Burn & Shock Trauma Institute, Department of Surgery, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
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ABSTRACT |
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We evaluated the
dependency of neutrophil O
burn; rat; polymorphonuclear neutrophil; protein kinase C signaling; platelet-activating factor blockade; Lyn blockade; extracellular signal-regulated kinase 1/2 blockade
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INTRODUCTION |
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POLYMORPHONUCLEAR
NEUTROPHILS (PMNs) play a frontline defensive role following
inflammation and injury (38). Several previous studies
have shown that blood neutrophils harvested from
burn/trauma-injured hosts, in the early stages of the host
injury, generate effector responses of higher magnitude than those
produced by neutrophils from uninjured hosts. The excessive effector
responses in the injured host may emanate from an injury-induced
priming of the neutrophils in vivo. Such priming of neutrophils with
burn injury could be due to one or more of the known mediators
generated during the course of burn injury. These mediators bind to
their receptors to initiate a series of G protein-mediated
intramembrane signaling events followed by activation of
Ca2+-dependent and -independent signaling pathways. Whereas
activation of the Ca2+-dependent pathway involves the
-subunit of Gi protein (10), the
Ca2+-independent pathway is presumably mediated by
Gi protein's
-subunit-linked stimulation of protein
tyrosine kinases such as Lyn (3). Although the activation
of these Ca2+-dependent and -independent signaling pathways
stimulates neutrophils to produce various effector responses
(43), a prior priming of the neutrophils by inflammatory
agents such as TNF-
or granulocyte macrophage colony-stimulating
factor (GM-CSF) through the activation of Gi
protein-independent pathways is known to result in an effector response
of a much greater magnitude (17). Inflammatory mediators activating Gi protein-linked pathways may also mediate
prior priming of neutrophils (40, 43).
Neutrophil activation by a number of agonists involves both Ca2+ signaling and sequential tyrosine phosphorylation of proteins including members of the Src family protein tyrosine kinase (PTK), such as Lyn, and mitogen-activated protein kinase (MAPK), such as ERK1/2 (13). The ERK p42/44 MAPK is a member of distinct serine/threonine kinase pathways in PMNs that relay signals from surface receptors to elicit cellular responses such as transcription, chemotaxis, and phagocytosis (4, 16, 27). Both platelet-activating factor (PAF) and formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) cause activation of p42/44 MAPK in PMNs and have been implicated in regulating multiple PMN functions, including NADPH oxidase activity and chemotaxis (16). The importance of PTK and MAPK pathways has been substantiated by the results of recent in vitro studies demonstrating that both tyrosine kinase and MAPK inhibitors abrogate several neutrophil responses, including oxidative burst (1, 9, 23). These studies support the hypothesis that tyrosine phosphorylation via PTK and MAPK plays an active functional role in the regulation of neutrophil activation under inflammatory conditions such as trauma, burn, and sepsis.
This study followed three major lines of investigations. First, to
obtain information on the molecular identity of the kinases participating in burn-induced neutrophil activation, we focused on the
Src family of tyrosine kinase, Lyn, and the MAPK, ERK 1/2, which could
particularly affect neutrophil oxidant production after burns
(25, 41). Second, we compared the involvement of the above
signaling pathways in the burn-induced modulations in
O
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MATERIALS AND METHODS |
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Animals. Adult male Sprague-Dawley rats weighing 250-275g were obtained from Harlan (Indianapolis, IN). The rats were acclimatized in the animal quarters for 3 days before their use. The care of animals was in accordance with the guidelines set forth by Loyola University Chicago Medical Center Animal Care and Use Committee.
Thermal injury protocol. The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (45 mg/kg body wt). The hair on the animals' backs was clipped. The animals were then placed in a supine position in a plastic template that exposed 30% of the total body surface area. In the sham group, the exposed backs were immersed for 10 s in a room temperature water bath. In the burn group, full-thickness scald burns were inflicted by immersing the back of the animal in 95°C water for 10 s. Rats were quickly dried after the exposure to hot water to avoid additional injury. The animals in each group were resuscitated with a 10-ml intraperitoneal injection of normal saline. The animals were housed in cages after thermal injury and killed at 24 h postinjury after being anesthetized with pentobarbital sodium (45 mg/kg) and exsanguinated through a cardiac puncture.
Neutrophil signaling blockers. Tyrphostins (also known as AG compounds) inhibit PTKs by binding to the substrate binding site. The dose and route of administration for each of the inhibitors of neutrophil signaling were used as determined by previous studies. PTK inhibitor AG556 has been successfully used in vivo by other researchers (6, 19, 28, 36); the dose of 5 mg/kg was originally determined by Sevransky et al. (36). They also determined the half-life of AG556 (9.2 ± 4.5 min immediately and 7.4 ± 4.6 min after 6 h) in their multiorgan failure study in a canine Escherichia coli peritonitis model. Similarly, Brenner et al. (6) used the same dose of AG556 once a day by intraperitoneal injection in mice. Jarrar et al. (19) used the same dose of AG556 intraperitoneally in a trauma-hemorrhage model of rats. Rice et al. (34) and Busse et al. (7) used AG1478 in vivo in a rat model of pulmonary fibrosis and in retinoblastoma tumors in mice, respectively. Diltiazem at micromolar concentrations, such as are expected to prevail in the circulation of rats administered with 2 mg/kg diltiazem, can inhibit a plasma membrane receptor-gated Ca2+ channel (36) and thus decrease Ca2+ influx. Moreover, other investigators have found PAF-16 antagonist to be efficacious in in vivo studies (11, 29, 42). Tokumura et al. (42) calculated that the IC50 for PAF-16 antagonist was 40 nM. Our rat is ~230 g, and the volume of distribution is about 161 ml (70%). We used 10 times the IC50, i.e., 400 nM; to achieve this concentration, we injected PAF-16 antagonist at the dose of 90 µg/rat.
Administration of neutrophil signaling blockers in vivo. Tyrphostins AG556 and AG1478 were purchased from Calbiochem (San Diego, CA). AG556 was originally dissolved in dimethyl sulfoxide (DMSO; 10,000 µM: 5 mg/1.49 ml) diluted with Hanks' balanced salt solution (HBSS) and given subcutaneously (5 mg/kg body wt) in a volume of 0.100 ml. AG1478 was dissolved in DMSO and injected intraperitoneally (5 mg/kg body wt) in a final volume of 1.0 ml. PAF-16 antagonist (0.35 mg/kg body wt; Calbiochem, La Jolla, CA) was dissolved in 1.0 ml of normal saline per animal and injected intravenously in a single bolus. As described in previous studies (35), the Ca2+ channel blocker diltiazem hydrochloride (Sigma, St. Louis, MO) was administered intravenously as a single bolus of 1.0 ml to rats at 2 mg/kg body wt. AG556, AG1478, diltiazem, or PAF-16 antagonist were administered to the animals 1 h before subjecting them to burn injury.
Preparation of blood neutrophils.
At euthanization, rats were anesthetized with pentobarbital sodium
(40-50 mg/kg ip) and blood (10-12 ml) was collected by cardiac puncture to heparinized syringes. Neutrophils were isolated from the heparinized blood by using the standard Ficoll-paque (Pharmacia) cell separation technique followed by dextran sedimentation and hypotonic red blood cell lysis. Neutrophils were then washed and
resuspended in HBSS buffer. Neutrophil preparations routinely contained
95% neutrophils, as identified by the Giemsa stain, and were found
to be ~98% viable by the trypan blue exclusion technique.
Stimulation of PMN with fMLP and lysate preparation. PMN obtained from sham and thermally injured rats were stimulated with fMLP (1 µM) for 180 s at 37°C. The stimulation was stopped by cell solubilization in a phosphorylation lysis buffer (PLB: 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 1 mM MgCl2, 10 mM Na4P2O7, 200 µM Na3VO4, 0.5% Triton X-100, and 10% glycerol) on ice for 45-50 min. Lysates were centrifuged at 10,000 rpm for 5 min at 4°C.
Immunoprecipitation. Lysates were incubated with monoclonal antibodies to Lyn p56/p53 protein (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, and then the mixture was incubated with protein G-Sepharose beads for another 2 h (18). These incubations were carried out at 4°C. The precipitates were washed three times in PLB without added glycerol.
In vitro kinase assay. Kinase assays were performed by using previously described methods (18). After the final wash, immune complexes were collected and washed twice with in vitro kinase buffer (50 mM Tris · HCl, pH 7.4, 10 mM MnCl2, and 0.1% Triton X-100). After these washes, kinase assays were performed by incubating immune complexes first with 5 µg/ml acid-treated enolase and then for 30 min with 10 µCi [32P]ATP. This incubation was carried out at room temperature (28°C). Samples were analyzed on SDS-PAGE (9%), and the proteins were transferred to the Immobilon membrane. Phosphoproteins were analyzed by autoradiography, and the intensity of the bands was assessed using densitometry.
Immunoblot analysis. Immunoblot detection of ERK1/2 was done with phospho-p44/42 MAPK (Thr202/Tyr204) purchased from Cell Signaling Technology (Beverley, MA). The amount of protein in each sample was quantitated with the Bio-Rad protein assay system using BSA standards. Cell lysates (5 × 106 cell equivalent/lane) were analyzed by electrophoresis on 10% polyacrylamide gels (12). The resolved proteins were electrophoretically transferred to Immobilon-P. After transfer, the membranes were first incubated at room temperature for at least 1 h in the blocking buffer consisting of 10% BSA in Tris-buffered saline (TBS; pH 7.5). Membranes were then incubated in ERK1/2 antibody at 1:200 dilution in 10 ml of primary antibody dilution buffer with gentle agitation overnight at 4°C. After the membrane had been rinsed five times with wash buffer containing 0.05% Tween 20 in TBS (pH 8), the membrane was incubated with horseradish peroxidase-conjugated (HRP) secondary antibody (1:2,000) and HRP-conjugated anti-biotin antibody (1:1,000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 h at room temperature. After five rinses with wash buffer, the transfers were color developed for 1 min by enhanced chemiluminescence (ECL; Amersham) Western blotting reagents. The relative intensity/content of an individual protein band was quantitated by densitometry (Hewlett-Packard, HP ScanJet Scanner). In each batch of experiments, blot intensities were measured in terms of densitometric units in sham and burn group rat neutrophils. Statistical analyses were carried out on values in densitometric units.
Isoluminol-enhanced luminometry.
Luminometry was used to measure O4 M isoluminol (Sigma) in
HBSS was added to individual wells of white polystyrene microtiter
96-well plates and allowed to equilibrate at 37°C for 30 min. Fifty
microliters of neutrophils at 5 × 106
ml
1 were then added, and the plate was incubated at
37°C. After a 15-min equilibration period, background readings were
recorded on an MLX analyzer every 5 min for 20 min until a steady value was obtained. The cells were then stimulated with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml). The kinetics of O
Fura 2 Ca2+ imaging. Neutrophils suspended in HBSS were loaded with 10 µM fura 2-AM (Molecular Probes, Eugene, OR) for 1 h at room temperature. A drop of neutrophil suspension (100 µl) was placed on a 1-µm-thick coverslip and examined with the ×40 oil-immersion objective of an inverted Nikon Microscope. Computerized fura 2 ratio imaging was then performed with the aid of MetaFluor software (Series 4.5; Universal Imaging, West Chester, PA) and the associated hardware, including a SenSys charge-coupled device camera (Photometrics) and a Metaltek shutter. With the use of this setup, real-time fluorescence images were generated by exposing cells to alternating 340- and 380-nm excitation wavelengths and automatically collecting associated fura 2 emissions through a 505-nm band-pass filter (24). The following steps were followed for each cell sample. First, cells were focused for optimal fluorescence fura 2 signal and background correction was set up based on an adjacent blank part of the coverslip. Second, a field of cells was chosen, and its 340-nm, 380-nm, and ratio images were optimized. Third, a computer-controlled series of 30 subsequent images was acquired at 20-s intervals. Of the 30-point image series, the first 5 images were kept as baseline images, because cellular stimulation with 1 µM fMLP was administered in vitro after the 5th point (100 s). Fourth, acquired data were then used to generate 340/380 ratio images and curves for analysis and presentation.
Statistical analysis. All statistical analyses were carried out using the Statistical Package, Social Sciences Software Program (SigmaStat version 2.0; SPSS, Chicago, IL). To determine inter- and intragroup differences between variables, a one-way repeated-measures ANOVA, followed by a pairwise multiple comparison procedure (Tukey's post hoc test), was performed. The statistical analysis of the different experimental groups included the comparison of sham-burned, day 1 postburn, and inhibitor-treated burned rats in n animals. Statistical significance was assigned at P values <0.05.
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RESULTS |
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Burn-induced activation of PTK-Lyn.
In an initial series of experiments, we examined whether PTK-Lyn is
modulated by burn-induced PMN activation. Figure
1 shows immunoblot and blot densitometric
analyses of Lyn autophosphorylation of neutrophils as well as the
ability of Lyn to phosphorylate other proteins (kinase activity),
evaluated by examining phosphorylation of enolase. There was no
detectable Lyn autophosphorylation or its kinase activity in
unstimulated neutrophils from sham or burn animal groups (data not
shown). Stimulation of neutrophils with fMLP (1 µM) from day 1 postburn or sham rats with fMLP caused significant elevations in
both Lyn autophosphorylation and the kinase activity. The fMLP-caused
autophosphorylation and kinase activity as determined by densitometric
analysis were 2.5 times higher in the burn animals than in the sham
group (Fig. 1).
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Effect of PTK inhibitor AG556 on PMN O
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Effect of PTK inhibitor AG556 on PMN intracellular
Ca2+ mobilization.
Figure 4A shows digitized
Ca2+ images from a representative sham, burn, and
burn-treated rat, and Fig. 4B demonstrates digitized fluorescence ratios (F340/F380)
representing the intracellular Ca2+
concentration ([Ca2+]i) obtained in
neutrophils. The frequency of cells showing pseudocolors of blue and
green, representing unstimulated resting cells, seemed to be comparable
in the sham, burn, and AG556-treated burn animals. Upon stimulation
with fMLP, a significant number of sham rat neutrophils transformed
into cells with pseudocolors in the yellow and red range, corresponding
to an image ratio around 1.3. The fMLP stimulation of burn rat
neutrophils caused a transformation into the pseudocolor red and white
range, corresponding to image ratios >2. Imaging of individual
neutrophils confirmed that fMLP-induced elevations in the burn group
were markedly higher than for cells in the sham group. Figure
4B shows the elevation in the digitized fluorescence ratios
as a function of time following fMLP stimulation of neutrophils from
sham and burn rats with and without AG556 treatment. The Ca2+ image analyses did not show a measurable effect of
treatment with AG556 on the basal [Ca2+]i or
fMLP-mediated [Ca2+]i elevation in
the sham animals. These analyses in burn rat neutrophils also did not
show any effect of AG556 treatment on fMLP-mediated [Ca2+]i responses.
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Effect of Ca2+ channel blocker on
burn-induced Lyn activation.
To determine the contributions of intracellular Ca2+
mobilization in burn-induced upregulation of Lyn, we treated the
animals in vivo with the Ca2+ channel blocker diltiazem and
determined Lyn activation in PMNs. The dose of diltiazem was chosen as
reported previously to block Ca2+ mobilization in the
neutrophil (35). The results indicated that Lyn
autophosphorylation as well as enolase phosphorylation by Lyn in
neutrophils from diltiazem-pretreated burn rats (day 1 postburn) was unaffected (Fig. 5).
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Role of Ca2+ channel blocker in
burn-induced PMN O
Phosphorylation/activation of p42/44 MAPK in burn-induced PMNs.
One of the early events of fMLP signal transduction in neutrophils is
the phosphorylation of intracellular proteins, including ERK1/2 MAPK
protein. The activation of ERK protein follows dual phosphorylation of
discrete threonine and tyrosine residues. We investigated activation of
p42/44 MAPK by fMLP via Western blot analyses of whole cell extracts
with antibodies to dually phosphorylated (Thr202/Tyr204) p42/44 MAPK (8).
PMNs were stimulated with 1 µM fMLP for 5 min, and whole cell lysates
were subjected to SDS-PAGE and immunoblotting. Similar aliquots of PMN
samples taken from different experimental groups were probed for p42/44
kinase to examine differential phosphorylation patterns. No dual
phosphorylation was detected in the PMNs without stimulation with fMLP
(data not shown). Phosphorylation of ERK1/2 (Fig.
6A) was significantly
(P < 0.05) elevated on day 1 postburn neutrophils compared with phosphorylation of this protein
in neutrophils from sham animals. Identical aliquots from the same PMNs
employed above were probed with a monoclonal antibody to p42/44 MAPK.
No differences in the amounts of immunoreactivity were visualized among
any of the samples (data not shown).
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Attenuation of burn-induced phosphorylation/activation of the p42/44 MAPK by AG1478. To determine whether p42/44 block administered to animals specifically affected the burn-induced activation of the MAPK, we pretreated animals with these agents before performing the sham and burn procedures. PMNs from sham and burn animals with and without MAPK blocker treatments were stimulated with fMLP. The treatment of sham animals with AG1478 (ERK blocker) did not effect ERK1/2 expression in PMNs from sham animals. However, AG1478 treatments of burn animals significantly downregulated ERK1/2 activation in burn animal PMNs (Fig. 6A).
Effect of PTK inhibitor AG556 on burn-induced phosphorylation/activation of the p42/44 MAPK. To determine whether blockade of a signal upstream to MAPK, i.e., a tyrosine kinase (3), could cause the inhibition of MAPK ERK1/2, we treated the animals with PTK blocker AG556 and assayed PMNs for the expression of ERK1/2 activity. We found that the inhibitor of upstream tyrosine kinase signaling, AG556, blocked burn-induced elevations of ERK1/2 activity (Fig. 6B).
Prevention of burn-induced enhancement in PMN O
Effects of Ca2+ channel blocker on
burn-induced phosphorylation of p42/44 MAPK.
To investigate the role of blocking intracellular Ca2+
release on burn-induced activation of neutrophil MAPK, we treated the animals with the Ca2+ channel blocker diltiazem.
Phosphorylation of p42/44 MAPK with fMLP stimulation of PMNs was
unaffected by diltiazem pretreatment of the burn animals (Fig.
7). These results suggest that
phosphorylation/activation of ERK MAPK was independent of intracellular
Ca2+ mobilization after burn injury.
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Effect of PAF-16 antagonist on Lyn.
Figure 8 shows immunoblot and blot
densitometric analyses of the Lyn autophosphorylation of neutrophils as
well as the ability of Lyn to phosphorylate other proteins (kinase
activity) as evaluated by examination of phosphorylation of enolase.
There was no detectable Lyn autophosphorylation or its kinase activity
in unstimulated neutrophils from sham or burn animal groups (data not
shown). The stimulation of neutrophils from day 1 postburn
or sham rats with fMLP caused significant elevations in both Lyn
autophosphorylation and the kinase activity. The fMLP stimulation of
both autophosphorylation and kinase activity was five times higher in
the burn animals than in the sham group as determined by densitometric
analysis (Fig. 8). The pretreatment of rats with PAF-16 antagonist
(0.35 mg/kg) had no significant effect on Lyn autophosphorylation by neutrophils in the sham animal group. However, Lyn autophosphorylation as well as enolase phosphorylation activity in neutrophils from PAF-16
antagonist-pretreated burn rats (day 1 postburn) was
significantly (P < 0.05) lower than that in the
untreated day 1 postburn rat neutrophils. The
autophosphorylation and kinase activities in the PAF-16
antagonist-pretreated day 1 postburn rats were comparable to
those in the neutrophils from sham groups.
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Prevention of burn-induced enhancement in PMN O
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DISCUSSION |
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The present study demonstrates an enhancement in
O
A contribution of our study is that PTK and ERK1/2 blockers, which have hitherto been utilized primarily as agents efficient in blocking signaling pathways in cells under in vitro conditions, can be administered to animals to produce selective signaling blockade in vivo. Such administrations of blockers to produce cell signaling modulations in vivo could modulate various, and perhaps all, cell systems in the body. Although this possibility cannot be ruled out, it is reasonable to postulate that blockers would exert inhibitory effects only on cell systems in which the targeted signaling pathways would be selectively activated under inflammatory conditions such as prevailing after burns. There is evidence that PMNs are among the cell systems in which MAPKs (ERK and p38) are activated during early stages after burn injury (2).
We found that both Lyn autophosphorylation and enolase phosphorylation
by Lyn with burn injury was blocked completely by the PTK blocker
AG556. This finding indicated a maximal inhibitory effect on this Src
kinase pathway activated during burn injury. Because we also found that
PMN O
Our findings that Lyn protein kinase is essentially involved in
fMLP-induced upregulation of O and
GM-CSF (17). Whether PTK activation occurs in a
Gi-dependent or -independent manner, it does seem to
proceed via Ca2+-independent signaling pathway.
The upregulation of PMN O or GM-CSF (17). Activations of PMNs could be
achieved via mediators such as fMLP, C5a, leukotriene B4,
and/or PAF (33) subsequent to priming of TNF-
or
GM-CSF. PAF action on PMN proceeding through its seven-transmembrane
domain receptor could involve both the turn-on of the Ca2+
pathway (5) and the tyrosine phosphorylation of cellular
proteins (10) such as phosphatidylinositol 3-kinase
(PI3K), MAPKs, and phospholipases A2, C, and D, all of
which play a role in the O
In summary, our studies demonstrate that Ca2+-independent
activations of PTK-Lyn and MAPK-ERK1/2 play an important role in the enhanced O
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Shafeeq Khan, Mehdi Khan, Farah Haque, and Amit Goyal for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants GM-53235 and GM-56865 (to M. M. Sayeed) and R21-AA-12901-01A1 (to M. A. Choudhry).
Address for reprint requests and other correspondence: M. M. Sayeed, Dept. of Surgery, Loyola Univ. Chicago Medical Center, 2160 South First Ave., Maywood, IL 60153 (E-mail: msayeed{at}lumc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 20, 2002;10.1152/ajpcell.00114.2002
Received 12 March 2002; accepted in final form 11 June 2002.
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