McKnight et al., 2005; Irani et al., 2008).
More recently, antibodies against the N-methyl-D-aspartate sub-
type of ionotropic glutamate receptors [N-methyl-D-aspartate re-
ceptor (NMDAR) antibodies] have been reported, predominantly
in young women who developed a subacute-onset encephalop-
athy, commonly associated with a prominent movement disorder
and frequently an underlying ovarian teratoma (Dalmau et al.,
2007), although two recent reports suggest a much higher inci-
dence of non-paraneoplastic cases in children (Dale et al., 2009;
Florance et al., 2009). Removal of the teratoma combined with
immunotherapy resulted in substantial recovery, but in the minor-
ity of patients without a tumour, recovery appeared to be less
impressive (Dalmau et al., 2007, 2008). In those studies, the
age range was wide, but only 9% of the patients were males.
The full clinical spectrum associated with NMDAR antibodies is
likely to widen with increasing recognition. Here we describe an
assay suitable for both serum and CSF detection of the antibodies,
modified from that of Dalmau et al. (2008), which allowed us to
identify 50 patients in the UK and Europe in 2008. Clinical details
were gathered on 44 patients and allowed us to correlate serum
NMDAR antibodies with clinical outcomes using a novel quantita-
tive fluorescent assay, and to demonstrate the detailed temporal
progression of clinical and paraclinical features. Our observations
emphasize the importance of early intensive immunotherapies and
suggest that the neurological disease occurs in two main stages.
Materials and methods
Ethical approval for this study was from the Oxfordshire Regional
Ethical Committee A (07/Q1604/28). Serum and CSF samples were
referred for testing by clinicians, mainly from the United Kingdom and
Europe. Standardized clinical questionnaires (Supplementary data),
patient information sheets and consent forms were distributed to the
clinicians referring the first 50 NMDAR-antibody-positive samples from
a total of 450 sent for testing. We requested details of clinical features,
investigation results and serial modified Rankin scores (Graus et al.,
2001). Clinicians were asked to document and detail the timing of
specific features such as higher cognitive dysfunction, psychiatric, seiz-
ure and movement disorders: we have maintained the categorizations
as provided by the clinicians. Eight of the patients have been described
in case reports (Schimmel et al., 2009; Zandi et al., 2009; Davies
et al., 2010). All data were analysed using GraphPad Prism 5 and
the tests used are mentioned in the figures and tables. Thirty-six ques-
tionnaires were returned, and eight were completed from clinic letters,
emails or telephone conversations.
Cell-based assay for antibodies binding
EX-XO451-M02) and NMDAR2B subunit (NR2B, IMAGE clone
number 8322670) complementary
in vitro mutagenesis to match the respective GenBank ID consensus
sequences NM_000832 (GRIN1-1a) and BC113618 (GRIN2B). The
NR2B subunit was subcloned from pCRXL-TOPO into the mammalian
NMDAR1 subunit(NR1, Genecopoeiacataloguenumber
Brain 2010: 133; 1655–1667S. R. Irani et al.
expression vector pcDNA3.1hygro(–) using a BamH1/Not1 restriction
digest. An enhanced green fluorescent protein (EGFP)-tagged human
NR1-1 splice variant was also generated. To create the expression
construct for this, the C-terminal stop codon was removed, an NheI
restriction site generated and the coding sequence for EGFP inserted
with a GT(GS)4 linker between the C-terminus of the NR1-1 and the
beginning of the EGFP.
For the cell-based assay, human embryonic kidney (HEK293) cells
were grown on glass coverslips in Dulbecco’s modified Eagle’s medium
with 10% foetal calf serum and penicillin, streptomycin and ampho-
tericin. After 24h, cells were transfected, using polyethylenimine and
glucose, with untagged-NR1 and NR2B cDNA at a ratio of 3:1. An
EGFP expression vector was co-transfected to visualize cells taking-up
cDNAs. To prevent cytotoxicity as a result of glutamate in the medium
activating the NMDARs, cells were supplemented with 500mM keta-
mine 16h post-transfection. Live cells were incubated with patient sera
(1:20) or undiluted CSF for 1h before fixation (3% formaldehyde)
followed by 35min incubation with Alexa Fluor 568 anti-human im-
munoglobulin G (IgG). Cells were subsequently washed three times in
phosphate buffered saline and mounted on slides in fluorescent
mounting medium (DakoCytomation, Cambridge, UK) containing
DAPI (40,60-diamidino-2-phenlindoledichloride, 1:1000). They were
visualized using a fluorescence microscope with a MacProbe v4.3
digital imaging system and the binding scored on a scale from 0–4
by two independent observers (Supplementary Fig. 1A; as in Leite
et al., 2008; Waters et al., 2008). All positive samples were retested
on muscle-specific-kinase or glycine ?1 receptor transfected cells to
exclude non-specific binding to the human embryonic kidney cells.
For subclass experiments, cells were incubated with isotype-specific
mouse antibodies and subsequently with an Alexa Fluor 568
anti-mouse IgG. For immunoabsorption, the sera (1:20 dilution) were
preincubated with 5million trypsinized EGFP-NR1-expressing human
embryonic kidney cells for 30min at 4?C before testing for binding
For detection of deposited complement on transfected cells, the
live cells were incubated with heat-inactivated sera (nine NMDAR-
antibody positive and eight healthy individuals) and fresh human
plasma, as a source of complement (as in Leite et al., 2008; Waters
et al., 2008). After 30min at 37?C the cells were kept on ice, fixed
with 3% formaldehyde, incubated with rabbit antibodies against C3b
(DAKO; 1:500), or polyclonal rabbit anti-C5b9 (membrane attack
complex; kind gift of Prof BP Morgan; 1:100) and Alexa Fluor 568
anti-rabbit IgG (1:1000). Binding to the cells was scored as positive or
negative by a blinded observer (PW).
Fluorescent immunoprecipitation assay
for antibodies to NMDAR
Human embryonic kidney 293 cells were transfected as above with
NR2B and postsynaptic density 95 (PSD95, Open Biosystems, Image
Consortium cDNA clones, USA) cDNA expression constructs and with
the NR1-EGFP tagged expression construct in a ratio of 1:1:1. PSD95
was used because it improved NR1-EGFP expression in preliminary
studies (data not shown). Cells were solubilized using a buffer con-
taining 1% digitonin. A protease inhibitor cocktail (Sigma-Aldrich, UK)
was added to this buffer immediately before use. The supernatant was
analysed using antibodies to NR1 (Millipore, USA), NR2B (kind gift
from Prof F Anne Stephenson, School of Pharmacy, University of
London) and PSD95 (Neuromab, USA), and analysis of EGFP fluores-
cence in fractions following sedimentation on a continuous sucrose
gradient (Waters et al., 2008). For serological studies, 25ml of serum
was incubated with 250ml of solubilized extract overnight at 4?C, as
described previously (Waters et al., 2008). Protein-A beads were
rotated with the antigen-antibody mixture for 2h at room tempera-
ture. The beads were washed and the captured fluorescence was
measured using a fluorescent plate reader (Gemini XS, Molecular
Probes). Results were expressed as relative fluorescent units precipi-
tated by 25ml serum and are reported as fU.
Detection of NMDAR antibodies
We initially used three assays for the detection of NMDAR anti-
(Dalmau et al., 2007; Niehusmann et al., 2009). Rat brain sections
were incubated with undiluted CSF to look for hippocampal
‘neuropil’ binding (Fig. 1A). To confirm the neuronal cell surface
specificity of the antibodies and, thus, their likely clinical relevance,
we incubated primary cultures of hippocampal neurons with sera
and CSF to observe binding to the surface of live neurons
(Fig. 1B). We established an immunofluorescent cell-based assay
(Fig. 1C) for detection of NMDAR antibodies, modified from that
described previously (Dalmau et al., 2007). Specifically, we em-
ployed a 3:1 ratio of NR1:NR2B cDNA (with small amounts of
EGFP cDNA) as this gave better results with positive sera
(Supplementary Fig. 1B) and the NR1 subunit is thought to con-
tain the main epitope (Dalmau et al., 2008). To observe binding
only to the extracellular domain of the NR1/NR2B, we did not
permeabilize the cells and applied serum diluted 1:20 and CSF
undiluted. Using a semi-quantitative approach similar to that em-
ployed for binding of antibodies to other cell surface antigens
(Leite et al., 2008; Waters et al., 2008), the binding was scored
visually from 0 (no binding) to 4 (very strong binding) by two
independent observers. All positive results were retested, and
also checked for non-specific binding to the cells using human
embryonic kidney cells transfected with other antigens (see
‘Materials and methods’ section). Examples of different scores
are shown in Fig. 1C and Supplementary Fig. 1A. Sera from 32
healthy individuals and from 138 disease controls (with other
forms of autoimmune encephalopathy, chronic psychosis, multiple
sclerosis, lupus, opsoclonus–myoclonus, herpes encephalitis or seiz-
ures) gave values of51 (range 0–0.5, mean 0.01; median 0) (data
We found the cell-based assay to be most sensitive and, as it is
specific for NMDAR antibodies, it was used for testing the 450
sera and 35 CSF samples sent for NMDAR-antibody testing over
the period of study (results shown in Supplementary Fig. 1C). The
scores of 14 paired samples from NMDAR-antibody-positive pa-
tients are shown in Fig. 1D; one CSF was negative while the
paired serum was positive (the remaining 21 paired samples
were negative in both CSF and serum). Overall serum at 1:20
was found to be slightly more sensitive than undiluted CSF
(Fig. 1D), and many patients were diagnosed on the basis of
the serum antibody alone.
NMDAR-antibody encephalitisBrain 2010: 133; 1655–1667 |
When we serially diluted the 14 available paired samples to find
detection endpoints, NMDAR-antibody titres were between 6 and
450 times higher (mean 13.5) in serum than CSF (Fig. 1E). Except
in one patient, there was clear evidence of intrathecal synthesis of
NMDAR antibodies (median 21.7, range 0–118, n=10 pairs;
Supplementary Table 1).
NMDAR antibodies were found to be predominantly of the
complement-fixing IgG1 subclass (Fig. 1F) and were able to de-
posit complement C3b (9/9 NMDAR antibodies; 0/8 control sera)
and C9neo, the membrane attack complex (6/9 patients, 0/8 con-
trols), on NMDAR-transfected human embryonic kidney cells
(Fig. 1G), as expected for this IgG subclass.
A quantitative assay for antibodies
against the NR1 subunit
In order to measure NMDAR-antibody levels more quantitatively,
we established a fluorescent immunoprecipitation assay similar to
that reported for AQP4 antibodies (Waters et al., 2008). We
found the best expression of EGFP-NR1 occurred when it was
co-expressed with NR2B and PSD95. The cells were solubilized
in a buffer containing 1% digitonin, and the EGFP-NR1 could
be immunoprecipitated by anti-NR1 antibodies but not by
anti-NR2Bor anti-PSD95 antibodies
(Supplementary Fig. 2A) with a peak corresponding to 280kDa.
This suggested that the major component is a dimer of NR1-EGFP
(predicted size 274kDa) rather than a tetramer containing both
NR1 and NR2B.
Eighty-four percent of the cell-based assay-positive sera immu-
noprecipitated the EGFP-NR1-dimers at levels greater than the
mean+3SD (970fU) of the healthy control sera (Fig. 2A),
and the values correlated with results of the cell-based assay
(Fig. 2B, P50.0001, r=0.86). However, the fluorescent immuno-
precipitation assay was negative in seven of the sera tested and,
therefore, was used only for quantitative serial estimations on in-
dividual patients (see below). Because eight of the cell-based assay
scores were relatively low (1 or 1.5), including five negative by
fluorescent immunoprecipitation assay, we re-analysed the binding
to NMDAR (Supplementary Table 2). All eight sera were again
Figure 1 Detection and characterization of NMDAR antibodies. (A) NMDAR-antibody-positive CSF shows a hippocampal ‘neuropil’
binding pattern on rat brain sections. (B) NMDAR-antibody-positive serum IgG (green) showing surface binding to primary cultures of live
hippocampal neurons identified by the neuronal marker microtubule associated protein 2 (MAP2, in red, within merged lower image;
600? magnification). (C) Cell-based assay for NR1/NR2B (NMDAR) antibodies, using transfected human embryonic kidney (HEK293)
cells identified by EGFP (enhanced green fluorescent protein) cDNA co-transfection (green), shows surface binding of patient sera
(anti-IgG, red). This sample was scored as ‘4’ by visual observation (600? magnification). (D) Cell-based assay scores from 14 paired
CSF-serum samples; note one CSF was negative. (E) Values from end-point titrations of the 14 sera and positive paired CSFs in the
cell-based assay; note the different vertical scales and that serum levels of NMDAR antibodies were higher than CSF levels. Two of the
data pairs overlapped and their values have been slightly adjusted so that the points are visible. Asterisk indicates one data pair could not
be plotted as the CSF was negative (D). (F) NMDAR antibodies were found to be predominantly of the IgG1 subclass (n=11). (G)
NMDAR-antibody-positive sera (NMDAR-Ab) but not control sera (HC) were able to deposit complement C3b and C9neo, the membrane
attack complex, on human embryonic kidney cells expressing NMDAR (1000? magnification).
Brain 2010: 133; 1655–1667S. R. Irani et al.
positive for binding to NR1/NR2B transfected cells, but they also
bound to cells expressing NR1-EGFP alone, and the binding was
substantially reduced by prior absorption of the sera with cells
expressing NR1 (Supplementary Fig. 2B). These results confirmed
the specificity of the antibodies for NMDAR and for the NR1
(Dalmau et al., 2008), but we continue to use the NR1/NR2B
combination for diagnostic assays in order not to miss any possible
binding to NR2B.
Patients with positive NMDAR
Overall, 50 of the 450 referred sera were positive for NMDAR
antibodies with scores between 1 and 4. Of the 44 patients
from whom clinical data were obtained (88%), 10 were identified
retrospectively from sera sent over the preceding three years for
other antibody tests. The remaining 34 presented over the first
10 months of 2008. Samples were referred from centres in the
UK (n=28), Germany (n=8), the rest of Europe (n=6) and else-
where (n=2). Interestingly, 8 of the 28 patients (29%) seen in the
UK were non-Caucasian (two Chinese and one each from
Pakistan, Malaysia, Nigeria, India, Iraq and Singapore).
Using assays in routine clinical use, all NMDAR-antibody-
positive sera were negative for antibodies against Hu, Yo, Ri,
CV2, Ma/Ma2, amphiphysin, glutamic acid decarboxylase and
voltage-gated calcium channels. Two patients had low levels of
voltage-gated potassium channel antibodies (197 and 241pM,
normal5100pM) and four had anti-thyroid peroxidase antibodies
(137–416, normal 532IU/ml).
Demographics and tumour association
The ages at onset, stratified for sex and for presence or absence of
tumours, are shown in Fig. 2C. Among 44 patients, 31 were
females (70%; ages ranging from 2 to 49 years, median
22 years). These had all undergone intensive whole body/pelvic
imaging, but strikingly there were only eight female patients with
ovarian teratomas (26%; ages between 20 and 35 years), and no
other tumours in females. Among the 13 males (age range 4–59,
median 23 years) there was only one tumour, a recurrence of a
previously-treated Hodgkin’s lymphoma at 49 years of age. Of the
23 females and 12 males without detectable tumours, 10 (eight
females, two males) were under 18 years at presentation. The
youngest were a 2-year-old girl and a 4-year-old boy. It is possible
that tumours may still be found, but all have been negative to
Figure 2 Quantitative analysis of NMDAR antibodies and subgroups of NMDAR-antibody-positive patients. (A) The fluorescent
immunoprecipitation assay. NR1-specific commercial antibodies (anti-NR1) precipitated large amounts of EGFP-NR1 which was not bound
by NR2B-specific (anti-NR2B) or PSD95 (anti-PSD95) antibodies (data from three experiments). Eighty-four percent of 44
NMDAR-antibody-positive patients (as determined by the cell-based assay) precipitated EGFP-NR1 at levels greater than the mean plus
three standard deviations (mean+3SD) of results from 20 healthy controls (HCs). (B) There was a strong correlation between the
NMDAR-antibody levels determined by the fluorescent immunoprecipitation assay and by the cell-based assay (r=0.86, P50.0001;
Spearman rank correlation). (C) The ages at disease onset of the 44 NMDAR-antibody-positive patients shown as male and female
paraneoplastic (PN), non-paraneoplastic (NPN), and children (none of whom had tumours detected). (D) The results of the fluorescent
immunoprecipitation assay showed the NMDAR-antibody levels were higher in paraneoplastic when compared to all non-paraneoplastic
cases (P=0.0017, Mann-Whitney U-test). fU=relative fluorescence units precipitated by 25ml of serum.
NMDAR-antibody encephalitis Brain 2010: 133; 1655–1667 |
date on whole body CT (35 cases), pelvic ultrasound (13 cases),
pelvic MRI (eight cases), whole body PET (14 cases) and
often more than one imaging modality (26 cases) (follow-up
3.6-121 months, median 16 months).
Clinical features of 44 patients
The full description (summarized in Table 1) relates to the 44 pa-
tients, out of the first 50 identified, on whom detailed clinical data
were provided by the referring neurologists. Between 1 and
21 days (median 7) before the onset of neurological disease, 11
(25%) patients, including two adults with ovarian teratomas and
seven children, developed an infectious episode. Commonly the
prodrome consisted of an upper-respiratory tract infection (n=6;
one mycoplasma IgM, two anti-streptolysin-O antibodies), diar-
rhoeal illness (n=2; one with Campylobacter jejuni IgM), one
meningitic presentation, and one infected mole. One 13-year-old
female received diphtheria/tetanus/pertussis vaccination one day
prior to seizure onset. Prodromal headaches or fever were seen in
nine of these patients (20%), but more commonly headache and
fever occurred after the onset of seizures or neuropsychiatric fea-
tures (see below for detailed time course).
As described by Dalmau et al. (2007, 2008), the most common
presenting features included seizures, confusion, amnesia, behav-
ioural changes and psychosis. In our series only 8 of 44 patients
presented to a psychiatrist, and all were managed by neurologists.
Rarer presenting features included hyperacusis, deafness, ataxia
and dystonia. The most distinctive clinical features occurred later
and included involuntary choreoathetoid orofacial movements,
tachy- or bradycardia and a spontaneous fall in conscious level;
central hypoventilation occurred in only seven patients. In Table 1,
we describe these clinical features divided into six categories with
the percentage of patients exhibiting each feature, divided into
early and later categories (see below).
Most patients progressed to a severe clinical syndrome and
required admission to intensive care. However, three patients
had relatively mild syndromes, and four patients, two males
(both aged 23) with 4-year histories of drug-resistant temporal
lobe epilepsy, and two females (aged 17 and 33 years) who pre-
sented with an acute-onset of complex partial status epilepticus,
had had minimal or no cognitive involvement and did not develop
any movement disorders or other features consistent with the later
stages of the disease. None of these four patients had a malig-
nancy detected and they had some of the lower NMDAR-antibody
levels (cell-based assay scores 1–2; for details see Supplementary
Comparison of patient subgroups
Quantitative antibody titres on serum samples are shown in
Fig. 2D. The fluorescent immunoprecipitation assay values were
higher in the paraneoplastic than in the non-paraneoplastic pa-
tients (P=0.0017, Mann–Whitney U-test; Table 2). There were
also a few clinical features that were less evident in the
non-paraneoplastic compared with the paraneoplastic patients
(Table 2). Of the 44 patients, 10 including eight females, were
under age 18 at onset. The most common presenting features in
children were seizures and behavioural changes. All children
Table 1 Typical clinical features in 44 NMDAR-antibody positive patients
Higher cognitive dysfunction 40/44 (91%): confusion 29, behavioural changes 20, amnesia 14, dysphasia 13
Psychiatric 34/44 (77%): hallucinations 22, psychotic 20, agitation 18, depressive 12, anxiety 10, obsessive 1
Seizures 36/44 (82%): generalized 33, complex partial 16, simple partial 12
Spontaneous reduction in conscious level 20/44 (45%)
Movement disorder 39/44 (89%): choreoathetoid 30 (orofacial 27, upper limbs 22, lower limbs 10), parkinsonian 13, rigidity 10, myoclonus 7,
oculogyric crises 3, opisthotonus 3, startle 2
Dysautonomia 32/44 (72%): tachy/brady-cardia 22, hyperhidrosis 12, persistent pyrexia 10, central hypoventilation 7, labile/high blood
pressure 6, hypersalivation 4, pseudoobstruction 3, cardiac asystole 2
Total numbers of patients/total number (%). Anxiety was found as an isolated feature, without psychosis or depression, in only two cases. Data were provided by the
referring neurologists. Although shown as early and later, there were some individuals in whom this distinction was not evident. Data from individual patients are shown
in Fig. 6 and Supplementary Fig. 3.
Table 2 Clinical features that differed in frequency between paraneoplastic and non-paraneoplastic patients
Paraneoplastic, n=9 (%) Non-paraneoplastic, n=35 (%) P-valuea
Initial NMDAR-antibody level (mean and range)b
Spontaneous reduction in conscious level
Relapses were found in 10 of the non-paraneoplastic cases compared with none of the paraneoplastic cases, but this difference did not reach significance.
a Fisher’s exact test (two-tailed) was used for the comparisons (P-values have not been corrected for multiple comparisons).
b Measured using fluorescent immunoprecipitation assay with solubilised enhanced green fluorescent protein-tagged NR1 subunit.
Brain 2010: 133; 1655–1667S. R. Irani et al.
developed neuropsychiatric features and 80% developed a move-
ment disorder, not obviously different from the adult patients. The
incidence of preceding infections was, however, higher in children
(P=0.0008) and none had detectable tumours.
CSF analysis revealed lymphocytosis in only 30/44 (68%) of pa-
tients. CSF lymphocytosis was absent in samples from 35 days
after symptom onset (Fig. 3A), except in those who underwent
a clinical relapse (data not shown). By contrast, CSF-specific
(unmatched) oligoclonal bands were only present in 9% at first
CSF sampling but appeared later in the disease course in another
43% (see below).
Brain imaging was normal in 39/44 (89%) at initial MRI and
remained normal in 34/44 (77%; Fig. 3B). The few imaging
abnormalities were in the hippocampi (n=4) or within white
matter regions (n=6) on T2/fluid attenuated inversion recovery
sequences. One non-paraneoplastic male with long-standing
drug-resistant epilepsy showed hippocampal sclerosis at 4-year
follow-up. Results of brain PET were limited, but it was abnormal
in all three patients tested, with frontotemporal, occipital and cere-
bellar hypermetabolism reported at times when the MRI was
discharges in 22/44 (50%) patients, usually early during the
course of the disease, whereas generalized slowing in the slow
theta or delta range was found in 35/44 (80%) patients, generally
later during the disorder. Serial EEGs from one additional patient
with NMDAR antibodies are shown in Fig. 3C, demonstrating the
transition of epileptiform potentials to generalized slowing occur-
ring between 24 and 42 days, followed by normalization at
Clinical outcomes and serial estimations
of antibodies against the native NR1
To measure clinical outcomes, the modified Rankin score was re-
ported by the attending clinicians at peak of disease severity and
at least two further time points. 37 of 44 (84%) of the patients
Figure 3 Imaging, cerebrospinal fluid and EEG results. (A) The extent of CSF lymphocytosis (45cells/mm3, red dots) observed at different
time points across all patients. Thirt-two percent of patients had persistently negative CSF lymphocytosis (55 cells/mm3, blue dots). (B)
MRI was commonly normal (white) and abnormalities were mainly restricted to white matter tracts (n=6, red) and hippocampi (n=4,
blue) both at initial (median Day 1) and subsequent (median Day 25) imaging. (C) Serial electroencephalograms (EEGs) of a 17-year-old
non-paraneoplastic female NMDAR-antibody-positive patient, not within our cohort of 44 cases. Bipolar transverse EEG recordings are
shown in the scheme below. (a) EEG was normal after the patient had experienced a generalized convulsion. From Day 21–24, she
suffered from frequent complex partial seizures; (b) EEG showed frequent, in part long-lasting frontal epileptiform spike-wave activity;
(c–d) subsequently, there was continuous diffuse high-amplitude slowing without epileptiform potentials. From Day 39 on, no further
seizures were observed. On Day 42, anti-epileptic therapy consisted of phenytoin (blood level: 25.4mg/ml), phenobarbital (29.7mg/ml)
and lorazepam (2mg daily dose). Glucocorticosteroids were instituted on Day 38. From Day 56, the patient recovered; and (e) normal EEG
appearances recurred in sections in between abnormally high waves.
NMDAR-antibody encephalitisBrain 2010: 133; 1655–1667 |
had a score of 5 [severe disability with total dependency requiring
constant attention (Graus et al., 2001)] at peak of disease, and
average duration of hospitalization was 160 days (range 16–850).
Thirty-five of the patients were treated with immunotherapy:
plasma exchange (13), cyclophosphamide (4), rituximab (2),
azathioprine (1) and mycophenolate mofetil (1), or a combination
of the above (23).
Six examples of serial NMDAR antibodies and modified Rankin
scores are shown in Fig. 4 and corresponding clinical vignettes are
given in the Supplementary data. Overall there was a good
correlation within a patient between the level of serum NMDAR
antibody and the modified Rankin scores. Two patients who did
well had substantial contemporaneous falls in NMDAR-antibody
levels (Fig. 4A and B), whereas two who died had very high ab-
solute antibody levels (410000fU) that persisted despite immuno-
therapy (Fig. 4C and D). One of these was a patient with an
ovarian tumour (Fig. 4C) that was not removed until Day 180.
Two patients who were first diagnosed during a relapse (Fig. 4E
and F) are discussed below.
The clinical outcomes in all 44 patients are shown in Fig. 5A.
Overall, there was no difference in the outcomes between
However, within the paraneoplastic group, there was a correlation
and childhood cases.
between improvement and time to oophorectomy (r=?0.8,
P=0.02, Spearman rankcorrelation,
Dalmau et al., 2008). Importantly, within the non-paraneoplastic
group, those patients administered no immunotherapy or only
treated after 40 days, did significantly less well than those treated
before 40 days (Kruskal–Wallis, P50.0001; Dunn’s multiple com-
parisons P50.05). There was a trend (P=0.12, Mann–Whitney
U-test) towards better outcomes when glucocorticosteroids were
combined with at least one other immunotherapy rather than
given alone (Fig. 5B; additional immunotherapies are listed in
the figure legend). Overall, there was a correlation between clin-
ical improvement and reduction in NMDAR antibodies for all
25 patients on whom sequential samples were available (Fig. 5C;
r=0.54, P=0.005, Spearman rank correlation).
data not shown;see
Importantly, 10 of 35 non-paraneoplastic patients experienced
relapses (two to four relapses; time between relapses 3 months
and 6 years) following some improvement after a previous epi-
sode. These were aged between 17 and 44 years (median 25.5
years, two males and eight females) and did not have an occult
tumour after at least 14 months of careful follow-up. NMDAR
antibodies were detected in all six patients who had samples
Figure 4 (A–F) Effects of immunotherapy [plasma exchange (downward arrow), glucocorticosteroids (solid lines) and intravenous im-
munoglobulins (upward arrow)] and oophorectomy (asterisk) on NMDAR-antibody titres [fluorescent units precipitated (fU), black] and
on clinical outcomes (modified Rankin scores, red). Death is indicated by ‘dagger’. Age, sex and neoplasm status (paraneoplastic=PN,
non-paraneoplastic=NPN) are shown within each figure. (A) and (B) show reductions in NMDAR antibodies correlating with clinical
outcome after successful immunotherapy, although antibodies persist in (B). (C) and (D) show two patients who died with very high
NMDAR-antibody levels persisted throughout the illness despite immunotherapies (C and D) and late oophorectomy (C). (E) and (F) show
patients with clinical relapses after no immunotherapy (E) or only 3days of intravenous glucocortocosteroid therapy (F) during their first
episode. Both patients showed clinical and serological improvement after immunotherapy administered during their second episode. In (F),
the dotted line represents unknown titres between episodes. More detailed vignettes of these cases are available in the Supplementary
data. MRS=modified Rankin score.
Brain 2010: 133; 1655–1667 S. R. Irani et al.
stored from their previous clinical episode(s). Two examples of
relapsing patients, both diagnosed during their second episode
after limited immunotherapy had been given in the first episode,
are shown in Fig. 4E and F. Overall, during their first episodes, five
relapsers received no immunotherapy, three were only adminis-
tered 3–5days of intravenous glucocorticosteroids and two re-
lapses occurred immediately after glucocorticosteroid withdrawal.
By contrast, most paraneoplastic patients remained severely af-
fected until tumour removal (one improved spontaneously).
Time course of the clinical features
We were struck by the time course of the presenting features and
the paraclinical investigations, as shown in Fig. 6A. There was a
striking uniformity in the median values of the times at which the
seizures, psychiatric and cognitive disorders first appeared, usually
within the first few days, whereas the median values for the later
features of dysautonomia, movement disorders and fall in con-
sciousness were delayed by ?10–20 days. Data from individual
cases are shown in Supplementary Fig. 3. In the majority, there
were at least 5days between the onset of each of the early fea-
tures and the subsequent development of the later features. In
addition, whereas headache or fever associated with a known in-
fection (upper respiratory tract infection or diarrhoeal) preceded
the onset of neurological symptoms by around 5days, headache
or fever without evidence of infection showed a similar time
course to the later features.
To see whether the paraclinical investigations showed a similar
dichotomy, we plotted all the available results for each patient
against time after onset over the first 120 days (Fig. 6B) (although
these data included a variable number of time points from differ-
ent individuals, the data compare the presence or absence of a
particular finding at each time point). Of most interest, was the
early appearance of CSF lymphocytosis compared to the later
absence of CSF lymphocytosis (P=0.0007), contrasting with the
early absence of CSF-specific oligoclonal bands compared to their
later presence (P=0.0067) (data for individual cases shown in
Supplementary Fig. 4). There were also clear differences in the
Figure 5 Clinical outcomes and therapeutics. (A) The change in modified Rankin score (MRS) at 120 days after onset of symptoms
divided into subgroups. (B) The improvement in modified Rankin scores at 120days after onset of symptoms in non-paraneoplastic
patients according to administration of no immunotherapy (No IT), any immunotherapy after 40 days (IT440 days), or before or at 40
days (IT?40 days). The other immunotherapies were intravenous lg (n=4), plasma exchange (n=2), intravenous lg and plasma exchange
(n=3) or cyclophosphamide (n=1). (Kruskal–Wallis, P50.0001; ***Dunn’s multiple comparison test P50.0001 for no IT or IT440 days
compared to IT?40 days. For those administered IT at ?40 days, there was a trend towards better outcomes when steroids plus other
immunotherapies were given (Steroids alone v. Steroids+other IT; Mann Whitney, P=0.02). (C) The correlation between percentage
change in NMDAR-antibody levels determined by fluorescent immunoprecipitation assay and the corresponding change in modified
Rankin scores over the time the two samples were taken (r=0.54, P=0.005; n=25). Symbols as in (A).
NMDAR-antibody encephalitisBrain 2010: 133; 1655–1667 |
median times at which epileptiform discharges (P=0.0002)
occurred compared to generalized slowing, and the few cortical
MRI changes were earlier than the equally uncommon subcortical
MRI changes (P=0.04).
This first predominantly European series of NMDAR-antibody
encephalitis broadens the published demographic and clinical fea-
tures, details the timing of different clinical features and describes
treatment responses of paraneoplastic and non-paraneoplastic
cases. The condition was first identified in 2007 (Dalmau et al.,
2007). In 100 cases described by Dalmau et al. (2008) the major-
ity were female (91%) and 59% had ovarian tumours. By con-
trast, only 9/34 adult patients we studied had tumours, and 11/34
of the adults and 2/10 of the children were male. Here we further
characterized the pathogenic potential of the antibodies and show
that serial NR1-antibody levels correlate with clinical severity over
time within individuals and across the cohort. Moreover, early
immunotherapy appeared to be important in improving outcomes,
reducing NMDAR-antibody levels and protecting against relapses,
which occurred in 23%. By careful analysis of the temporal pro-
gression of the clinical features, we found consistent time-lags of
10–20 days between the early/presenting features and the later
features that were accompanied by switches in the CSF, MRI and
EEG findings, suggesting that the neurological disease occurs in
two distinct clinical and neuropathological stages.
For diagnosis, we used a sensitive cell-based assay similar to
those we developed for measuring antibodies to other cell surface
antigens (Hutchinson et al., 2008; Leite et al., 2008; Waters et al.,
2008). The higher proportion of non-paraneoplastic cases in this
study compared to the previous report (Dalmau et al., 2008) may
reflect the sensitivity of our cell-based assay for serological screen-
ing, and the wide referral base of the clinical neuroimmunology
service in Oxford, UK. Perhaps partly for this reason, some of the
patients had less characteristic syndromes. For instance, relatively
low antibody binding scores were found in four adults who pre-
sented with seizures and did not progress further (see also one
patient in Niehusmann et al., 2009). One of these subsequently
developed hippocampal sclerosis, suggesting that NMDAR antibo-
dies may be another cause of limbic encephalitis (Graus et al.,
2008) that can progress to hippocampal sclerosis (Bien et al.,
Figure 6 Temporal progression of clinical features and investigations. (A) The times of first appearance of the main clinical features for
each patient, up to 120days after onset of neurological symptoms. (B) The times of all CSF pleocytosis or oligoclonal bands, and all
abnormal EEG or MRI findings. Median and interquartile ranges are shown. P-values in B represent Mann–Whitney U-tests for each
blue-red pairing. OCB=CSF-specific oligoclonal bands. Percentages (bracketed) show the frequency of each feature or investigation
within the total NMDAR-antibody patient cohort.
Brain 2010: 133; 1655–1667S. R. Irani et al.
Twenty-three percent of our cases overall were children aged
between 2 and 17, confirming the increasing numbers of children,
often without tumours, who develop this antibody (Florance et al.,
2009), some of whom had been given a previous diagnosis of
encephalitis lethargica (Dale et al., 2009). Since some of the fea-
tures and investigations overlap with those of other disorders, such
as Hashimoto’s encephalitis (Castillo et al., 2006) and the paedi-
streptococcal infection (PANDAS, Church et al., 2003), the pheno-
type associated with this antibody may expand in both children
For serial measurements on sera we used immunoprecipitation
of solubilized native NR1-EGFP dimers, but this assay would need
further refinement for use in serological diagnosis or on CSF. An
enzyme-linked immunosorbent assay (Dalmau et al., 2008) has
been used successfully for serum and CSF studies, although at
present we feel that the cell-based assay offers the best sensitivity
and specificity for diagnosis. Using our NR1-EGFP immunoprecipi-
tation assay, the nine paraneoplastic patients, eight of whom were
females aged between 20 and 35 years with ovarian teratomas,
had higherserum NR1-antibody
paraneoplastic patients and did well after early tumour removal
(as in Dalmau et al., 2008). Follow-up in the non-paraneoplastic
patients, who accounted for 80% of our cohort, extend previous
data (Dalmau et al., 2008; Florance et al., 2009) that reported a
less good response to immunotherapy in the non-paraneoplastic
patients. Our best outcomes were in those patients treated early
(?40 days), and given glucocorticosteroids and at least one other
immunotherapy, whereas 9/10 of the relapsing patients received
insubstantial or no immunotherapy during their previous epi-
sode(s). In some cases, positive NMDAR antibodies were still pre-
sent after treatment of the first episode when only partial clinical
remission had been obtained (two examples are shown in Fig. 4).
This, together with the finding of high and persistent antibody
levels in the three patients who died, and reduction of antibody
titres in patients who improved, suggests that the condition needs
to be treated more intensively and may need long-term mainten-
ance immunosuppression. This contrasts with voltage-gated potas-
sium channel antibodies which, in general, fall rapidly following
similar immunotherapy (Thieben et al., 2004; Vincent et al.,
2004), although occasionally patients do relapse (Irani and
Vincent, unpublished observations). With an average hospital
stay of 160 days, long periods of ventilation and multiple infec-
tious complications, this condition is now becoming a recognized
and relatively frequent cause of admission to intensive care
(Davies et al., 2010).
The idea that this condition could be ‘multi-stage’ was sug-
gested previously on the basis of four female patients (Iizuka
et al., 2008), but the results of our detailed analyses of the
timing of clinical features and investigations combine to support
a model of the disease occurring in two main stages. The com-
monest presenting features (seizures, confusion, amnesia and
psychosis) seen in 41/44 patients are likely due to cortical tem-
poral lobe dysfunction, as frequently seen in classical limbic en-
cephalitis, and the same applies to the psychotic features
(Harrison, 1999; Janssen et al., 2009; Minatogawa-Chang et al.,
2009). Clinically, there was a time-lag of 10 to 20 days between
the onset of these early features (cognitive, psychiatric, seizure
syndromes, CSF lymphocytosis, epileptiform discharges and the
few cortical MRI lesions), and the later appearance of basal gang-
lia and brainstem-localised features (movement disorders, fall in
level of consciousness, dysautonomia, CSF oligoclonal bands, gen-
eralized slowing or intermittent rhythmic delta activity on EEG, and
some subcortical MRI abnormalities). Fever and headache with an
obvious infection did precede the neurological features in some
cases, but fever and headache without any apparent infection
were found later in the disease course, when they might have a
subcortical, or perhaps hypothalamic origin. Similarly, whereas
hypersomnia was an early feature, insomnia occurred later, possi-
bly reflecting generation by different localisation and mechanisms
These clinical observations raise many questions regarding the
aetiology and the pathogenic mechanisms of this disorder. Firstly,
the relatively high proportion of non-Caucasians (but mainly UK
resident) individuals in our cohort suggests that there may be
human leucocyte antigen or other genetic factors involved in dis-
ease susceptibility. Secondly, although numbers of paraneoplastic
patients were small, there were no apparent differences in the
timing of the CSF, imaging or EEG findings between the paraneo-
plastic and non-paraneoplastic patients (data not shown) and in all
patients, high serum:CSF ratios of NMDAR antibodies suggest that
the antigenic stimulus begins initially in the periphery rather than
in the CNS. Clearly NMDAR expression by the ovarian tumours
provides the antigenic stimulus in young paraneoplastic females
(Dalmau et al., 2007; Tuzun et al., 2009), and this strong anti-
genic stimulus may account for the higher antibody levels. In the
more frequent non-paraneoplastic cases, it is possible that occult
tumours may have been eliminated by the immune surveillance
mechanisms that generated the neurological syndrome, or are still
present as has been reported even in some patients who improved
spontaneously (Iizuka et al., 2008; Shindo et al., 2009). But in
general, for the non-paraneoplastic patients, one needs to
invoke some other antigenic stimulus. In our study, preceding in-
fections were common in children, but only two developed their
infectious prodrome sufficiently long before the onset of neuro-
logical symptoms (i.e.410days) to be consistent with the infection
generating a novel cross-reacting IgG antibody (one reported in
Schimmel et al., 2009) as clearly demonstrated, for instance, in
patients with Guillain–Barre ´ syndrome (Willison and Yuki, 2002).
Dalmau et al. (2008) reported a higher incidence of preceding
infections, but they did not perform such a detailed chronological
It seems likely that the infectious prodrome, when it occurs,
represents an inflammatory event. In many cases this may be
associated with CSF lymphocytosis, which was detected within
the first few days after onset of neurological symptoms, and is
seen frequently in paraneoplastic neurological syndromes (e.g.
Psimaras et al., 2010). Between them, these could be responsible
for a temporary and/or localized disruption of the blood-brain
barrier, allowing antibodies to gain entry to the CNS, possibly in
the temporal lobe where many of the presenting features may be
localised. It is possible that the CSF lymphocytosis might partly
represent an influx of antigen-specific T and B cells (data on
lymphocyte subsets is unfortunately not available); the intrathecal
NMDAR-antibody encephalitisBrain 2010: 133; 1655–1667 |
synthesis of NMDAR IgG1 antibodies (Tuzun et al., 2009) is likely
to require antigen-specific T cell help. Alternatively, our data
showing that the antibodies can activate deposition of both
C3b and C5b9 (membrane attack complex) on NMDAR express-
ing cells would be consistent with direct damage and/or
complement-dependent chemotaxis being responsible for a subse-
quent CSF lymphocytosis. Complement deposition was not found
at 3 and 4 months, respectively, in two post-mortem studies
(Tuzun et al., 2009) but might be important in the early stages.
Clearly further studies on serum and CSF taken at different stages
of the disease, including looking for specific cellular immune re-
sponses, combined with more detailed and investigative imaging,
could help unravel the early events but, since the presenting fea-
tures are not disease-specific, a high suspicion for this condition in
young adults and early serological and CSF referral will be import-
ant for these studies.
The next questions relate to where and how the antibodies act.
It has already been shown that NMDAR-antibody-positive CSF
and purified IgG can lead to internalization of NMDARs in hippo-
campal neurons in culture (Dalmau et al., 2008) and this may play
a major role in vivo, particularly if complement levels in the par-
enchymal extracellular fluid are low. From the time-course we
describe, it seems likely that the early features, accompanied by
epileptiform discharges and sometimes cortical MRI abnormalities,
are due to dysfunction of cortical neurons, perhaps by a combin-
ation of reduced NMDARs and complement-mediated damage.
But why the later features do not occur at this time is not clear.
A recent case series proposed that the involuntary movements
seen in NMDAR-antibody encephalitis were the result of reduced
corticostriatal disinhibition of autonomous central pattern gener-
ators in the striatum (Kleinig et al., 2008), but it is difficult to see
why this would occur with such a delayed time-course. It may be
that the secondary intrathecal synthesis of NMDAR antibodies and
development of oligoclonal bands are required before there is
more global cerebral involvement with predominant subcortical
dysfunction, slow-wave discharges and white matter lesions. An
attractive hypothesis is that, concurrent with the later appearance
of unmatched CSF oligoclonal bands, intrathecal antigen spreading
with production of different antibody specificities occurs; such
non-NMDAR antigen immunization might then be responsible
for some of the later features. In support of this concept, the
four patients restricted to the early, likely cortical, stage, with pre-
dominant epilepsy, never developed oligoclonal bands and only
one had CSF lymphocytosis. Further clinical investigations and ex-
perimental studies in active and passive transfer models should
help unravel some of these challenging questions.
We are very grateful to Ms P Pettingill for her help and to all of
the patients and the clinicians who provided the clinical data and
additional samples: Dr M Douglas, Dr R Etti (Birmingham, UK),
Dr T Hughes, Dr H Morris (Cardiff, UK), Dr J Miller (Newcastle, UK),
Dr M Manford, Prof D Menon, Dr P Molyneux (Cambridge,
UK), Dr L Costelloe (Dublin, UK), Dr A Bhattacharya, Dr G Davies,
Dr S Slaught, Dr R Walker (London, UK), Dr J Ealing, Dr G Vassallo
Prof R Gregory, Dr Y Hart, Dr T McShane (Oxford, UK),
Dr Murrigan (Southampton, UK), Dr M Hoppe, Dr J Penzien,
Dr B Reuter, Dr M Wolff (Germany), Prof U Aguglia (Italy),
Prof E Wilder-Smith (Singapore), Dr A Jiminez Huete (Spain),
Dr A Rossetti (Switzerland). We thank Prof F Anne Stephenson
(School of Pharmacy, University of London) for the anti-NR2B
antibody, and Prof B Paul Morgan for the anti-C5b9 antibody.
National Institute for Health Research (NIHR), Department of
Health, UK (S.R.I.), the Oxford Biomedical Research Centre
(P.W. and A.V.), a Wellcome Trust funded OXION studentship
(K.B.), the Medical Research Council, UK (D.B. and S.M.), a
European Federation of Neurological Society fellowship (L.Z.)
and an Eastern Region Neurosciences Training Fellowship (M.S.Z.).
Conflict of interest: AV and the University Department of Clinical
Neurology in Oxford receive royalties and payments for antibody
assays. The authors report no other conflict of interests.
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