Beruflich Dokumente
Kultur Dokumente
Medicine, Division of Infectious Diseases, and Pharmacology, University of North Carolina, Chapel Hill, North Carolina, USA; and
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Institute of Medical Microbiology, University Hospital of Mnster, Mnster, Germany
RECEIVED JANUARY 12, 2012; REVISED JULY 9, 2012; ACCEPTED JULY 23, 2012. DOI: 10.1189/jlb.0112014
ABSTRACT
The Staphylococcus aureus pore-forming toxin PVL is
most likely causative for life-threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of
PVL on human neutrophils is already well established, the
PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we
used different types of human leukocytes (neutrophils,
monocytes, macrophages, lymphocytes) to investigate
cell-specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL-sensitive
cells, we identified the binding of the subunit LukS-PV as
the critical factor for PVL-induced cytotoxicity, which was
followed by binding of LukF-PV. LukS-PV binds to
monocytes, macrophages, and neutrophils but not to
lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release
Introduction
Abbreviations: -toxin-hemolysin, ASCapoptosis-associated speck-like
protein, BMDMbone marrow-derived macrophage, CA-MRSA
community-acquired methicillin-resistant Staphylococcus aureus, CTSB
cathepsin B, DAMPdanger-associated molecular pattern, EVempty
vector, HKSAheat-killed Staphylococcus aureus, HMGB1high-mobility
group protein B1, LTAlipoteichoic acid, LukF-PVsubunit F (F for fast)
of Panton-Valentine leukocidin, LukS-PVsubunit S (S for slow) of
Panton-Valentine leukocidin, MFImean fluorescence intensity, MRP8/
14myeloid-related protein complex 8/14, NLRP3nucleotide-binding domain and leucine-rich repeat-containing gene family, pyrin domain containing 3 protein, PAMPPathogen associated molecular pattern,
PRRpattern recognition receptors, PSMphenol soluble modulins (expressed by Staphylococcus aureus), PVLPanton-Valentine leukocidin
(Staphylococcus aureus pore-forming toxin), shshort hairpin,
shASCmutscrambled sequence with base content equal to short hairpin
apoptosis-associated speck-like protein, shGFPshort hairpin RNA against
GFP, siRNAsmall interfering RNA, zYVAD-fmkz-Tyr-VAD-fmk
The online version of this paper, found at www.jleukbio.org, includes
supplemental information.
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Ethics statement
Taking of blood samples from humans and animals and cell isolation were
conducted with approval of the local ethics committee (Ethik-Komission der
rztekammer Westfalen-Lippe und der Medizinischen Fakultt der WestflisA
chen Wilhelms-Universitt Mnster; Az. 2008-034-f-S). Human blood samples
were taken from healthy blood donors, who provided written, informed consent for the collection of samples and subsequent neutrophil isolation and
analysis. All animals were handled in strict accordance with good animal practice and animal keeping, according to European guidelines (86/609/EWG),
and in strict accordance with the German Protection of Animal Act (Deutsches
Tierschutzgesetz). Taking of blood samples was supervised by the veterinary
office of Mnster (Veterinramt der Stadt Mnster; Az. 39.32.7.1).
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Stimulation of phagocytes
Primary monocytes, macrophages, or neutrophils were cultured on 24-well or
six-well plates and subjected to PVL for 15 min16 h at 37C under 5%
(monocytes) or 7% CO2 (all other cell types). Where indicated, 100 ng/ml
ultrapure LPS from Escherichia coli (InvivoGen, San Diego, CA, USA; serotype
0111:B4), 25 M caspase-1 inhibitor zYVAD-fmk (Enzo Life Sciences, Germany), 30 M CTSB inhibitor CA-074Me (Enzo Life Sciences), or 130 mM
KCl was added to the incubation medium. For the costimulation experiments,
HKSA from strain 6850 (80C for 30 min), LTA (Sigma-Aldrich, St. Louis,
MO, USA), MRP8, or S100A12, manufactured at our institute [38, 39], was
used. After the incubations, culture media were collected for analysis of their
cytokine contents, and cells were washed and lysed for RNA analysis or Western blots.
out FCS) were harvested and precipitated with TCA, protein pellets were resuspended in SDS-PAGE buffer. Cell lysates were prepared for Western blot analysis as described earlier [42]. Western blot analysis was performed by standard
procedures with the following antibodies: human anticleaved-IL-1 antibody
(2021S; Cell Signaling Technology, Danvers, MA, USA) was used at 1/1000
dilution, anti-IL-1 antibody (R&D Systems) was used at 1/500 dilution, and
-actin antibody (Sigma-Aldrich Chemie GmbH) was used at 1/500 dilution.
Cell lysates of THP1-knockdown cells were analyzed with human anti-ASC
(AL177; Enzo Life Sciences), dilution 1/1000; anti-NLRP3 (Cryo-2; Enzo Life
Sciences), dilution 1/1000; anti-CTSB (Enzo Life Sciences), dilution 1/1000;
and anti-HMGB1 (4C3; Abcam, Cambridge, MA, USA), dilution 1/1000.
Quantitative RT-PCR
After stimulation, RNA was isolated from the cells, and expression of IL-1
and TNF- RNA was analyzed by real-time RT-PCR, as described previously
[42]. Each measurement was set up in duplicate, and three independent experiments were performed. After normalization to the endogenous housekeeping control gene GAPDH, the relative expression was calculated. The primers
used for PCR analysis were: IL-1 forward, 5=-GCGGCCAGGATATAACTGACTTC-3=, and IL-1 reverse, 5=-TCCACATTCAGCACAGGACTCTC-3=; TNF
forward, 5=-CTTCTCGAACCCCGAGTGAC-3=, and TNF reverse, 5=-TGAGGTACAGGCCCTCTGATG-3=; GAPDH forward, 5=-TGCACCACCAACTGCTTAGC3=, and GAPDH reverse, 5=-GGCATGGACTGTGGTCATGAG-3=.
Western blotting
IL-1 processing was analyzed in a serum-free culture medium sample corresponding to 2 106 cells. After 3 h of treatment supernatants (McCoys with-
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THP1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI, 10% FCS. shRNAs for knockdown of ASC (shASC), NLRP3
(shNLRP3), CTSB, or an EV and shASCmut and shGFP as negative controls
were stably introduced using retrovirus as described previously [4345]. Efficient knockdown of targeted proteins was confirmed by Western blotting. To
assess effects of knockdown, cells were plated at 106 cells/ml. Equal amounts
of LukS-PV and LukF-PV were added to yield a final concentration of 210
nM, and cells were incubated for 24 h. Cell supernatants were collected and
assayed for IL-1 levels. Results represent the average plus sd of triplicate wells
and are representative of three independent experiments.
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Mice
Eight- to 12-week-old C57BL/6 mice were obtained from Harlan Winkelmann (Borchen, Germany). Animals were maintained under specific pathogen-free conditions and according to the institutional guidelines in the animal facility of the University of Mnster, Institute of Immunology.
Generation of BMDMs
BMDMs were differentiated in vitro from BM cells, as described previously
[47]. Briefly, to generate macrophages, BM cells were cultured in DMEM
(Invitrogen, Karlsruhe, Germany) containing 2 mM L-glutamine (Invitrogen), 1 mM nonessential amino acids (Invitrogen), 100 mg/ml penicillin/
streptomycin (Biochrom, Berlin, Germany), 10% heat-inactivated FCS (Biochrom), and 50 ng/ml M-CSF for 6 days.
Stimulation of BMDMs
Macrophages (1106/ml) were seeded into 96-well tissue-culture plates
(Greiner Bio One, Frickenhausen, Germany) and treated with different
stimuli: 1 g/ml ultrapure LPS from E. coli (InvivoGen; serotype 0111:B4),
4 g/ml PVL, and 20 M nigericin. After 6 h, supernatants were harvested,
and cytokine production was determined using CBA technology (BD Biosciences), according to the manufacturers instructions. CBA FlexSets were
used for IL-1 and TNF.
Statistical analysis
Statistical analyses were performed by using Prism 5.0 software (GraphPad
Software, La Jolla, CA, USA). Analyses between two groups were performed
by using an unpaired two-tailed Students t test. Comparisons among three
or more groups were performed by using one-way ANOVA, followed by
Dunnetts multiple means test for comparing all columns versus control or
by Bonferronis multiple means test for comparing all pairs of columns.
Differences were considered statistically significant at a P value of 0.05.
All experiments were done three times or more.
RESULTS
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Figure 1. Cytolytic effects and binding of purified PVL to human leukocytes. Primary bloodderived monocytes (A), macrophages (B), neutrophils (C), and lymphocytes (D; 11060.5
ml1 cells) were incubated with increasing doses
of PVL [0.04 0.4 g/ml (110 nM)]. Cells were
stimulated for 1, 3, or 16 h, respectively, or with
LukS-PV and LukF-PV for 16 h and then were
washed and stained with PI, and cell death was
measured by flow cytometry. Statistical differences between control and stimulated cells were
determined by Students t test. Leukocytes
[monocytes (E), macrophages (F), neutrophils
(G) and lymphocytes (H)] (11060.5 ml1
cells) were incubated with untreated and FITCconjugated LukS and/or LukF (1.0 g/ml) for 1 h and washed, and binding of toxin was quantified by flow cytometry. Rate of binding was calculated by the MFI shift between untreated control cells and toxin-treated cells. The values represent the mean sd of at least three independent experiments. Statistical differences were determined by ANOVA comparing the MFI between control and treated cells (*P0.05; **P0.01; ***P0.001).
PVL-induced inflammasome activation. Cleavage of caspase-1 after PVL treatment was confirmed by measuring the p20 subunit
in the supernatant of PVL- or nigericin-treated cells. Caspase-1
levels increased fast in a time-dependent manner (Fig. 4B). Our
hypothesis was strengthened further by using stable THP-1-derived cell lines with expression of the inflammasome components
NLRP3 or ASC knocked down by expression of shRNAs targeting the mRNA encoding those proteins. Western blot analysis
indicates that levels of NLRP3 and ASC proteins are reduced by
90% in these cells (Fig. 4C). Testing the effect of PVL on both
cell lines revealed a strong reduction of IL-1 secretion when
compared with control cell lines with intact expression of NLRP3
and ASC (Fig. 4C). The functionality of the model was confirmed
by treatment with LPS and ATP (Supplemental Fig. 1A). Interest-
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Figure 2. Human phagocytes respond to PVL by IL-1 secretion and release of DAMPs. Primary monocytes (A and D),
macrophages (B and E), and neutrophils (C and FH; 11060.5 ml1 cells) were incubated with PVL (0.04 0.4 g/
ml) for 3 h. Ultrapure LPS (100 ng/ml) was used as a costimulant for the primary cells and was added 30 min before
PVL treatment. Concentrations of IL-1, TNF-, MRP8/14, and S100A12 were subsequently determined from cell culture supernatants. The inset in A verifies the presence of mature 17 kDa IL-1 and absence of pro-IL-1 in cell culture
supernatants (SN) of primary monocytes, as well as the induction of intracellular pro-IL-1 (IC) by Western blotting.
The values represent the means sd from at least three experiments. Statistical differences were determined by Students t test (*P0.05; **P0.01; ***P0.001 compared with untreated cells).
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used the DAMPs MRP8, the active subunit of the MRP8/14 complex, and S100A12, which are released by PVL-damaged neutrophils (Fig. 2G and H), to preactivate monocytes. The stimulation
with DAMPs and PAMPs alone was not sufficient to induce IL-1
secretion, whereas the addition of PVL leads to massive release of
IL- (Fig. 7A). By contrast, the activation with PAMPs and
DAMPs induced TNF- secretion in monocytes, which was not
further increased by PVL (Fig. 7B). All costimulants were capable
of inducing IL-1 gene expression (Fig. 7C), leading to increased
production of pro-IL-1, which resulted in enhanced processing
and a strong IL-1 release after PVL stimulation. Therefore,
DAMPs and PAMPs are capable of amplifying IL-1 release by
priming monocytes before activation by PVL.
sterile-filtered bacterial supernatants from overnight cultures. Culture media from strain TM300 PVL induced rapid cell death
within 1 h (Fig. 6C) and secretion of IL-1 (Fig. 6D), whereas
supernatants from the control strain TM300 did not show any
effects.
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DISCUSSION
The pathogenic mechanisms of S. aureus necrotizing infections
that lead to massive inflammation and tissue destruction are
largely unknown. According to epidemiological data [12] and to
results from an in vivo model in rabbits [22], PVL could be identified as a crucial, responsible factor. Yet, the exact molecular
mechanisms that are induced by PVL at the cellular level are not
fully elucidated. Previous work largely focused on PVL-induced
cytotoxicity of human neutrophils, whereas there is binding to
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Figure 4. Mechanism of PVL-induced IL-1 secretion. Primary monocytes (11060.5 ml1 cells; A) were
incubated with 0.04 g/ml PVL in the absence or presence of KCl (130 mM), CTSB inhibitor CA-074Me
(30 M), or caspase-1 inhibitor zYVAD-fmk (25 M). After the incubation, IL-1 (average response to PVL,
690 pg/ml) was analyzed by ELISA. The values represent the means sd from at least three experiments.
The inset in A verifies the inhibition of processing and secretion of mature 17 kDa IL-1 and the absence
of pro-IL-1 in cell culture supernatants of LPS-primed primary monocytes (51062.5 ml1 cells) and
the presence of intracellular pro-IL-1 by Western blotting. Statistical differences were determined by
ANOVA comparing PVL-treated cells with PVL-treated cells inhibitors (***P0.001). Further, cells were
treated with PVL (0.04 g/ml), and p20 caspase-1 subunit was analyzed by ELISA in the supernatant after
different time-points; the K-ionophore nigericin (20 M) served as a positive control. Statistical differences were determined by Students t test (*P0.05; **P0.01; ***P0.001 compared with untreated cells; B). THP-1-derived cell lines (C) stably transduced with shRNA expressing retrovirus were treated with 0.08 or 0.4 g/ml PVL, and cell culture supernatants were analyzed for IL-1 after 24 h. The
shRNAs are directed to knock down expression as follows: EV, negative control; shASCmut, negative control; shASC, shRNA directed against ASC protein; shNLRP3, shRNA directed against NLRP3. Knockdown of ASC and NLRP3 and expression of pro-IL-1 were confirmed by Western blot. Results
represent the means sd of triplicate wells and are representative of three independent experiments. Primary monocytes (51062.5 ml1 cells) were
treated with LukS or LukF or both for 3 h, and cell culture supernatants were analyzed for IL-1 or TNF- (D). Induction of IL-1 and TNF- RNA was
determined by RT-PCR (E) and expressed as n-fold compared with untreated control. The values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA comparing PVL-treated cells to control (*P0.05; **P0.01; ***P0.001).
Therefore, the PVL-induced proinflammatory effect on monocytes could play an important role to initiate an infection and to
attract further immune cells, such as neutrophils and lymphocytes, which are usually present in large numbers during necrotizing infections. In our experiments, we demonstrate that PVL is
capable of inducing activation of caspase-1 through the NLRP3
inflammasome. Pore formation of PVL leads to K-efflux and the
consecutive activation of CTSB, which mediates programmed necrosis and activation of NLRP3. By performing siRNA silencing
experiments, we confirmed the NLRP3 receptor as the PVL-responsive element in monocytes. Previous work revealed that S.
aureus, lacking -toxin or - or -hemolysin, was still capable of
activating IL-1 secretion, indicating that other staphylococcal
factors cause inflammasome activation as well [33]. Our findings
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Figure 5. PVL-mediated K-efflux leads to CTSB activation and pyronecrosis. THP-1-derived cell
lines (A), stably transduced with shRNA expressing retrovirus, were treated with 0.4 g/ml PVL,
and cell culture supernatants were analyzed for IL-1 after 24 h. The shRNAs are directed to
knockdown expression as follows: EV, negative control; shGFP, negative control; shCTSB#1 and
#2, two shRNAs directed against CTSB. Knockdown of CTSB and the presence of ASC and NLRP3 were confirmed by Western blot. Results represent
the means sd of triplicate wells and are representative of three independent experiments. Statistical differences were determined by ANOVA comparing knockdown cells with control (***P0.001). CTSB activity was measured via degradation of the fluorescent Magic Red CTSB substrate (ImmunoChemistry Technology). Magic Red CTSB substrate was added to primary monocytes (11060.5 ml1 cells) treated with PVL (0.4 g/ml), -toxin (10
g/ml), or nigericin (20 M) for 15 min, with or without pretreatment with KCl (130 mM), CA-074Me (30 M), or zYVAD-fmk (25 M) (B). Degradation of the Magic Red substrate was measured via flow cytometry on a BD FACSCalibur. Analysis of the data was accomplished with detection of the optimal Magic Red substrate fluorescence emission in fluorescence-2 on a logarithmic scale. The values represent the means sd from three experiments. Statistical differences were determined by ANOVA comparing the MFI between control and treated cells (*P0.05; **P0.01). Primary monocytes (11060.5 ml1 cells; C) were incubated with 0.04 g/ml PVL for 3 h in the absence or presence of CTSB inhibitor CA-074Me (12.5100 M),
KCl (32.5130 mM), or caspase-1 inhibitor zYVAD-fmk (25 or 100 M). LDH was measured in the supernatants, and cells were washed and stained with
PI. The values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA comparing PVL-treated
cells with PVL-treated cells inhibitors (**P0.01; ***P0.001).
Figure 6. The cytotoxic effect and induction of IL-1 secretion of TM300 and its supernatant are dependent on PVL expression. Primary monocytes (51062.5 ml1 cells) were incubated with 10 MOI of bacteria (A and B) for 3 h or for 1 h with bacterial supernatants (C and D), which
were prepared from overnight cultures and used for stimulating cells (10% vol/vol) or were left untreated. In this experiment, the heterologous
expression strain TM300 PVL and its WT strain were used. After incubation of the cells with bacterial supernatants or whole bacteria, cells were
washed and stained with PI for analysis by flow cytometry. IL-1 was measured in the cell supernatants by ELISA. The values represent the means sd
of at least three independent experiments. Statistical differences were determined by Students t test (***P0.001 compared with TM300).
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masome-dependent protein IL-18 [57] is released by PVL stimulation. In contrast to pro-IL-1, pro-IL-18 is constitutively expressed
in cells, independently of a primary stimulus [58], but it shares
the same effects of IL-1 and is especially involved in T cell activation [59].
With respect to IL-1 secretion, primary stimuli, such as LPS,
induce synthesis of pro-IL-1 and may provide signals that enhance the effect of the secondary stimulus [50]. In this work, we
demonstrate that PVL does not require a LPS-priming step, as
PVL alone was sufficient to induce pro-IL-1 and activate
caspase-1 and mature IL-1 release. PVL treatment is capable of
inducing IL-1 gene expression, independent from an early
NF-B activation, and might be a result of an intermediate step
by released DAMPs (e.g., MRPs) or cytokines. Nevertheless, priming with LPS enhanced IL-1 release significantly. As LPS is restricted to Gram-negative bacteria, we tested other factors that are
Figure 8. Induction of IL-1 and TNF- secretion by staphylococcal components. Primary monocytes (11060.5 ml1 cells) were incubated with
PVL (0.04 g/ml), -toxin (10 g/ml), Protein A (10 and 100 g/ml),
and PSM1 and -2 (25100 g/ml) for 3 h or were left untreated; the
K-ionophore nigericin (20 M) served as positive control. After incubation, cells were washed and stained with PI for flow cytometry (A), and
cell culture supernatants were analyzed for IL-1 or TNF- by ELISA (B).
The values represent the means sd from at least three experiments.
Statistical differences were determined by ANOVA (*P0.05; **P0.01;
***P0.001 compared with control).
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Figure 9. PVL has no effect on murine BMDMs. BMDMs were prestimulated with ultrapure LPS (1 g/ml) for 6 h and then incubated further
with PVL (4 g/ml) for 30 min or costimulated for 6 h with LPS and PVL. Thirty minutes of treatment with nigericin served as a positive control.
After incubation, cells were washed and stained with PI (A), and cell culture supernatants were analyzed for IL-1 (B) or TNF- (C) by CBA. The
values represent the means sd from at least three experiments. Statistical differences were determined by ANOVA (**P0.01 compared with
control). BMDMs were incubated with unlabeled and FITC-conjugated LukS and/or LukF (1.0 or 4.0 g/ml) for 1 h and washed, and binding of
toxin was quantified by flow cytometry. Rate of binding was calculated by the MFI shift between untreated control cells and toxin-treated cells. The
values represent the mean sd of at least three independent experiments (D).
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Figure 10. Proposed mechanism of PVL-induced inflammasome activation. PVL leads to K-efflux and CTSB activation, which consecutively
activates the inflammasome and induces pyronecrosis. Activated
caspase-1 processes IL-1 and IL-18. All steps can be blocked by specific inhibitors.
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PVL induces cell death in monocytes, macrophages, and neutrophils, which is most likely a strategy of the pathogen to
overcome the cellular innate immune system. In addition, our
findings regarding priming of phagocytes for PVL responses by
endogenous DAMPs may be of relevance for the tissue damage
associated with overwhelming disease activity during PVLdriven inflammation and may thus represent a novel molecular target for future adjuvant therapy to appropriate antibiotic
use in severe S. aureus infections.
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AUTHORSHIP
D.H., J.R., G.P., and B.L. conceived of and designed the experiments. D.H., L.G., V.M., N.N., D.J.T., K.M., T.V., D.F., S.N.,
J.A., J.A.D., and P.M.B. performed the experiments. D.H. and
B.L. analyzed the data and wrote the manuscript. J.R. and G.P.
initiated the research, supervised the program, and analyzed
and discussed data.
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ACKNOWLEDGMENTS
This work was supported by Deutsche Forschungsgemeinschaft
of Germany (DFG) grants (HA3177/2-1 and SFB1009) to B.L.,
a German Federal Ministry of Education and Research
(BMBF) grant (Flu-Bak Signaling) to B.L., and grants from the
Interdisciplinary Centre for Clinical Research (Lf2/030/10
and Ro2/004/10) to B.L. and J.R. at the University of Mnster. D.H. was supported by an Innovative Medizinische Forschung (IMF) grant of the University of Mnster (HO220912).
P.M.B. is an awardee of the University of North Carolina STD
and HIV Training Program (U.S. National Institutes of Health
T32AI007001). J.A.D. is supported by the Burroughs Wellcome
Fund through the Career Award for Medical Scientists and
U.S. National Institutes of Health (AI088255). We thank Melanie Saers, Heike Berheide, Brigitte Schuhen, and Michaela
Brck for excellent technical assistance.
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KEY WORDS:
Panton-Valentine leukocidine monocytes macrophages IL-1 and
IL-18 expression inflammasome
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