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Plasminogen Activator Inhibitor Type-1-Deficient Mice Have an Enhanced IFN- Response to Lipopolysaccharide and Staphylococcal Enterotoxin B¹

Rosemarijn Renckens^2,*,, Jennie M. Pater^*, and Tom van der Poll^*,

^* Center for Infection and Immunity Amsterdam, and Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

	Abstract

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Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1^–/–) mice demonstrated strongly elevated plasma IFN- concentrations after injection of either LPS or SEB. In addition, PAI-1^–/– splenocytes released more IFN- after incubation with LPS or SEB than Wt splenocytes. Both PAI-1^–/– CD4⁺ and CD8⁺ T cells produced more IFN- upon stimulation with SEB. LPS-induced IFN- release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN- release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN- release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.

	Introduction

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Mediators of the fibrinolytic system have been associated with functions besides their classical fibrin-dissolving properties (1, 2). Plasminogen activator inhibitor type-1 (PAI-1)³ is the main inhibitor of the fibrinolytic system. By inactivating both urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), PAI-1 inhibits activation of the key-enzyme plasmin and subsequent fibrin degradation. However, PAI-1 has also been implicated in several other processes and diseases by actions that are not or only partially related to its capacity to inhibit plasminogen activation, including wound healing, tumor angiogenesis, rheumatoid arthritis, fibrosis, metabolic disturbances, glomerulonephritis, cell migration, bleomycin-induced lung injury, asthma, and nasal allergy.

During severe infection or sepsis, the systemic inflammatory response is almost invariably associated with a strong rise in circulating PAI-1 levels. In sepsis patients, PAI-1 concentrations predict lethality in a very sensitive manner (3, 4). Moreover, a (4G/5G) promoter deletion/insertion polymorphism in the PAI-1 gene has been found to influence the risk of the development of septic shock, and to be associated with a poor outcome in patients with meningococcal sepsis (5, 6). Although these observations suggest that PAI-1 plays a functional role in the inflammatory response during severe infection, such a role has not been established thus far. One can hypothesize that PAI-1 influences the innate immune response by inhibition of mediators of the fibrinolytic system. For instance, plasmin can activate the p38 MAPK signaling pathway in monocytes (7, 8), and activation of this pathway was recently shown to be of key importance for the inflammatory response to endotoxin in humans (9, 10). Furthermore, in vitro, plasmin was demonstrated to stimulate the release of cytokines and other inflammatory mediators by different cell types (7, 11, 12, 13). Moreover, PAI-1 inhibits uPA, which can enhance LPS-induced cytokine expression in vitro and in vivo (14), and studies using genetically modified mice have implicated uPA as an important regulator of inflammatory responses to bacterial and other stimuli (15, 16).

LPS, present in the outer membrane of Gram-negative bacteria, plays a pivotal role in triggering inflammatory responses during Gram-negative sepsis. Staphylococcal enterotoxin B (SEB) is a product of Staphylococcus aureus, which stimulates both APCs and T cells in vivo (17, 18). We investigated the role of PAI-1 in cytokine responses to LPS and SEB-induced inflammation in vivo, using PAI-1 gene-deficient (PAI-1^–/–) mice. We show that PAI-1 deficiency is associated with a strongly enhanced LPS and SEB-induced IFN- release in vivo and in vitro by a mechanism that does not depend on its interaction with tPA, uPA, or its receptor uPAR.

	Materials and Methods

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Animals

PAI-1^–/–, uPA^–/–, uPAR^–/–, and tPA^–/– mice, all backcrossed to a C57BL/6 genetic background, and C57BL/6 wild-type (Wt) mice were purchased from The Jackson Laboratory. Eight-week-old female mice were used for all experiments. All experiments were approved by the institutional animal care and use committee of the Academic Medical Center (Amsterdam, The Netherlands).

LPS and SEB-induced inflammation in vivo

A total of 200 µg of LPS (Escherichia coli 055:B5; Sigma-Aldrich) or 100 µg of SEB (Sigma-Aldrich) was injected i.p. in 200 µl of sterile NaCl 0.9%.

Sample harvesting

At the time of sacrifice, mice were first anesthetized by i.p. injection of 0.07 ml/g FFM mixture (Fentanyl (0.315 mg/ml)-Fluanisone (10 mg/ml) (Janssen), Midazolam (5 mg/ml) (Roche)). Next, blood was drawn via direct heart puncture with a sterile syringe, and transferred to tubes containing heparin; plasma was prepared by centrifugation at 1400 x g for 10 min at 4°C, after which aliquots were stored at –20°C.

Assays

Murine PAI-1 was measured by ELISA (Korida). TNF-, IL-6, MCP-1, IL-10, IL-12p70, IFN-, IL-5, IL-4, and IL-2 were measured by cytometric bead array (CBA) multiplex assay (BD Pharmingen) in accordance with the manufacturer’s recommendations. The detection limit of all cytokines was 2.5–5.0 pg/ml.

Ex vivo splenocyte stimulation

Single-cell suspensions were obtained from Wt and PAI-1^–/– mice by crushing spleens through a 40-µm cell strainer (BD Pharmingen). Erythrocytes were lysed with ice-cold isotonic NH₄Cl solution (155 mM NH₄Cl, 10 mM KHCO₃, 100 mM EDTA (pH 7.4)), and the remaining cells were washed twice with RPMI 1640 (BioWhittaker Europe). Splenocytes were suspended in medium (RPMI 1640 with L-glutamine, 5% autologous serum, 5% antibiotic-antimycotic (Invitrogen Life Technologies), seeded in 96-well round-bottom culture plates at a cell density of 1 x 10⁶ cells/well in triplicate, and stimulated with medium, 10 ng/ml LPS or 10 µg/ml SEB in an end-volume of 200 µl. Supernatants were harvested after a 48-h incubation at 37°C in 5% CO₂, and cytokine levels were analyzed by CBA.

Ex vivo peritoneal macrophage stimulation

Peritoneal macrophages were harvested from Wt and PAI-1^–/– mice by washing the peritoneal cavity with 5 ml of sterile saline. Collected cells were allowed to adhere to 96-well tissue-culture plates (10⁵ cells/well) for 2 h at 37°C, after which nonadherent cells were removed by rinsing with medium. More than 95% of the cells were peritoneal macrophages, as identified by cytospin preparations stained with modified Giemsa stain. Macrophage monolayers were stimulated with medium, 10 ng/ml LPS or 10 µg/ml SEB in an end-volume of 200 µl for 48 h at 37°C; then supernatants were aspirated, and cytokine levels were analyzed by CBA.

FACS analysis

For FACS analysis, splenocytes suspensions obtained from Wt and PAI-1^–/– mice were washed with FACS buffer (PBS supplemented with 0.5% BSA, 0.01% NaN₃, and 100 mM EDTA) and resuspended in 150 µl of FACS buffer. Immunostaining for cell surface molecules was performed for 30 min at 4°C, using directly labeled Abs against CD3, CD4, CD8, NK1.1, CD11b, CD11c, F4/80, and GR1. All Abs were used in concentrations recommended by the manufacturer (BD Pharmingen). To correct for nonspecific staining, an appropriate control Ab (rat IgG2; BD Pharmingen) was used. For the ex vivo stimulation experiments, splenocytes were seeded in 24-well tissue-culture plates at a cell density of 1 x 10⁶ cells/well in duplicate, and stimulated with medium or 10 µg/ml SEB in an end-volume of 2 ml. After 1 h of incubation at 37°C in 5% CO2, the protein transport inhibitor brefeldin A (2 µg/ml; Sigma-Aldrich) or medium was added to the wells. Cell cultures were incubated at 37°C in 5% CO₂ for 48 h, after which cells were washed with FACS buffer and resuspended in FACS buffer. Cells were fixed with 4% formaldehyde and permeabilized using 100 µl of Cytofix/Cytoperm (BD Pharmingen) for 20 min at 4°C. Next, cells were stained for intracellular (IC) IFN- (BD Pharmingen) for 30 min at 4°C, after which they were washed and resuspended in FACS buffer for FACS analysis (using FACSCalibur; BD Biosciences).

Statistical analysis

Data are expressed as means ± SEM, unless indicated otherwise. Comparison between time curves were conducted using two-way ANOVA, with Bonferroni post hoc tests. Comparisons between groups were conducted using the Mann-Whitney U test. A p value of <0.05 was considered to represent a statistically significant difference.

	Results

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PAI-1 is up-regulated by LPS and SEB

To confirm PAI-1 production in Wt mice in our models, we measured PAI-1 levels in plasma before and at 4 and 8 h after i.p. injection of LPS (200 µg) or SEB (100 µg). Baseline plasma PAI-1 concentrations were 5 ± 0.3 ng/ml. At 4 and 8 h after LPS administration, PAI-1 levels were significantly increased compared with baseline values (512 ± 34 and 669 ± 54 ng/ml, respectively, both p < 0.05). SEB injection also resulted in an elevation of plasma PAI-1 concentrations at 4 and 8 h postinjection (18 ± 2.3 and 31 ± 2.3 ng/ml, respectively, both p < 0.05 vs baseline).

The LPS-induced cytokine response

To investigate whether PAI-1 deficiency influences LPS-induced cytokine production, we measured plasma levels of TNF-, IL-6, MCP-1, IL-10, IL-12p70, and IFN- at various time points up to 48 h after i.p. injection of LPS. At 48 h, all cytokine levels were back to baseline in both groups of mice, therefore only results obtained during the first 24 h are shown. The plasma concentrations of all cytokines measured showed a profound rise after LPS injection (Fig. 1). Plasma TNF-, MCP-1, and IL-10 levels were not different between Wt and PAI-1^–/– mice. In Wt mice, plasma IL-6 levels showed a peak at 2 h postinjection; in contrast, in PAI-1^–/– mice IL-6 levels peaked after 4 h and were still significantly higher than in Wt mice at 8 h after injection. IL-12p70 levels peaked at 4 h after LPS in both groups of mice, but levels were much higher in PAI-1^–/– mice compared with Wt mice. The plasma concentrations of IFN- peaked at 8 h after LPS injection in both groups. However, IFN- levels were strongly increased in PAI-1^–/– mice compared with Wt mice.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Effect of PAI-1 deficiency on LPS-induced cytokine release. TNF-, IL-6, MCP-1, IL-10, IL-12p70, and IFN- were measured in plasma at several time points after i.p. injection of LPS (200 µg). Results are expressed as means ± SE of eight mice per strain at each time point. *, p < 0.05, and **, p < 0.001 vs Wt mice at the same time point.

Having found that PAI-1 deficiency is associated with strongly enhanced LPS-induced IFN-, IL-6, and IL12p70 release, we decided to investigate the cytokine response to a polyclonal T cell activator. Therefore, we measured plasma levels of TNF-, IL-6, MCP-1, IL-10, IL-12p70, IFN-, IL-5, IL-4, and IL-2 at 4 and 8 h after i.p. injection of SEB in Wt and PAI-1^–/– mice (Fig. 2). We chose these two time points because they were found to be representative of superantigen-induced cytokine release in vivo; in particular, IFN- reaches peak levels at 8 h after SEB administration (17, 19). The SEB-induced cytokine response was less strong than the LPS-induced cytokine response, although all cytokines with the exception of IL-4 displayed elevated plasma concentrations after SEB administration. TNF- levels were similar in the PAI-1^–/– mice and Wt mice. In line with the LPS-induced IL-6 response, SEB-induced IL-6 levels were higher in PAI-1^–/– mice than in Wt mice at both time points. MCP-1 concentrations were higher in PAI-1^–/– mice at 4 h after SEB administration. IL-10, IL-12p70, IL-5, and IL-2 concentrations were similar between both genotypes. In line with the results obtained after LPS injection, IFN- levels were markedly elevated in PAI-1^–/– mice when compared with Wt mice at both 4 and 8 h after SEB injection (Fig. 2).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Effect of PAI-1 deficiency on SEB-induced cytokine release. TNF-, IL-6, MCP-1, IL-10, IL-12p70, IFN-, IL-5, and IL-2 were measured in plasma at 4 and 8 h after i.p. injection of SEB (100 µg). Results are expressed as means ± SE of eight mice per strain at each time point. *, p < 0.05 and **, p < 0.001 vs Wt mice at the same time point.

PAI-1^–/– splenocytes release more IFN- during ex vivo stimulation

In an attempt to determine which cell populations in PAI-1^–/– mice show a changed cytokine response and to determine which cytokine changes are influenced directly by PAI-1 deficiency, we stimulated splenocytes and peritoneal macrophages harvested from Wt and PAI-1^–/– mice that had not received LPS or SEB in vivo with LPS, SEB, or culture medium for 48 h ex vivo, and measured TNF-, IL-6, IL-12p70, IFN-, IL-5, IL-4, and IL-2 levels in the supernatants. The cellular distribution of spleens obtained from PAI-1^–/– and Wt mice did not differ (Table I). None of these cytokines was detectable in the supernatants of cell cultures incubated with medium only. Stimulation with either LPS or SEB resulted in TNF-, IL-6, and IFN- release by splenocytes (Fig. 3), whereas IL-12p70, IL-5, IL-4, and IL-2 remained undetectable. LPS and SEB-induced TNF- and IL-6 levels were similar in the supernatants of Wt and PAI-1^–/– splenocytes. However, in line with the in vivo findings, IFN- release by PAI-1^–/– splenocytes stimulated with either LPS or SEB was significantly increased. Peritoneal macrophages only released detectable levels of TNF- and IL-6 upon stimulation with LPS or SEB; these concentrations did not differ between Wt or PAI-1^–/– macrophages (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. PAI-1–/– splenocytes produce more IFN- in vitro. TNF-, IL-6, and IFN- were measured in the supernatants of splenocyte cultures after 48 h of incubation with either LPS (10 ng/ml) or SEB (10 µg/ml). Results are expressed as means ± SE of six mice. *, p < 0.05 and **, p < 0.01 vs Wt mice.

CD4⁺ and CD8⁺ T cells are involved in the stronger IFN- response in PAI-1^–/– mice

To determine which cell types are involved in the production of IFN- in response to SEB and to examine whether they were influenced by PAI-1 deficiency, we measured the percentage of cells positive for IC IFN- in splenocyte cultures after 48 h of SEB stimulation and differentiated between CD3⁺CD4⁺, CD3⁺CD8⁺, and NK1.1⁺ cells by FACS analysis (Fig. 4). In these studies, we focused on SEB-induced IFN- production because this stimulus had proved to be a far more potent IFN- inducer than LPS (see Fig. 3). The percentage of IFN- positive T cells was low, and there was no increase in IC IFN- after SEB stimulation in Wt CD4⁺ T cells; in contrast, in PAI-1^–/– cultures, we did find an increase in the percentage of IFN--positive CD4⁺ T cells that was significantly different from Wt CD4⁺ T cells. CD8⁺ T cells showed a rise in IC IFN- in both Wt and PAI-1^–/– cells; however, the percentage was significantly higher in PAI-1^–/– cultures. Finally, NK-cells also showed a rise in the percentage of IC IFN--positive cells after SEB stimulation; although PAI-1^–/– NK cells showed a higher percentage with positive IC IFN- staining, the difference with Wt cells did not reach statistical significance. These data show that both CD4⁺ and CD8⁺ T cells from PAI-1^–/– mice produced more IFN- upon SEB stimulation in vitro.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. PAI-1–/– CD4+ and CD8+ T cells produce more IFN- in vitro. Splenocytes were stimulated with SEB (10 µg/ml) for 48 h. The percentage of cells that positively stained for IC IFN- was measured by FACS analysis. Results are expressed as means ± SE of six mice. *, p < 0.01 vs Wt mice.

uPA, uPAR, or tPA deficiency does not influence LPS-induced IFN- release

A major function of PAI-1 in the fibrinolytic system is inhibition of the plasminogen activators uPA and tPA. Therefore, we were interested to determine the role of uPA and the uPA receptor and tPA in IFN- release induced by LPS. To address this issue, uPA^–/–, uPAR^–/–, and tPA^–/– mice were i.p. injected with LPS and after 8-h plasma IFN- concentrations were measured, i.e., the time point at which plasma IFN- reached peak levels in this model. None of the mice displayed an altered IFN- response when compared with Wt mice (Fig. 5).

View larger version (9K): [in this window] [in a new window]   FIGURE 5. uPA, uPAR, or tPA deficiency does not influence LPS-induced IFN- release. Plasma IFN- levels were measured 8 h after i.p. injection of LPS (200 µg) in uPA–/–, uPAR–/–, tPA–/–, or Wt mice. Results are expressed as means ± SE of eight mice per group. Differences between groups were not significant.

	Discussion

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LPS has been used extensively to obtain insight in the mechanisms contributing to systemic inflammation during sepsis. Bolus injection of LPS into humans results in a strong rise in plasma PAI-1 concentrations peaking after 4 h (10, 21). In this study, we used the LPS challenge model to obtain a first insight in the role of PAI-1 in systemic release of cytokines. We first confirmed PAI-1 release after LPS administration to Wt mice. When we subsequently found elevated IFN- concentrations in LPS-challenged PAI-1^–/– mice, we decided to investigate whether this observation could be reproduced after administration of a polyclonal T cell activator. For this model we used SEB, a superantigen produced by S. aureus implicated in nonmenstrual toxic shock syndrome (18). Indeed, SEB injection elicited PAI-1 secretion into the circulation, and higher IFN- levels were detected in PAI-1^–/– mice. Of note, not all cytokine responses were similarly affected by PAI-1 deficiency in the LPS and SEB models. These differences might be explained by the fact that LPS and SEB activate different cell types (20). LPS primarily activates mononuclear cells (20). SEB can bind directly to regions of the class II MHC molecule that are outside the physiological MHC haplotype-restricted Ag-binding groove, which eventually results in activation of both APCs and SEB-reactive V8⁺ T cells (17). Such differences in cell activation likely contributed to the differential effects of PAI-1 deficiency on IL-12p70 and MCP-1 release. In particular, the different impact of PAI-1 deficiency on IL-12p70 release is important. IL-12p70 is a strong inducer of IFN- production (22); the fact that IFN- release was enhanced in PAI-1^–/– mice after both LPS and SEB injection, whereas IL-12p70 was increased only after LPS injection strongly suggests that the effect of endogenous PAI-1 on IFN- release does not rely on IL-12p70. This notion is further supported by the fact that PAI-1^–/– splenocytes released more IFN- upon stimulation with either LPS or SEB under conditions in where IL-12p70 release remained undetectable.

Mice with a targeted deletion of the gene-encoding uPA have been found to mount a reduced type 1 response upon pulmonary infection with the opportunistic yeast Cryptococcus neoformans, as reflected by lower IFN- levels in bronchoalveolar lavage fluid (16). In addition, lung mononuclear cells and regional lymph node cells obtained from infected uPA^–/– mice released less IFN- upon Ag-specific stimulation in this model (16). These findings led us to hypothesize that PAI-1 may facilitate a type 2 response through inhibition of uPA. However, this appeared not to be the case: not only uPA^–/– mice, but also uPAR^–/– and tPA^–/– mice demonstrated an unremarkable IFN- response upon injection of LPS. These data suggest that the effect of endogenous PAI-1 is not mediated by an effect on uPA/uPAR or tPA.

Although our study was in progress, Sejima et al. (23) reported on the consequences of PAI-1 deficiency in a model of nasal allergy. In this latter investigation, OVA-sensitized mice demonstrated increased PAI-1 levels in nasal washings upon intranasal OVA challenge; this locally produced PAI-1 contributed to a Th2 response as reflected by Th1-biased responses in sensitized PAI-1^–/– mice characterized by reduced OVA-specific circulating IgE and elevated plasma IgG2a levels, as well as lower IL-4 and IL-5 and higher IFN- concentrations in nasal lavage fluid (23). Our present findings extend and are partly in line with these results: naive PAI-1^–/– mice displayed enhanced systemic IFN- release after i.p. injection of either LPS or SEB, and these in vivo observations could be reproduced using PAI-1^–/– splenocytes in vitro. However, in contrast to the results obtained by Sejima et al. (23), we did not find a diminished release of the type 2 cytokine IL-5 in the SEB model; whereas IL-4 could not be studied because even in Wt mice, plasma IL-4 remained undetectable after SEB administration. Nonetheless, these data together suggest that PAI-1 inhibits IFN- release under markedly different conditions, thereby identifying a novel biological activity of this protein. Further studies are warranted to unravel the mechanisms by which PAI-1 influences IFN- production.

Inhibition or elimination of IFN- has been reported to reduce LPS-induced lethality in mice (24, 25, 26). In contrast, pretreatment with recombinant IFN- increased LPS-induced lethality (24). Thus, it is possible that PAI-1 deficiency influences survival during endotoxemia. We considered survival studies beyond the scope of the current investigations, because our study focused on the regulation of IFN- production by PAI-1 and because many other factors besides IFN- have been implicated as mediators of LPS toxicity and lethality. In addition, in this respect, it should be noted that one study reported that IFN- receptor-deficient mice sensitized with D-galactosamine were protected against LPS-induced lethality, whereas the toxicity evoked by high-dose LPS in the absence of D-galactosamine sensitization was similar in IFN- receptor-deficient and Wt mice (26). Of note, the influence of endogenous PAI-1 on IFN- production could be of relevance to different conditions and diseases besides sepsis in which IFN- has been found to play a role, including allergy (see above), viral, and parasitic infections, and IC infections such as tuberculosis.

Enhanced PAI-1 release is a consistent part of the systemic inflammatory response syndrome induced by sepsis or administration of bacterial products. In this study, we demonstrate for the first time that elevated PAI-1 concentrations are functionally important for attenuating IFN- release after injection of LPS or SEB. These results further exemplify the complex role of PAI-1 in the host response to severe infection, which reaches far beyond its classical role as an inhibitor of fibrinolysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by a grant from the Netherlands Heart Foundation (2001B114; to R.R.).

² Address correspondence and reprint requests to Dr. Rosemarijn Renckens, Center for Experimental and Molecular Medicine, Academic Medical Center, Room G2-132, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: r.renckens{at}amc.uva.nl

³ Abbreviations used in this paper: PAI-1, plasminogen activator inhibitor type-1; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; SEB, staphylococcal enterotoxin B; Wt, wild type; CBA, cytometric bead array; IC, intracellular.

Received for publication May 8, 2006. Accepted for publication September 11, 2006.

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