suppression of aortic atherosclerosis in cholesterol...

Suppression of Aortic Atherosclerosis inCholesterol-fed Rabbits by Purified Rabbit Interferon

Alan C. Wilson, Robert G. Schaub, Randee C. Goldstein, and Peter T. Kuo

The effectiveness of rabbit Interferon In suppressing atherosclerosis was evalu-ated In rabbits fed a diet containing 1% cholesterol. Ten male New Zealand Whiterabbits received Intramuscular Injections of 1 million units of Interferon twice a week,while a control group of 10 rabbits received Injections of buffer. Both groups hadaverage serum cholesterol levels of over 2000 mg/dl during the 8-week experimentalperiod. Interferon treatment resulted In no significant hypolipidemlc effect orchanges In llpoproteln composition. Atherosclerotic lesions In aortas were quantifiedboth macroscopically and microscopically. Interferon treatment decreased thegrossly visible lesion area significantly from 25±4% to 8 ± 1 % (mean±SEM, p<0.005)compared to the untreated group. Microscopic analysis of serial cross-sections ofaortic segments revealed significant (p<0.01) reductions In both lesion size andfrequency in the Interferon-treated group. Electron microscopy also showed thatInterferon treatment reduced the pathological effects of cholesterol feeding. Tissueanalysis showed that total aortic cholesterol was reduced by 28% by Interferontreatment, while the aortic phosphollpid concentration was Increased by 25%. Thepossibility exists that the interferon preparation used contained other biologicalresponse modifiers and that the observed effects may be totally unrelated withInterferon. These results suggest that the mechanism of atherosclerosis suppressionIn these cholesterol-fed rabbits Is not related to the lowering of serum cholesterol butmay be associated with Inhibition of lesion Initiation.(Arteriosclerosis 10:208-214, March/April 1990)

A therosclerotic lesions develop as the result of theinterplay between the multiple risk factors associ-

ated with Increased incidence of atherosclerosis, the cellsof the arterial wall, and the circulating blood cells.1 In-creased understanding of these interactions is critical todeveloping new approaches to prevention and therapy.

Since the discovery of interferon as an antiviral agent in1957, interest has grown in its nonantiviral activities,2

particularly in its use in cancer therapy.3 Interferonsexhibit a wide range of effects, each depending on thetype of interferon and the nature and state of differentia-tion of the target cell. Interferons are involved in theinhibition of cell growth and proliferation, regulation of theexpression of specific genes, modulation of cell differen-tiation, and activation of certain cell types in the immunesystem, for example, macrophages and natural killercells.4

The Interferon inducers, polyinoslnic-polycytidylicacid and 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone(U-54,461), have been shown to suppress atherosclero-sis in rabbits.5 It is important to establish whether the

From the University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School, New Brunswick, NewJersey, and the Atherosclerosis/Thrombosis Untt, The UpjohnCompany, Kalamazoo, Michigan.

This work was supported in part by grante from the RobertWood Johnson Medical School and The Upjohn Company.

Address for reprints: Alan C. Wilson, Ph.D., Division of Cardio-vascular Diseases and Hypertension, Department of Medicine,UMDNJ-Robert Wood Johnson Medical School, New Bruns-wick, NJ 08903.

Received October 28,1988; revision accepted October 20,1989.

anti-atherogenic effect is a direct effect of these drugs oris mediated by interferon. The purpose of the presentstudy was to evaluate the anti-atherogenic effectivenessof the administration of purified rabbit interferon in thecholesterol-fed rabbit model. The results presented inthis report support the findings of the inducer studies andmay suggest novel approaches to the pharmacology ofatherosclerosis control.

MethodsInterferon

Rabbit interferon (lot 83016, titer 1.5x106 IRU/ml, spe-cific activity 1.3x107 IRU/mg) purified from RK13 rabbitkidney cultures stimulated with para-influenza-1 virus wasobtained from LEE BioMolecular Research Laboratories,San Diego, CA, The titer was assayed by dye uptake assayand normalized to International reference units (IRU)against a National Institutes of Health rabbit interferonstandard.8 The interferon titer was confirmed by indepen-dent assay at The Upjohn Company. Because of the cellculture source, there is a possibility of the presence ofother biological response modifiers in the material.

Interferon NomenclatureUnlike the mouse and human interferons, which are

designated alpha or beta based on various physical prop-erties, no information is yet available concerning the subdi-vision of rabbit interferon into alpha or beta components.

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INTERFERON AND ATHEROSCLEROSIS Wilson et al. 209

Animals and DietAfter being assigned into two hypercholesterolemic-

response matched groups, 20 New Zealand White malerabbits (weight, 3 to 4 kg) were fed an atherogenic diet7

(1% cholesterol, 4% peanut oil supplied by ICN Nutri-tional Biochemicals, Cleveland, OH) for 8 weeks. Eachrabbit in the interferon-treated group received injectionsof 1X108 IRU of rabbit interferon twice a week intramus-cularly in alternate rear legs. This route of administrationwas chosen because interferon is rapidly cleared afterintravenous injection (t!£=13 minutes), while fairly stablelevels of serum interferon are maintained for at least12 hours after intramuscular injection.8 The rabbits in theuntreated cholesterol-fed group were handled similarlyexcept that the injections consisted of sterile 0.01 Mphosphate buffer, pH 7.2. A third group of 10 rabbits wasmaintained on regular chow as normal controls. Thestudies involving experimental animals were In accord-ance with institutional guidelines.

Experimental Procedure

Body weight and food consumption were recordedtwice weekly. At 4 and 8 weeks, the animals were fastedfor 16 hours, and blood samples for blood chemistry,interferon, and lipoprotein analysis were withdrawn fromthe marginal ear veins into plastic tubes on ice and wereallowed to clot; the serum was collected. The blood forplatelet studies was collected into plastic tubes contain-ing citrate (final concentration, 3.8%). Upoproteins wereseparated from serum by sequential preparative ultracen-trtfugatlon after adjustment of solvent densities to 1.006,1.019, and 1.063 g/ml with KBr,9 and the cholesterol andtriglyceride contents of serum and lipoprotein fractionswere determined by the methods described in the UpldResearch Clinics Program Manual.10

Postmortem Studies

After sacrifice, samples of liver, spleen, kidney, andadrenal tissue were taken for pathological examination.The whole aorta from the aortic valve to the iliac bifurca-tion was removed intact from each animal and wasopened longitudinally along the anterior side. The adven-titJa and adhering adipose tissue were removed, and thewet weight of the aorta was determined. Samples (3 mmdiameter) for light and electron microscopic examinationwere punched out from identical sites in the aortic arch(distal to the origin of the coeliac trunk) and the thoracicaorta (distal to the fifth intercostal arteries) and were fixedIn 2% glyceraldehyde in Tyrode's buffer. The remainderof the aorta was fixed in 10% buffered formaldehyde, andthe lipid-rich lesions on the surface of the aorta werestained with 1% Sudan IV.11 After tracing on an overlaidclear plastic sheet, the aortic surface area and its lipid-staining lesions were quantitated by an electronic graph-ics calculator (Model 1224, Numonics, Lansdale, PA).The punch samples were freeze-sectioned and stainedfor neutral lipid with oil red O. A Zeiss Videoplan imageanalysis unit was used for computer-assisted morpho-metric planimetery of lesions in 137 of these serialfreeze-sections. The stained lesion area in each section

Table 1. Effect of Interferon Treatment on SerumUpld and Upoproteln Concentrations In Rabbits Fed aCholesterol Diet

Treatment group

Serum cholesterol(at start of study)

Serum cholesterol(after 8 weeks)

Triglyceride(after 8 weeks)

VLDL cholesterol(d< 1.006)

IDL cholesterol(d=1.006-1.019)

LDL cholesterol(d=1.019-1.063)

HDL cholesterol(d> 1.063)

Buffer(mg/dl)

67±9

2259±232

295±158

1450±169

461 ±94

222±61

46±6

Interferon

63±4

2164±309

286±99

1328±222

442±43

196±55

41±11

The values are means±SE. n=10 for total cholesterol andtriglyceride, n=7 for ultracentrtfugal separations.

HDL=hlgh density lipoprotein, IDL=lntermedlate density lipo-protein, LDL=low density lipoprotein, VLDL=very low densitylipoprotein.

Purified rabbit Interferon (1x10* IRU) was Injected intramus-cularly twice a week for 8 weeks.

was expressed as a percent of the total vessel sectionarea (intima+media). Tissue sampling and macroscopicand microscopic area analyses were performed withoutknowledge of prior treatment to avoid subjective bias.

The aortas were dried by lyophilization and werestored at -20°C until analysis. Total lipid extracts12 wereprepared by cutting each aorta into small pieces andhomogenizing three times in methanol/chloroform/water(2:1:0.8, vol/vol/vol) in a tapered glass-glass homoge-nizer. Determinations of total, esterified, and free choles-terol and phospholipids were performed in duplicate afterthin-layer chromatography of the total lipid extracts. Analy-sis of DNA content was performed in duplicate on thedefatted, dry tissue by the method of Burton.13

Statistical Analysis

The statistical significance of between-group differ-ences was tested by one-way analysis of variance fol-lowed by Duncan's multiple range test by using theStatistical Analysis Systems computer program.14

Results

Serum LJplds

The serum of both groups of rabbits became milkywithin 4 weeks, and total serum cholesterol increasedfrom baseline levels of about 65 mg/dl to over 2000 mg/dland remained at this level throughout the experiment(Table 1). Serum triglyceride concentrations after8 weeks were in the range of 300 mg/dl in both thebuffer-treated and the Interferon-treated groups.

Ultracentrtfugal fractJonatJon revealed that the hyper-cholesterolemic response to the atherogenic diet waspredominantly in the density d<1.006 g/ml lipoprotein


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210 ARTERIOSCLEROSIS VOL 10, No 2, MARCH/APRIL 1990

Table 2. Effect of Interferon Treatment on Visible Upld Staining Lesions and Aorta CompositionIn Cholesterol-fed Rabbits

Aorta parameter

Grossly visible(aortic lesion area %)

Total C (mg/g, dry wt)

C ester (mg/g, dry wt)

PL (mg/g, dry wt)

DNA (mg/g, dry wt)

Dry weight (g)

Buffer

25.3±4.1

10.40±1.73

3.66±1.65

1.80±0.16

0.19±0.03

0.21 ±0.05

Treatment group

Interferon

7.6±1.3*

7.44±0.97

2.84±0.96

2.26±0.19

0.18±0.02

0.27±0.09

Normal

_

1.77±0.92

0.12±0.04—

—

0.14±0.03

The results are means±SEM, n«=10 in each group.Whole aortas from root of arch to iliac bifurcation were analyzed individually in duplicate as described In the

Methods section.C-cholesterol, DNA=deoxyribonuclelc add, PL-phosphollpld.*p<0.005, analysis of variance compared to buffer group.

fraction (Table 1), reflecting the accumulation of beta-VLDL18 Although there was slightly more cholesterol inall lipoprotein fractions from the buffer-treated group,interferon administration did not result in any significantreduction of the increased cholesterol concentration inany of the beta-lipoprotein fractions. There also were nosignificant differences in the mean food consumption andweight gain of the interferon-treated group compared tothe untreated cholesterol-fed group of rabbits.

Macroscopic and Chemical FindingsIn rabbits fed the atherogenic diet without interferon

treatment, grossly visible lipid-staining lesions covered amean 25.3% (range 12% to 43%) of the total aorticsurface (Table 2), mostly affecting the aortic arch. Inter-feron treatment significantly decreased the lipid stainingsurface affected by lesions to a mean of 7.6% (range 3%to 25%, p<0.005) compared to the untreated atheroscle-rotic group. Aortic dry weight and DNA content were notsignificantly different in the untreated or interferon-treatedgroups. Treatment with interferon resulted in a 28%decrease in the contents of esterifled and total choles-terol and a 25% increase in phospholipid content.

Microscopic AnalysisExamination of serial freeze-sections of thoracic aorta

and aortic arch from untreated cholesterol-fed rabbitsshowed markedly raised intima with large proliferations oflipid-laden "foam" cells and some pericellular lipid in theintjmal-medial layers. In rabbits treated with interferon,the lesions consisted of fewer layers of intimal foam cellsovertying an otherwise normal-looking arterial wall.

Statistical analysis indicated that interferon treatmentsignificantly reduced the amount of visible lipid accumu-lation (Table 3). In the aortic arch, a significant reductionin lesion area in the aortic wall from 24.5±4.2% to12.9±1.6% was found (p<0.01). In the thoracic aorta(which was less severely atherosclerotic than the arch), asmaller decrease in the lipid accumulation in the wall wasobserved from 6.9±1.5% to 5.1 ±1.4%. Pooling the datafrom both areas, the overall 46% reduction in lipid accu-mulation from 15.7+2.5% to 8.7±1.2% was found to besignificant (p<0.01) compared to the untreated group.

Table 3. Morphometric Analysis of MicroscopicUpld Accumulation In Serial Cross-sections of AorticArch and Thoracic Aorta Wall from Cholesterol-fedRabbits Treated with Interferon

Total lipid staining lesion area

Aortic section Buffer Interferon (%)

Arch (n=66) 24.5±4.2 12.9±1.6*

Thoracic (n=71) 6.9±1.5 5.1 ±1.4

Arch+thoraclc (n=137) 15.7±2.5 8.7±1.2*

The values are means±SE. n=number of serial freeze sec-tions of aorta examined.

Lesion area Is expressed as the sum of areas of the Individuallipid-staining lesions in each microscopic section x 10O/total sec-tion area.

*p<0.01, analysis of variance compared to buffer group.

IFN

Area (<*b)Figure 1. Frequency histogram of lesion (llpkj stain) size deter-mined as a percent of the section area by using a Zelss Vldeoplanimage analysis unit In 137 serial microscopic cross-sections ofartery wall In aortic arch (n=66) and thoracic aorta (n=71) fromInterferon-treated and buffer-treated cholesterol-fed rabbits.

A frequency plot (Figure 1) of the pooled data shows thatin interferon-treated rabbits there were significantly fewerlarge lesions (those covering more than 20% of the totalsection area) than in untreated rabbits. The largest lesions


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C DFigure 2. Transmission electron micrographs of lesion areas from interferon-treated (A and B) and untreated cholesterol-fed rabbits(C and D). Many of the foam cells In the Interferon-treated group appear to be In the early stage of development, with smalllipid-containlng vacuoles (arrows). Many of the cells are of smooth muscle cell origin (S). The foam cells In the untreated group are morecomplex, containing larger numbers of electron-translucent vacuoles (arrows), which distend the cells and increase their size. Morefoam cells of macrophage origin (M) are found in addition to those of smooth muscle cell origin (S). The origin of some foam cells Inthe untreated group could not be identified. Bars-2/im.

found In interferon-treated rabbits covered only 35% of triearea, while the largest lesions found in aortic sections fromuntreated rabbits covered over 70% of the section area

Transmission Electron MicroscopyExamination of electron micrographs from treated and

untreated rabbits confirmed and extended the observa-tions made by light microscopy.

In the interferon-treated rabbits (Figures 2A and 2B) lesslipid accumulation was apparent within the cells of thelesions, with smaller lipid-contalning vacuoles (arrows),and in some areas there was almost no lipid accumulation.Many of the cells were of smooth muscle origin (S) andappeared to be more "normal" in both their morphologyand size compared to the nontreated rabbits.

In the untreated animals, generally, the smooth musclecells and macrophages of the lesion had taken up largequantities of lipid (Figures 2C and 2D). Because of theamount of lipid that was deposited in some of the cells, itwas difficult to determine many of the important charac-teristic criteria of the cells, and morphologic identificationof the cell precursor was difficult These cells were alsolarger than normal intimal cells. A typical foam cell

(Figure 2D) in an intimal lesion from an untreated,cholesterol-fed rabbit had the features of a macrophage(M). There was no basal lamina; it had a regularly shapednucleus with peripherally clumped chromatin prominentGolgi apparatus and many lipid droplets.

Pathology of Other Organs andSerum Chemistry

Postmortem histologic studies of liver, kidney, spleen,and adrenals of each rabbit were performed to comparethe effect of Interferon administration. The livers of bothtreated and untreated groups of rabbits showed a rangeof mild to marked fatty changes, with little or no bilestasis, and with early signs of flbrosis and necrosis. Thereappeared to be no correlation between interferon-treatment and the severity of the hyperiipidemic changes.All the sections of kidney were normal. The spleensexhibited a few foamy histiocytes, again with no correla-tion with the interferon treatment. The adrenals of theuntreated cholesterol-fed rabbits showed necrosis andmany cholesterol clefts, while the interferon-treatedgroup appeared to be normally lipid-laden, with no cho-lesterol clefts and only mild necrosis. No significant effect


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of interferon treatment was detected by serum chemistrydeterminations of liver enzymes serum aminotrans-ferases (SGOT and SGPT), alkaline phosphatase, andgamma-glutamyttransferase.

Platelet ActivityPlatelets from the interferon-treated and untreated

cholesterol-fed rabbits were tested for sensitivity to ag-gregation in response to both arachidonic acid andcollagen. No effect of the interferon treatment could bedetected. Rabbit interferon added In vitro directly to themeasuring cuvettes had no effect on platelet aggrega-tion. In addition, washed platelets from hypercholester-olemic rabbits showed no differences in aggregabilitycompared to platelets from normolipidemic rabbits.

DiscussionA previous report5 described the inhibition of atherogen-

esis by interferon inducers in chdesterot-fed rabbits, but themechanism of action was unclear since no serumcholesterol-lowering effect was found. The irrterferon induc-ers might have acted by inducing interferon production atthe lesion site or at a distant site. Alternatively, the effectmight have been a direct one with no involvement ofinterferon at all. To clarify the mode of action, the aim of thisstudy was to determine whether the suppression could bereproduced by treatment with purified rabbit interferon.

Interferon treatment was found to decrease the devel-opment of grossly visible atherosclerotic plaques by anaverage of 70% In cholesterol-fed rabbits. This finding wassupported by results of morphometric analysis of serialsections of aortic wall, which showed a 47% decrease inlipid staining lesion area and a significant shift in the lesionsize-frequency distribution. These results indicate thatintramuscular administration of purified rabbit interferonwas effective in achieving substantial and significant sup-pression of atheromatous lesion formation in the aortas ofcholesterol-fed rabbits. The protection did not appear toarise from a hypolipidemic action, and the precise mech-anism of action remains unclear. There is a possibility thatthe interferon preparation used contained other biologicalresponse modifiers and that the observed effects may betotally unrelated with interferon. It is of interest that otherclasses of antJatherogenic agents, the calcium and cal-modulin antagonists, also accomplish their effect withoutlowering serum cholesterol levels.1617 The followingmechanisms may explain the observed results.

Mechanism of UpoprotelnsThe relationship between interferon treatment and llpo-

protein metabolism has been examined in healthy menby Cantell et al.18 and Enholm et al.19 Daily subcutaneousinjections of leukocyte interferon (3x10° IRU) for 1 weekdecreased the level of high density lipoproteln choles-terol, very low density plus low density lipoprotein cho-lesterol, and apolipoprotein A-l by about 15%. Simultane-ously the activity of both postheparin plasma lipoproteinlipase and hepatic lipase decreased by 40%. In patientsbeing treated for various carcinomas, Dixon et al.20 andMassaro et al.21 found similar effects with alpha-

interferon. With beta-interferon, the same authors re-ported a dose-related decrease in low density lipoproteinbut not high density lipoprotein, and an increase in verylow density lipoprotein.22

In the present study, interferon treatment for 8 weeksproduced no significant changes in the beta-iipoproteinfractions. A transient significant decrease in high densitylipoprotein cholesterol was detected in serum samplesdrawn at 4 weeks (data not shown). The relevance of thisdecrease to the present antJatherogenic effect is unclear,since decreased levels of high density lipoprotein havebeen associated with an increased risk of coronary heartdisease.23 While small differences were seen, it is unlikelythat cholesterol-lowering is responsible for the suppressionof atherosclerosis because serum cholesterol levels inexcess of 2000 mg/dl were found in both groups of rabbits.

Mechanism of Arterial Wall CellsCurrent models of atherogenesis implicate many factors

that modify arterial wall cells and circulating blood cells. Inthese various models, smooth muscle cells, endothelialcells, platelets, monocytes and their derived macrophagesall interact with one another and with serum lipoproteins.Interferon may affect one or more of these interactionsdirectly so as to modify cholesterol accumulation. It hasbeen suggested that gamma-interferon is responsible forsmooth muscle expression of la antigens during the re-sponse to arterial injury in rats.24 Studies in vitro withHeLa-S3 cells have shown that beta-interferon stimulatescholesterol and phospholipid synthesis but inhibits cho-lesterol ester synthesis, possibly by inhibition of low den-sity lipoprotein endocytosis.25 The same workers havedescribed the Inhibition of concanavalin A-stmulated cal-cium flux by beta-interferon.26 Alternatively, the influenceof interferon may be indirect, perhaps being mediatedthrough the action of prostanoids, since a distinct interre-lationship apparently exists between interferon and pros-taglandin metabolism.2726

Mechanism of Monocyte MacrophagesIn the cholesterol-fed rabbit, hypercholesterolemia is

accompanied by rapid development of arterial fattystreaks with foam cells of smooth muscle cell ormonocyte-macrophage origin.2930 In the earty stages ofthe formation of atheromatous lesions, macrophagesderived from circulating blood monocytes appear to actas scavenger cells, taking up /3-VLDL from the areas oflesion formation during hyperlipidemia, while their failureto do so results In the accumulation of lipid at the lesion.As the concentration of blood lipids and lipoproteins wasnot changed, the infiltration of lipoprotein cholesterolester must remain the same. The possibility, then, is thatinterferon increases the removal processes and/or mod-ifies the composition of aortic cell membranes to allow theaccommodation of part of the excess cholesterol ester.The capacity for cholesterol efflux from monocyte-derivedmacrophages from cholesterol-fed rabbits has beenshown to be significantly greater than that found inmacrophages from normocholesterolemic rabbits.31 Inaddition, it has been reported that interferon and polyan-ionic interferon inducers activate macrophages to in-


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creased phagocytosis and cholesterol ester accumula-tion.323334 Tamm et al.36 have summarized the effects ofinterferon on the cell membrane, endocytosis, and matrixprotein synthesis.35

The data presented here indicate that intramuscularinjection of rabbit interferon was effective in significantlyreducing the visible aortic atherosclerosis induced byfeeding rabbits a diet containing cholesterol and peanutoil. In this short-term study in rabbits response-matchedto cholesterol feeding, atherosclerosis suppression wasjudged objectively by "blinded" observers from the re-duction in macroscopic lesion area on the surface andthe decrease in area and size of microscopically analyzedlesions in the artery wall.

These reductions in atherosclerosis were achievedwithout any significant lowering of serum cholesterol orchanges in lipoprotein cholesterol distribution, addingsupport to the conclusion that other approaches as wellas lowering serum cholesterol may be protective againstatherogenesis. This study further suggests that it may bepossible to pharmacologically influence vascular cells toreduce lipid accumulation in the face of substantial hy-peiiipidemia. It should be noted that the severe sideeffects of interferon therapy rule against its probableutility in prophylaxis of atherosclerosis. Substantiation ofthe observed results and further studies in long-termmodels of atherosclerosis, such as the Watanabe herita-ble hypertipidemic rabbit, perhaps utilizing interferoninducers or other agents, will be necessary before clinicalapplications can be envisioned.

AcknowledgmentsWe thank John Teller for coordinating the study and Wendell

Wlerenga (both from The Upjohn Company) for interferon analy-ses, Elliott Krauss for pathology of organ sections, and RaphaelSchumert and Jeff Grill for able assistance with necropsy andplanimetry.

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Index Terms: atherosclerosis • rabbits • interferon


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A C Wilson, R G Schaub, R C Goldstein and P T KuoSuppression of aortic atherosclerosis in cholesterol-fed rabbits by purified rabbit interferon.

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1990 American Heart Association, Inc. All rights reserved.

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