Antibody to aquaporin-4 in the long-term
course of neuromyelitis optica
S. Jarius,1,2F. Aboul-Enein,3P.Waters,1B. Kuenz,4A. Hauser,5T . Berger,4W. Lang,6M. Reindl,4
A.Vincent1and W. Kristoferitsch3
1Neurosciences Group,Weatherall Institute of Molecular Medicine, and Department of Neurology, John Radcliffe Hospital,
University of Oxford,UK,2Division of Molecular Neuroimmunology, Department of Neurology,University of Heidelberg,
Heidelberg,Germany,3Department of Neurology, Sozialmedizinisches Zentrum Ost ^ Donauspital,Vienna,4Clinical
Department of Neurology, Innsbruck Medical University, Innsbruck,5Department of Laboratory Medicine,
Sozialmedizinisches Zentrum Ost ^ Donauspital,Vienna and6Department of Neurology, Hospital Barmherzige Brueder,
Correspondence to: Univ.-Doz. Dr Wolfgang Kristoferitsch, Department of Neurology, Sozialmedizinisches Zentrum
Ost- Donauspital, Langobardenstra?e122,1220 Vienna, Austria
Neuromyelitis optica (NMO) is a severe inflammatory CNS disorder of putative autoimmune aetiology, which
predominantly affects the spinal cord and optic nerves. Recently, a highly specific serum reactivity to CNS
microvessels, subpia and Virchow^Robin spaces was described in patients with NMO [called NMO^IgG
(NMO^immunoglobulin G)]. Subsequently, aquaporin-4 (AQP4), the most abundant water channel in the
CNS, was identified as its target antigen. Strong support for a pathogenic role of the antibody would come
from studies demonstrating a correlation between AQP4-Ab (AQP4-antibody) titres and the clinical course of
disease. In this study, we determined AQP4-Ab serum levels in 96 samples from eight NMO^IgG positive
patients (median follow-up 62 months) in a newly developed fluorescence-based immunoprecipitation assay
employing recombinant human AQP4.We foundthat AQP4-Ab serumlevels correlate with clinicaldisease activ-
ity, with relapses being preceded by an up to 3-fold increase in AQP4-Ab titres, which was not paralleled by a
rise in other serum autoantibodies in one patient.Moreover, AQP4-Ab titres were found to correlate with CD19
cellcounts during therapy with rituximab.T reatment with immunosuppressants such asrituximab, azathioprine
and cyclophosphamide resulted in a marked reduction in antibody levels and relapse rates.Our results demon-
strate a strong relationship between AQP4-Abs and clinical state, and support the hypothesis that these
antibodies are involved in the pathogenesis of NMO.
Keywords: Devic syndrome; neuromyelitis optica; longitudinally extensive transverse myelitis; NMO-IgG;
aquaporin-4 antibody; long-term follow-up
Abbreviations: Ab=antibody; AChR=acetylcholine receptor; AQP4=aquaporin-4; CNS=central nervous system;
FIPA=fluorescence based immunoprecipitation assay; FU=fluorescence units; IgG=immunoglobulin G; IVMP=intravenous
methylprednisolone; LETM=longitudinally extensive transverse myelitis; MRI=magnetic resonance imaging;
NMO=neuromyelitis optica;TG=thyroglobulin;TPO= thyroid peroxidase
Received April 26, 2008. Revised and Accepted September1 , 2008
Neuromyelitis optica (NMO) is a severe inflammatory
central nervous system (CNS) disorder of putative auto-
immune aetiology, which predominantly affects the spinal
cord and optic nerves (Wingerchuk et al., 1999, 2006;
de Seze et al., 2002). Recently, a highly specific serum
reactivity to CNS microvessels and subpia was described in
patients with NMO [called NMO–immunoglobulin G
(NMO–IgG)] (Lennon et al., 2004). Subsequently, the
same group identified aquaporin-4 (AQP4), the most
abundant water channel in the CNS, as the target antigen
(Lennon et al., 2005). Both findings have been confirmed
doi:10.1093/brain/awn240 Brain (2008),131, 3072^3080
? 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which
permits unrestricted non-commercialuse, distribution, andreproductionin anymedium, provided the original work is properlycited.
by others (Jarius et al., 2007; Takahashi et al., 2007; Paul
et al., 2007). There is increasing evidence that NMO–IgG/
AQP4-Ab (antibody) contributes to the pathogenesis of the
disease (Lucchinetti et al., 2002; Wingerchuk et al., 2007;
Jarius et al., 2008). Sites of intralesional AQP4 loss were
histopathologically found to correlate with sites of immu-
noglobulin and complement activation (Misu et al., 2007;
Roemer et al., 2007), and the antibody is predominantly
IgG1 subclass and activates complement after binding to
extracellular epitopes in vitro (Hinson et al., 2007; Waters
et al., 2008). Support for a pathogenic role of the antibody
would come from studies demonstrating correlation of
AQP4-Ab titres and clinical course.
In the present study, we assessed AQP4-Ab in NMO
patients with long-term follow-up using a newly developed
immunoprecipitation assay employing enhanced green
fluorescent protein (EGFP)-tagged recombinant human
AQP4 (Waters et al., 2008).
Patients and Methods
Caucasian origin diagnosed with either isolated longitudinally
extensive transverse myelitis (LETM) (n=2) or LETM and optic
neuritis (n=6) were retrospectively evaluated for AQP4-Abs.
NMO–IgG testing was done by the Mayo Medical Laboratories
(Lennon et al., 2004). Six patients fulfilled Wingerchuk’s revised
diagnostic criteria (Wingerchuk et al., 2006); the two patients with
remitting LETM are part of the NMO-spectrum, a broader clinical
syndrome than originally described (Wingerchuk et al., 2007). No
history of disease outside the optic nerve or spinal cord was
present at onset. Extra-opticospinal MRI lesions were detectable in
two patients at disease onset and in five of eight patients (71%)
later in the disease course. Disease followed a relapsing course in
all patients. Median follow-up was 62 months (range 33–114).
Seven patients were female, one male. Median age at onset was
45 years (range 14–59). Serum samples were stored at ?80?C until
testing. The clinical course was retrospectively evaluated without
knowledge of the AQP4-Ab test results. The study was approved
by the institutional review boards of the City of Vienna and the
Innsbruck Medical University, and patients’ consent was obtained
in all cases.
AQP4-Abs were assessed in a fluorescence based immunopre-
cipitation assay (FIPA) as described in detail elsewhere (Waters
et al., 2008). Briefly, 25ml of each serum was incubated with 250ml
of an extract from human embryonic kidney cells transfected with
EGFP-tagged M1- and M23-human AQP4. The IgG was then
precipitated using Protein A sepharose beads, washed thoroughly,
and the amount of EGFP–AQP4 bound by antibody detected by
counting the green fluorescence [arbitrary fluorescence units (FU)]
at 512nm (excitation 472nm; cut-off 495nm) on a fluorescence
plate reader (SpectraMAx Gemini XS, Molecular Devices, CA,
USA). Results were given as FU precipitated by each serum sample
under standard conditions. The mean + 3 SD from 10 healthy
control samples was 63 FU. Acetylcholine receptor (AChR)
antibodies were detected by a commercially available radio-
peroxidase (TPO) and thyroglobulin (TG) were detected by
two commercially available chemoluminescence immunoassays
(Immunlite 2000 system; DPC-Buehlmann, Salzburg, Austria).
The proportion of CD19-positive cells among total lymphocytes
was established by standard flowcytometric analysis of whole-
blood samples using a Cytomic FC 500?cell counter (Beckman
Fullerton, CA, USA) and IOTest?CD19 PC7 conjugated antibody
(Immunotech S.A., Marseille, France) (Pat. 1, 3 and 4) or a
FACScan (BD Biosciences, NJ, USA) and tritest CD45/CD3/CD19
antibodies (BD Biosciences, NJ, USA) (reference range: 0.1–0.5 ?
109cells/l or 6–19% of the total lymphocyte number) (Pat. 2).
The protocols for flowcytometric analysis are approved for
10/patient; range 7–18) from eight patients previously
found to be NMO–IgG positive. Ninety-five out of 96
samples samples were positive for AQP4-Ab. AQP4-Ab
values varied between 61 and 1091 FU (median 302; cut-off
63). Detailed results are shown in Figs 1–3.
wasdetermined in96 samples(median
AQP4-Ab serum levels in relapse and
AQP4-Ab was determined in 20 samples from eight patients
obtained at onset of relapse. Median AQP4-Ab levels in
these samples were higher (607 FU; range 198–1091 FU)
when compared with samples taken during remission
(median 221; range 61–761; n=57) (P50.0001, Mann
Whitney test; Fig. 1A), and maximum AQP4-Ab levels in
relapse were significantly higher than nadir values during
the following remission period in paired samples (P50.001;
Wilcoxon’s matched-pairs rank sum test; Fig. 1B). The
19/20 (95%) samples taken during relapse yielded results
exceeding the overall median AQP4-Ab levels found during
remission. In five cases, samples taken within 100 days
prior to onset of relapse were available, demonstrating that
NMO attacks are preceded by a marked rise in AQP4-Ab
levels (Fig. 2A–E). Antibody values rose by 124–294%
(median 192) within 48–99 days (median 85), correspond-
ing to a median increase of around 20% per week prior to
Conversely, AQP4-Ab was detectable in 56/57 samples
from eight patients during remission (430 days from
relapse onset), but the titres were low (52?cut-off) in
eight samples and slightly under cut-off in one (Fig. 2I).
The 50/57 remission samples (88%) yielded results below
the overall median AQP4-Ab value found during relapse.
Increase in AQP4-Ab levels during relapse
is not paralleled by a rise in other
In one patient with pre-existing autoimmune myasthenia
AQP4-Ab in the long-term course of NMOBrain (2008),131, 3072^30803073
determined in parallel with antibodies to AChR, TPO and
TG. While AQP4-Ab levels increased during two relapses by
42% and 67% compared with the first available value, all
other autoantibodies titres had declined at that time
(AChR-Ab by 54.1%; TPO-Ab 32%; TG-Ab 45.7%)
(Fig. 3). Moreover, AQP4-Ab levels rose by 49.8% over
the observation period of 2323 days, while AChR-, TPO-
and TG-Ab concentrations declined by 90%, 94.2% and
94.3%, respectively, under therapy with cyclophosphamide.
While AChR-, TPO- and TG-Ab titres correlated well over
time (r2=0.9, 0.95 and 0.99, respectively; P50.005), no
correlation of AQP4-Ab levels with any of these auto-
antibodies was found (r250.04).
Prompt and rapid decline of AQP4-Ab
levels under therapy
obtained within 100 days (median 37; range 6–92) from
onset of relapse. AQP4-Ab testing demonstrated a prompt
and marked decline of serum levels under therapy with
steroids and immunosuppressants in all cases. Treatment
regimens included intravenous methylprednisolone (IVMP)
in combination with azathioprine, dexamethasone, ritux-
imab, mitoxantrone or prednisolone. AQP4-Ab levels
decreased by 4–19% (median 8%) per week; when taking
into account only those samples that were taken within the
first month after relapse, a decremental rate of 9.3% per
week was found. In two patients treated with a combination
of azathioprine and prednisolone following IVMP for acute
relapse, further samples were obtained during remission,
demonstrating constantly low serum levels over more than
350 and 500 days, respectively.
Correlation of CD19 counts and AQP4-Ab
levels under treatment with rituximab
CD19 cell numbers declined after treatment with rituximab
following a protocol proposed by Cree and co-workers
(Cree et al., 2005). They reappeared after 251, 258, 265, 272
and 350 days, respectively, following the last application of
rituximab, with slight rises in cell counts being associated
with a strong increase in AQP4-Ab values (Fig. 2A, day
3168; Fig. 2B, day 1392). Although application of the drug
was followed by a prompt and marked decline in titres
(51–90%; P=0.02, Wilcoxon’s matched-pairs rank sum
test; Fig. 4), AQP4-Ab remained detectable during therapy
with rituximab in 29/30 samples. Moreover, AQP4-Ab was
still positive despite the cell numbers being below the
detection limit in 16/17 samples. No correlation between
CD19 cell count and AQP4-Ab values was found in those
patients not treated with rituximab (data not shown).
Reduced relapse rate under
Relapse rates in patients treated with rituximab are given in
Table 1; median relapse rate was 2.3/year (1.55–2.79) before
and 0.51/year (0.46–1.04) after initiation of therapy.
Although the overall relapse rate declined under rituximab,
at least one relapse occurred in each patient while under
therapy (Fig. 2A–C and I). In three of four patients relapses
occurred 260, 311 and 364 days, respectively, from last
infusion (Fig. 2A–C). Relapses were preceded or paralleled
by reoccurrence of CD19 cells and an up to 3-fold rise in
Fig.1 AQP4-Ab levels during relapse and remission. (A) Median
AQP4-Ab levels from 57 samples stratified according to disease
activity (P50.0001; Mann Whitney test). (B) Maximum AQP4-Ab
serum levels from11samples taken during relapses in six patients
and Nadir values during subsequent remission (P=0.001; Wilcoxon
matched-pairs signed-rank test).
3074Brain (2008),131, 3072^3080 S. Jarius et al.
Fig. 2 AQP4-Ab levels,CD19 cell counts (% of total lymphocytes), relapses and immunosuppressive treatment over time in eight
patients with NMO.Time points are selected to illustrate the relationship between AQP4-Ab, relapses and therapies. (A and J) Pat.1;
(B) Pat.3; (C) Pat.2; (D and H) Pat. 7; (E) Pat. 8; (F) Pat. 6; (G) Pat. 5; (I) Pat. 4. See results section for details. ^=AQP4-Ab serum
=CD19 cell counts; =clinical relapse;=intravenous methylprednisolone;
=dexamethasone; =rituximab; =cyclophosphamide;
eod=every other day.
AQP4-Ab in the long-term course of NMO Brain (2008),131, 3072^30803075
AQP4-Ab levels in these cases. Therapy intervals were
shortened in two of them resulting
further attacks (Fig. 2A and B). In another patient with
elevated AQP4-Ab levels prior to application, rituximab
could not prevent clinical attacks 27 and 99 days later
In the only patient undergoing long-term treatment
1295 days (=2.82/year) prior to initiation of therapy but
only one within 1610 days (=0.23/year) while on therapy
(50mg/day orally; later reduced to 50mg eod; Fig. 2G).
Long-term treatment with mitoxantrone was tried in three
patients but resulted in a reduction of clinical attacks in
only one of them (Table 1). However, relapse free periods
prior to any long-term immunosuppressive therapy were
found to last up to 40 months (median 308.5 days;
interferon beta was not paralleled by decrease in relapse
rate (Table 1).
in absence of
Suspension of azathioprine is followed
by increase in AQP4-Ab levels and
In three patients, therapy with azathioprine was maintained
for more than the maximum latency period of six months
(268, 235 and 673 days) and interrupted later in the disease
course. Azathioprine resulted in a marked decline in relapse
rate in two of them, and a mild decline in one (Table 1).
While 10 relapses occurred within 2199 days prior to
therapy, only two relapses occurred within 2212 days under
therapy. Annualized relapse rates prior to and under
treatment are given in the Table 1.
Interruption of therapy was, however, followed by
clinical relapse (34, 65 and 181 days after suspension,
respectively) and increase of AQP4-Ab values (2.2-, 1.4-
and 7.2-fold, respectively) in all cases (Fig. 2D, F, H and J).
Relapses occurred despite sustained therapy with low dose
prednisolone (tapered off to 5mg/day one month before
Fig. 2 Continued.
3076Brain (2008),131, 3072^3080S. Jarius et al.
relapse) in one patient (Fig. 2D). In the other patients,
prednisolone was interrupted 155 and 302 days, respec-
tively, prior to interruption of azathioprine without causing
clinical deterioration or relapse (Fig. 2F, H and J).
High AQP4-Ab levels are not always
associated with clinical relapse
Despite the observations above, a confirmed and consider-
able rise in AQP4-Ab levels (284%, 149% and 95%,
respectively, compared with last Nadir value), in three
patients was not followed by clinical relapse (Fig. 2C, F
and G). In one of them, rituximab-induced B-cell deple-
tion interrupted any further increase of antibody levels
(Fig. 2C). In the two other patients, AQP4-Ab levels rose
respectively, when reducing the dosage of cyclophospha-
mide or azathioprine without resulting in clinical deteriora-
tion or relapse (Fig. 2F and G). Despite the increasing
AQP4-Ab levels, the relapse rate declined under therapy
with cyclophosphamide to one relapse within 1610 days
compared with 10 relapses within 1295 days before therapy
In this study, we provide quantitative data on AQP4-Ab in
the long-term course of NMO. Our finding that relapses in
NMO is preceded by a rise in serum AQP4-Ab levels
strengthens the case for the antibody being involved in the
pathogenesis of the disease. Although the correlation found
in our study does not prove a causal relationship on its
own, it is consistent with recent evidence from histopatho-
logical and immunological studies that indicate a direct
contribution of AQP4-Ab to tissue damage in NMO
(Hinson et al., 2007; Misu et al., 2007; Roemer et al., 2007;
Fig. 3 Increase in AQP4-Ab levels during relapse is not paralleled by a rise in other autoimmune autoantibodies. ^=AQP4-Ab;
=AChR-Ab; =TG-Ab;=TPO-Ab; =clinical relapse;
Fig. 4 AQP4-Ab serum levels before and after (values refer to
Nadir values) eight applications of rituximab in four patients with
NMO/LETM (P=0.0156; Wilcoxon matched-pairs rank sum test).
AQP4-Ab in the long-term course of NMOBrain (2008),131, 3072^30803077
Jarius et al., 2008; Waters et al., 2008). This is in contrast to
other antibody-associated autoimmune diseases of the CNS
(such as paraneoplastic neurological disorders), in which
the antibodies are considered mere diagnostic markers with
no well accepted role in the mechanisms of lesion
AQP4-Ab was detectable in serum during relapse as well
as during remission, both in untreated patients and in
almost all samples obtained under immunosuppressive
therapy, suggesting that AQP4-Ab testing can be of
diagnostic relevance independently of disease activity or
Shortly before relapse, AQP4-Ab levels rose rapidly
(?20% per week) and markedly (up to ?290%) in all
cases studied. In addition, in one of our patients, we found
that AQP4-Ab levels rose selectively during clinical attack
despite immunosuppressive treatment, while three other
auto-antibodies, unrelated to NMO, declined at the same
time or remained low. These findings argue in favour of the
increase in AQP4-Ab levels being specific in this patient
and against it being simply part of a general increase in B-
cell activity during relapse.
Interestingly, however, no general threshold value for
triggering clinical relapse was found, but serum levels
detected during relapse differed widely both intra- and
inter-individually. Although all attacks studied were paral-
leled by a rise in AQP4-Ab levels, in a minority of samples
titres during remission were found to be higher at some
time-point than titres found during relapse in the same
patient or other patients. While in some cases, low titres
were associated with clinical relapse, high titres were not
paralleled by clinical disease activity in some patients
treated with immunosuppressants. These observations are
similar to those in myasthenia gravis, an accepted antibody-
mediated neurological disease, and do not argue against the
hypothesis that the antibodies are pathogenic. However,
they indicate that the presence of AQP4-Ab alone may not
be sufficient to cause disease; other factors, for instance,
disease-specific T cells, raised cytokines, unspecific stimula-
tion by exogenous triggers or damage to the blood brain
barrier, might be required to initiate or cause tissue
damage. Our results do suggest, however, that AQP4-Ab
will generally be of use as a marker of disease activity over
time in individual patients.
Initiation of immunosuppression was apparently fol-
lowed by a marked and rapid decline of both AQP4-Ab
levels and flare rates in all of our patients. However, taking
into accountthe well knownlatencyof actionof
T able1 Relapse rates under therapy with various immunosuppressants
Relapses before initiation of therapyRelapses after initiation of therapy
Pat.35/545 days3.35/year 2/395 days 1.84/year
d. Azathioprine (+prednisolone)
Pat.63/638 days1.72/year 0/710 days
and 0/313 days
Pat.7 4/197 days 7 .41/year
Pat.8 3/1293 days 0.85/year
Pat.5 10/1295 days2.82/year 1/1610 days0.23/year
f. Interferon beta
Pat.2 10/1091days3.34/year 2/190 days3.84/yearc
#/"=change40.5 relapses/year, $=change5=0.5 relapses/year.
aPatients were treated with various immunomodulatory and/or immunosuppressive agents prior to rituximab.bAfter interruption of
therapy, which had resulted in one new relapse.cAllowing for six months latency of action (otherwise 2/370 days or1.98/year).
3078Brain (2008),131, 3072^3080S. Jarius et al.
azathioprine and cyclophosphamide this prompt response
was probably rather caused by IVMP, which was applied as
treatment for acute relapse. Importantly, however, antibody
azathioprine and prednisolone over an observation period
of up to 500 days with no further clinical attacks, while
interruption of azathioprine resulted in clinical relapse and
an increase of antibody levels within 1–6 months. These
findings are in accordance with results from a small
Japanese study, reporting a marked and sustained decline
of AQP4-Ab titres in two patients undergoing treatment
with azathioprine and prednisolone with no relapse over 6
and 11 months, respectively (Takahashi et al., 2007).
Cyclophosphamide (50mg/day) resulted in a long-lasting
relapse-free interval in one of our patients, with 10 relapses
within 1295 days (2.82/year) prior to initiation of therapy
but only one within 1610 days (0.23/year) under therapy.
However, a continuous yet slightly oscillating increase in
AQP4-Ab titres was found after dose reduction, which was
not followed by clinical relapse after 956 days. This again
indicates that the antibody might not be sufficient to cause
clinical relevant damage on its own, but further players
might be involved in the pathogenesis of NMO.
While azathioprine (and prednisolone) was most effective
in lowering the relapse rate in our patients (Table 1),
treatment with rituximab, a therapeutic monoclonal anti-
body causing temporary B-cell depletion (Kazkaz and
Isenberg, 2004), resulted in the most pronounced decline
in AQP4-Ab levels. Nonetheless, AQP4-Ab did not fall
below cut-off in almost all samples obtained under
rituximab. Moreover, AQP4-Ab remained positive despite
the CD19 cell counts being below the detection limit. There
are two possible explanations for this finding: Although
rituximab causes complete depletion of CD19-positive
peripheral B cells, it does not affect plasma cells. Second,
although rituximab interrupts the steady flow of new
plasma cells from differentiating B cells, the so-called long-
life plasma cells, some of which may survive for the lifespan
of the host, could maintain specific antibody production
over long periods. Our findings are in line with previous
studies demonstrating a decline of specific autoantibodies
under therapy with rituximab in such well recognized
autoimmune disorders such as Graves disease, immune
(Levesque and St Clair, 2008).
It is important to notice that the duration of action
varied considerably among patients treated with rituximab.
CD19 cells reappeared in our patients 250–350 days after
last infusion. Fixed therapy intervals might thus not be
advisable, but application intervals might have to be
adapted individually based on CD19 counts.
numbers was found to be sufficient to induce an increase
in AQP4-Ab values, and reoccurrence of CD19 cells was
associated with a high relapse risk. CD19 counting might
thus be an alternative to AQP4-Ab testing in those patients.
or pemphigus vulgaris
However, B cells remained detectable in all patients not
treated with rituximab, with no correlation between CD19
counts and AQP4-Ab serum values.
There are some obvious limitations of this study,
including the small number of patients analysed, the
possibility of unintentional selection bias and the wide
variety of treatments regimens used. These result from its
retrospective design, the rarity of the disease investigated,
and the short availability of AQP4-Ab testing. However, we
believe that the large number of samples investigated, the
long observation periods in each patient, and the detailed
clinical data adds to the growing body of evidence for a
significant relationship between AQP4-Ab levels and clinical
In conclusion, this study shows that AQP4 antibodies are
higher overall in patients during relapse than during
remission, demonstrates a correlation between rises in
antibody levels and clinical attacks, and illustrates the
decline of AQP4-Ab levels during various immunosuppres-
sive therapies which were associated with reduced relapse
rates. Although only four patients were treated with
rituximab, there were marked falls in AQP4 antibodies
correlating with the changes in CD19 positive cells, however
the duration of action of rituximab was quite variable
between patients. Overall, these findings strengthen the case
for the role of AQP4-Ab in the pathogenesis of NMO/
LETM. Larger studies are now warranted to affirm and to
extend our findings in a prospective and controlled setting.
European Neurological Society (ENS) (to S.J.); European
Committee for Treatment and Research in Multiple
Sclerosis (ECTRIMS) (to S.J.); University of Cologne
(Prof. R. Voltz), Germany (to S.J.); NIHR Biomedical
Research Centre Programme (to P.W. and A.V.). The
funding sources had no role in study design; collection,
analysis or interpretation of data; writing of the paper or
decision to submit it for publication.
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