1 Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, School of Medicine, University of California, Davis, California 95616; and 2 National Institute for Food and Nutrition Research, Rome, Italy
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ABSTRACT |
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Cigarette smoking is known to contribute to inflammatory diseases of the respiratory tract by promoting recruitment of inflammatory-immune cells such as neutrophils and perhaps by altering neutrophil functional properties. We investigated whether acrolein, a toxic unsaturated aldehyde found in cigarette smoke, could directly affect neutrophil function. Exposure of freshly isolated human neutrophils to acrolein markedly inhibited spontaneous neutrophil apoptosis as indicated by loss of membrane asymmetry and DNA fragmentation and induced increased neutrophil production of the chemokine interleukin-8 (IL-8). Acrolein (1-50 µM) was found to induce marked activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs), and inhibition of p38 MAPK activation by SB-203580 prevented acrolein-induced IL-8 release. However, inhibition of either ERK or p38 MAPK did not affect acrolein-dependent inhibition of apoptosis. Acrolein exposure prevented the activation of caspase-3, a crucial step in the execution of neutrophil apoptosis, presumably by direct inhibition of the enzyme. Our results indicate that acrolein may contribute to smoke-induced inflammatory processes in the lung by increasing neutrophil recruitment and reducing neutrophil clearance by apoptosis.
aldehyde; inflammation; interleukin-8; caspase-3
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INTRODUCTION |
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CIGARETTE SMOKING is a major causative factor in the development of inflammatory lung diseases such as chronic bronchitis and emphysema (22, 42). In addition, secondhand or environmental tobacco smoke exposure, especially in children, is associated with a number of respiratory problems, including an increased risk of severe asthma (52). Although numerous chemical components of cigarette smoke (CS) have the potential to contribute to the development of lung disease, reactive aldehydes such as acrolein appear to play an important role in CS lung toxicity and in the effects of CS on inflammatory-immune processes (16, 21, 23, 27). Acrolein (CH2=CH---CHO) is a volatile, highly toxic unsaturated aldehyde found at up to 90 ppm in mainstream CS (25). In addition to being a component of both indoor and outdoor air pollution (25, 31), acrolein can also be formed endogenously during lipid peroxidation (46), neutrophil myeloperoxidase-catalyzed amino acid oxidation (2), and metabolism of the anticancer drug cyclophosphamide (31).
One line of evidence suggesting that aldehydes play an important role in CS lung toxicity is the fact that many cellular effects of CS can be replicated using aldehydes alone. For example, inhibitory effects of CS on fibroblast function, which may interfere with normal lung repair processes, are also caused by acrolein or acetaldehyde (6). Furthermore, either CS, acetaldehyde, or acrolein can induce tracheal epithelial cells to produce the neutrophil chemoattractant interleukin-8 (IL-8) (36), thus promoting respiratory tract inflammatory processes. Several studies have reported effects of acrolein on alveolar macrophage function, including inhibition of phagocytosis (32), increased rates of cell death, and altered cytokine production (30). Furthermore, acrolein and crotonaldehyde as well as filtered CS can inhibit the respiratory burst production of superoxide in rat alveolar macrophages and in human neutrophils (37, 53). However, the exact contribution of acrolein to inflammatory processes in vivo is still unclear.
Although they are necessary components of the host defense system, neutrophil-derived oxidants and proteases are also able to contribute to host tissue injury and are believed to play a harmful role in smoking-associated lung diseases (39, 42, 51). Neutrophil apoptosis is a key step in the resolution of inflammation (10, 13, 17, 45), and proinflammatory cytokines such as IL-8 and granulocyte-macrophage-colony stimulating factor (GM-CSF) can regulate such apoptotic pathways (1, 5, 9, 26, 29). Environmental factors such as CS-related aldehydes may affect such regulatory mechanisms and thereby alter ongoing inflammatory processes. The possibility that acrolein affects respiratory inflammation through direct modulatory effects on extravasated neutrophils has to date received little attention.
We hypothesized that acrolein can directly alter neutrophil functional properties and thus influence the outcome of lung inflammation by affecting critical signal transduction pathways in neutrophils, such as mitogen-activated protein kinase (MAPK) pathways. Stimulation of the extracellular signal-regulated kinase (ERK; also known as the p42/p44 MAPK) and/or the p38 MAPK is known to be involved in many functional responses in neutrophils, including IL-8 production (34), respiratory burst activation (28, 38), and regulation of constitutive (spontaneous) apoptosis (1, 14). We investigated the effects of CS-derived acrolein on neutrophil cytokine production and overall neutrophil lifespan in relation to changes in specific signal transduction pathways and observed that exposure of human neutrophils to acrolein inhibits spontaneous apoptosis and induces neutrophil production of the proinflammatory cytokine IL-8. Such alterations in the normal regulation of neutrophil function by acrolein may play some role in the development of chronic respiratory infections and inflammatory diseases that are associated with cigarette smoking and exposure to air pollution.
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METHODS |
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Neutrophil isolation. Human neutrophils were isolated from freshly drawn heparinized venous blood from healthy nonsmoking volunteers, as described previously (8). Briefly, blood cells were centrifuged over a Ficoll-Hypaque solution (LSM, ICN Biomedicals, Aurora, OH), and the major cell pellet containing granulocytes and erythrocytes was mixed with an equal volume of 5% dextran (average molecular weight ~450,000; Sigma, St. Louis, MO) to allow sedimentation of erythrocytes. The upper layer was collected and washed, the remaining erythrocytes were removed by hypotonic lysis, the remaining granulocytes were counted, viability was assessed by trypan blue exclusion, and the granulocytes were resuspended in a Krebs-Ringer buffer with glucose [KRBG; (in mM): 137 NaCl, 4.9 KCl, 0.5 CaCl2, 1.2 MgSO4, 5.7 sodium phosphate, and 5.5 glucose, pH 7.4]. This procedure consistently resulted in neutrophil suspension with >95% viability and at least 95% purity.
Acrolein treatment and sample preparation. Neutrophils were suspended at a density of 2 × 106 cells in 1 ml of KRBG in Eppendorf tubes. After a brief preincubation at 37°C, acrolein (Aldrich Chemical, Milwaukee, WI) was added at a final concentration of 1-50 µM (equivalent to 0.5-25 fmol/cell). The acrolein doses used are based on reported concentrations in mainstream or secondhand smoke [100 nmol/puff and up to 20 nmol/l, respectively (11, 31)], which upon inhalation could result in micromolar levels at the airway surface. To study specific signaling pathways, the following pharmacological inhibitors were added 10-15 min before acrolein treatment: SB-203580 (Calbiochem, La Jolla, CA), PD-90859 (Calbiochem), and diphenyleneiodonium chloride (DPI; Sigma). Cells were incubated in KRBG for 30 min after acrolein treatment, after which the cells were pelleted by centrifugation (10 min at 250 g) and resuspended in serum-free RPMI 1640 cell culture medium (Life Technologies, Gaithersburg, MD) without acrolein or pharmacological inhibitors. Given its reactivity and volatility, virtually all of the added acrolein can be expected to be depleted during the 30-min incubation, e.g., by reaction with cellular GSH, especially at low concentrations (23). After further incubation for 4-12 h at 37°C, cells were collected by centrifugation for determination of apoptosis.
For SDS-PAGE and IL-8 secretion experiments, neutrophils were placed in six-well tissue culture plates at a density of 4 × 106 cells in 2 ml KRBG/well. After acrolein addition, the cells were incubated for 20 min at 37°C; the buffer was aspirated; the adherent neutrophils were collected in 100 µl of RIPA lysis buffer (250 mM NaCl, 1.5 mM MgCl2, 50 mM HEPES, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 10% glycerol, 1% Triton X-100, and 10 µg/ml of aprotinin and leupeptin), incubated for 30 min at 4°C for optimal cell lysis, and centrifuged (10 min at 14,000 g) to remove insoluble debris; and the supernatants were frozen until used. When neutrophils were incubated for longer time periods, the incubation buffer was replaced with serum-free RPMI 1640 medium 30 min after addition of acrolein. Incubation media were collected at various time points and were stored atMeasurements of neutrophil apoptosis. An early phenomenon of apoptosis is loss of membrane asymmetry and exposure of phosphatidylserine on outer cell membranes, which can be detected by cell labeling with annexin V (49). The assay was performed using an annexin V-FITC apoptosis detection kit (PharMingen, San Diego, CA) according to the manufacturer's directions. Briefly, cells were washed in cold PBS and resuspended in assay buffer, and a 100-µl aliquot was withdrawn for analysis. After addition of annexin V-FITC (which labels exposed phosphatidylserine) and the nuclear stain propidium iodide (which stains permeable cells), the sample was incubated 15 min in the dark and then analyzed on a Becton Dickinson (San Jose, CA) FACScan flow cytometer.
Neutrophil apoptosis was also determined by characteristic fragmentation of DNA, which was isolated and treated as described by Herrmann et al. (19). Neutrophils treated and incubated in 2-ml Eppendorf tubes as described above were pelleted by centrifugation, washed with PBS, and resuspended in 125 µl of 10 mM Tris and 1 mM EDTA, pH 8.0 (TE buffer). An equal volume of lysis buffer (5 mM Tris, 20 mM EDTA, and 0.5% Triton X-100, pH 8.0) was added, and the tubes were incubated for 30 min at 4°C, after which the samples were centrifuged for 15 min at 14,000 g to precipitate intact chromatin and other cellular material. The supernatant was retained, and DNA was precipitated by addition of 1 ml of cold ethanol and 50 µl of 5 M NaCl. After an overnight incubation atDetermination of lactate dehydrogenase release. As a general measure of neutrophil viability, we determined the release of lactate dehydrogenase (LDH) in the medium at various time points after acrolein addition. After collection of the incubation medium, neutrophils were lysed by addition of 0.5% Triton X-100 in PBS. One milliliter of 5% BSA in PBS was added as a stabilizer to both medium and lysate fractions, and samples were stored at 4°C and analyzed within 48 h. LDH activity in lysate and medium was measured spectrophotometrically using a Sigma in vitro toxicology assay kit, and results are expressed as the percentage of the total LDH activity released in the medium.
Formation of neutrophil IL-8. Neutrophil IL-8 secretion was measured in medium at various times after exposure to acrolein using ELISA (Biosource, Camarillo, CA) and quantitated using standards of authentic IL-8 and are expressed as picograms per 106 cells.
SDS-PAGE and Western blotting. Cell lysates were mixed with 6× reducing sample buffer (125 mM Tris · HCl, pH 6.8, 6% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.1% bromphenol blue), and equal amounts of cellular protein, determined according to Bradford (Bio-Rad, Hercules, CA), were loaded on 4-12% gradient Tris-glycine gels (Novex, San Diego, CA) for electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes and immunoblotted with antibodies against phosphotyrosine (clone PY20; Upstate Biotechnology, Lake Placid, NY), p38, phospho-p38, ERK1/2, phospho-ERK1/2 (New England Biolabs, Beverly, MA), c-Jun NH2-terminal kinase (JNK) 2, and phospho-JNK (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies were detected using horseradish peroxidase-linked secondary antibodies (Sigma) and were visualized with enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Analysis of cellular GSH. Cellular levels of GSH were measured by analysis of cell lysates by HPLC with fluorescence detection after precolumn thiol derivatization with monobromobimane (Calbiochem, San Diego, CA), as described previously (48).
Caspase-3 activity assay. Cellular caspase-3 was measured in neutrophil lysates based on cleavage of a synthetic caspase-3 substrate (Ac-DEVD-pNA; Calbiochem). Neutrophils were collected by centrifugation and lysed in RIPA buffer, and lysates were separated into aliquots in a half-volume 96-well plate and incubated for 15 min at 37°C. After addition of the caspase-3 substrate, absorbance increase was monitored at 405 nm, and the rate of absorbance increase was compared with standard incubations with recombinant caspase-3 (Calbiochem).
Statistical analysis. Results are shown as representative experiments or as means ± SD of two to six experiments. Statistical comparisons were made using the two-tailed Student's t-test, and differences were considered significant at P < 0.05.
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RESULTS |
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Acrolein inhibits spontaneous neutrophil apoptosis.
Flow cytometry analysis of annexin V-FITC- and propidium iodide-stained
neutrophils demonstrated that treatment of neutrophils with 10 µM
acrolein reduced the percentage of early apoptotic cells after
4 h incubation from ~25 to <5%. Accordingly, the relative amounts of both live cells and necrotic (propidium iodide-positive) cells were both increased (Fig. 1).
Concentrations of acrolein below 5 µM did not significantly affect
spontaneous apoptosis, whereas higher acrolein concentrations
(up to 50 µM) inhibited apoptosis and resulted in increased
necrosis (Fig. 1C). Similar findings were observed after
longer incubation periods. After 12 h of incubation after acrolein
treatment, the percentage of apoptotic cells was reduced from
76.8% in untreated cells to 67.6 and 52.4% in neutrophils treated
with 5 and 10 µM acrolein, respectively (n = 2).
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Acrolein stimulates IL-8 production by neutrophils.
Because acrolein has previously been reported to augment IL-8 release
by tracheal epithelial cells (36), we investigated its
potential effects on neutrophil IL-8 production using ELISA. Indeed,
neutrophil exposure to 10 µM acrolein resulted in increased secretion
of IL-8 over a period of 4-8 h (Fig.
4).
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Acrolein activates MAPKs in neutrophils.
To explore the potential mechanisms by which acrolein affects
neutrophil apoptosis and cytokine production, we investigated the potential activation of MAPK signaling pathways. Indeed, exposure of freshly isolated human neutrophils to acrolein (10-50 µM)
resulted in a marked increase in tyrosine phosphorylation of several
proteins. Thus we studied the specific tyrosine kinase pathways that
might be activated. Activation of MAPKs such as p38 occurs by dual
phosphorylation on threonine and tyrosine (40) and can be
assessed by the use of antibodies specific for the dual-phosphorylated
form of the protein. On the basis of Western blotting with a
phospho-specific p38 antibody, p38 was found to be activated 20 min
after neutrophils were treated with >1 µM acrolein (Fig.
5A). This activation of p38
appears to persist for at least 4 h (data not shown). Similarly, addition of acrolein to neutrophils also resulted in activation of the
ERK MAPK, determined using a phospho-specific ERK antibody (Fig.
5B). We also investigated the potential activation of JNK using a phospho-specific JNK antibody but found no evidence for JNK
activation by acrolein at concentrations up to 50 µM. Thus acrolein
activated both ERK and p38 MAPK pathways in neutrophils but not JNK.
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Neutrophil apoptosis is not prevented by inhibition of NADPH oxidase. Because oxidants generated by the phagocyte NADPH oxidase have been implicated in neutrophil apoptosis (3, 20, 24), and acrolein is known to inhibit NADPH oxidase activation (37, 53), we used the NADPH oxidase inhibitor DPI to investigate the effect of oxidase inhibition on neutrophil apoptosis. The percentage of apoptotic cells after 4 h of incubation as measured by annexin V flow cytometry averaged 34.2 with no DPI, 33.4 with 10 µM DPI, and 33.3 with 50 µM DPI (n = 2), indicating that, under our conditions, NADPH oxidase inhibition did not block apoptosis.
Depletion of intracellular GSH by acrolein.
On the basis of the known reactivity of ,
-unsaturated aldehydes
such as acrolein with thiols (18, 35), it is likely that
acrolein mediates its effects primarily by depleting cellular GSH or by
modifying protein cysteine residues. Indeed, neutrophil exposure to
acrolein caused rapid and dramatic depletion of GSH as measured 20 min
after acrolein addition. Neutrophil GSH levels in untreated cells
averaged 14.2 ± 2.8 nmol/mg protein, and this was reduced to
9.7 ± 2.6 and 0.8 ± 1.1 by treatment with 1 and 10 µM
acrolein, respectively (n = 4).
Neutrophil caspase-3 activity is inhibited by acrolein.
Spontaneous apoptosis of neutrophils involves the activation of
the cysteine protease caspase-3 (12), and this caspase is susceptible to redox regulation because it contains a sensitive cysteine residue. Thus caspase activity might be affected indirectly by
acrolein-induced GSH depletion and changes in cellular redox status or
by direct alkylation of the active-site cysteine residue by acrolein.
Indeed, neutrophil exposure to 10 µM acrolein completely prevented
the gradual increase of neutrophil caspase activity over 8 h of
incubation (Fig. 6A).
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DISCUSSION |
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The major finding of the present study is that the
,
-unsaturated aldehyde acrolein is capable of markedly inhibiting
the constitutive apoptotic pathway in human neutrophils. In
addition, acrolein was found to activate MAPK pathways that mediate
many neutrophil functions and caused an increase in neutrophil IL-8 production. Previously, acrolein was also found to increase IL-8 production by tracheal epithelial cells (36); thus, it
appears plausible that acrolein can contribute to sustained and
exacerbated neutrophilic lung inflammation. When neutrophils were
treated with up to 10 µM acrolein, the inhibition of
apoptosis was accompanied by an increase in viability. Higher
acrolein doses also inhibited apoptosis but appeared to
decrease neutrophil viability while promoting necrotic cell death.
Because neutrophil apoptosis is a critical step in the
resolution of inflammation (10, 13, 17, 45), the
inhibition of neutrophil apoptosis by acrolein may greatly
increase the potential for host tissue injury by prolonging the
lifespan of neutrophils or by shifting the mode of cell death from
apoptosis to necrosis.
In contrast to our results, some investigators have reported increased apoptosis in response to cell exposure to acrolein. For example, acrolein causes apoptosis, measured by DNA fragmentation, in isolated human alveolar macrophages (30). In fact, we have observed that acrolein appears to stimulate apoptosis in the human lung epithelial cell line HBE1 (unpublished results). On the other hand, acrolein was found to be cytotoxic but not to increase apoptosis in several tumor cell lines (41). These inconsistent findings may be the result of differences in the apoptotic pathways in different cell types. Unlike most cell types, neutrophils are nonproliferating cells that are programmed to spontaneously undergo apoptosis within a short time after maturation (50). Also, neutrophil apoptosis may have some biochemical differences from apoptosis in other cells. Some of the proteins involved in the apoptotic pathway in many cell types, such as the commonly assayed caspase substrate poly(ADP-ribose) polymerase, are absent in neutrophils (43, 50).
The cellular effects of acrolein are believed to be due primarily to its ability to react rapidly with reduced thiols such as GSH (18, 25). In fact, of the low molecular weight aldehydes abundant in CS (acrolein, formaldehyde, acetaldehyde, and crotonaldehyde), acrolein is by far the most potent at depleting cellular GSH (35). Indeed, the acrolein-induced activation of MAPK, stimulation of IL-8 secretion, and inhibition of apoptosis appeared to coincide with the depletion of cellular GSH, which suggests that the effects of acrolein may be the result of redox changes of critical protein cysteine residues secondary to GSH depletion or to direct oxidation/alkylation of protein thiol residues that may occur along with or subsequent to the loss of GSH. In previous studies, depletion of GSH and other cellular thiols has been correlated with apoptosis in a number of cell types (4, 44); thus, it seems paradoxical that acrolein can both rapidly deplete GSH and inhibit apoptosis in neutrophils. However, the relationship between GSH depletion and apoptosis is not clear, and GSH depletion itself may not be sufficient to trigger apoptosis. In fact, it has been proposed that apoptosis involves an efflux of reduced GSH from the cell (15, 47); thus, depletion of cellular GSH may be a consequence rather than a cause of apoptosis.
There are several potential mechanisms by which acrolein might inhibit neutrophil apoptosis. First, acrolein increases the secretion of IL-8, which is known to delay neutrophil apoptosis (26, 29). However, we found that the p38 MAPK inhibitor SB-203580 blocked IL-8 release but did not affect apoptosis, suggesting that the inhibitory effects of acrolein on apoptosis are independent of IL-8 release. Second, reactive oxygen species generated by the phagocyte NADPH oxidase are known to promote neutrophil apoptosis (3, 20, 24). Thus the inhibitory action of acrolein on neutrophil apoptosis might be related to the ability of acrolein to inhibit neutrophil oxidant production (37, 53). However, we were not able to inhibit neutrophil apoptosis under our conditions with the NADPH oxidase inhibitor DPI, suggesting that inhibitory effects on oxidant production are not responsible for our findings. Third, acrolein might directly affect the activation of caspases such as caspase-3, critical events in the execution of neutrophil apoptosis (12). These enzymes are known to contain a nucleophilic active-site cysteine residue and can be regulated by oxidative or nitrosative stress via direct modification of the active site thiol (7, 33). Indeed, our results suggest that acrolein was capable of directly inhibiting caspase-3 activity, possibly by alkylation of its cysteine residue.
Needless to say, our model is quite simplistic compared with the in vivo situation during respiratory inflammation in which neutrophils are responding to an environment containing diverse cytokines, bacterial products, lipid mediators, and other stimuli. There may be important differences in the regulation of survival and apoptosis between peripheral blood neutrophils and extravasated neutrophils in the lung, since neutrophil adhesion and/or the action of local inflammatory mediators are generally believed to increase neutrophil lifespan by delaying constitutive apoptosis (1). However, our results indicate that acrolein, originating from either endogenous or exogenous sources, has the potential of altering inflammatory processes by activating specific neutrophil signaling pathways and changing neutrophil properties. In fact, it can be estimated that, during cigarette smoking or inhalation of environmental tobacco smoke, acrolein concentrations at the airway surface may be as high as 80 µM (11), similar to or higher than the concentration used in our experiments. Because persistent neutrophilic inflammation is a common feature of smoking-related lung diseases, it seems plausible that CS-derived aldehydes such as acrolein, by stimulating production of proinflammatory cytokines and by interfering with the normal apoptotic process in neutrophils, could contribute to amplified and more chronic inflammatory processes. Further investigation is clearly warranted to examine whether such modulating properties of acrolein or similar reactive aldehydes indeed contribute to the development of lung inflammation in vivo.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Tatsuya Okamoto for helpful suggestions and assistance in preparing the manuscript.
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FOOTNOTES |
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This work was supported by University of California Tobacco-Related Disease Research Program Grant 7RT-0167 and National Heart, Lung, and Blood Institute Grants HL-60812 and HL-07013.
Address for reprint requests and other correspondence: A. van der Vliet, CCRBM, Dept. of Internal Medicine, 1121 Surge I, Univ. of California, Davis, CA 95616 (E-mail: avandervliet{at}ucdavis.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.
Received 7 December 2000; accepted in final form 10 April 2001.
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