Vol. 14, No. 6 Printed in U.S.A.

INFECTION AND IMMUNITY, Dec. 1976, p. 1276-1283 Copyright ©O 1976 American Society for Microbiology

Microbicidal Mechanisms of Human Granulocytes: Synergistic Effects of Granulocyte Elastase and Myeloperoxidase or Chymotrypsin-Like Cationic Protein H. ODEBERG* AND I. OLSSON Department ofInternal Medicine, University ofLund, Lund, and Dalby Comrntunity Health Research Centre, Dalbv, Sweden

Received for publication 6 May 1976

The antibacterial activity of a myeloperoxidase (MPO)-glucose oxidase system was found to be greatly increased by granulocyte elastase, present in azurophil granules of human neutrophils. The MPO-H.,O.,-mediated killing of both Escherichia coli and Staphylococcus aureus was potentiated by granulocyte elastase at an acid pH, whereas at pH 7.4 only killing of E. coli was potentiated. The potentiating effect of elastase was not dependent on the enzymatic properties of the protein since it was not abolished by heating, which destroys the enzymatic activity. A peptide chloromethyl ketone elastase inhibitor abolished both the elastolytic activity and the potentiating effects on MPO-H.O.,-mediated bacterial killing. The antibacterial activity of chymotrypsin-like cationic protein of human neutrophils was also potentiated by elastase. Other degradative enzymes isolated from human granulocytes, e.g., collagenase and lysozyme, did not potentiate MPO-H.,O.,-mediated or cationic protein-dependent bacterial killing. The present study indicates that a neutrophil constituent, elastase, which is not microbicidal by itself, can initiate sublethal changes that render some microorganisms more susceptible to the action of microbicidal agents like MPO and chymotrypsin-like cationic protein. After ingestion into the phagocytic vacuole of granulocytes, microorganisms are exposed to several antimicrobial systems, both oxygen dependent and oxygen independent (18). Among the oxygen-dependent systems, the myeloperoxidase (MPO)-H..O., system is the most potent (17, 36, 37), but H.,O., itself (12), superoxide (6), hydroxyl radicals (16), and singlet oxygen (1) have been implicated in antimicrobial reactions. The oxygen-independent systems include lysozyme (5), lactoferrin (20), phospholipase (39), and granular cationic proteins (29, 40-42). Granular cationic proteins, which are abundant in some species, e.g., rabbits (42), have also been demonstrated in human granulocytes to consist of a number of related components (34) with bactericidal activity against a variety of microorganisms (29). These proteins have a chymotrypsin-like esterase activity (30), but the microbicidal effects are generally independent of the chymotrypsin-like activity since heating destroys enzymatic but not microbicidal activity. The contribution of the various antimicrobial systems to the overall bactericidal activity of granulocytes has not been clarified. The relevance of a defective oxidative metabolism is exemplified by chronic granuloma-

tous disease, in which the H.,O.2 formation is abolished (4), leading to severe infections. Some organisms are, however, also killed in chronic granulomatous disease leukocytes (35) as well as in leukocytes under anaerobic conditions (27). Although MPO is a highly potent microbicidal agent when combined with H.,O.. and a halide (19, 36), the genetic deficiency of this protein usually does not lead to excessive infections, indicating that alternative mechanisms operate that may be oxygen or non-oxygen dependent (17, 21). The present study was undertaken mainly to test the hypothesis that interaction of different agents in the phagocytic vacuole results in a potentiating of microbicidal effects. Investigators in our laboratory have succeeded in purifying several neutrophil granular constituents, e.g., MPO, (33), lysozyme, and chymotrypsinlike cationic protein (34), which were used in the present study. Included also are the granulocyte-degradative enzymes elastase (32) and collagenase (31), representing main constituents of human neutrophils but generally not considered as bactericidal agents. For the continuous generation of H.,O., glucose oxidase was used. The combined action of the various


VOL. 14, 1976


agents was studied at both neutral and acid pH, since the pH conditions prevailing in the phagocytic vacuole of human granulocytes were reported to be maintained at or near neutrality for significant periods of time after phagocytosis (26). MATERIALS AND METHODS Microorganisms. Staphylococcus aureus 502A and a clinical isolate from urine of Escherichia coli (O group 06) were used in the bactericidal assays. S. aureus was maintained on CCY medium (2) solidified with 1.5% agar (Difco), andE. coli was cultured on blood agar. Stock cultures are passed every 2nd and 4th weeks. Granulocyte constituents. MPO (33), elastase (32), collagenase (31), lysozyme, and chymotrypsinlike cationic protein (34) were purified from granules of human leukocytes as previously described. Glucose oxidase was purchased from Hughes and Hughes Ltd, Essex, England. The glucose oxidasecatalyzed production of H202 was assayed in the same medium used for bactericidal assays, using a Clark electrode to estimate 02 consumption. Bactericidal assays. The bactericidal assays were performed essentially as described previously (29). Bacteria were grown in antibiotic medium 3 (Difco) at 37°C overnight. To obtain bacteria in logarithmic growth phase, 0.1 to 0.2 ml of the overnight broth culture was transferred to 5 ml of fresh antibiotic medium 3 and incubated at 37°C. The optical density of the broth cultures was followed at 628 nm, and the organisms were harvested when an optical density of 0.5 to 0.6 was reached. After being washed twice in calcium-free Krebs-Ringer phosphate buffer (KRP), pH 7.4, containing 0.1% gelatin, the bacteria were added to the incubation medium. The incubation mixture consisted of about 10" bacteria, various concentrations of granulocyte constituents, 67 mM NaCl, 3.5 mM NaHCO3, 4.2 mM KCl, 1.0 mM CaCl2, 0.32 mM MgSO4, 0.39 mM MgCl2, 0.27 mM Na2HPO4, 0.35 mM KH2PO4, 4.4 mM glucose, 16 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.4, and 0.1% gelatin in 0.5 ml. When incubations were at pH 5.5, NaHCO3 and HEPES buffer were omitted and substituted for by 16 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.5. Incubations were in plastic tubes (12 by 75 mm; Falcon Plastics, Division of BioQuest, Oxnard, Calif.) during end-over-end rotation at the rate of 20 rpm at 37°C. For determination of the number of surviving bacteria by the pour plate method, aliquots of 20 to 200 ul were removed at various time intervals and diluted in 8 ml of KRP containing 0.1% gelatin. Samples were mixed with antibiotic medium 3 containing 1.5% agar, and the number of colony-forming units was determined after overnight incubation. Binding of elastase to bacteria. Incubations were essentially as described for the bactericidal assays. After 30 min the tubes were centrifuged in a swingout rotor at 3,200 x g for 30 min, and the uppermost 0.1 ml of the supernatant was removed for assay of elastase, using the single radial immunodiffusion


method of Mancini et al. (25) after addition of 0.3% (wt/vol) cetyltrimethylammonium bromide in 0.01 M phosphate buffer, pH 7.0. The centrifugation procedure cleared the supernatant of more than 99% of the bacteria, and the concentration of elastase determined thus represents the unbound protein. Inactivation of elastase. Elastase, 50 uM, and Ac-Ala-Ala-Pro-AlaCH2Cl (38), 500 ,uM, were incubated in KRP, pH 6.5, with 2% methanol for 60 min at 37°C. The incubation mixture was dialyzed once against 100 ml of pH 6.5 KRP and twice against pH 7.4 KRP before assay of its bactericidal activity. Elastase inactivation was also performed by heating at 90°C for 10 min in a sealed glass ampoule. Elastase activity was determined by using elastin agarose as previously described (32). Heated elastase showed no residual elastolytic activity, and the elastase inhibitor caused a more than 95% reduction of the elastolytic activity.


The effects on E. coli and S. aureus of increasing amounts of MPO and H.,O.,-generating glucose oxidase are compared in Fig. 1. Since a continuous generation of H202 is typical of phagocytizing granulocytes, it was preferred to the addition offixed amounts of H,O.,. The H202 production increased in a linear fashion with 40 100 0

0) 0.

ioj 4)



~bi Ql



OS 1


5 tO 20


1001 40 o



0. 1


0 w



di i0oi 2 5 10 205 0 GLUCOSE OXIDASE pg/mi FIG. 1. Microbicidal effect of the MPO-glucose oxidase system on S. aureus (above) and E. coli (below). Incubations were carried out for 60 min at pH 7.4 with various concentrations of glucose oxidase (abscissa) (0), together with 5 (I), 10 (II), 20 (11), 40 (IV), and 200 (V) ,Ag of MPO/ml (-). Ordinate: Surviving organisms as percentage of control.




increasing amounts of glucose oxidase in the actual range of enzyme concentrations used (results not shown). S. aureus was highly sensitive to H2G2 itself since the number of colonyforming units was extensively reduced with 0.5 to 1.0 ,ug of glucose oxidase per ml (generating about 10 nmol of H.,O.,/min) in the absence of MPO. E. coli, on the other hand, was rather insensitive to H2GO.; only a slight reduction of surviving bacteria was found with as much as 25 ,ug of glucose oxidase per ml (generating about 300 nmol of H.,O./min). S. aureus was also more sensitive than E. coli to the action of the MPO-glucose oxidase system. MPO in excess of 20 ,ug/ml gave a reasonable killing of E. coli, whereas S. aureus was killed with MPO in excess of 5 to 10 ,ug/ml. It must be emphasized that the microbicidal effect of MPO was dependent on the concentration of glucose oxidase used (Fig. 1). The rate of H.,O., production, as well as the MPO concentration, relevant to the local conditions in the phagocytic vacuole is difficult to calculate. Figure 1 demonstrates, however, that the MPO-mediated microbicidal activity was inhibited at a certain ratio between MPO and glucose oxidase, e.g., 20 ,ug of MPO and 0.25 gg of glucose oxidase per ml (corresponding to production of 4 nmol of H2G2/

min) for S. aureus and 40 ,ug of MPO and 4 ,ug of glucose oxidase per ml (corresponding to production of 50 nmol of H202/min) for E. coli. By increasing the MPO concentration and/or the glucose oxidase concentration, the inhibition was overcome. The microbicidal effect of the MPO-glucose oxidase system was higher at pH 5.5 than at pH 7.4 (Fig. 2 and 3). The kinetics of the bacterial killing by the MPO-glucose oxidase system are also demonstrated. The initial rate was low compared with the killing rate during the latter phase of incubation (Fig. 2 and 3). Preincubation with glucose oxidase before addition of MPO and organisms did not change the kinetics of killing. Substitution of glucose oxidase with H2G.2 (1 x 10-4 to 3 x 10-4 M) gave a linear relationship between the log number of surviving bacteria and time, but killing was much less efficient. By increasing the H.,O, concentration, the MPO-mediated bacterial killing was inhibited (results not shown). For studies on synergistic effects of neutrophil constituents, experimental conditions were designed in which suboptimal concentrations of various agents were combined. The addition of 20 ,ug of granulocyte elastase per ml highly potentiated the action on E. coli of the MPOglucose oxidase system at both pH 7.4 and pH

pH 5.5

107 10 18 -a;,


pH 7.4 0

q %



E 1-








4)d E




103 30







FIG. 2. Potentiating effect of granulocyte elastase on the microbicidal effect of the MPO-glucose oxidase system against E. coli as a function of time. Incubations were carried out at pH 7.4 (left) and pH 5.5 (right) with 1.25 (V), 2.5 (M), 10 (0), 20 (0), and 30 (A) mg of MPO/ml. The glucose oxidase concentration was 0.66 (pH 7.4) or 0.125 (pH 5.5) ,ug/ml. Unbroken lines show results from incubations with the MPO-glucose oxidase system and elastase (20 pAgl ml), and broken lines show incubations with elastase omitted. During the incubation, the number ofE. coli slightly increased in controls without MPO-glucose oxidase and elastase and with elastase alone. With glucose oxidase alone, the number of bacteria was



10@ L










FIG. 3. Potentiating effect of granulocyte elastase on the microbicidal effect of the MPO-glucose oxidase system against S. aureus as a function of time. Incubations were carried out at pH 7.4 (left) and 5.5 (right) with 5 (0),10 (0), and 20 (A) pg ofMPO/ml. The glucose oxidase concentration was 0.125 pAg/ml.

Unbroken lines show results from incubations with the MPO-glucose oxidase system and elastase (20 pgl ml), and broken lines show incubations with elastase omitted. During the incubation, the number of S. aureus did not change in control incubations without MPO-glucose oxidase and elastase, with elastase alone, or with glucose oxidase alone.


VOL. 14, 1976

5.5 (Fig. 2). Granulocyte elastase itself had no microbicidal effect. The potentiating effect of elastase increased the initial rate of killing, whereas the killing rate during the latter phase of incubation was not significantly changed. The microbicidal effect of the MPO-glucose oxidase system on S. aureus was also increased by elastase at pH 5.5 but not at pH 7.4. In fact, an inhibition of bacterial killing was caused by elastase at pH 7.4. The microbicidal effect of the chymotrypsinlike cationic protein was potentiated by the addition of granulocyte elastase (Fig. 4). The reduction of colony-forming units of S. aureus with 12.5 ,ug of cationic protein per ml at pH 7.4 was increased by the addition of 10 to 20 jig of granulocyte elastase per ml. Potentiation was not shown at pH 6.0, but the microbicidal activity of the cationic protein was low at this pH. Similar results were obtained for E. coli by





using 50 ,ug of cationic protein and 20 to 40 ,ug of elastase per ml (Fig. 4). The microbicidal effect at pH 7.4 and 5.5 on E. coli and S. aureus of the MPO-glucose oxidase system was unchanged by 20 ,tg of lysozyme or 20 ,ug of collagenase per ml (results not shown). Neither did lysozyme and collagenase potentiate the microbicidal effect at pH 7.4 of25 ,ug (S. aureus) and 50 ,ug (E. coli) of chymotrypsin-like cationic protein per ml. The combined microbicidal effects at pH 7.4 of the chymotrypsin-like cationic protein and MPO-glucose oxidase are shown in Fig. 5. Cationic protein at 50 ,ug/ml gave only a slight reduction of the number of colony-forming units of E. coli. Bacterial killing of the MPO-glucose oxidase system was not affected. S. aureus was more sensitive to the action of cationic protein thanE. coli, since 12.5 ,tg/ml gave a reasonable killing that was almost linear with time. With combination of the two microbicidal systems, most of the initial killing of S. aureus was caused by cationic protein, whereas MPO-glucose oxidase was most efficient during the latter part of incubation. Synergistic effects were not found either during the initial or during the latter part of incubation. Furthermore, experi-











5. Combined antibacterial effect against E. en~~~~~~~~ x coliFIG. and S. aureus of the MPO-glucose oxidase system 90 the chymotrypsin-like cationic protein. Incuba30 60 120 and tions were carried out at pH 7.4 with 10 (0), 20 (-),


FIG. 4. Potentiating effect of granulocyte elastase on the microbicidal effect of the chymotrypsin-like cationic protein against E. coli and S. aureus. Incubations were carried out at pH 7.4 with 50 (E. coli) and 12.5 (S. aureus) pg of cationic protein per ml (-). Unbroken lines show incubations with cationic protein and 10 (I), 20 (1), and 40 (III) pg of elastase per ml, and broken lines show incubations with elastase omitted. Results are also shown for controls in the absence of cationic protein and elastase (O --- 0) and with elastase alone (0-0).

and 30 (A) pg of MPO/ml and 0.66 (E. coli) and 0.125 (S. aureus) pg ofglucose oxidase per ml. Unbroken lines indicate incubations with the MPO-glucose oxidase system and 50 (E. coli) or 12.5 (S. aureus) pg of cationic protein per ml, and broken lines indicate incubations without cationic protein. (0) Incubations with cationic protein alone. The number of E. coli slightly increased and the number of S. aureus was unchanged in controls in the absence of MPO-glucose oxidase and cationic protein. With glucose oxidase alone, the number of both organisms was unchanged.



ments carried out at pH 5.5 did not show synergistic effects (results not shown). To determine whether the elastase-mediated potentiation of the MPO-glucose oxidase system is due to enzyme activity, heating of elastase (90°C for 10 min) and incubation with elastase inhibitor were carried out. Both procedures abolished the elastolytic activity. Inhibitor-treated elastase had no potentiating effect on the microbicidal activity of the MPO-glucose oxidase system. Heated elastase, however, potentiated bacterial killing in the same manner as untreated enzyme (Table 1). The results indicate that the potentiating effect of elastase does not depend on its elastolytic activity. The binding of elastase to E. coli and S. aureus at pH 7.4 and 5.5 is shown in Fig. 6. The unspecific binding to the incubation tube showed saturation at a concentration of approximately 4 to 6 ,ug/ml. The binding curve for E. coli leveled off when approximately 8 ,tg/ml was bound (8 ,ug/I1.5 x 108 bacteria). The results indicate a binding of approximately 10' elastase molecules per bacterium. Binding of granulocyte elastase to S. aureus could not be demonstrated at pH 7.4 or 5.5.

I)ISCUSSION Our findings demonstrate that granulocyte elastase, which by itself has no antimicrobial activity, potentiates the effects of both oxygendependent (e.g., MPO-H.,O.2) and oxygen-independent (e.g., chymotrypsin-like cationic protein) granulocyte microbicidal systems. Other TABLE 1. Effect of heating (experiment 1) and elastase inhibitor (experiment 2) on the potentiating effect of elastase on bacterial killing by the MPOglucose oxidase system" Supplements None MPO + GO MPO + GO + elastase (20 ,ig/ ml) MPO + GO + heated elastase (20 ,g/ml) MPO + GO + inhibitor-treated elastase (20 ,ug/ml) MPO + GO + inhibitor-treated elastase (40 ug/nml) Elastase (20 ,ig/ml) Heated elastase (20 ,tg/ml) Inhibitor-treated elastase (40

E 20 pH 5.5

z 0


Expt 1 2.6 x 106 6.4 x 105 4.3 x 10'

Expt 2 2.8 x 106 1.1 x 106 1.4 x 105

7.8 x 104







E 20

Viable cell count (organisms/ml)

pH 74 0)~~~~~~





u1 0i


1.1 x 106

1.1 x 106 2.5 x 106 2.4 x 106

2.5 x 106 2.7 x 106

)Ug/ml) The incubation mixture consisted of 2 x 106 E. coli, 30 ,ug of MPO, and 0.66 ,ug of glucose oxidase (GO)/ml (generating approximately 8 nmol of H2O,/min.). Incubation period was 20 min. The experimental procedures are detailed in Materials and Methods. "

neutral proteases like granulocyte collagenase showed no effect under the present conditions. Elastase is a major constituent of azurophil granules of human neutrophils (7). The same granule type is also the exclusive site of MPO and chymotrypsin-like cationic protein (K. Ohlsson, I. Olsson, and J. K. Spitznagel, in preparation), which gives these components the opportunity to interact when simultaneously liberated into the phagocytic vacuole. The more or less selective effect of elastase during the initial phase of bacterial killing is important as regards in vivo conditions, since microorganisms normally succumb shortly after ingestion. Human neutrophil elastase has been purified (14, 32) and shown to consist of three isoenzymes. Potentiating effects on microbicidal systems were found with all three isoenzymes. Although granulocyte elastase has been implicated in tissue damage associated with inflammation (13), its function within neutrophils is not known, but it probably participates in digestion of phagocytized microorganisms. Re-




FIG. 6. Binding of elastase to E. coli (0) and S. aureus (0) as a function of the elastase concentration. Binding to laboratory utensils (incubation tubes) is also indicated (A). The concentration of E. coli was 1.5 x 108/ml (pH 7.4) and 1.4 x 108/ml (pH 5.5), and the concentration of S. aureus was 1.5 x 101/ml (pH 7.4) and 0.7 x 108/ml (pH 5.5).

VOL. 14, 1976



cent studies have indicated that granulocyte vivo. Some correlation was found between dielastase plays a supplementary role in the minished microbicidal activity and abnormally digestion of bacterial cell walls by human leu- increased [14C]glucose oxidation of neutrophils kocytes (15). The potentiating effect on microbi- in cases of chronic granulocytic leukemia (28), cidal killing shown in this work was not due to which may be due to inhibition of the MPOthe enzymatic activity of elastase, since heat- H2G2 system by excess H2G.[. Acid extracts of rabbit heterophil granules ing destroyed the elastolytic but not the antibacterial activity. The elastase inhibitor abol- have been shown to kill bacteria in vitro (9). ished the potentiating effect of bacterial killing, This activity depends on a family of highly which may have been due to inhibition of bind- cationic proteins (43). Cationic proteins with ing to the bacterial surface. The granulocyte similar electrophoretic mobility and antibacteelastases are cationic proteins with a high elec- rial properties were found in human neutrotrophoretic mobility toward the cathode, and phils (29, 34). These proteins possessed chymobinding to elastin is due to electrostatic interac- trypsin-like activity, but the microbicidal activtions between positively charged groups of elas- ity was generally independent of the enzymatic tase and carboxyl groups of elastin (8). Similar activity since this was abolished by heating, electrostatic interactions may facilitate absorp- which generally did not affect microbicidal tion of elastase on bacteria, which normally are properties. The fungicidal properties of identinegatively charged (11). Binding of elastase cal proteins have been shown, and it has been could distort ionic bonds, which are important suggested that these account for the ability of for the stability of the bacterial envelope (23) MPO-deficient and chronic granulomatous disand render the bacteria more sensitive to the ease granulocytes to kill certain fungi (22). activity of various microbicidal systems. Even When chymotrypsin-like cationic proteins were if binding of elastase to S. aureus was not combined with the MPO-glucose oxidase sysdemonstrated, minute amounts may have been tem, no potentiating was found, indicating that these microbicidal systems operate independabsorbed and responsible for potentiation. The microbicidal potential of the MPO-me- ently. On the other hand, the present results diated system is considerable (18). It cannot, demonstrate that granulocyte elastase amplihowever, be ruled out that the microbicidal fies the action of the chymotrypsin-like cationic effects of the MPO-H202 system observed in protein. The approach in our previous work (29) was studies using crude MPO preparations were in part due to a potentiation of the peroxidative to identify and characterize components of the activity resulting from contamination with granulocytes that affect microorganisms, which hopefully will lead to an understanding of moelastase. The concentrations of MPO and H2G2 that lecular events underlying microbial killing are relevant to in vivo conditions in phagocytic during phagocytosis. Clearly several neutrovacuoles are not known. The concentration of phil agents exist (18), interacting in a complex MPO is approximately 8 ,g/0l granulocytes fashion in the phagocytic vacuole. Some of this (28), and the maximal rate of H2.,0 production complexity may be explored by the present apof phagocytizing granulocytes is approximately proach, in which highly purified neutrophil 1.5 nmol/106 granulocytes per min (10). Since constituents are allowed to interact. The pres106 granulocytes occupy about 1 pLI (3), these ent results derimonstrate that certain neutrophil figures correspond to an MPO concentration of constituents, e.g., elastase, which is not microbicidal by itself, nevertheless can initiate about 8 mg per ml of granulocytes and an H2.,G production of 1.5 ,umol/ml of granulocytes per sublethal changes, rendering some microorgamin. These calculations indicate that the MPO nisms more susceptible to the action of known concentrations (1.25 to 40 ,ug/ml) and rate of microbicidal agents like MPO and chymotrypH2G2 production (2.5 to 8 nmol/ml per min) used sin-like cationic protein. On the basis of this work, we suggest that in the present study may exist in the phagocytic vacuole. The microbicidal activity of the bacterial killing in granulocytes can be the reMPO-H202 system has been reported to be in- sult of the synergistic action of different antimihibited at high concentrations of H02G (24); this crobial systems, oxygen dependent as well as was confirmed in the present study. The inhibi- oxygen independent. tion is overcome if either the rate of H2.G2 production or the concentration of MPO is inACKNOWLEDGMENTS creased. Since the concentrations of MPO and The technical assistance of M. B. Hakansson, A. H202 in the phagocytic vacuole are unknown, it Lundqvist, and M. Andersson is greatly appreciated. cannot be determined whether inhibition by This study was supported by the Unit for Community H202 of the MPO-H2G2 system will also occur in Care Sciences of the National Board of Health and Social




Welfare at Dalby, the Swedish Cancer Society, and the Medical Faculty, University of Lund.

20. 1.







8. 9.


11. 12.

13. 14. 15. 16.

17. 18.


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Microbicidal mechanisms of human granulocytes: synergistic effects of granulocyte elastase and myeloperoxidase or chymotrypsin-like cationic protein.

Vol. 14, No. 6 Printed in U.S.A. INFECTION AND IMMUNITY, Dec. 1976, p. 1276-1283 Copyright ©O 1976 American Society for Microbiology Microbicidal Me...
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