Curr Rheumatol Rep (2011) 13:440448

DOI 10.1007/s11926-011-0201-y

Synovial Tissue Heterogeneity and Peripheral

Blood Biomarkers
Serena Bugatti & Antonio Manzo & Michele Bombardieri & Barbara Vitolo &
Frances Humby & Stephen Kelly & Carlomaurizio Montecucco & Costantino Pitzalis

Published online: 17 August 2011

# Springer Science+Business Media, LLC 2011

Abstract Rheumatoid arthritis is characterized by multiple

pathobiological processes and heterogeneous clinical phenotypes. Not surprisingly, the inflamed synovium harbors
an equally complex pathology. This includes variability in
infiltrating and resident cell populations, spatial arrangements, and cellcell interactions, as well as gene expression
profiles. Remarkable progress in our understanding of the
many facets of tissue heterogeneity has been facilitated by
the increasing availability of patients material and the
development of advanced research technologies. The next
challenge is to capitalize on the large amount of data
generated to elucidate the specific pathogenic pathways
disparately activated in different patients and/or different
phases of the disease. When tissue pathology can be
reliably explored through noninvasive circulating biomarkers, then the circle will be closed. We attempt to
highlight key advances in the understanding of synovial
tissue heterogeneity in rheumatoid arthritis and summarize
novel perspectives in synovial biomarker discovery in
relation to peripheral blood.
Keywords Rheumatoid arthritis . Synovial membrane .
Synovial tissue . Heterogeneity . Peripheral blood .
Biomarker
S. Bugatti : A. Manzo : B. Vitolo : C. Montecucco
Division and Laboratory of Rheumatology,
University of Pavia School of Medicine,
IRCCS Policlinico San Matteo Foundation,
Piazzale Golgi 2,
27100 Pavia, Italy
M. Bombardieri : F. Humby : S. Kelly : C. Pitzalis (*)
William Harvey Research Institute, Barts and the London School
of Medicine and Dentistry, Queen Mary University of London,
Charterhouse Square,
London EC1M 6BQ, England, UK
e-mail: c.pitzalis@qmul.ac.uk

Introduction
Tissue pathobiology has provided invaluable advances
in standard clinical practice in most medical specialties.
In the context of rheumatic diseases, typical and not
comprehensive examples include Sjgrens syndrome
and lupus nephritis, in which the microscopic observation of the target tissue has become part of the
diagnostic work-up [1, 2], provides relevant prognostic
information [3, 4], and may influence therapeutic decisions [5].
Compared with these examples and many other clinical
settings, in rheumatoid arthritis (RA) we still face the
challenge of a crucial discrepancy between the large
amount of data generated from synovial tissue analysis
and the thus far little applicability in routine clinical care.
Indeed, synovial pathology at present does not fit in
classification or diagnostic criteria [6, 7] or provide
foolproof disease prognostication [79]. Paradoxically,
despite several biological drugs having entered the therapeutic armamentarium of RA, and many more in development, rarely has the clinical efficacy been consistently
correlated with specific mechanisms of action at the
synovial tissue level [1013].
The difficulty in integrating synovial tissue research into
comprehensive patient management in RA matches with
the many unresolved aspects of the disease. Our understanding of the pathogenic complexity of the disorder is
incomplete, and heterogeneous pathways are likely to play
diverse roles in different patients and clinical phases.
Furthermore, although the synovial tissue clearly stands at
the epicentrum of joint pathology, it is currently unknown
whether disease-specific mechanisms might be generated
elsewhere, such as in secondary lymphoid organs or bone
marrow [14, 15]. Finally, the possible clinical and pathological overlapping between RA and other chronic inflam-

Curr Rheumatol Rep (2011) 13:440448

matory polyarthritides endorses the concept that synovial

tissuebased evidence probably should be more informative
at the disease subtype level rather than at enforcing rigid
diagnostic definitions. These same limitations, however,
further fuel expectations from the field of synovial tissue
investigation. Learning from other disciplines such as
oncology, integration of the cellular and molecular
heterogeneity that characterizes the synovial lesion in
RA with clinical and imaging phenotyping of different
disease subsets at different stages may deliver more robust
tools for the identification of specific pathogenic pathways, as well as predictive algorithms for prognosis and
therapeutic response to be translated into personalized
management of RA. In this context, we focus on new and
renewed aspects of synovial tissue variability and discuss
how they can eventually improve our understanding of the
biological and clinical heterogeneity with which we are
dealing in RA. We also review recent works exploring
whether tissue pathology may be reflected in peripheral
blood. This is done with the ambition of maximizing our
possibilities to deconvolute disease mechanisms and
develop new clinical indicators through noninvasive,
repeatable, and widely accessible approaches, with the
ultimate goal of identifying biomarkers in the blood
representative of tissue pathology.

Setting the Stage for Evaluating Synovial Tissue:

What is the Clinical Utility?
Propaedeutic to any consideration of the value of tissuebased information, several questions need to be addressed:
1. Is the synovial tissue accessible at every stage of the
disease and in every joint?
2. Is examination of a limited area of the tissue representative of the synovium in the whole joint?
3. Is examination of one joint representative of other
inflamed joints?
4. Can synovial tissue heterogeneity be captured and
quantified reliably?
The development and improvement of mini-invasive
techniques for synovial tissue acquisition, such as smallbore arthroscopy and ultrasound-guided biopsy [16], have
definitively enabled synovial sampling from the earliest
phases of the disease. These procedures also can be safely
performed in small joints, such as in the metacarpophalangeals [17, 18], although tissue quantity (and occasional
quality) in the latter case may limit more extensive
analyses. Nonetheless and very positively, small-bore
arthroscopy promises to yield adequate sampling even in
quiescent joints [19], thus extending synovial tissue
accessibility to preclinical [20] and remission phases.

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Despite the degree of histologic variation within a single

joint, representative measures of several cellular and
molecular parameters of inflammation may be obtained by
examining a limited number of independent samples
(standardized as six to eight) [16]. Furthermore, notwithstanding the need for further validation, it has been
suggested in studies with paired synovial biopsy from
wrist and knee joints that the gross inflammatory picture
is almost comparable [17], thus making synovial specimens from a single joint representative of the patients
overall tissue subtype. Reliable quantification of synovial
cellular and molecular heterogeneity is allowed via
manual and computer-assisted approaches whose strong
interobserver agreement has been proven extensively [21].
Additionally, single-gene and large-scale expression profiling through real time quantitative polymerase chain
reaction and microarray analysis on synovial tissue
samples enables investigators to capture considerable and
consistent variability among different patients [22, 23]. All
together, synovial tissue investigation in RA thus appears
to accomplish feasibility, accuracy, and reproducibility.
Major drawbacks that need to be considered in light of
still some variability in the published literature and the
potential for clinical translation, however, include the
fact that synovial biopsy, although mini-, remains an
invasive procedure and requires some technical skills
and facilities that might ultimately influence the outcome. As discussed below, these and other concerns are
fueling recent debates on the utility of focusing research
on potential synovial biomarkers in comparison with
more accessible compartments (eg, the peripheral
blood).

Heterogeneity of Individual Synovial Cell Populations

Morphologic characteristics of the inflamed synovium in
RA include resident cell proliferation, increased vascularization, and infiltration of innate and adaptive
immune cells, such as monocytes, natural killer cells,
mast cells, T cells, B cells, plasma cells, and dendritic
cells. Disappointingly, none of these features is pathognomonic for RA [6], nor do they help in distinguishing
different clinical phases [24, 25] or anti-citrullinated
protein antibody (ACPA)-positive versus ACPA-negative
disease phenotypes [26]. Within such stereotyped response
(namely the presence of the same types of immune and
stromal cells) implying qualitative homogeneity, virtually
every histopathological marker shows high degrees of
quantitative interpatient variability. Furthermore, the activation status of each cell population may vary considerably. Not surprisingly, synovial biomarker discovery over
the past few years largely focused on the possible biological

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and clinical correlates of such quantitative and functional

differences.
At present, robust evidence indicates that synovial
macrophageskey effector cells in the pathobiological
cascade of RAbear informative attributes of the degree
of joint involvement. Indeed, the number of CD68+
macrophages in the synovial sublining strongly correlates
with local disease activity [24] and systemic inflammation
[6, 27]. More importantly, the extent of macrophage
infiltration is sensitive to change, with significant reductions being observed following administration of active
antirheumatic therapies [28], but not following ineffective
treatment [29] or placebo [30]. As a consequence, analysis
of synovial tissue macrophage variations is being increasingly adopted in early-phase clinical trials to reduce the size
and duration of the studies. However, the same characteristics that make synovial macrophages an accurate indicator
of some disease processesthat is, their dynamic correlation
with the levels of ongoing inflammationintrinsically limit
their possible long-term prognostic value. Not surprisingly,
although macrophages are an important source of local
osteoclastogenic cytokines [31] and they themselves may
serve as osteoclasts precursors [32], the association
between the degree of synovial macrophage infiltration
and the development of bone erosions at follow-up appears
controversial [8, 9].
Another cell population that has historically attracted
great interest are fibroblast-like synoviocytes (FLS), the
most abundant cell type of tissue stroma. In addition to
setting up the structural scaffold that shapes the synovial
microenvironment, FLS actively participate in key pathogenic pathways through local production of chemokines,
mediators of inflammation and tissue damage. Fibroblasts
phenotypic and functional profile in RA uniquely shows the
imprint of tissue heterogeneity among different patients.
Microarray analysis has identified two main subgroups of
FLS based on their gene expression pattern. FLS from
highly inflamed synovium display a transforming growth
factor-/activin Ainducible signature characteristic of
myofibroblasts, while insulin-like growth factorregulated
genes predominate in FLS from tissues with low inflammation [33]. Heterogeneous transition from healthy FLS to
smooth muscle actin-positive myofibroblasts, shown to
express matrix degrading proteases [34], may contribute
to the explanation for the interindividual variations in the
invasive behavior of FLS recognized among RA patients
[35]. Furthermore, given the propensity of myofibroblasts
to produce cytokines and chemokines that orchestrate intratissue migration and positioning of circulating lymphocytes
[36], functional differences of FLS may also modulate the
degree and pattern of leukocyte infiltration in RA synovia,
and thus the quality of overall tissue inflammation. In line
with this concept, we recently showed that smooth muscle

Curr Rheumatol Rep (2011) 13:440448

actin-positive, CD45- spindle-shaped stromal cells within

synovial lymphocytic aggregates are the source of the
T-cell/dendritic cell chemoattractant CCL21 [37]. Similarly,
although phenotypic characterization is lacking, FLS from
lymphocyte-rich tissues also express CXCL12 and interleukin
(IL)-7, which are involved in immune cell retention and
lymphoid-like microanatomic organization [38, 39]. If such
molecular variations reflect patient-specific fingerprints, then
extensive gene profiling of FLS may turn out to be an
invaluable platform for disease subsetting according to
different pathotypes and phenotypes [40].

Heterogeneity of Synovial Patterns of Cell Infiltration

The tremendous complexity of the pathophysiologic cascade
of RA makes single-cell analysis unlikely to explain fully the
heterogeneous behavior of the disease. As every biological
event is the result of sophisticated interactions among
different cellular and molecular players in the context of
spatially structured microenvironments, then specific pathogenic processes of RA could be better disclosed through
examination of the topographic arrangement that shapes
local cellcell contacts.
It has long been recognized that the histomorphology of
RA synovium allows the distinction of different leukocyte
infiltration patterns. Mononuclear cells have been classically
described as randomly distributed within the synovial
sublining or spatially grouped into follicular clusters [41].
In our hands, based on a series of 103 consecutive RA
patients, the two patterns do not appear mutually exclusive;
rather, they significantly overlap, with each characterized
by variable intensity (Fig. 1). Nevertheless, in some tissues,
large and densely distributed mononuclear cell aggregates
strikingly predominate, whereas in some others, they are
nearly undetectable despite considerable tissue inflammation. Diffuse sublining infiltration is mainly composed of
scattered T lymphocytes and monocytes. In contrast,
mononuclear cell aggregates bear together intermixed T
and B cells, which can segregate into compartmentalized
T and B cell areas, as well as macrophages and dendritic
cells. Remarkably, B cells are virtually restricted to
follicular structures, their scattered extrafollicular distribution nearly insignificant. Thus, B lymphocytes appear
to be the only conventional cell population in RA
synovitis that shows both quantitative and qualitative
heterogeneity, as a variable proportion of tissuesthose
devoid of consistent aggregational phenomenacompletely
lack B cells.
A characteristic feature of synovial T-cellB-cell clusters
is their possible further progression into follicular, noncapsulated structures sharing some morphologic features
with secondary lymphoid organs, with detectable high-

Curr Rheumatol Rep (2011) 13:440448

443

Fig. 1 Heterogeneity of the mononuclear cell microarchitecture in

rheumatoid synovitis. Hematoxylin- and eosin-stained sections of
synovium samples from representative rheumatoid arthritis patients
show heterogeneous combinations of different levels of diffuse

cellular infiltrate (13, vertical axis) and aggregational organization

(03, horizontal axis), leading to the formation of follicle-like
structures in some patients. (Original magnification 100)

endothelial venules, a vestige of the lymphoid stromal

reticular network, and CD21+ germinal centers [37, 42].
The process of immune cell organization within discrete
lymphoid microenvironments in the context of chronically
inflamed tissues, also known as lymphoid neogenesis, has
received a great deal of attention over the past 10 years due
to its possible pathophysiologic significance, and has
already been reviewed in detail by our group [14]. Here
we summarize recent data attempting to define the
biological and clinical translation of lymphoid patterns
heterogeneity in RA.
The close interaction among immune and resident cells
within synovial follicles virtually creates the conditions that
maximize reciprocal activation and effector functions.
Experiments with human synovium xenotransplanted into
severe combined immunodeficiency (SCID) mice indicate
that synovial B cells are required for major histocompatibility complex class IIdependent T-cell activation in situ
[43]. Accordingly, tissues enriched in lymphoid aggregates
display the highest levels of the T-cellderived cytokines
interferon- and IL-2 [44, 45], and we recently presented
data demonstrating that the progressive increase in quantitative expression of these cytokines closely matches the
degree of synovial B-cell infiltration [46]. In turn, synovial

B cells consistently express activation-induced cytidine

deaminase (AID), a key enzyme controlling somatic hypermutation and class-switch recombination of the immunoglobulin genes [46, 47], and selected B-cell clones are
continuously activated in situ and locally differentiate into
plasma cells [48].
Because RA synovia harvested from different patients
show wide variations in the size and density distribution of
lymphoid aggregates, a question that therefore arises is
whether such heterogeneity translates into specific pathological and phenotypic differences. Depending on the
histologic criteria adopted to identify lymphocytic aggregates (B cells only/T cells only/B+T cells) and the cutoff
values set for defining lymphoid neogenesis (aggregates
size, specific lymphoid-like morphologic features), different
clinical studies yielded partially conflicting conclusions on
the diagnostic and prognostic meaning of different
lymphoid patterns. Whereas previous work highlighted a
possible role for synovial B-cell and plasma cell quantitative infiltration in predicting future diagnosis of RA in
early arthritis [49], a very recent report failed to confirm
disease-specific diagnostic and prognostic properties of
lymphocyte aggregatesevaluated through CD3+ T-cell
expressionin arthritis patients with disease duration of

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less than 12 months [7]. Similarly, variable associations

with disease severity in established RA in terms of
therapeutic burden and radiographic damage arise from
different studies focusing on heterogeneous aspects of the
process of lymphocyte aggregation and organization [50
52]. Again, depending on how the synovial aggregates are
categorized, this may lead to opposite conclusions with
regard to their capacity for predicting clinical response to
tumor necrosis factor (TNF) inhibitors [52, 53]. Collectively,
these data would tempt us to conclude that different
patterns of lymphocyte arrangement within the RA synovium are nothing more than serendipitous facets of local
inflammation, irrelevant in terms of pathobiological events
and clinical consequences. However, it appears that the
more selectively we look at specific B-cellcentered
processes, the more unfavorable outcomes we capture.
Given the unique biology of synovial B cells as core
elements of lymphoid follicles, investigating synovial
architecture through morphologic and functional analysis
of B-cell responses might eventually open novel perspectives in the field of lymphoid tissue reactions in arthritis. In
keeping with this conceptual framework, follicle-like
structures in the inflamed meninges of patients with
progressive multiple sclerosis analyzed through B-cell
markers were recently shown to associate with a gradient
of cortical neurodegeneration and a more aggressive course
of the disease [54].

Curr Rheumatol Rep (2011) 13:440448

inflammation-driven joint remodeling in RA arises from the

uncoupling between destructive and reparative processes [55].
Accordingly, actively inflamed synovial tissues display
increased RANKL (receptor activator of nuclear factor-B
ligand) [56] and decreased osteoprotegerin expression [57]
upon immunohistochemical evaluation.
Microarray analysis also reveals the imprint of tissue
heterogeneity in relation to the pattern of synovial lymphoid infiltration. Patients with ectopic lymphoid follicles
characteristically show enhanced expression of secondary
lymphoid organ constitutive chemokines and of the IL-7
pathway [39]. Hierarchical clustering based on the pattern
of expression of lymphoid organogenesis genes was
recently adopted to dissect variations in intragraft immunologic responses in chronic kidney rejection [58]. Importantly, it was demonstrated that the complete recapitulation
of secondary lymphoid organ ontogenic programs results in
the generation of fully functional ectopic germinal centers
that allow for the efficient maturation of B cells, triggering
an aggressive intragraft immune response associated with
shorter survival of the graft. A similar quantitative approach
applied to RA could allow better understanding of the
pathobiological and clinical outcomes associated with
synovial lymphoid neogenesis and B-cell autoimmune
responses, overcoming the limitations of subjective morphologic assessments highlighted earlier.

Synovial Tissue Biomarkers in Peripheral Blood

Heterogeneity of Synovial Transcription Profiling
Although considered the gold standard, morphology of the
target tissue alone, as well as single-cell functional profiling
is becoming increasingly insufficient in complex diseases
such as RA, whose pathogenic mechanisms upstream of
clinical phenotypes cannot be reduced in a linear fashion,
but rather arise from large numbers of pathways being
simultaneously activated. By allowing comprehensive
identification of differentially expressed genes, transcription
profiling of whole synovial tissues through DNA microarray technology has emerged as a very powerful method
for gaining insight into the molecular heterogeneity of
the disease.
Seminal works by van der Pouw Kraan [22] have
revealed considerable heterogeneity among RA patients.
Two molecularly distinct forms of RA tissues could be
identified, one characterized by genes involved in inflammation and adaptive immune response, and the other
characterized by a low-inflammation gene signature reminiscent of that of tissues from patients with osteoarthritis.
Remarkably, genes involved in tissue repair were not
expressed in tissues enriched in lymphocyte-related
transcripts, which falls in line with the concept that

Several problems need to be considered when attempting to

unravel the multiple pathological and clinical facets of RA
exclusively at the synovial tissue level and in a single joint.
As previously mentioned, facilities and skills for synovial
biopsy are still restricted to a minority of rheumatology
centers worldwide. Furthermore, as is the case of every
invasive procedure that is not part of a routine diagnostic
work-up, patients may refuse the procedure or its repetition
over time. Synovial-based research is still largely confined
to situations in which tissue becomes available, which may
not coincide with the very early phases of the disease.
Additionally, synovial tissue is more easily accessible in
larger joints such as the knee, possibly accounting for
selection bias. The knee joint is only affected in a subset of
early RA patients generally characterized by a more
aggressive disease course. Also, it is currently not known
whether the quality and intensity of disease-specific
mechanisms, particularly bone remodeling, within the knee
joint fully overlap with those activated in small joints of the
hands and feet, which are more commonly involved and
more frequently eroded. Finally, there may be a conceptual
problem dealing with the intrinsic nature of RA in that
although the disease has a clear predilection for the joint, it

Curr Rheumatol Rep (2011) 13:440448

still entails systemic immunologic abnormalities generated

elsewhere (lung, secondary lymphoid organs) [14, 59, 60]
and indicated by their presence in the preclinical (presynovial) phase of the disease [61]. To overcome these and
many other possible concerns, a novel frontier of research
in arthritis aims at investigating potential biomarkers of
tissue pathology that can be reliably captured in more
accessible compartments, such as the peripheral circulation.
A clue to the practicability of comparative synovial
systemic studies comes from the renowned association
between the degree of synovial inflammation, as measured
through quantitative/semiquantitative sublining macrophage
infiltration, and systemic biomarkers of inflammation, such
as erythrocyte sedimentation rate and C-reactive protein [6,
27]. Agreement between local and systemic inflammation
appears unsatisfactory, however, as no correlation is found
for tissue and serum levels of several proinflammatory
cytokines, including TNF- and IL-1 [62].
The identification of two RA tissue subtypes highly
divergent in terms of immune cell activation and tissue
remodeling [22] recently encouraged the same investigators
to analyze paired synovium and peripheral blood samples
through extensive transcriptional profiling in search of
suitable peripheral biomarkers representative of tissue
heterogeneity [63]. Disappointingly, no statistically significant differences in peripheral blood gene expression profiles
could be highlighted between the two tissue groups. As
discussed by the authors, the analysis of a single joint may
not be fully representative of the inflammatory status of the
other involved joints, leading to inconsistent matching with
systemic patterns. Additionally, using whole blood with
variable mixtures of cells may have led to inclusion of
irrelevant cell profiles, such as the polymorphonucleates
(virtually absent from synovial tissue), while missing
relevant transcripts of rare cell types potentially increased
(clonal expansion) in the synovium. Analyses on selected
purified subpopulations of cells may provide more valuable
information. A good example of this is provided by the
recent publication of McKinney et al. [64] that demonstrates that the capacity of predicting relapse in antineutrophil cytoplasmic antibodyassociated vasculitis could be
revealed when using the transcriptional profiling of purified
CD8+ T cells, thus avoiding the confounding influences of
unseparated cells.
An emerging peripheral blood biomarker of tissue
inflammation is CXCL13, a lymphoid chemokine critical
for B-cell recruitment and functional organization of
peripheral lymphoid tissues. CXCL13 expression is increased
in peripheral blood mononuclear cells from RA patients
compared with healthy donors [65], and in the plasma using
proteomic profiling [66]. In addition, recent data obtained in
different pathological contexts indicate that peripheral blood
mononuclear cells in acute renal rejection upregulate

445

CXCL13 expression in association with poor graft histology

and detrimental prognosis [67, 68]. Although synovial
CXCL13 has been shown to correlate with accumulation of
CXCL13 protein in the serum [69], it remains to be
demonstrated whether systemic expression of this chemokine
simply represents an additional nonspecific inflammatory
marker or rather identifies a definite pattern of synovial
lymphoid cell infiltration. Nonetheless, these authors
reported that serum CXCL13 correlates with synovial
CXCL13 measured at a single joint, and that within the
synovium, CXCL13 expression is highly correlated with
markers of synovitis. Thus, although larger, prospective,
confirmatory studies are needed, there may be opportunities
to identify blood biomarkers that reflect synovial and
disease subtypes.

Conclusions
Despite remarkable advances in our understanding of the
complexity of synovial tissue pathobiology in RA, a great
deal of work needs to be done to better understand how the
cellular and molecular heterogeneity of the synovial lesion
fits with specific pathogenic pathways and results in
specific clinical phenotypes of the disease.
Just as much work remains to be done to fully
understand the intimate mechanisms that lead to tissue- and
joint-specific localization following the preclinical phase,
with evidence of breakage of tolerance at different sites and
circulating autoantibodies that can be present prior to disease
for years without apparent harm. Importantly, synovial tissue
biopsy studies need to be carried out in early arthritis
populations in patients nave to therapeutic intervention that
can modify the pathological processes in a diverse fashion at
an individual patient level. This may be related not only to
the potential synovial heterogeneity at the beginning of the
disease between different individuals but also to their
predetermined capacity to variably respond to the same
and/or different drugs (pharmacogenomic response).
Furthermore, interpretation of the high degree of
variability that characterizes single-cell populations and
their activation status, cell systems, and gene expression
signatures within the synovial environment requires further
definition and validation in large, well-powered cohorts.
Nevertheless, the increased availability of patients material
and the development of multiparameter research approaches
forebode fast progress in the field of synovial tissue
analysis in RA. Furthermore, although they are in their
infancy, comparative studies on peripheral blood biomarkers look promising. The identification of accurate
indicators of joint pathology that can be accessed easily and
demonstration of their clinical utility have the potential to
make an enormous impact on research as well as clinical

446

practice by, for example, helping with patient stratification

in different therapeutic response groups.
Disclosure No potential conflicts of interest relevant to this article
were reported.

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