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Rheumatology 2006;45:20–25
Advance Access publication 27 September 2005
doi:10.1093/rheumatology/kei111
Review Article
Use and significance of anti-CCP autoantibodies
in rheumatoid arthritis
A. J. W. Zendman, W. J. van Venrooij and G. J. M. Pruijn
One of the most common autoimmune diseases is rheumatoid
arthritis (RA), affecting 0.5–1% of the population. This systemic
disease is marked by chronic inflammation of synovial joints,
which leads to destruction of cartilage and bone and eventually
to disability of the patient [1]. Though not directly lifethreatening, RA severely affects the quality of life of a patient
and also has major economic consequences for society.
Therefore, every attempt should be made to prevent the erosive
processes to occur. Currently, the classification of RA relies
mainly on the criteria described by the American College of
Rheumatology (ACR) [2]. These criteria, originally formulated
50 yr ago and last adjusted in 1987, are based mainly on clinical
parameters. Since these parameters are often only sufficiently
fulfilled when the damaging effects of the inflammatory process
are already in progress, this set of criteria is not very suitable for
the early diagnosis of RA [3].
The key to early recognition of autoimmunity lies within
the humoral immune system. Since blood samples are taken
from clinic-visiting (pre-)patients, screening for serological
RA-indicators can be performed quite easily. In the ACR
criteria for RA one serological marker is included: rheumatoid
factor (RF). The RF autoantibody system, directed against the Fc
part of immunoglobulin (Ig) G molecules, has had a central role
in the diagnosis and prognosis of RA during recent decades [4]
because RF can be detected in the majority of RA patients.
However, it is becoming more and more clear that the presence
of RF is not restricted to patients with RA, but that it can also
be detected in subsets of patients suffering from other diseases
and even in a percentage of healthy (especially elderly)
individuals [5]. The resulting lack of specificity for RA can
lead to confusion and unwanted treatment. The shortcomings of
the RF test have kept the search for more specific RA markers
alive. Most autoantibody systems described during recent
decades have failed to mature into mainstream tests for RA
because of low sensitivity, lack of specificity or technical
inconvenience, as reviewed previously [6–8]. The only antibody
system that combines good sensitivity with superior specificity
for RA is that targeting citrullinated epitopes. This review will
focus mainly on the diagnostic potential of the second-generation
anti-cyclic citrullinated peptide (CCP) test (CCP2) for RA. Next,
we will address the prognostic ability of the anti-CCP test. The
presence of these antibodies early in disease development opens
a window of opportunity for early custom-tailored treatment
of RA. Finally, we will review the effect of disease treatment on
anti-CCP levels and briefly go into the putative functional role
of these antibodies in the chronic aspects of RA.
From APF to CCP2
The autoantibody system most specific for RA known to date
is that directed to citrullinated antigens. The citrulline moiety,
which is the essential part of the antigenic determinant in these
antigens, is post-translationally generated by peptidylarginine
deiminases (PAD; EC 3.5.3.15) [9]. RA autoantibodies against
citrullinated antigens have been detected and used for diagnostic
purposes for many decades via the well-known antiperinuclear
factor (APF) [10] and antikeratin antibody (AKA) tests [11].
Supported by the fact that APF and AKA share many features,
as reviewed previously [12], and are reactive with native filaggrin,
these autoantibodies are now designated as antifilaggrin
antibodies (AFA) [13]. Using the laborious and inconvenient
immunofluorescence assay, roughly 50% of RA sera can be
scored AFA-positive. A key finding was the discovery that the
reactivity of these AFA was completely dependent on the presence
of citrulline residues (present in mature filaggrin but not in
profilaggrin) [14, 15]. Since then, two approaches for detecting
autoantibodies to citrullinated epitopes have been taken: a
protein-based and a peptide-based approach.
Screening for citrulline-specific RA reactivity has been performed with several proteins, including both purified naturally
occurring citrullinated proteins and in vitro-citrullinated proteins.
For these purposes, mainly filaggrin, fibrinogen and myelin basic
protein have been used. Although most of these proteins
are arginine-rich, there is obviously a limit to the number
of citrullinated epitopes associated with a certain protein. A
complication of the use of natural antigens is that it is difficult
to obtain reasonable amounts in sufficient purity in a reproducible way. Batch-to-batch variation also compromises standardization when in vitro citrullination is used to generate the
antigen. Insufficient purity of the antigen lowers the specificity
of the test because reactivities directed to other components
(e.g. the PAD enzyme, the non-citrullinated part of the antigen,
other contaminants) may be detected as well. The use of proper
(non-citrullinated) controls is therefore very important, as indeed
has been noted by Vittecoq and colleagues [16]. Interestingly,
autoantigenicity of the PAD protein has recently been described,
but this reactivity is not specific for RA [17]. However, despite
these limitations several studies have successfully improved the
sensitivity of AFA detection while maintaining specificity levels
[18]. Using various technologies, assays with various sources of
filaggrin have been developed, allowing sensitivities up to 60%
[16, 19, 20]. Using citrullinated fibrinogen, Nielen and colleagues
[21] reported a similar sensitivity (56%) for a cohort of early
arthritis patients.
Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
Received 2 June 2005; revised version accepted 9 August 2005.
Correspondence to: A. J. W. Zendman, Department of Biochemistry, NWI 161, Radboud University Nijmegen, PO Box 9101, 6500 HB, Nijmegen,
The Netherlands. E-mail: [email protected]
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ß The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
Diagnostic potential of CCP2 test for RA
The use of synthetic citrullinated peptides for anti-citrullinated
protein antibody detection can overcome many of the
complicating factors of the protein-based approach. Synthetic
peptide production and purification is cheap and easily
standardized, and via peptides one can synthesize an unlimited
pool of defined epitopes. Furthermore, citrulline residues can
be incorporated during synthesis of the peptides, leading to a
homogeneous preparation of citrullinated molecules. A major
breakthrough came with the development of an enzyme-linked
immunosorbent assay (ELISA) that used filaggrin-based citrullinated peptides [14]. The reactivity of RA sera was completely
dependent on the citrulline residue(s) present, since the same
peptides in which the citrulline was replaced by another amino
acid were not antigenic. The variation in reactivity patterns
against different citrullinated peptides clearly showed that the
anti-CCP response in RA is polyclonal. When a filaggrin-based
cyclic peptide (cyclization increased the sensitivity) was applied
in the first generation anti-CCP (CCP1) test, a sensitivity of
68% was obtained with very high specificity for RA (98%) [22].
Though better than the protein-based methods, the sensitivity
was not as high as that of the routinely used RF test. Because
filaggrin is not present in the synovium, dedicated libraries
of citrulline-containing peptides were screened with RA sera to
select for superior epitopes. This culminated in the CCP2 test,
which displays a sensitivity of up to 80% without loss of
specificity.
Anti-CCP2 autoantibodies as diagnostic markers
Diagnostic markers of disease ideally fulfil three requirements:
(i) good sensitivity, to detect a high percentage of patients;
(iii) good specificity, to limit false-positive results as much as
possible; and (iii) early presence, to facilitate early diagnosis.
Over the last decade many studies have investigated the
diagnostic performance of the anti-CCP test. Those using the
CCP1 test have been reviewed by van Boekel and colleagues [6].
Increasing data on the improved [23] second-generation
anti-CCP test show that the CCP2 test result is a good
diagnostic parameter for (early) RA.
Sensitivity/specificity
The first large cohort studies of anti-CCP2 as a diagnostic
marker showed that anti-CCP2 combines RF-like sensitivity with
almost absolute specificity for RA [5, 24, 25]. These multicentre
studies showed that anti-CCP2 antibodies, just like RF, are
present in about 80% of established RA patients. In the healthy
control group and the non-RA disease controls, the CCP2
test was only positive in maximally 1 and 5%, respectively.
The corresponding percentages of the RF (over 10% of healthy
controls and more than 20% of disease controls) were markedly
higher [5, 25]. Several recent independent studies confirmed these
sensitivity/specificity data for CCP2 [26–30]. Using a cohort of
549 RA patients, the study of Suzuki and colleagues [26] clearly
showed higher discriminative ability for the CCP2 test than for
RF test. The reported sensitivity of 65% (at 96% specificity)
by Dubucquoi and colleagues [27] was found in a group of
RA patients that included several patients with recent onset of
disease. The sensitivity in their established RA patient group was
77%. Overall, the observed intercohort variations in these studies
might be explained by differences in the characteristics of the
patients that were included. One study reported a somewhat
lower specificity for RA of about 90% with the CCP2 test [31],
but this could have been caused by the fact that the juvenile RA
(JRA) group included in this study might have contained several
adult RA patients with an early onset of disease, as discussed
previously [32]. The JRA patients had a high average age (31 yr)
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and often longstanding disease (21 yr on average), mostly
resulting in erosions (87%). A striking example of the diagnostic
performance of the CCP2 test is the recent study by de Rycke
and colleagues [28]. Setting the specificity values for RA at
98.5% resulted in a sensitivity for RF of only 12.8% compared
with 73.7% for anti-CCP2.
Taken together, these studies show that the anti-CCP2 test
at least equals the RF level for sensitivity, but combines this
with far better specificity. The fact that around 40% of
RF-seronegative patients appear to be anti-CCP-positive
substantiates the additional diagnostic potential of CCP [29, 33].
The anti-CCP test also enables clinicians to effectively
distinguish RA patients from other arthritic diseases in cases
where the RF is not always discriminative. One of the first
examples of such a role in differential diagnosis comes from
patients with erosive systemic lupus erythematosus (SLE).
Mediwake and colleagues [34] showed that anti-CCP (in this
case CCP1) can be used to distinguish RA patients from
SLE patients who present with erosive polyarthritis, which is
often accompanied by RF seropositivity. Another disease that
can readily be misdiagnosed because it often reveals RA-like
arthropathies is chronic hepatitis C virus (HCV) infection, which
is often accompanied by a positive RF. Wener et al. [35] reported
a good discriminative ability of anti-CCP2 over RF in a group
of randomly selected HCV patients (44% RFþ, none CCP2þ).
These data were confirmed by Bombardieri and colleagues [36].
Whereas RF was detected in 15% of the HCV patients (37% in
case of joint involvement), no anti-CCP2 positivity was seen
in these patients. The value of anti-CCP for use in differential
diagnosis was also shown when comparing RA patients with
polymyalgia rheumatica patients [37]. Taken together, these
data clearly outline the diagnostic strength of the anti-CCP test
for RA.
Recently, some papers have reported anti-CCP positivity in
arthritic diseases that share certain features with RA [38–40]. In
palindromic arthritis (PR), a percentage of anti-CCP positivity
(56%) similar to that of patients with early RA (55%) was found
[38]. PR is a relapsing from of arthritis that shares many features
of RA. In addition to the observed anti-CCP reactivity, many of
these sera are also RF-positive (30–60%). Moreover, as is the
case for RA, PR is associated with the presence of the MHC
shared epitope (odds ratio of 2.9) [41]. The current assumption
is that PR is an abortive form of RA that in a percentage of
patients may develop into RA. The presence of RF in PR has
already been shown to be associated with the development of
chronic disease [42]. Regarding the prognostic significance of
anti-CCP positivity in PR and the possible development of RA,
additional studies with larger cohorts and longer follow-up are
needed.
In patients with primary Sjögren’s syndrome, anti-CCP
reactivity was detectable in 8% of the 134 patients tested [39].
For another RA-like disease, psoriatic arthritis, the group of
de Rycke [40] found anti-CCP positivity in 8% of the 192
patients tested. This reactivity, confirmed with an additional test
using home-made citrullinated peptides, was higher than could
be ascribed to simple overlap with RA. These studies make clear
that, especially in RA-like arthritic disease entities, further
clinical follow-up is needed to establish whether the anti-CCP
positivity predicts the development of RA.
Early presence and predictive potential
Because RA patients at their first visit to the clinician often do
not fulfil the criteria for the diagnosis/classification of RA, an
early detectable, highly predictive marker would greatly help the
clinician in reaching a diagnosis. Obviously, the sensitivity and
specificity of such a marker should be as high as possible.
Recently, two studies, both making use of dated samples from
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A. J. W. Zendman et al.
RA patients who were former blood donors, reported the
presence of anti-CCP antibodies prior to the appearance of the
first clinical symptoms of arthritis [43, 44]. Samples from 72
blood donors were characterized by Nielen and colleagues [43]
for positivity of IgM-RF or anti-CCP1. Both serological markers
were detectable long before the disease became clinically overt.
In some patients, anti-CCP1 was found up to 14 yr prior to the
first clinical symptoms of disease. The same was found for 39%
of the patients 5.3 yr (median) before the first visit to the clinic.
IgM-RF was also found in predisease samples, but not as far
back (up to 10 yr) and in a smaller percentage of patients (23%
at a median of 3.3 yr). The second study, with a similar set-up,
detected anti-CCP2 and RF up to 10 yr before clinical disease
in predisease blood samples of 83 RA patients. Anti-CCP2
positivity gradually increased in the years prior to the first
clinical symptoms and had reached a positivity of 70% at the
time the patients visited the rheumatology clinic for the first
time. The sensitivity for detecting RF autoantibodies in these
predisease samples was slightly less than for anti-CCP2 [44].
From these studies it is clear that the production of anti-CCP
and RF autoantibodies is an early process in RA development,
and that their presence is predictive for the development of this
disease.
Recent data from several longitudinal studies confirm the
predictive ability of anti-CCP2 for RA development [16, 45, 46].
van Gaalen and colleagues [45] used serological markers to
predict which of the patients attending an early arthritis clinic,
who were classified as undifferentiated arthritis (UA), would
progress to RA within the next few years. Follow-up data of
318 patients with UA clearly showed the predictive potential of
anti-CCP autoantibodies for the development of RA. After 1 yr
of follow-up, 75% of the UA patients who were anti-CCP2positive at baseline had already progressed to RA. This
percentage increased to 93% after 3 yr (odds ratio 38). Of the
UA group who were anti-CCP2 negative at baseline, only 25%
were classified RA after 3 yr. Similar results were reported by
Vittecoq and colleagues [16]. In their cohort of 314 early arthritis
patients, 90% of the anti-CCP2-positive patients were classified
as RA patients at the 1-yr follow-up. Thus, the combined
early presence and predictive ability of anti-CCP may find an
important clinical application in the design of treatment
strategies.
Anti-CCP2 autoantibodies as a prognostic marker
Various studies have addressed the prognostic value of anti-CCP
antibodies. Though the anti-CCP test has only recently become
widely available, several studies have already demonstrated its
ability to predict the erosiveness of developing RA. Most of
these studies used the CCP1 test and the results of these have
been discussed previously [32, 47]. An increasing number of
studies with anti-CCP2 confirm the prognostic potential. First, in
a cohort of 379 early RA patients, Forslind and colleagues [48]
showed that anti-CCP2 positivity at baseline (55%) predicts
radiological damage and progression at 2 yr follow-up. Similar
results were obtained by Kastbom and colleagues [49]. In their
study, CCP2 positivity at baseline predicted disease activity at
the 3-yr follow-up. In addition, Rönnelid et al. [50] showed that
anti-CCP2-positive early RA patients developed worse clinical
disease and greater radiological damage within a few years in
comparison with anti-CCP2-negative patients.
The prognostic ability of the anti-CCP test is often
complemented by other disease parameters. With respect to
the presence of the shared epitope MHC class II molecules, this
was reported by independent studies of Berglin et al. [51], van
Gaalen et al. [52], de Rycke et al. [28] and Lindqvist et al. [53].
The study by de Rycke and colleagues also showed that
anti-CCP2 positivity, unlike RF, did not correlate with the
presence of extra-articular manifestations [28]. Raza and
colleagues [54] reported that the combination of anti-CCP2
and RF positivity was the best prognosticator for the
development of persisting RA in a patient group with very
early synovitis. Anti-CCP2 levels are also associated with the
development of bone erosions in RA [55]. On the other hand,
Gossec et al. [56] reported that there was no significant
correlation between the absence of anti-CCP antibodies and
remission of early RA.
The conclusion from these studies is that a positive anti-CCP
test appears to predict the development of erosive RA and that
this predictive value complements that of RF. Together with
additional individual data (e.g. the presence of shared epitope,
family members with RA), this may lead to a very high
probability that erosive RA is developing.
Effect of therapy on anti-CCP status
Among the most effective drugs for the treatment of RA at
present are those targeting the pro-inflammatory TNF-. TNF-inhibiting agents like infliximab can reduce disease activity and
delay radiographic progression of RA [57]. However, side-effects,
such as an increased risk of infections and the induction
of autoantibodies (e.g. antinuclear antibodies or anti-doublestranded DNA antibodies), have also been reported [58].
The determination of autoantibody levels during the course of
treatment (anti-CCP, RF) may give some clues regarding the
effectiveness of the treatment and the role of these antibodies
in the disease process.
In the past year, several papers have reported effects on
RA-associated markers, including anti-CCP, in patients treated
with disease-modifying anti-rheumatic drugs (DMARDs) [mainly
methotrexate (MTX)] with [59–63] or without [50, 64] infliximab
(summarized in Table 1). Bobbio-Pallavicini and colleagues [59]
studied autoantibody profiles during long-term (78 weeks)
combination treatment with infliximab and MTX. Though
treatment resulted in a significant decrease in disease activity
scores, no changes in the percentages of patients who were
positive for anti-CCP2 or IgM-RF were observed. Titres of RF,
on the other hand, were significantly reduced, unlike those of
anti-CCP. These results were confirmed by several other studies,
although follow-up periods varied between the individual studies
(Table 1). In the study of Nissinen and colleagues [60], anti-CCP
(measured by the anti-CCP1 test) did not change during 6 weeks
of follow-up, though 60% of the patients had a significant
clinical response. IgM-RF levels were somewhat decreased in
the first weeks of therapy. With follow-up periods of 22 and
30 weeks, respectively, Caramaschi and colleagues [63] and
de Rycke and colleagues [62] also observed a decrease in RF
levels but not of anti-CCP. In contrast to the previous studies,
Alessandri and colleagues [61] did find a small but significant
decrease in anti-CCP levels at week 24 of treatment in the
patients with clinical improvement. This effect, also observed for
RF, was dependent on infliximab, since reductions in anti-CCP
levels were not observed after treatment with MTX alone.
Nevertheless, DMARD-only therapy can result in a significant
(>25%) reduction in both anti-CCP and RF in about 50% of
patients [64]. In this last study, effective treatment correlated
with a reduction of RF. Recently, Rönnelid and colleagues [50]
showed that treatment with sulphasalazine, but not other
DMARDs, resulted in a drop in anti-CCP levels, but this
decrease occurred only in the first year of follow-up and did not
correlate with clinical indicators.
From these studies, it is clear that the anti-CCP autoantibody
system is different from the RF system with respect to response
to treatment. Clearly, investigating the functional importance of
anti-CCP in the progression of RA demands other approaches
specifically aimed at decreasing this humoral response.
Diagnostic potential of CCP2 test for RA
23
TABLE 1. Features of longitudinal studies on anti-CCP status during RA treatment
Study
Reference
Treatment
Cohort
Follow-up
Outcome
Bobbio-Pallavicini
Nissinen
Caramaschi
de Rycke
Alessandri
Mikuls
[59]
[60]
[63]
[62]
[61]
[64]
Infliximab/MTX
Infliximab/DMARDs
Infliximab/MTX
Infliximab/MTX
Infliximab/MTX
DMARDs
39
25
27
62
43
208
78 weeks
6 weeks
22 weeks
30 weeks
24 weeks
2 yr
Rönnelid
[50]
DMARDs
379
5 yr
No effect on anti-CCP levels (87% responders)
No effect on anti-CCP levels (60% responders)
No effect on anti-CCP levels (74% responders)
No effect on anti-CCP levels (100% responders)
Decrease in anti-CCP in patients with clinical improvement
Small anti-CCP reduction in 50% of patients;
no association with response
Anti-CCP stable; only small drop in 1st year
(not correlating with disease)
Putative functional implications for anti-CCP
Because anti-CCP autoantibodies are so specific for RA, one
wonders what they can tell us about the aetiology of the disease.
To answer that question we need to know more about (synovial)
protein citrullination and the PAD enzymes responsible for
the conversion of peptidylarginine to peptidylcitrulline. The
five isoforms of human PAD show a tissue-specific expression
profile [9]. It has been shown that PAD enzymes are present
in the inflamed synovium and that their activity is regulated at
the transcriptional and translational levels [65, 66]. In addition,
these enzymes require relatively high Ca2þ concentrations, about
100 times higher than normally present in the cytosol of a living
cell. Interestingly, citrullination occurs primarily in dying cells.
Indeed, during inflammation, when many cells die by apoptosis
or necrosis, one can detect citrullinated proteins, both in animal
models of inflammation and in the inflamed synovia of RA and
non-RA patients. Paradoxically, the presence of citrullinated
proteins in most cases does not lead to the generation of anticitrullinated protein antibodies [67, 68]. This phenomenon might
be related to the genetic background of the patient. It has been
known for some time that there is a rather strong correlation
between RA and certain HLA-DR alleles, particularly HLADRB1*0401 and HLA-DRB1*0404. These alleles contain the
so-called shared epitope (SE) motif. Hill and colleagues [69]
showed that the conversion of arginine to citrulline increased the
affinity of a peptide for binding to HLA-DRB1 and can lead to
activation of CD4þ T cells in DR4-transgenic mice. These results
thus indicate that the production of anti-citrullinated protein
antibodies is dependent on the presence of certain susceptibility
genes for RA. The fact that the observed odds ratio of 66.8 for
the combination of anti-CCP and SE HLA gene carriage is over
two times the value of the multiplication of the corresponding
single odds ratios (anti-CCP, 15.9; SE, 2.4) also suggests that
these factors are functionally associated [51]. Very recently it was
shown that patients who were homozygous for SE alleles and
seropositive for anti-CCP antibody presented with an increased
rate of joint destruction compared with patients carrying only
one or none of these alleles [52]. Also, these results suggest that
the production of these antibodies stimulates the ongoing
inflammation in RA.
These data have led to a relatively simple model for the
development of RA, depicted in Fig. 1 and discussed previously
[70, 71]. In individuals with a genetic predisposition for RA, an
inflammation of the joint, in itself innocent, leads to infiltration
of inflammatory cells (monocytes, granulocytes) that contain
PAD enzymes. After activation, these cells will die via apoptosis
and will be cleared (removed) by phagocytosing cells. However,
when there is massive apoptosis, or a (genetic) defect in the
clearance system, some apoptotic cells may become necrotic,
thereby releasing citrullinated proteins (histones, vimentin) and
PAD enzymes. The PAD enzymes can subsequently citrullinate
synovial proteins, such as fibrin. In 99% of individuals this
will be the end of the story. In the 1% of individuals who
FIG. 1. Hypothesized cycle of RA chronicity.
are able to present fragments of the citrullinated proteins to
the T cells via certain MHC class II molecules, a B-cell
response to citrullinated antigens will be generated, resulting in
the formation of immune complexes which will stimulate the
inflammatory process by upregulation of pro-inflammatory
cytokines. New monocytes and granulocytes will enter the
synovium, will be activated, die, and stimulate a new flare of
inflammation. The repetition of this process over a number of
years, eventually accompanied by fresh traumas or environmental events that stimulate inflammation, will finally lead to a
chronic inflammation which can develop into the disease we
know as rheumatoid arthritis. In this way, anti-CCP contributes
to the perpetuation of joint inflammation and thereby to the
chronicity and severity of RA.
Future perspectives
Currently available therapies for RA, nicely reviewed by Smolen
and Steiner [72], are mainly anti-inflammatory and unable to
cure the disease. At best these therapies are able to slow down
the extent of swelling and erosive damage. More insight obtained
over recent years suggests that combination therapies given
early in the disease have the greatest therapeutic potential [73].
To achieve the ultimate goal of curative treatment, it is crucial
to identify RA patients before joint damage occurs. The fact
that the CCP2 test is now widely available will speed up
these studies, as shown by the burst of data on CCP in the
literature. Knowledge about the citrullinome (total repertoire
of citrullinated proteins), the application of array-based
screening technologies [74] and use in automated assays [75]
will undoubtedly further increase the diagnostic potential of
these autoantibodies.
A.Z. received a grant from the Netherlands Organisation for
Scientific Research. G.P. and W.V. are cofounders and shareholders of ModiQuest b.v. W.V. serves as a consultant to EuroDiagnostica b.v. and Axis shield.
24
A. J. W. Zendman et al.
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