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Anti-NR2 antibodies, blood-brain barrier, and cognitive dysfunction

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Abstract

Cognitive dysfunction (CD) is one of the most common neuropsychiatric manifestations of systemic lupus erythematosus (SLE). In animal models, antibodies to NR2 subunit of N-methyl d-aspartate receptor (anti-NR2) cause memory impairment, but only with blood-brain barrier (BBB) disruption or intrathecal administration. Several studies have failed to find association of aNR2 with CD, but none have assessed BBB integrity. S100B, an astrocyte-specific protein, has been used as biomarker of BBB disruption in traumatic brain injury and some neurodegenerative disorders. Antibodies to this immunologically privileged protein (anti-S100B) might indicate preceding BBB disruption. We hypothesized that aNR2 antibody is pathogenic in SLE patients only with BBB disruption. Demographic, clinical, and laboratory data was collected from patients with SLE. Total throughput score (TTS) of the Automated Neuropsychological Assessment Metrics (ANAM) was used as primary outcome measure. CD was defined as TTS < 1.5 SD below an age-, sex-, and race-matched RA population mean. Serum was analyzed by established ELISA techniques. Fifty-seven patients were evaluated and 12 had CD. Age, ethnicity, and family income were significantly different between the two groups (p < 0.05). In a multiple regression model adjusting for other variables, no significant effects of anti-NR2, S100B, or anti-S100B on TTS were found. Even at high levels of S100B and anti-S100B, no significant influence of anti-NR2 on TTS was found. The anti-NR2 was not associated with CD in SLE even in context of potential BBB disruption. This suggests that, if pathogenic, these antibodies may be produced intrathecally.
1 23
Clinical Rheumatology
Journal of the International League of
Associations for Rheumatology
ISSN 0770-3198
Volume 35
Number 12
Clin Rheumatol (2016) 35:2989-2997
DOI 10.1007/s10067-016-3339-1
Anti-NR2 antibodies, blood-brain barrier,
and cognitive dysfunction
Gaurav Gulati, Philip H.Iffland, Damir
Janigro, Bin Zhang & Michael E.Luggen
1 23
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ORIGINAL ARTICLE
Anti-NR2 antibodies, blood-brain barrier,
and cognitive dysfunction
Gaurav Gulati
1
&Philip H. Iffland II
2
&Damir Janigro
2
&Bin Zhang
3
&
Michael E. Luggen
1
Received: 10 February 2016 /Revised: 17 June 2016 /Accepted: 21 June 2016 / Published online: 29 June 2016
#International League of Associations for Rheumatology (ILAR) 2016
Abstract Cognitive dysfunction (CD) is one of the most com-
mon neuropsychiatric manifestations of systemic lupus erythe-
matosus (SLE). In animal models, antibodies to NR2 subunit of
N-methyl D-aspartate receptor (anti-NR2) cause memory impair-
ment, but only with blood-brain barrier (BBB) disruption or
intrathecal administration. Several studies have failed to find as-
sociation of aNR2 with CD, but none have assessed BBB integ-
rity. S100B, an astrocyte-specific protein, has been used as bio-
marker of BBB disruption in traumatic brain injury and some
neurodegenerative disorders. Antibodies to this immunologically
privileged protein (anti-S100B) might indicate preceding BBB
disruption. We hypothesized that aNR2 antibody is pathogenic in
SLE patients only with BBB disruption. Demographic, clinical,
and laboratory data was collected from patients with SLE. Total
throughput score (TTS) of the Automated Neuropsychological
Assessment Metrics (ANAM) was used as primary outcome
measure. CD was defined as TTS < 1.5 SD below an age-,
sex-, and race-matched RA population mean. Serum was ana-
lyzed by established ELISA techniques. Fifty-seven patients
were evaluated and 12 had CD. Age, ethnicity, and family in-
come were significantly different between the two groups
(p< 0.05). In a multiple regression model adjusting for other
variables, no significant effects of anti-NR2, S100B, or anti-
S100B on TTS were found. Even at high levels of S100B and
anti-S100B, no significant influence of anti-NR2 on TTS was
found. The anti-NR2 was not associated with CD in SLE even in
context of potential BBB disruption. This suggests that, if path-
ogenic, these antibodies may be produced intrathecally.
Keywords Anti-NR2 antibody .Anti-S100B antibody .
Blood-brain barrier (BBB) .Cognitive dysfunction .Lupus .
Neuropsychiatric lupus .S100B protein .Systemic lupus
erythematosus (SLE)
Introduction
Cognitive dysfunction (CD) is one of the most frequent neu-
ropsychiatric manifestations of systemic lupus erythematosus
(SLE) [1,2]. The reported prevalence has varied widely de-
pending on the method of assessment, definition employed,
population studied, and controls used. The largest study to
date by Murray et al. involving 694 subjects estimated the
prevalence of CD in an unselected lupus population at 15 %
[3]. CD is associated with significant work disability, impaired
functional capacity, and diminished quality of life through
disruption of multiple cognitive domains [48].
The pathophysiology of CD in SLE is poorly understood.
The N-methyl D-aspartate (NMDA) receptor is a glutamate
receptor in the brain and is responsible for synaptic plasticity
and memory [9]. The NR2 subtype has been found in partic-
ularly high density in the hippocampus [10]. In the hippocam-
pus, the calcium-gated NMDAR is responsible for the neuro-
nal plasticity and long-term potentiation (LTP) required for
memory and learning [11]. Continuous excitation of the re-
ceptor leads to high influx of calcium, with an initial excitato-
ry response followed by neuronal apoptosis [12].
*Gaurav Gulati
gaurav.gulati@uc.edu
1
Division of Immunology, Allergy and Rheumatology, Medical
Sciences Building (MSB), University of Cincinnati College of
Medicine, 231 Albert Sabin Way, ML 0563, Cincinnati, OH 45267,
USA
2
Department of Biomedical Engineering, Cleveland Clinic Lerner
Research Institute, Cleveland, OH 44195, USA
3
Department of Biostatistics & Epidemiology, Cincinnati Childrens
Hospital and Medical Center, Cincinnati, OH 45229, USA
Clin Rheumatol (2016) 35:29892997
DOI 10.1007/s10067-016-3339-1
Author's personal copy
Diamond et al. established that some murine monoclonal
anti dsDNA antibodies cross-react with the NR2 receptor and
caused neuronal cell death when injected into mouse brains
presumably due to excessive excitation [13]. This antibody is
directed specifically against the BDWEYS^amino acid se-
quence (Asp-Trp-Glu-Tyr-Ser) which is common to both mu-
rine and human NR2 subunits [9,13]. The same effects were
noted when human SLE sera containing high titers of anti-
dsDNA with cross-reactivity to anti-NR2 was injected into
mouse brains. However, this antibody was not pathogenic
when injected systemically unless the blood-brain barrier
(BBB) was disrupted [14].
There have been multiple studies on human anti-NR2 levels
and its relationship to CD [9]. The results have been conflicting
[9]. Most studies, which have examined the correlation of pe-
ripheral blood antibody and CD, have been negative.
Interestingly, all studies that have examined CSF have reported
positive associations. This would suggest that potentially path-
ogenic anti-NR2 antibodies are either produced intrathecally or
else, as in mice, that disruption of the BBB is required for their
CNS pathogenicity. Increased permeability of the BBB has
been noted in previous studies on NPSLE [9,15], and steroid
administration has been shown to lower CSF antibody concen-
tration [16]. However, previous studies evaluating the effects of
serum anti-NR2 levels on cognitive performance have not con-
sidered the role of the BBB in their analysis.
S100B protein is a glial-specific protein expressed by
endothelial-lining mature astrocytes that help form the BBB
[17,18]. It is an established biomarker for acute damage to the
BBB and has been studied in a variety of chronic neurological
diseases [18]. Three studies have examined the relationship of
S100B and NPSLE in general and have found conflicting
results [1921]. There have been no studies that have specif-
ically addressed the relationship of S100B and CD [1921].
Disruption of the BBB may be transient in response to
infections, trauma, drugs, or toxins [22]. Transient disruption
may be sufficient to permit entrance of pathogenic antibodies
into the CNS and yet not be detectable by S100B or other
direct measures of BBB integrity performed at a single point
in time. To identify intermittent disruption, it would be neces-
sary to longitudinally evaluate these markers, especially at
potentially critical times. Alternatively, since S100B is immu-
nologically privileged [18,19], if released into the systemic
circulation, it may induce autoantibody formation. Limited
work has been done in this area, but anti-S100B antibodies
have been found in some chronic neurological diseases like
multiple sclerosis and Parkinsonsdisease[23] and as well as
in some patients with Alzheimersdisease[24]. Anti-S100B
antibodies have also been studied in football players, and cor-
relations were found between the number of head Bhits^and
MRI changes and the titers of the antibody [22]. There have
not been any studies to date evaluating the role of anti-S100B
antibodies and cognitive function in SLE patients.
Therefore, the purpose of our study was to evaluate the
relationship of peripheral blood anti-NR2 antibodies with
CD in the context of BBB integrity. Our study is the first to
attempt to assess this complicated relationship.
Methods
Patient selection
We recruited willing patients from three clinical settingsthe
University of Cincinnati Medical Center SLE clinic, and the
faculty academic and community-based private practices.
Inclusion criteria for our study were adult SLE patients fulfill-
ing the ACR classification criteria, clinically stable disease
activity for at least 4 weeks prior to the study with no medi-
cation changes for that period. The University of Cincinnati
Institutional Review Board approved this research.
We collected demographic information including age, gen-
der, ethnicity, family income, occupation, and educational at-
tainment. We also ascertained clinical status which included
comorbidities, medications, SLE classification criteria met,
SLE disease activity index (SLEDAI-2K), SLE chronic dam-
age index (SLICC), and routine lab data by chart review [25,
26]. Finally, we assessed psychological state with Becks
Depression Inventory (BDI) and the Functional Assessment
of Chronic Illness (FACIT) Therapy Fatigue assessment [27,
28]. Health-related quality of life was also measured with the
Short Form 36 (SF-36) [29].
Patients underwent analysis of cognitive function using the
Automated Neuropsychologic Assessment Metrics (ANAM), a
validated and frequently used tool for cognitive assessment in a
number of conditions including SLE [30,31]. The ANAM is a
self-administered, computer-based test that takes about a half an
hour to complete, requires minimal training to administer, and
has been successfully used in mixed ethnic populations [32]. It
was designed to assess the cognitive domains similar to tradition-
al neuropsychological testing but does not need a highly trained
professional to administer and is less time intensive. The
ANAM4 includes simple reaction time (SRT) and eight subtests
that measure short-term and long-term recall, learning, working
memory, sustained attention, logical reasoning, and mathematical
and visual-spatial processing. The SRT is the time required to
depress the mouse button after seeing a symbol flashed onto the
computer screen. It measures neuromuscular efficiency (which
may be affected by arthritis, tendonitis, neuropathy, etc.) and
permits adjustment for this in subsequent analyses. For each
subtest, a number of parameters are reported, including accuracy,
mean and median response times, standard deviations of re-
sponse times, and throughput. The throughput is the number of
correct responses divided by the time required for the correct
responses. The total throughput score (TTS) is the sum of the
throughput scores for each of the eight domains. Patients were
2990 Clin Rheumatol (2016) 35:29892997
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categorized as having cognitive dysfunction if their TTS was
more than 1.5 SD below the mean of an age-, sex-, and race-
matched rheumatoid arthritis control population on two assess-
ments, a minimum of 6 months apart [30].
We collected serum samples from the patients at the time of
their cognitive assessments. These were aliquoted and frozen
at 80 °C until analyzed as a group.
Biomarker assays
Anti-NR2 antibody COSTAR 3690 plates (Fisher
Scientific) with 96 wells (25 μL per well) coated with
the DWEYSVWLSN decapeptide (20 μg/mL) in PBS
(phosphate-buffered saline) were used. FBS/PBS was
added at 50 μL/well at 37 °C. Serum was diluted in
3 % FBS/PBS. Diluted serum sample was then added
(25 μL/well). This was then incubated at 37 °C for an
hour and subsequently washed with PBS 0.05 %Tween-
20 (PBS-T) six times. Labeled secondary antibody was
added in 3 % FBS/PBS in 1:1000 dilutions (25 μL/
well).Itwasthenre-incubatedat3Cfor1hand
subsequently washed with PBS-T six times. It was then
developed at 25 °C using one tablet AP substrate
(SIGMA) for 5 mL of water solution containing
MgCl
2
(0.001 M final) and NaHCO
3
(0.05 M final),
50 μL/well. Serum anti-NR2 antibody levels were mea-
sured by ELISA using the 10 amino acid peptide se-
quence BDWEYSVWLSN^described by DeGiorgio
et al. previously, and the Optical Density (O.D.) values
measured were reported [13]. The samples were run in
triplicate and a standard curve was created.
S100B protein The S100B measurements were performed
using a commercially available ELISA kit (96 wells, anti-
human S100B, Diasorin, Stillwater, MN) according to the
manufacturers instructions and was read using a multiplate
fluorescent reader. Fluorescent signals were converted into
nanograms per milliliter according to standard curve
calibration.
Anti-S100B antibody The measurement of serum S100B au-
toantibodies was performed based on an ELISA method de-
scribedbyMarchietal.[22]. First, 96-well plates were coated
with a PBS solution containing S100B protein (human brain,
catalog number-559291, EMD Chemicals). Optimization of this
ELISA was achieved by testing two concentrations of S100B
protein (1 or 5 μg per well). No significant differences were
observed at these two concentrations of S100B coating. A coat-
ing solution of 1 μg per well was used. An S100B monoclonal
antibody (catalog number: Q86610M, Meridian Life Science
Inc.) was used as a standard to allow for the conversion of ab-
sorbance unit (AU) values into concentrations (μg/mL) or titer
(number of dilutions). Standard curves were obtained with
100 μL of serially diluted S100B monoclonal antibodies. Plates
were coated overnight at 4 °C with S100B protein (1 μg/well).
Wells were then washed three times with PBS. Subsequently,
100 μLofa1%BSAblockingsolutionwasaddedineachwell
and incubated for 2 h at room temperature. Wells were then
washed three times with 200 mL of PBS containing 0.05 %
Tween-20. Serum samples and standards were added and incu-
bated for 1 h at room temperature. Samples were aspirated and
wells washed three times using 200 μL of PBS containing
0.05 % Tween-20. A secondary antibody solution of 200 mL
of horseradish-peroxidase (HRP) goat anti-mouse IgG and
200 mL of HRP goat anti-human was added to the standards
and serum samples, respectively. After 1-h incubation at room
temperature wells, were washed three times with 200 μLofPBS
containing 0.05 % Tween-20. Finally, 100 μL of OPD solution
was added and the reaction incubated for 30 min at room tem-
perature. The reaction was stopped by adding 100 μLof2.5M
sulfuric acid. Samples were analyzed using an ELISA plate read-
er at 490 nm. A standard curve was created for anti-S100B
antibodies, and variability of the assay was assessed and found
to be within acceptable ranges [22].
Statistical analysis
Data entry was done on Microsoft Excel® spreadsheets
and analyzed using SAS 9.3® software [33]. Patient
population was characterized using descriptive statistics.
The association between CD status and the categorical
variable was evaluated by chi-square test or Fishers
exact test, and the continuous variables were compared
by either Studentsttest or Mann-Whitney test between
patients with and without CD. Initial evaluation of the
relationship between TTS and serum anti-NR2 antibody,
serum S100B protein levels, and serum anti-S100B
levels was done by Spearman correlation. To assess
the potential influence of BBB disruption on the corre-
lation of the TTS and anti-NR2 antibody, patients were
also divided into tertiles of S100B and anti-S100B and
a similar analysis was performed. In order to adjust for
the influence of independent variables (like age, ethnic-
ity, education, disease activity, medications, etc.) on
TTS, a multiple regression model was utilized with in-
teraction terms for anti-NR2 and S100B and anti-NR2
and anti-S100B. Some of the variables were not normal-
ly distributed, and logarithmic transformation was used
to correct for this. Candidate variables were first identi-
fied by a forward stepwise procedure. Logistic regres-
sion was also performed to identify independent vari-
ables influencing the presence of CD.
Preliminary estimates of power with case/control ratio of
approximately 1:4 would suggest that 50 subjects total would
be required to achieve an 80 % power to detect a 1 SD differ-
ence (0.07 od) in anti-NR2 at the α= .05 level.
Clin Rheumatol (2016) 35:29892997 2991
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Results
Patient characteristics
For our study, we enrolled a total of 57 patients with SLE.
Table 1summarizes the patient demographics and clinical
parameters including medication use. The mean age of pa-
tients was 49.9 years, 93 % were women, and a majority
was African-American (56.1 %). Education was limited with
36.8 % having 12 years and 45.6 % had a family income of
less than 20,000 USD a year. Disease activity measured by the
SLEDAI-2K was low (mean ± SD 3.6 ± 3.4), while the mean
chronic damage index (SLICC) score was substantial at 2.75
(SD = 2.4). Patient pain described on a 0 to 100 visual ana-
logue scale (VAS) was 40.0, while overall global assessment
by the patient on a similar scale was 53.7. Depression on
average was mild with a mean score of 16.2 (±12.3), but
35 % were affected to a moderate or severe extent. Fifty-
four percent were on prednisone, but only about 16 % were
on doses exceeding 20 mg. The mean prednisone use was
7.1 mg/day (SD 8.4). Approximately, 50 % were taking other
immunosuppressive medications including methotrexate, my-
cophenolate, and azathioprine (Table 1).
Using the above definition of CD, we identified 12 patients
who were cognitively impaired. Eleven of the 12 were evalu-
ated to 2 occasions at least 6 months apart and had persistent
CD. The remaining patient has yet to be reevaluated.
Comparing those with CD to those without CD, we found
the differences in age, family income, and ethnicity to be
statistically significant (p< 0.05). All other parameters includ-
ing disease duration, education, SLICC, FACIT and SLEDAI
scores, patientsglobal assessment and pain assessment, and
medication use were not statistically different between the two
groups (Table 1).
Serum anti-NR2 antibody, S100B protein, and anti-S100B
antibody levels
We compared the anti-NR2 antibody levels between patient
groups with and without CD and did not find a statistical
Tabl e 1 Patient demographics
and biomarkers: dichotomized by
CD
Va r i ab l e A l l s ub j e c t s ( n= 57) CD (n= 12) No CD (n = 45)
Age (years (SD)) 49.9 (11.2) 54.9 (8.8) 48.5 (11.5)*
Caucasians (%) 36.8 8.3 44.4*
Family income < USD 20 k (%) 45.6 75.0 37.8*
Females (%) 92.9 83.3 95.6
Education < 12 years (%) 36.8 50.0 33.3
SLE disease duration (years) 13.1 (10.1) 17.5 (13.9) 12.0 (8.6)
SLEDAI (mean (SD)) 3.6 (3.4) 3.2 (4.3) 3.8 (3.3)
SLICC (mean (SD)) 2.75 (2.4) 3.4 (2.1) 2.4 (2.4)
Pain (100 mm VAS patient) 40.0 (28.2) 49.6 (29.1) 36.2 (27.7)
Global assessment (100 mm VAS patient) 53.7(22.6) 58.8 (22.6) 52.3 (22.7)
BDI II (depression) (mean (SD)) 16.0 (12.3) 17.6 (10.2) 14.4 (12.6)
FACIT (fatigue) (mean (SD)) 24.6 (13.5) 26.1(10.8) 23.3 (14.3)
APL positive (%) 38.6 25.0 42.2
Current prednisone use (%) 53.7 54.5 53.9
Prednisone > 20 mg/day (%) 15.8 25.0 13.3
Immunosuppressant use (%) 48.2 36.4 51.2
Plaquenil use (%) 70.0 50.5 74.4
Warfarin (%) 14.8 18.2 14.0
Aspirin (%) 38.9 27.3 41.9
Antidepressants (%) 31.5 36.4 30.2
Opioid (%) 25.9 45.5 20.9
NSAID (%) 22.2 18.2 23.3
Serum anti-NR2 antibody (SD) 0.41 (0.24) 0.45 (0.19) 0.40 (0.26)
Serum anti-S100B antibody (SD) 0.57 (0.11) 0.58 (0.09) 0.57 (0.11)
Serum S100B protein (SD) 0.09 (0.05) 0.09 (0.05) 0.09 (0.05)
VAS visual analogue scale, CD cognitive dysfunction, SLE systemic lupus erythematosus, SLEDAI SLE disease
activity index, SLICC SLE chronic damage index, BDI Becks Depression Inventory, FA C I T Functional
Assessment of Chronic Illness Therapy, APL antiphospholipid, NSAID nonsteroidal anti-inflammatory
*Significant difference between the CD and no CD groups (p<0.05)
2992 Clin Rheumatol (2016) 35:29892997
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significant difference (p> 0.05). The same was true with re-
gard to the serum S100B protein levels and anti-S100B anti-
body levels between the two groups. The correlations of TTS
with anti-NR2 antibody levels, serum S100B protein levels,
and anti-S100B antibody levels were not significant either
(Table 2).
Next, we classified subjects into tertiles stratified according
to levels of S100B and anti-S100B, and then examined the
relationship of the anti-NR2 antibody levels with TTS (Figs. 1
and 2). Again, no statistically significant associations were
found (p> 0.05). To adjust for the influences of age, ethnicity,
family income, and other potentially important variables on
cognitive function, we used a multiple regression model in a
forward stepwise manner with TTS as the dependent variable.
Because S100B and anti-NR2 were not normally distributed,
they were log transformed. The other variables in the model
conformed to the assumption of normality. The results are
shown in Table 3. The only statistically significant indepen-
dent variables were age, ethnicity, throughput of simple reac-
tion time (SRT), and opioid use. The levels of anti-NR2 anti-
body, S100B protein levels, and anti-S100B antibody levels
did not significantly affect TTS. Because it is thought that
anti-NR2 antibody may be deleterious only when there is ev-
idence of BBB disruption, we performed the multiple regres-
sion using interaction terms (anti-NR2 and S100B levels and
anti-NR2 and anti-S100B antibody levels) which evaluated
the influence of anti-NR2 at high and low levels of these
markers of BBB disruption. Again, no consistent and statisti-
cally significant effect of anti-NR2 on TTS was found
(Table 3). Finally, we performed a logistic regression to model
CD (as defined previously). Apart from age, ethnicity, and
family income, we found no statistically significant associa-
tions (data not shown).
Discussion
There is persuasive animal data linking cross-reactive anti-
DNA/anti-NR2 antibodies and apoptotic neuronal cell death
in mice [13].Studiesofanti-NR2antibodiesintheCSFdem-
onstrate a consistent relationship between the anti-NR2 anti-
body levels and cognitive function [16,34,35].
However, previous studies looking at serum anti-NR2 an-
tibodies have led to conflicting results with most reporting no
significant association, although Brunner et al., studying pe-
diatric patients longitudinally, were able to find a correlation
of changes in anti-NR2 antibody with changes in some mea-
sures of cognition [36]. These differences in results are per-
haps due to differences in biomarker assay techniques, defini-
tion of CD and tools used to measure cognitive function, and
possibly differences in patient populations [9,31,3642]. The
other explanation is that the antibody is produced intrathecally
and never enters the systemic circulation [9]. Or, alternatively,
as in the animal models, the antibody could be produced sys-
temically but is pathogenic only if there has been disruption of
Tabl e 2 Correlation analysis between biomarkers and TTS
Comparison groups Correlation coefficient pvalue
TTS versus anti-NR2 0.059 0.6616
TTS versus S100B 0.161 0.2302
TTS versus anti-S100B 0.011 0.9334
S100B versus anti-S100B 0.132 0.3276
Spearmans correlation coefficient
a
100
200
3
400
500
-0.1
Total Throughput Score (TTS)
0
00
0.1 0.3
An-NR2 Ab Level
0.5 0.7
r = -0.030
p = 0.9035
0.9
b
c
100
200
300
500
-0.1
Total Throughput Score (TTS)
100
200
300
400
500
Total Throughput Score (TTS)
0
400
0.1
0
0
0.3
0.2
An-NR2 Ab Level
0.4
An-NR2 Ab Level
0.5
0.6
0.7
0.8
r = 0.037
p = 0.8810
r = -0.113
p = 0.6568
0.9
1
Fig. 1 Tertiles of S100B protein levels: anti-NR2 antibody versus TTS.
(acrepresent first, second, and third tertiles of serum S100B, respectively)
Clin Rheumatol (2016) 35:29892997 2993
Author's personal copy
the BBB. Our study was designed to assess this latter
possibility.
We chose to examine the serum levels of S100B, an astrocyt-
ic protein normally not found in peripheral blood, as an indicator
of current or recent disruption. It has been demonstrated to be a
reliable and valid measure of BBB disruption in other acute and
chronic neurologic diseases [24]. S100B protein has also been
used to assess patients with NPSLE in three studies. Schenatto et
al. examined the role of S100B protein in their study on NPSLE
patients and found S100B levels in serum to be double in
NPSLE (median 0.164 ng/mL, inter-quartile range 0.113 to
0.332) than the non-NPSLE patients (0.062 ng/mL, 0.026
0.109) or controls (0.088 ng/mL, 0.0130.124), and these differ-
ences were statistically significant (p< 0.001). CSF was not
evaluated [20]. Subsequently, Yang reported on 157 patients,
of which 65 had NPSLE, and found significant differences in
both their serum and CSF-based samples [21], while Fragoso-
Loyo, studying the utility of serum S100B levels in CNS in-
volvement in SLE patients, found no differences in their
NPSLE group, either in serum or in CSF [19]. In this last study,
no patients were reported to have CD as their NPSLE manifes-
tation. And, in the studies by Yang et al. and Schenatto, very few
patients had CD and these were not analyzed separately [1921].
Since disruption of the BBB could occur intermittently
and/or could have occurred previously and still have permitted
significant CNS injury, we measured antibodies against
S100B in serum as a potential indicator of previous BBB
disruption. As an immunologically privileged protein,
S100B might initiate an immune response in susceptible indi-
viduals if released in significant concentrations into the pe-
ripheral blood. That autoantibody might persist for months,
if not longer. There have been a few previous studies which
have examined antibodies to S100B in other chronic neuro-
logical disorders and head trauma [2224]. Poletaev and col-
leagues looked at patients with multiple sclerosis and
Parkinsons(N= 26 and 20, respectively) and found the prev-
alence of these antibodies to be significantly higher than con-
trols (p< 0.001) [23]. Gruden et al. studied the anti-S100B
antibody levels in Alzheimers patients (N= 48) and found
that the levels were significantly higher in early cases with
moderate dementia; the same was not true for early cases with
mild dementia or advanced cases [24]. Marchi and colleagues
have demonstrated that half of the football players they
a
100
200
300
400
500
-0.1
Total Throughput Score (TTS)
0
0.1 0.3
An-NR2 Ab Level
0.5 0.7
r = 0.2240
p = 0.3216
0.9
b
c
100
200
300
400
500
-0.1
Total Throughput Score (TTS)
100
200
300
400
500
Total Throughput Score (TTS)
0
0.1
0
0
0.3
An-NR2 Ab Level
0.2 0.4
An-NR2 Ab Level
0.5
0.6
0.7
0.8
r = 0.075
p = 0.7664
r = -0.409
p = 0.0823
0.9
1
Fig. 2 Tertiles of anti-S100B Ab levels: anti-NR2 antibody versus TTS.
(ac represent first, second, and third tertiles of serum anti-S100B,
respectively)
Tabl e 3 Multiple regression analysis
Parameter Parameter
estimate
SE t
value
p
value
Intercept 341.34 120.17 2.84 0.007
Log (anti-NR2) 71.54 70.23 1.02 0.314
Log (S100B) 22.91 54.40 0.42 0.676
Anti-S100B 56.45 150.70 0.37 0.710
Log (anti-NR2)
a
Log (S100B) 40.71 65.66 0.62 0.639
Log (anti-NR2)
a
Anti-S100B 4.38 73.21 .06 0.953
Log (S100B)
a
anti-S100B 49.82 73.21 1.59 0.119
Simple reaction time
(throughput)
0.600 0.17 3.43 0.001
Age (per year increase) 0.008 0.002 3.71 0.001
Opioid use 82.93 22.05 3.76 0.001
Caucasian ethnicity 76.60 22.27 3.44 0.001
Income level (low) 15.89 23.86 0.67 0.509
a
Dependent variable: total throughput score (TTS); method: forward
stepwise multiple regression
a
Interaction between two variables such that the effect of one depends on
the level of the other
2994 Clin Rheumatol (2016) 35:29892997
Author's personal copy
studied with repetitive head trauma had increased levels of the
antibody (p< 0.05) which correlated with serum S100B levels
and MRI changes [22]. But, there have been no previous stud-
ies evaluating anti-S100B antibodies in patients with SLE.
In our study, we found significant association of the TTS, a
measure of global cognitive function, with age, ethnicity, sim-
ple reaction time (SRT), and opioid use as discussed above.
We were, however, unable to find a significant relationship
between the anti-NR2 antibody and CD despite using bio-
markers of both recent and remote BBB disruption. We select-
ed our study population from three differentsources to make it
more representative of the general SLE population and to try
to minimize selection bias. We used a validated and reproduc-
ible test of cognitive performance, the ANAM, for our study.
We used an age, sex, and race-matched control population
of RA patients in order to define CD. In doing so, we were
able to control for the potential influence of a chronic, debil-
itating, and painful condition on cognition. Most studies have
not used disease controls and may have misclassified subjects
as cognitively impaired as a consequence of SLE when, in
fact, their dysfunction was due to other non-immunologic fac-
tors such as fatigue, anxiety, fear, pain, and depression. We
also required persistence of CD for at least 6 months in order
for a subject to be so classified. This was done to identify the
subset of patients of greatest interestthose with persistent or
progressive disease and to exclude those with self-limited
dysfunction who may not need specific intervention.
We also collected detailed information on potential covar-
iates that might impact cognitive performance including edu-
cation, income, disease activity and severity, medication use,
pain, fatigue, and depression. We adjusted for these potential
confounders in our multivariate analyses.
Our study has limitations. We examined a relatively small
patient population, especially of those who were cognitively
impaired. However, power calculations suggest that this is not
a major problem. We utilized used a cross-sectional design for
efficiency. However, CD may be a slowly progressive or an
intermittently progressive phenomenon, or it may have oc-
curred years previously and left no trace of the responsible
pathogenic influence. We would not be able to detect these
time-dependent processes reliably, only a prospective study
would be able to do so.
In addition, the biomarkers we used to identify BBB dis-
ruption may not be sufficiently sensitive or specific for this
process. There is very little information currently available
comparing these biomarkers to others that might be employed
in this context. Our choice was based upon limited informa-
tion that is available. Better biomarkers may exist.
The other explanation for the lack of association of periph-
eral anti-NR2 antibody and CD is that it is only the intrathe-
cally produced anti-NR2 antibody that is pathogenic and pe-
ripheral antibody is irrelevant. The ideal study design would
have included measurement of CSF levels of anti-NR2
antibody, Q albumin, and IgG index. This would have provid-
ed more definitive information to answer this question.
However, we felt that sampling of CSF would have been
invasive and not felt to be feasible in our patient population.
However, if the intrathecal anti-NR2 antibody does produce
neuronal cell damage and death, this might lead secondarily to
disruption of the BBB and leakage of the antibody into the
peripheral circulation enabling its detection. If reliable surro-
gate biomarkers of BBB disruption were available, then the
combination might serve as a less invasive diagnostic test.
Further work is needed to understand cognitive dysfunc-
tion in SLE. A larger population of SLE patientsneeds to be
studied in order to account for the multiplicity of variables that
could potentially influence cognitive function. A prospective
study design with multiple sampling times and multiple bio-
markers measured longitudinally will be necessary to help
unravel the web of cause and effect. Moreover, future studies
should also assess the possibility that CD is the result of a
multistep process with potentially different pathogenic factors
operating at each of the steps.
Acknowledgments The project was supported by the National Center
for Advancing Translational Sciences of the National Institutes of Health
[Award Number UL1TR000077] (Bin Zhang, Ph.D.) and P30AR047363
(Susan Thompson, Ph.D.). The content is solely the responsibility of the
authors and does not necessarily represent the official views of the NIH.
This work would not have been possible without the cooperation and
enthusiastic support of Drs. J. Lawrence Houk, Yolanda Farhey, Avis
Ware, and David Greenblatt who encouraged their patients to participate
in this study and Susan D. Thompson, PhD, and her expert staff at the
Cincinnati Childrens Hospital Medical Center who processed, stored,
and managed all serum samples. We are also most grateful to Dr. Betty
Diamond and her lab at the Center for Autoimmune and Musculoskeletal
Diseases at The Feinstein Institute for Medical Research, Manhasset, NY,
for the performance of the anti-NR2 antibody assays. We also would like
to thank Mr. Vikram Puvenna in the Department of Biomedical
Engineering at the Cleveland Clinic Lerner Research Institute for his
technical expertise and assistance with the S100B and anti-S100B assays.
Compliance with ethical standards
Disclosures None.
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... [7] Consequently, (transient) BBB dysfunction in NPSLE may lead to inflammatory mediators, such as plasma cells, accessing the cerebrospinal fluid and cerebrum and produce intrathecal antibodies. [7] Studies on the correlation between cerebrospinal fluid markers of BBB function in humans (such as Q albumin [8][9][10][11][12][13][14], S100B [15], anti-S100B [15]) and psychiatric NPSLE manifestations yielded contradictory results. In these studies with contradictory results, Q albumin was compared between NPSLE patients and SLE patients (without known NPSLE) or patients with a different neurologic J o u r n a l P r e -p r o o f disease. ...
... [7] Consequently, (transient) BBB dysfunction in NPSLE may lead to inflammatory mediators, such as plasma cells, accessing the cerebrospinal fluid and cerebrum and produce intrathecal antibodies. [7] Studies on the correlation between cerebrospinal fluid markers of BBB function in humans (such as Q albumin [8][9][10][11][12][13][14], S100B [15], anti-S100B [15]) and psychiatric NPSLE manifestations yielded contradictory results. In these studies with contradictory results, Q albumin was compared between NPSLE patients and SLE patients (without known NPSLE) or patients with a different neurologic J o u r n a l P r e -p r o o f disease. ...
... No correlation with depression or cognitive dysfunction was found. [15] Three studies found disruption of the BBB in a subgroup of patients presenting with neuropsychiatric symptoms according to the ACR classification. [8,10,13] A considerable number of methodologically similar (N=8) of studies found evidence for the intrathecal synthesis of several different antibodies in NPSLE patients with psychiatric manifestations [8,10,13,14,[16][17][18][19], while only one study showed no such association [9]. ...
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Introduction The aim of this narrative review is to provide an overview of the literature on the possible immunologic pathophysiology of psychiatric manifestations of neuropsychiatric systemic lupus erythematosus (NPSLE). Methods A systematic search on PubMed was conducted. English studies with full text availability that investigated the correlation between blood-brain barrier (BBB) dysfunction, intrathecal synthesis of antibodies, antibodies, cytokines, chemokines, metalloproteinases, complement and psychiatric NPSLE manifestations in adults were included. Results Both transient BBB-dysfunction with consequent access of antibodies to the cerebrospinal fluid (CSF) and intrathecal synthesis of antibodies could occur in psychiatric NPSLE. Anti-phospholipid antibodies, anti-NMDA antibodies and anti-ribosomal protein p antibodies seem to mediate concentration dependent neuronal dysfunction. Interferon-α may induce microglial engulfment of neurons, direct neuronal damage and production of cytokines and chemokines in psychiatric NPSLE. Several cytokines, chemokines and matrix metalloproteinase-9 may contribute to the pathophysiology of psychiatric NPSLE by attracting and activating Th1-cells and B-cells. Discussion This potential pathophysiology may help understand NPSLE and may have implications for the diagnostic management and therapy of psychiatric NPSLE. However, the presented pathophysiological model is based on correlations between potential immunologic etiologies and psychiatric NPSLE that remain questionable. More research on this topic is necessary to further elucidate the pathophysiology of NPSLE.
... New technologies have enabled the quantification of nanomolar concentrations of brainderived biomarkers in blood, allowing a minimally invasive diagnosis of brain damage and neurodegenerative processes [167]. The appearance of NVU molecules in blood has been reported for neurodegenerative diseases [168,169], seizures [170][171][172][173][174], neurologic manifestations of systemic disease [175], traumatic brain injury (TBI) [176][177][178], psychiatric diseases [179] and brain tumours [180,181]. ...
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... Increased Q-albumin is seen when the blood-brain barrier is leaking, and albumin passes from blood into the CSF, followed by increased passage of other proteins, including antibodies, cytokines and other bioactive molecules. Disrupted integrity of the blood-brain barrier has lately been advocated as an important and necessary step for neuropsychiatric SLE to develop [33], although some have questioned this [19,34]. In this study, there was a tendency towards increasing Q-albumin increased NfL levels in the SLE patients, but not pSS. ...
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A wide range of patients with systemic lupus erythematosus (SLE) suffer from cognitive dysfunction (CD) which severely impacts their quality of life. However, CD remains underdiagnosed and poorly understood. Here, we discuss current findings in patients and in animal models. Strong evidence suggests that CD pathogenesis involves known mechanisms of tissue injury in SLE. These mechanisms recruit brain resident cells, in particular microglia, into the pathological process. While systemic immune activation is critical to central nervous system injury, the current focus of therapy is the microglial cell and not the systemic immune perturbation. Further studies are critical to examine additional potential therapeutic targets and more specific treatments based on the cause and progress of the disease.
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To the Editor: We read with great interest the editorial1 by David A. Isenberg related to our article, “Development and Assessment of Users’ Satisfaction with the Systemic Lupus Erythematosus Disease Activity Index 2000 Responder Index-50 Website”2 (S2K … Address correspondence to Dr. M.B. Urowitz, Centre for Prognosis Studies in the Rheumatic Diseases, Toronto Western Hospital, Room 1E-409, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. E-mail: m.urowitz{at}utoronto.ca
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Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease, which leads to the damage of multiple tissues and organs, including the central and peripheral nervous system. Due to the variety of neurological and psychiatric symptoms of lupus in 1999 the American College of Rheumatology (ACR) distinguished 19 neuropsychiatric syndromes that may appear in SLE and set specific criteria to diagnose them (Table I). The involvement of the nervous system in SLE occurs relatively often and neuropsychiatric syndromes correlate with severe disease progression and contribute to worse prognosis. This article presents the central nervous system (CNS) syndromes met in SLE included in the 1999 ACR classification. The latest European League Against Rheumatism (EULAR) recommendations for diagnosis and management of neuropsychiatric lupus are also presented.
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Neurological and psychiatric disorders are common in patients with systemic lupus erythematosus (SLE). While several pathogenetic mechanisms are thought to be involved, among of the most challenging and best investigated are antibodies against the N-methyl-D: -aspartate (NMDA) receptor subtypes 2a and 2b (anti-NR2 antibodies). This review summarizes the most relevant mechanisms for neuropsychiatric (NP) involvement in SLE (NPSLE) with special emphasis on the role of anti-NR2 antibodies and provides an overview of published articles on anti-NR2 antibodies and brain involvement as of May 2011. In mice, neuronal cell death occurs when anti-NR2 antibodies gain access to the brain, either by injection directly into the brain, or by systemic immunization and abrogation of the blood-brain barrier. Either impaired memory and hippocampal atrophy, or emotional disturbances and atrophy of the amygdala follow, seemingly dependent on the method used for disruption of the blood-brain barrier. Recent studies indicate that the effect of anti-NR2 antibodies is dose dependent; at low concentrations they alter synaptic function; at higher concentrations they can cause neuronal cell death by apoptosis. An association between anti-NR2 antibodies and NPSLE has been confirmed in 6 out of 13 human studies; the manifestations are primarily of diffuse cerebral character.