Download Th17 Cell Frequency and IL-17 Concentration Correlate

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Th17 Cell Frequency and IL-17 Concentration
Correlate With Pre- and Postoperative Pain
Sensation in Patients With Intervertebral Disk
Degeneration
Wen Zhang, MM; Lin Nie, MD, PhD; Ying-jun Guo, MM; Lei-xiang Han, MB; Xia Wang, MB;
Hua Zhao, MD; Ying-guang Han, MM; Yuan-qiang Zhang, MM; Lei Cheng, MD, PhD
abstract
Full article available online at Healio.com/Orthopedics
Numerous studies have revealed the presence of T helper 17 (Th17) cells in pathologic intervertebral disk (IVD) tissues and the contribution of Th17-associated cytokines to the development of this disease. However, the pre- and postoperative
changes in the proportion of Th17 cells and the concentration of IL-17 in the peripheral blood of patients with IVD degeneration are not clear. The levels of Th17
frequency and the interleukin-17 (IL-17) concentration in peripheral blood from
patients and volunteers were examined by flow cytometry and by enzyme-linked
immunosorbent assay (ELISA), respectively. The clinical results were evaluated using the visual analogue scale (VAS). These results were subjected to a correlation
analysis. Compared with the normal controls, the proportion of Th17 cells and the
concentration of IL-17 were significantly increased preoperatively in patients with
IVD degeneration. Postoperatively, the levels of Th17 cells and the expression of
IL-17 were dramatically decreased. The correlation analysis of the VAS pain scores,
Th17 cell frequency, and IL-17 concentration, including the pre- and postoperative
levels and the changes induced by the surgery, revealed a positive correlation. The
authors’ results explain the contribution of Th17 cells and IL-17 to the pain sensation experienced by patients with IVD degeneration. These 2 factors may be good
indicators for the evaluation of the surgical outcome of patients with lumbar disk
herniation.
The authors are from the Department of Orthopedics (WZ, LN, YG, LH, XW, HZ, YH, YZ, LC), Qilu
Hospital, Shandong University, Jinan; and the Department of Orthopedics (WZ), Qingdao Central Hospital, Qingdao, Shandong, China.
Drs Wen Zhang and Nie contributed equally to this work and should be considered as equal first
authors.
The authors have no relevant financial relationships to disclose.
This study was supported by a grant from the Natural Science Foundation of Shandong Province
(ZR2013HM095) and Ph.D. Programs Foundation of Ministry of Education of China (20110131120079).
Correspondence should be addressed to: Lei Cheng, MD, PhD, Department of Orthopedics,
Qilu Hospital, Shandong University, No 107, Wen Hua Xi Rd, Jinan, Shandong 250012, China
([email protected]).
Received: August 26, 2013; Accepted: December 27, 2013; Posted: July 11, 2014.
doi: 10.3928/01477447-20140626-62
JULY 2014 | Volume 37 • Number 7
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D
egeneration of the human intervertebral disk (IVD) is a widespread and debilitating disorder
characterized by pronounced anatomic
and biological changes in a process
that is strongly associated with aging.1
Low back pain in humans, which is an
increasingly common and costly health
problem, is mainly caused by IVD degeneration.2 Bobechko and Hirsh3 demonstrated that the nucleus pulposus,
which is the largest avascular tissue in
vivo, is recognized as a foreign antigen,
and an autoimmune response producing
inflammation and sustained lumbocrural
pain is induced once the immune system
is prominently exposed to this antigen.
Furthermore, Macnab4 reported that the
numbness experienced by patients with
low back pain is caused only by compression of the disks and that the main factor
leading to pain is inflammation. Subsequently, various proinflammatory and
inflammatory cytokines, such as nitric
oxide (NOx), prostaglandin E2 (PGE2),
interleukin-6
(IL-6),
interleukin-8
(IL-8), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin1b (IL-1b),5,6 have been found within
and around herniated disks.
T helper 17 (Th17) cells are a new
T-cell lineage characterized by the production of IL-17 and other abundant
proinflammatory cytokines, including
TNF-α, IL-22, and IL-26.7 Gabr8 demonstrated that IL-17 is expressed in degenerated IVD tissues and showed that
the production of NOx, PEG2, and IL-6
is up-regulated by the addition of Th17associated cytokines to the culture medium of nucleus pulposus cells. Furthermore, recent work by the authors’
group revealed that the levels of Th17
lymphocytes and IL-17 are increased in
degenerated IVD tissues.9 The correlation analysis of the visual analogue scale
(VAS) pain scores, PGE2, Th17 frequency, and levels of IL-17 suggested that the
increase in the number of Th17 cells and
the overexpression of IL-17 contribute
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to the low back pain experienced by patients with lumbar disk herniation.9 The
authors have also provided a potential
explanation for the involvement of the
CCL20-CCR6 system in the migration
of Th17 cells to degenerated IVD tissues
and explained the contribution of Th17associated cytokines to the development
of degenerated disks.10
However, all of the results discussed
above were obtained from samples collected preoperatively. The changes in the
Th17 cell frequency, the expression of
IL-17, and the positive correlation of these
factors with the VAS scores postoperatively have not been reported. In the current
study, the authors aimed to discover the
variation in the Th17 cell frequency, the
expression of IL-17, and the VAS scores
and the interrelationship between the preand postoperative variables in patients
with protruded and herniated IVD.
Materials and Methods
Patients and Controls
A total of 80 patients with lumbar
disk degeneration (median age, 45 years;
range, 29 to 66 years) and 40 healthy
adults (median age, 27 years; range, 19
to 40 years) from the Qi Lu Hospital of
Shandong Province in China were enrolled in this study. Patients were divided
into 2 groups based on the radiology results and the clinical findings during surgery: the protrusion group (intact annulus
fibrosus; n=40) and the extrusion group
(ruptured annulus fibrosus; n=40). Some
of the control volunteers experienced
strained lumbar muscles during the course
of the study, but the radiology results determined that these patients did not exhibit disk degeneration.
Early on the morning preoperatively,
4 mL of fasting blood was drawn from
patients using a heparin sodium anticoagulant tube. As a control, 4 mL of fasting blood was drawn from the healthy
volunteers. All of the patients had had
microendoscopic diskectomy surgery for
just 1 segment of the lumbar vertebra.
The patient selection criteria were the
following: (1) having typical sciatica; (2)
positive for Lasegue’s sign; (3) reduction
in the height of the degenerated disk as
revealed by spinal radiography, reduced
signal of the involved nucleus pulposus
as determined by T2-weighted magnetic
resonance imaging, and no signs of calcification on the computed tomography
scan; and (4) no tuberculosis, rheumatoid
arthritis, or a recent history of glucocorticoid use. The determination of the disk
degeneration grade was made according
to the Pfirrmann grading system based on
the magnetic resonance imaging signal intensity, disk structure, distinction between
nucleus and annulus, and disk height,11
as shown in the Table. The experimental
protocol was approved by the Shandong
University Human Subjects Institutional
Review Board, and an informed consent
form was signed by each patient prior to
the use of his or her blood samples.
Pain Assessment
The visual analogue scale (VAS) was
used to evaluate the level of low back pain
experienced by patients diagnosed with
degenerated IVD, and this scale was applied 1 day preoperatively and again 2
weeks postoperatively. The discomfort
experienced by the control subjects during the study was also assessed using this
scale (Table). In every case, the level responsible for the symptomatology of the
spinal cord was determined through clinical symptomatology, physical examination, and magnetic resonance imaging of
the lumbar spine.
Flow Cytometry
Heparinized peripheral whole blood
(400 µL) and an equal volume of Roswell
Park Memorial Institute 1640 medium
were incubated for 5 hours at 37°C and 5%
CO2 in the presence of 25 ng/mL phorbol
myristate acetate (PMA) (LK-CS0001;
LiankeBio, Hangzhou, China), 1 µg/mL
ionomycin (LK-CS0002; LiankeBio),
and 1.7 µg/mL monensin (LK-CS0004;
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LiankeBio). After incubation, the cells
were stained with FITC-conjugated antihuman CD8 (11-0086-41; eBioscience,
San Diego, California) and PE-Cy5.5conjugated anti-human CD3 (35-003641; eBioscience) monoclonal antibodies
for 15 minutes at room temperature in
the dark. The anti-human CD3 and CD8
monoclonal antibodies were used to delimit the CD4+ T cells because CD4 was
down-modulated when the cells were activated by PMA. After surface staining,
the cells were fixed and permeabilized
using the FIX&PERM Kit (LK-GAS003;
LiankeBio) and were then stained with
PE-conjugated anti-human IL-17A monoclonal antibody (12-7178-41; eBioscience). Isotype controls were used to obtain
the correct compensation and to confirm
the antibody specificities. The stained
cells were analyzed by flow cytometry using a FACScan cytometer equipped with
the CellQuest software (BD Bioscience
Pharmingen, San Jose, CA).
Enzyme-linked Immunosorbent Assay
(ELISA)
The serum concentrations of IL-17 in
patients with IVD disease were determined
using the Human IL-17 Quantikine ELISA
kit (eBioscience, BMS2017) according
to the manufacturer’s protocol. All of the
procedures were conducted at room temperature, and the mean absorbance of the
standards and samples was determined
from duplicates. The color reaction was
assayed at 450 nm using a Varioskan flash
multifunction plate reader (Thermo Fisher
Scientific, Waltham, Massachusetts).
Statistical Analysis
Data
are
expressed
as
the
means±standard deviations (SDs). The
statistical evaluation of the flow cytometry
and ELISA data was performed by oneway analysis of variance with a post hoc
analysis. A bivariate correlation was used
to determine the relationships between the
VAS scores, the frequency of Th17 cells,
and the levels of IL-17. All tests were per-
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Table
Summary of Characteristics in the Study Population
Characteristic
Protrusion Group
No. of patients
Extrusion Group
Control Group
40
40
40
39.63±6.91
50.58±8.09
26.80±4.19
Age range, y
29-55
35-66
19-40
Male/female, No.
20/20
22/18
20/20
III
IV
II
Preoperative
6.44±1.25
7.85±0.92
0.57±0.53
Postoperative
2.17±0.86
2.73±1.15
Mean±SD age, y
Average degeneration grade
Mean±SD VAS
Abbreviation: VAS, visual analogue score.
formed using SPSS version 16.0 software
(SPSS, Inc, Chicago, Illinois). Differences with a P value less than .05 were
considered statistically significant.
Results
Changes in the Th17 Levels
Representative flow cytometry data
are shown in Figure 1. The lymphocyte (Figure 1A) and CD3+ T cell
(Figure 1B) subsets were gated. The proportion of Th17 cells (CD8-IL-17+) was
determined from the CD3+ cell subset
(Figures 1C-G). The statistical analysis
of the flow cytometry data obtained preand postoperatively for the protrusion and
the extrusion groups is shown in Figures
1H and 1I, respectively. Compared with
the healthy controls (1.002%±0.337%),
the percentage of peripheral Th17 cells
was significantly increased in the 2
patient groups (2.231%±0.380% and
4.490%±0.671%, respectively; P<.0001).
Compared with the preoperative results,
the frequency of circulating Th17 cells
in the 2 groups postoperatively was significantly decreased (1.113%±0.194% vs
2.231±0.380% and 1.836%±0.145% vs
4.490%±0.671%, respectively; P<.001).
These results indicate that the frequency
of Th17 cells in the peripheral blood of
patients with degenerated IVD was significantly increased preoperatively and
that this frequency was significantly decreased postoperatively.
Changes in IL-17 Serum Levels
The IL-17 expression followed a
pattern similar to that observed in the
analysis of the Th17 cell frequency. The
statistical analysis of the IL-17 ELISA
data obtained for the protrusion and the
extrusion groups is shown in Figures
2A and 2B, respectively. Compared with
the level of IL-17 in the healthy controls
(1.317±0.731 pg/mL), the levels of IL-17
were significantly higher in the 2 groups
of patients with degenerated IVD preoperatively (5.994±1.587 and 13.444±1.655
pg/mL, respectively; P<.001). The level
of IL-17 in the 2 groups of patients with
degenerated IVD were significantly decreased postoperatively (2.574±0.469
and 5.058±1.093 pg/mL, respectively;
P<.001). These results indicate that the
levels of IL-17 in the peripheral blood of
patients diagnosed with degenerated IVD
were significantly increased preoperatively and that the IL-17 levels significantly
decreased postoperatively, similarly to
the changes observed in the frequency of
Th17 cells.
Changes in the VAS Scores
The statistical analysis of the VAS
data obtained for the protrusion and the
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are shown in Figure 4A. A positive correlation was found between the VAS pain
scores, the Th17 cell frequency, and the
IL-17 concentration: Th17 proportion vs
IL-17 concentration (r=0.723, P<.01),
Th17 proportion vs VAS scores (r=0.805,
P<.01) and IL-17 concentration vs VAS
scores (r=0.675, P<.01). These results
indicate that Th17 cells may be the chief
source of IL-17 in the peripheral blood of
patients with IVD degeneration and that
the levels of Th17 cells and the expression of IL-17 are positively related to the
intensity of the pain experienced by the
patients.
Figure 1: T helper 17 frequency in representative LDH patients and controls pre- and postoperatively. The
lymphocytes (A), CD3+ T-cell subsets (B), and proportion of Th17 cells (C) are shown. The proportions of
Th17 cells from Group P (protrusion group) preoperatively (D), Group P postoperatively (E), Group E (extrusion group) preoperatively (F), and Group E postoperatively (G) are shown. *Significantly different from
the control (P<.0001). #Significantly different from the preoperative frequency (P<.001). Abbreviations:
CD3 PE-cy5.5, the cells were stained with PE-cy5.5-conjugated anti-human CD3 monoclonal antibody;
CD8 FITC, the cells were stained with FITC-conjugated anti-human CD8 monoclonal antibody; FSC-H, a
forward angle scattering, general representative of cell volume; IL-17, interleukin-17; IL-17 PE, the cells
were stained with PE-conjugated anti-human IL-17A monoclonal antibody; LDH, lumbar disk herniation;
SSC-H, a lateral angle scattering, general representative of cell particle degree; Th17, T helper 17.
extrusion groups is shown in Figures 3A
and 3B, respectively. Compared with the
healthy controls (0.570±0.525), the pain
intensity experienced by the 2 patient
groups preoperatively, as represented
by the VAS scores, was significantly increased preoperatively (6.440±1.247 and
7.850±0.925, P<.0001). The VAS scores
for the protrusion and the extrusion
groups significantly decreased postoperatively (2.170±0.857 and 2.730±1.151,
respectively) compared with the preoperative scores. Combined with the flow cy-
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tometry and ELISA results, this outcome
suggests that there may be a correlation
between the amount of Th17 cells, the
IL-17 concentration, and the pain intensity.
Correlation of VAS Pain Scores
With Th17 Cell Frequency and IL-17
Concentration Preoperatively
The Pearson correlation coefficients
for the correlations of the VAS pain
scores with the Th17 cell frequency and
the IL-17 concentration preoperatively
Correlation of VAS Pain Scores
With Th17 Cell Frequency and IL-17
Concentration Postoperatively
The Pearson correlation coefficients
for the correlation of the VAS pain scores,
the Th17 cell frequency, and the IL-17
concentration postoperatively are shown
in Figure 4B. A positive correlation was
found between the VAS pain scores, the
Th17 cell frequency, and the IL-17 concentration: Th17 proportion vs IL-17 concentration (r=0.551, P<.01), Th17 proportion vs VAS scores (r=0.536, P<.01),
and IL-17 concentration vs VAS scores
(r=0.507, P<.01). The results revealed
that the proportion of Th17 cells, the
concentration of IL-17, and the VAS pain
scores all decreased postoperatively and
that a positive correlation existed between
these factors.
Correlation of the Changes Observed
in the VAS Pain Scores, Th17 Cell
Frequency, and IL-17 Concentration Due
to Surgery
The changes in the VAS pain scores,
Th17 cell frequency, and IL-17 concentration were obtained by subtracting the postoperative results from the preoperative results. The Pearson correlation coefficients
for the correlation of the changes in the
VAS pain scores, Th17 cell frequency,
and IL-17 concentration postoperatively
are shown in Figure 4C. A positive cor-
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relation was found between the changes in
the VAS pain scores, Th17 cell frequency,
and IL-17 concentration: changes in the
Th17 proportion vs changes in the IL-17
concentration (r=0.729, P<.01), changes
in the Th17 proportion vs changes in the
VAS scores (r=0.732, P<.01), and changes in the IL-17 concentration vs changes
in the VAS scores (r=0.536, P<.01). These
correlation results provide insight into the
roles that Th17 cells and IL-17 cytokines
play in the intensity of the pain experienced by patients with degenerated IVD.
The concentration of Th17 cells and the
expression of IL-17 may serve as a predictive value of the pain sensation postoperatively.
Figure 2: Changes in the interleukin-17 (IL-17) serum levels in patients with degenerated intervertebral
disks due to surgery. The expression level of IL-17 was significantly higher in Group P (protrusion group)
(A) and Group E (extrusion group) (B) compared with the control group, and it was markedly decreased
in both groups postoperatively. *Significantly different from the control (P<.001). #Significantly different
from the preoperative level (P<.001).
Discussion
In the current study, the authors used
flow cytometry and ELISA to demonstrate that the frequency of circulating
Th17 cells and the concentration of IL-17
are significantly increased in the peripheral blood of patients with IVD degeneration preoperatively. Postoperatively,
the proportion of Th17 cells and the concentration of IL-17 were dramatically decreased. To investigate whether the pain
sensation is related to the proportion of
Th17 cells and the IL-17 concentration,
the authors analyzed the correlations between the VAS pain scores, the Th17 cell
frequency, and the IL-17 concentration,
including the pre- and postoperative levels
and the changes induced by the surgery.
The results from this study indicate that
a positive correlation exits between the
Th17 cell frequency, the IL-17 concentration, and the VAS pain scores in patients
with IVD degeneration both pre- and postoperatively.
As is well known, Th17 cells are a new
T-cell subset characterized by the production of IL-17, which is a proinflammatory
cytokine capable of abundantly inducing
other inflammatory cytokines, including
TNF-α, IL-22, and IL-26.7 Since the discovery of this T-cell subset, many studies
have revealed that Th17 cells play a major
JULY 2014 | Volume 37 • Number 7
Figure 3: Changes in the visual analogue scale (VAS) scores in patients with degenerated intervertebral
disks due to surgery. The pain sensation was significantly higher in Group P (protrusion group) (A) and
Group E (extrusion group) (B) compared with the control group, and it was dramatically decreased in both
groups postoperatively. *Significantly different from the control (P<.0001). #Significantly different from
the preoperative scores (P<.001).
role in the pathogenesis of many immunemediated diseases, including psoriasis,12
rheumatoid arthritis,13 inflammatory bowel disease,14 and asthma.15 In the current
study, the authors confirmed that the number of Th17 cells and the expression of
IL-17 are both markedly increased in the
peripheral blood of patients with IVD degeneration and that a positive correlation
exists between these factors. The authors
speculated that Th17 cells are responsible
for the increased IL-17 concentration and
that the proliferation of these cells is posi-
tively correlated with the VAS pain scores
experienced by patients with IVD degeneration.
Previous studies have proposed that
disk herniation is an immune-mediated
inflammatory process based on the particular anatomical structure.16 According to
immunologists and Burnet’s clonal selection theory,3 the nucleus pulposus, which
is the largest avascular tissue in vivo, exists in an immunoprivileged state and cannot be recognized by the immune system
after it has been prominently exposed.
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Figure 4: Correlations between the visual analogue scale (VAS) scores, the T helper 17 (Th17) cell frequency, and the interleukin-17 (IL-17) concentration levels pre- and postoperatively and changes intraoperatively. The positive correlations are shown in A (a: r=0.723; b: r=0.805; c: r=0.675; P<.01), B (a:
r=0.551; b: r=0.536; c: r=0.507; P<.01), and C (a: r=0.729; b: r=0.732; c: r=0.536; P<.01), respectively.
Instead, the nucleus pulposus elicits an
immune response that stimulates autoimmunity and subsequently leads to chronic
inflammation and sustained lumbocrural
pain. In the lesions of extruded and herniated disks, noticeable elevations were observed in the levels proinflammatory cytokines, including NOx, PGE2, IL-6, and
IL-8,17 and increases were also observed
in the levels of cytokines that are important for the regulation of pathologies, including IFN-γ, TNF-α, and IL-1b.18 Coincidentally, Macnab4 reported that the
numbness experienced by patients with
degenerated IVD is caused by mechanical
compression only, whereas inflammation
is the main factor leading to pain.
In the current study, the authors confirmed that the Th17 proportion and the
IL-17 concentration are significantly in-
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creased in the 2 groups of patients preoperatively and that the levels were markedly decreased postoperatively; in addition,
this decrease was accompanied by a decrease in the VAS scores. Combined with
the previous results, the authors speculate
that a feedback loop exists that plays an
important role in the process of disk degeneration and lumbocrural pain sensation. Once the degenerated disks become
protruded or herniated, they can secrete
the chemotactic factor CCL20, which is
a specific ligand for CCR6 that is specifically expressed on the surface of Th17
cells. The CCL20/CCR6 system plays an
important role in the trafficking of Th17
cells to degenerated disks.10
The protruded or herniated disks then
activate the body’s immune response to
produce more Th17 cells and IL-17 cyto-
kines in the peripheral blood of patients
with IVD degeneration, and this effect
provides a rich source of Th17 cells that
can be trafficked into the local degenerated disks. After the Th17 cells are trafficked to the lesion areas via chemotaxis,
the cells can secrete many inflammatory
cytokines to yield a complex cytokine
milieu that includes the other cytokines
produced by macrophages and other inflammatory cells. This cytokine milieu
promotes the inflammatory process and
stimulates the production of CCL20,
which results in the trafficking of more
Th17 cells to the inflammatory lesion. The
result of the feedback loop is the formation of the cytokine milieu, which plays an
important role in lumbocrural pain. When
corrective surgery is performed on patients with IVD degeneration, the initiator
of the feedback loop (ie, the degenerated
disks) is removed and the loop is blocked;
thus, the cause of the compression and a
large portion of the inflamed tissues will
be removed. Furthermore, the activated
immune system is blocked, which results
in the marked decrease in the production
of Th17 cells and IL-17 postoperatively.
Ultimately, the lumbocrural pain experienced by patients is also significantly
improved.
To investigate whether pain sensation
is related to the production of Th17 cells
and the concentration of IL-17 cytokines
in the peripheral blood of patients with
IVD degeneration, the authors analyzed
the correlations between these 3 factors
pre- and postoperatively. The results revealed a positive correlation between the
VAS pain scores, the Th17 cell frequency,
and the IL-17 concentration, and these
positive correlations were obtained for
the pre- and postoperative levels and for
the changes induced by the surgery. Previous studies have reported that the TNF-α
levels are correlated with the VAS scores
6 weeks and 12 months postoperatively
but not preoperatively, which indicates
that the level of TNF-α can determine the
clinical outcome of patients with lumbar
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disk herniation after diskectomy.19 The results from the current study indicate that
the Th17 cell frequency and the IL-17 levels may be a better indicator for the evaluation of the surgical outcome of patients
with lumbar disk herniation.
In the near future, the immune-induced
inflammation may be reduced by either
blocking the biding of CCR6 to Th17 cells
during the initial process of IVD degeneration or reducing the CCL20 production by modifying the cytokine milieu in
the degenerated disks. These treatments
may prevent Th17 cell trafficking via the
CCL20/CCR6 chemotactic pathway and
thus have the potential to cure or prevent
inflammation and significantly improve
the lumbocrural pain experienced by patients with IVD degeneration. Although
the mechanisms responsible for chronic
lumbocrural pain are poorly understood,
this study may aid the prediction of the
clinical effectiveness of diskectomy surgery and the exploration of novel therapeutic approaches.
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