©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of 8-Epiprostaglandin F by Human Monocytes
DISCRIMINATE PRODUCTION BY REACTIVE OXYGEN SPECIES AND PROSTAGLANDIN ENDOPEROXIDE SYNTHASE-2 (*)

(Received for publication, November 28, 1995; and in revised form, January 30, 1996)

Domenico Praticó Garret A. FitzGerald (§)

From the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

F(2)-isoprostanes are free radical-catalyzed products of arachidonic acid. One of these compounds, 8-epiprostaglandin F (8-epi-PGF), is a mitogen and vasoconstrictor. We have shown that 8-epi-PGF, unlike other F(2)-isoprostanes, is a minor product of the prostaglandin endoperoxide synthase-1 (PG G/H S-1) expressed in human platelets (Praticó, D., Lawson, J. A., and FitzGerald, G. A.(1995) J. Biol. Chem. 270, 9800-9808). Human monocytes express PG G/H S-1 constitutively and exhibit regulated expression of PG G/H S-2. Induction of PG G/H S-2 by concanavalin A, the phorbol ester, phorbol 12-myristate 13-acetate, and bacterial lipopolysaccharide was confirmed with a specific antibody in monocytes pretreated with aspirin to inhibit PG G/H S-1. Induction of PG G/H S-2 by all three stimuli coincided with increased formation of prostaglandin E(2) (PGE(2)), thromboxane B(2) (TxB(2)), and 8-epi-PGF, but not of other F(2)-isoprostanes. Confirmation of PG G/H S-2 as the source of 8-epi-PGF formation was obtained by down-regulating the enzyme with dexamethasone; preventing protein synthesis with cycloheximide; and preventing synthesis of PGE(2), TxB(2), and 8-epi-PGF with the specific PG G/H S-2 inhibitor, L 745,337.

Monocytes also exhibit the facility to generate 8-epi-PGF in a free radical-dependent manner. Thus, stimulation with opsonized zymosan or coincubation with low density lipoprotein was unassociated with product formation. However, coincubation of low density lipoprotein with zymosan-stimulated human monocytes resulted in marked formation of 8-epi-PGF, but not of PGE(2) or TxB(2). Production of 8-epi-PGF coincided with that of thiobarbituric acid-reactive substances and lipid hydroperoxides, but was unaccompanied by PG G/H S-2 induction. Pretreatment of monocytes with the antioxidant, butylated hydroxytoluene or with superoxide dismutase, but not with L 745,337, suppressed formation of 8-epi-PGF, thiobarbituric acid-reactive substances, and lipid hydroperoxides.

In conclusion, human monocytes may form bioactive 8-epi-PGF either via free radical- or enzyme-catalyzed pathways. 8-Epi-PGF is a more abundant product of monocyte PG G/H S-2 than of platelet PG G/H S-1. Formation by inducible PG G/H S-2 must be considered as a source of this compound in vivo.


INTRODUCTION

Monocytes are thought to play a central role in atherogenesis(1) . They adhere to and transmigrate between endothelial cells(2) . Monocyte-derived macrophages have high affinity receptors for oxidized low density lipoprotein (LDL), (^1)and LDL ingestion results in their transformation into foam cells(3, 4) . Discharge of the contents of foam cells, themselves marked constituents of fatty streaks(5) , is thought to contribute to formation of atherosclerotic plaque(6) . Monocytes and macrophages exhibit the ability to transform arachidonic acid to prostaglandins and related compounds via the enzyme prostaglandin endoperoxide synthase (PG G/H S). There are two forms of this enzyme(7, 8) . Monocytes express PG G/H S-1 constitutively(9) , and PG G/H S-2 is up-regulated in response to cytokines, growth factors, and bacterial lipopolysaccharide (LPS) (9, 10, 11) . It is unknown what role, if any, prostanoids may play in atherogenesis, although they have been implicated in regulating cellular proliferation(12, 13) , vascular tone and permeability(14, 15) , and aspects of cholesterol metabolism(16) . Additional to its susceptibility to enzyme-catalyzed metabolism to biologically active compounds, arachidonic acid may be subject to free radical attack, leading to formation of prostaglandin isomers in situ in the cell membrane phospholipid(17, 18) . These isoprostanes may exert biological effects intra- or extracellularly. A prostaglandin F(2) isomer, 8-epi-PGF, induces vasoconstriction and cellular proliferation, effects that are prevented by thromboxane A(2) receptor antagonists(19, 20) . Recently, we have shown that this compound is also a minor product of PG G/H S-1 in human platelets(21) .

This study demonstrates that PG G/H S-2 may also form 8-epi-PGF. Interestingly, it is a more abundant product of this enzyme in monocytes than of the PG G/H S-1 isoform in platelets. Monocytes also retain the ability to form this compound in a free radical-catalyzed manner. Indeed, coincubation of monocytes with LDL results in a time-dependent formation of 8-epi-PGF, coincident with LDL oxidation.


EXPERIMENTAL PROCEDURES

Materials

LPS derived from Escherichia coli 026:B6, dexamethasone, cycloheximide, aspirin, zymosan, concanavalin A, phorbol 12-myristate 13-acetate (PMA), superoxide dismutase, butylated hydroxytoluene (BHT), leupeptin, Nonidet P-40, soybean trypsin inhibitor, and aprotinin were purchased from Sigma. Hanks' balanced salt solution, Dulbecco's phosphate-buffered saline, RPMI 1640 medium, fetal calf serum, L-glutamine, penicillin, and streptomycin were purchased from Life Technologies, Inc. [^4H(2)]TxB(2) and [^4H(2)]PGE(2) were obtained from Cayman Chemical Co., Inc. (Ann Arbor, MI). The internal standard used for 8-epi-PGF was the ^18O(2)-labeled compound derived from authentic 8-epi-PGF (Cayman Chemical Co., Inc.) using the technique described by Pickett and Murphy(22) . L 745,337 was kindly donated by Dr. Ian W. Rodger (Merck Frosst Canada Inc., Dorval, Quebec, Canada). Mouse polyclonal antibodies against PG G/H S-1 were a gift from Dr. W. L. Smith (Michigan State University). Mouse polyclonal antibodies prepared against the sequence of the carboxyl terminus (amino acids 580-598) of human PG G/H S-2 (COOH-NASSSRSGLDDINPTVLLK) were obtained as described previously (23) and kindly provided by Dr. J. Maclouf (INSERM, Unité 348, Paris, France).

Isolation and Stimulation of Human Monocytes

Mononuclear cells were obtained from fresh peripheral blood of healthy volunteers, who did not take any medication during the previous 2 weeks. Blood was subjected to Ficoll-Hypaque density gradient centrifugation as described by Boyum(24) . The mononuclear cell layer was recovered and washed with Hanks' balanced salt solution. The cells were resuspended in RPMI 1640 medium containing 10% inactivated fetal calf serum and EDTA for 15 min at 37 °C to remove platelets specifically adherent to the monocytes. They were washed twice in Hanks' balanced salt solution and resuspended at 1 times 10^6 ml in RPMI 1640 tissue culture medium supplemented with L-glutamine (220 mg/l) and antibiotics (streptomycin and penicillin). More than 94% of the cells were estimated to be viable based on trypan blue dye exclusion.

The cells were plated in 24-well multiplates and maintained at 37 °C in a temperature-controlled, humidified 95% air, 5% CO(2) incubator. The nonadherent cells were removed by washing the plates twice with Dulbecco's phosphate-buffered saline after 2 h of incubation, and the adherent cells were maintained in RPMI 1640 medium enriched with 5% inactivated fetal calf serum, L-glutamine, and antibiotics. The resultant harvested adherent cells routinely contained >92% monocytes as determined by morphology and staining for nonspecific esterase(25) . The contribution of PG G/H S-1 activity to eicosanoid production in response to the different stimuli was suppressed by pretreating the blood with aspirin (10 µg/ml) in the sampling syringe and at time 0. This was confirmed by product analysis. Isolated monocytes were incubated for 1, 4, 8, and 24 h in the absence and presence of LPS (10 µg/ml), concanavalin A (10 µg/ml), or PMA (100 nM). Fresh medium containing 10 µM arachidonic acid was added at each time point after removing the incubation medium; the supernatants were harvested after 30 min.

Concentrations of PGE(2), TxB(2), and 8-epi-PGF were determined by gas chromatography/mass spectrometry (GC/MS) as described previously(21, 26) . The contribution of induced PG G/H S-2 to formation of these products was elucidated by use of a protein synthesis inhibitor, cycloheximide(27) ; a specific inhibitor of this isoform of the enzyme, L 745,337(28) ; or dexamethasone, which has previously been shown to down-regulate PG G/H S-2(29) .

GC/MS Analysis of PGE(2), TxB(2), and 8-Epi-PGF

Briefly, a 30-m DB-1 capillary column was used for analysis of all products. The temperature program was 190-320 °C at 20 °C/min. The ions monitored were m/z 614 for TxB(2) and m/z 618 for [^2H(4)]TxB(2), m/z 624 for PGE(2) and m/z 628 for [^2H(4)]PGE(2), and m/z 695 for 8-epi-PGF and m/z 699 for 8-epi-[^18O(2)]PGF.

Lipoprotein Preparation

LDL was prepared according to previously described methods that minimize oxidation and exposure to endotoxin(30) . Each batch of LDL was assayed for endotoxin contamination by the Limulus amebocyte lysate assay. Final endotoxin contamination was always <0.02 unit/mg of LDL cholesterol. LDL was stored in 0.5 mM EDTA. Immediately before use, LDL was dialyzed at 4 °C against phosphate-buffered saline without calcium or magnesium in the dark. LDL was used at final concentration of 0.40 mg of protein/ml.

LDL Incubation with Human Monocytes

Human monocytes were washed with RPMI 1640 medium without serum, plated into 12-well tissue culture plates (1 times 10^6/ml/well), and co-cultured for 24 h with LDL at 0.40 mg of protein/ml in the presence or absence of opsonized zymosan (2 mg/ml) according to the method of Johnston(31) . LDL was extracted by adding 2 volumes of Folch reagent (chloroform and methanol at a 2:1 ratio) and base-hydrolyzed (1.0 M KOH) before quantitation as described above to determine the total 8-epi-PGF level in the supernatant.

Measurement of Lipid Peroxidation

Briefly, the presence of lipid oxidation products on LDL was detected spectrophotometrically by measuring the thiobarbituric acid-reactive substance (TBARS) levels monitored at 532 nm(32) . The lipid hydroperoxide levels were measured using the FOX 2 assay at 560 nm(33) .

Western Blot Analysis

Isolated human monocytes were lysed in ice-cold buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Nonidet P-40, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml aprotinin). The protein content was determined using a microbicinchoninic acid assay (Pierce) with bovine serum albumin as a standard. Twenty micrograms of total cell protein were analyzed by SDS-polyacrylamide gel electrophoresis. Acrylamide (8 and 4%) was used for the separating and staking gels, respectively. The resolved proteins were transferred onto nitrocellulose membranes. Blots were saturated overnight at 4 °C with a solution of 5% fat-free dried milk in Tris-buffered saline and incubated with either mouse polyclonal anti-PG G/H S-2 antibody (1 µg/ml) or specific polyclonal anti-PG G/H S-1 antibody for 2 h at room temperature. The membranes were then washed extensively with phosphate-buffered saline/Tween 20 and incubated with horseradish peroxidase-conjugated anti-mouse IgG at 1:2000 for 1 h at room temperature. Chemiluminescence substrates were used to reveal positive bands according to the manufacturer's instructions, and bands were visualized after exposure to Hyperfilm ECL (Amersham Corp.).

Statistical Analysis

Results are expressed as mean ± S.D. Statistical comparisons were made by using analysis of variance with subsequent application of Student's t test as appropriate.


RESULTS

PG G/H S-2-dependent Formation of 8-Epi-PGF

Incubation of human monocytes with 10 µg/ml LPS resulted in a time-dependent increase of prostaglandin production (Fig. 1). LPS did not significantly affect eicosanoid production at 1 h, but caused a statistically significant increase at 4, 8, and 24 h of incubation. Maximal stimulation of 8-epi-PGF, PGE(2), and TxB(2) at 24 h of incubation was in the concentration ratio of 1:5.3:200. To evaluate whether LPS-induced production of these compounds was dependent on induction of PG G/H S-2, we studied the effects of dexamethasone (2 µM) and cycloheximide (5 µg/ml). Both compounds markedly suppressed the production of the three prostaglandins (Table 1), coincident with suppression of the immunoreactive 72-kDa doublet recognized by the monoclonal anti-PG G/H S-2 antibody on Western blot analysis (Fig. 2). The band recognized by the anti-PG G/H S-1 antibody was unaltered by LPS, cycloheximide, or dexamethasone (data not shown). Confirmation that product formation was dependent on PG G/H S-2 induction was indicated by the specific inhibition of that enzyme. L 745,337 dose-dependently suppressed PGE(2), 8-epi-PGF, and TxB(2) with the same IC value (48 ± 10 nM). Unlike cycloheximide and dexamethasone, the effects of L 745,337 were not accompanied by down-regulation of PG G/H S-2 protein (data not shown). We observed a selective increase in a peak at m/z 695, comigrating with the 8-epi-PGF internal standard (m/z 699). It was unaccompanied by other peaks, presumably reflecting other F(2)-isoprostanes, and it was suppressed by cycloheximide (Fig. 3). These data are consistent with enzyme- rather than free radical-catalyzed formation of 8-epi-PGF under these conditions.


Figure 1: Time course of eicosanoid formation in human monocytes stimulated with LPS (10 µg/ml). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from four experiments.






Figure 2: Western blot analysis of PG G/H S-2 protein expression in monocytes treated with LPS (10 µg/ml). Isolated human monocytes (1 times 10^6/ml) were incubated with saline for 24 h (control (c)); with LPS alone for 1, 4, 8, and 24 h; with LPS + dexamethasone (D) for 24 h; and with LPS + cycloheximide (C) for 24 h. The figure is representative of three experiments.




Figure 3: Selected ion monitoring of 8-epi-PGF. The upper panel shows a peak at m/z 699 corresponding to authentic ^18O(2)-labeled internal standard. The center panel shows a peak (m/z 695) observed during LPS-stimulated human monocytes, corresponding to the retention time of authentic 8-epi-PGF. No other isoprostanes were present. The lower panel shows the suppression of that peak (m/z 695) when the sample was treated with cycloheximide.



Similar results were obtained with concanavalin A (10 µg/ml) and PMA (100 nM). Both compounds caused a time-dependent increase on 8-epi-PGF, PGE(2), and TxB(2). The respective ratios of product formed at 24 h of incubation were 1:5.3:160 for concanavalin A (Fig. 4) and 1:5:130 for PMA (Fig. 5). Again, both concanavalin A (Fig. 6A) and PMA (Fig. 6B) caused induction of PG G/H S-2 protein, which was suppressed, together with product formation, by cycloheximide and dexamethasone ( Table 2and Table 3). L 745,337 suppressed formation of concanavalin A (IC = 40 ± 6 nM), and PMA (IC = 35 ± 8 nM) stimulated formation of 8-epi-PGF, along with that of the other eicosanoids. The induced PG G/H S-2 protein was again unaffected by L 745,337.


Figure 4: Time course of eicosanoid formation in human monocytes stimulated with concanavalin A (10 µg/ml). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from five experiments.




Figure 5: Time course of eicosanoid formation in human monocytes stimulated with PMA (100 nM). The supernatant was assayed for prostanoid production by GC/MS at the indicated times. Values are reported as mean ± S.D. from four experiments.




Figure 6: Western blot analyses of PG G/H S-2 protein expression in monocytes treated with concanavalin A (10 µg/ml) or PMA (100 nM). A, isolated human monocytes were incubated with saline for 24 h (control (c)); with concanavalin A alone for 1, 4, 8, and 24 h; with concanavalin A + dexamethasone (D) for 24 h; and with concanavalin A + cycloheximide (C) for 24 h. This figure is representative of four experiments. B, monocytes were incubated with saline for 24 h (control (c)); with PMA alone for 1, 4, 8, and 24 h; with PMA + dexamethasone (D) for 24 h; and with PMA + cycloheximide (C) for 24 h. The figure is representative of three different experiments.







Free Radical-catalyzed Formation of 8-Epi-PGF by Human Monocytes

Activation of human monocytes with opsonized zymosan contrasted with the other stimuli in that it did not result in formation of 8-epi-PGF, PGE(2), or TxB(2). Similarly, coincubation of monocytes with LDL, a source of substrate for lipid peroxidation(30) , did not induce release of any of these products into the supernatant. Moreover, no significant changes in the levels of TBARS or lipid hydroperoxides were observed in human monocytes incubated with LDL in the absence of zymosan. This is in agreement with previous observations that activation is required for monocytes to oxidize LDL(34) .

Activation of human monocytes with opsonized zymosan in the presence of LDL caused a marked time-dependent increase in 8-epi-PGF, but not in PGE(2) or TxB(2). The increase in 8-epi-PGF was associated with an increase in TBARS and hydroperoxide levels (Table 4). This phenomenon was prevented by the oxygen free radical scavengers superoxide dismutase (300 units/ml) and BHT (20 µM), but not by L 745,337 (100 nM) (Fig. 7) or by the nonselective inhibitor of PG G/H S, aspirin (data not shown). The quantities of 8-epi-PGF formed under these conditions, in the presence of an excess of lipid (LDL) substrate, are not comparable with those formed in isolated stimulated human monocytes in the earlier experiments. In contrast to the data presented in Fig. 3, an array of peaks corresponding to F(2)-isoprostanes was evident in the supernatants of zymosan-stimulated human monocytes incubated with LDL (Fig. 8, center panel). One of these corresponds to the retention time of the 8-epi-PGF standard (m/z 699) (Fig. 8, upper panel). Pretreatment with antioxidants suppressed the peaks, reflecting free radical-catalyzed isoprostane formation under these experimental conditions (Fig. 8, lower panel). The retention time of authentic PGF is well away from that of the F(2)-isoprostanes. Finally, coincubation of zymosan-activated human monocytes with LDL did not result in induction of PG G/H S-2 protein (data not shown).




Figure 7: Formation of 8-epi-PGF during cell-mediated LDL oxidation. LDL (400 µg of protein/ml) was incubated with human monocytes stimulated with opsonized zymosan for 3 and 24 h. LDL was also incubated with zymosan-treated monocytes for 24 h in the presence of superoxide dismutase (SOD; 300 units/ml), BHT (20 µM), or L 745,337 (100 nM). Values are reported as mean ± S.D. from four experiments.




Figure 8: Selected ion monitoring of 8-epi-PGF. The upper panel shows a peak at m/z 699 corresponding to authentic ^18O(2)-labeled internal standard. The center panel shows a peak (m/z 695) corresponding to the retention time of authentic 8-epi-PGF. The array of peaks seen in the chromatogram most likely corresponds to F(2)-isoprostanes. The lower panel shows the reduction of the peaks (m/z 695) when the sample was incubated with butylated hydroxytoluene supporting the free radical-catalyzed origin of them.




DISCUSSION

8-Epi-PGF is an abundant isoprostane formed in response to free radical attack on arachidonic acid(17) . Urinary excretion of 8-epi-PGF is increased in syndromes putatively associated with increased oxidant stress in vivo, including paracetamol and paraquat poisoning(35) , cigarette smoking (36) , and vascular reperfusion(37) . We have previously shown that it is a minor product of the PG G/H S-1 expressed in human platelets(21) . However, this route of formation appears to contribute trivially to overall 8-epi-PGF biosynthesis, even in a setting such as chronic cigarette smoking(36) , in which platelet activation (38) is thought to coincide with increased oxidant stress.

Human monocytes contain PG G/H S-2 additional to the isoform expressed in platelets. This form of the enzyme is highly regulated by cytokines, LPS, hormones, and mitogenic factors(9, 10, 11) . It is thought to be the source of prostanoid production in inflammatory states (39) and, perhaps, in cancer(40) . The primary sequences of both human enzymes exhibit 60% similarity(8, 41) ; however, mutational analysis suggests that the active site of PG G/H S-2 is the more accommodating. For example, although aspirin is a relatively nondiscriminate PG G/H S inhibitor(42, 43) , inhibition of PG G/H S-2, but not of PG G/H S-1, by aspirin is associated with increased production of (15R)-hydroxyeicosatetraenoic acid, coincident with inhibition of prostanoid formation(44) . The present studies demonstrate that 8-epi-PGF may be formed in a PG G/H S-2-dependent manner. Using three independent stimuli, formation coincided in time with the kinetics of the de novo synthesis of PG G/H S-2. Furthermore, reduction in activity of the induced enzyme, by either cycloheximide or steroids, prevented 8-epi-PGF formation coincident with that of the conventional PG G/H S-2 products of these cells, PGE(2) and thromboxane A(2). A selective PG G/H S-2 inhibitor suppressed generation of all three products without preventing synthesis of the enzyme. PG G/H S-1 was not induced during stimulation of 8-epi-PGF generation in these experiments; thus, the contribution of the two enzymes to its production is clearly segregated.

Unlike the case with platelet PG G/H S-1, 8-epi-PGF is formed in greater abundance relative to the conventional products of PG G/H S-2 in monocytes. Thus, whereas maximal 8-epi-PGF formation in platelets is roughly that of TxB(2), maximal formation by PG G/H S-2 in monocytes is and of the corresponding production of PGE(2) and TxB(2), respectively, the most abundant conventional products of PG G/H S-2 in these cells. This observation raises the possibility that the mitogenic properties of 8-epi-PGF(20) may contribute to the role of PG G/H S-2 activation in syndromes of vascular proliferation(45, 46) . Selective inhibitors of this enzyme might be used in clarifying the utility of urinary 8-epi-PGF as an index of free radical generation in syndromes putatively associated with oxidant stress, in which PG G/H S-2 induction is possible. Although enzyme-catalyzed formation seems to be a feature of 8-epi-PGF, but not of other F(2)-isoprostanes, similar caution might be applied to estimates of ``F(2)-isoprostanes''(47) , of which 8-epi-PGF is an abundant constituent(48) .

Monocytes are the first example of cells in which both enzyme- and free radical-catalyzed formation of 8-epi-PGF have been demonstrated. Activation of monocytes with zymosan, in contrast to LPS, concanavalin A, or PMA, fails to induce PG G/H S-2 expression or 8-epi-PGF generation. However, when coincubation with LDL affords the availability of an abundant lipid substrate, zymosan-activated monocytes catalyze the formation of a substantial amount of 8-epi-PGF. This occurs coincident with production of TBARS and hydroperoxides, both indices of lipid oxidation, but not with either induction of PG G/H S-2 or generation of either PGE(2) or TxB(2). Under these circumstances, selective PG G/H S-2 inhibitors are ineffective in preventing 8-epi-PGF formation. Rather, antioxidants, such as superoxide dismutase or BHT, inhibit its production. Incubation of human monocytes with LDL in the absence of zymosan activation fails to stimulate 8-epi-PGF production.

Previous work has demonstrated that copper-induced oxidation of LDL (49) is associated with increased 8-epi-PGF generation. Whether it or indeed other arachidonic acid products contribute to the role of monocytes and their cellular derivatives in the process of atherogenesis remains to be determined.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Experimental Therapeutics, 901 Stellar-Chance Labs., University of Pennsylvania, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-6446; Fax: 215-573-9004; garret{at}spirit.gcrc.upenn.edu.

(^1)
The abbreviations used are: LDL, low density lipoprotein; PG G/H S-1 and S-2, prostaglandin endoperoxide synthase-1 and -2, respectively; LPS, lipopolysaccharide; 8-epi-PGF, 8-epiprostaglandin F; PMA, phorbol 12-myristate 13-acetate; BHT, butylated hydroxytoluene; PGE(2), prostaglandin E(2); TxB(2), thromboxane B(2); GC/MS, gas chromatography/mass spectrometry; TBARS, thiobarbituric acid-reactive substance(s).


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