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Keywords:

  • glioma antigen;
  • SOX6;
  • DNA vaccine;
  • CTL response;
  • antitumor immunity

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma. © 2008 Wiley-Liss, Inc.

Despite significant advances in modern microsurgery, radiotherapy and chemotherapy, the prognosis for patients with malignant gliomas remains poor.1 Thus, the development of new therapeutic strategies is required. Immunotherapy for gliomas is an attractive alternative treatment option because activated, antitumor immune cells have the potential to migrate into the central nervous system (CNS) and to selectively destroy malignant cells that have infiltrated into normal CNS tissues.2

The success in cancer immunotherapy is determined by the efficacy of the therapy in inducing an immune response against the tumor rejection antigen (Ag).3 It is thus important for inducing antitumor immunity to use more immunogenic tumor-associated Ag, because many tumors escape immunological destruction by down-regulation or loss of immunogenic epitopes.4–6 Therefore, vaccination with multiple epitopes will be advantageous over single epitope-based vaccines.7, 8 A DNA-based vaccine opens the possibility to combine multiple epitopes for cytotoxic T-lymphocyte (CTL) and helper T cell into one vaccine.9

We have identified a glioma antigen, SOX6, by serological screening of a testis cDNA library with glioma patients' sera. SOX6 expression is developmentally regulated and its expression in the adult was restricted to testis and glioma tissues. In addition, the specific IgG responses against SOX6 were frequently observed in glioma patients.10 Because of the restricted expression pattern and the high immunogenicity, SOX6 is an attractive antigen as a target for immunotherapy. In our study, we examine whether a SOX6 DNA vaccine could induce SOX6 specific CTL responses to destroy brain tumors in vivo.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines, antibodies and mice

The murine glioma cell line, GL261 [a primary mouse brain neoplasm cell line derived from the intracerebral implantation of a methylcholanthrene pellet in C57BL/6 mice (MHC haplotype H-2b)], and 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated fetal calf serum (IFCS) and penicillin-streptomycin (10% IFCS-DMEM). EL-4 [a mouse T-cell lymphoma cell line (H-2b)], was cultured in RPMI 1640 medium (Sigma-Aldrich) containing 10% IFCS, 50 μM 2-mercaptoethanol and penicillin-streptomycin (10% IFCS-RPMI).

The anti-CD4 hybridoma (clone GK1.5, rat IgG) and anti-CD8 hybridoma (clone 2.43, rat IgG) (kindly provided by Dr. S. Saito, Jikei University, School of Medicine, Japan) were grown as ascites in nude mice, and the MAb was purified from the ascites using a Protein G column (Amersham Biosciences AB, Uppsala, Sweden).11

Female C57BL/6 mice (6- to 8-week-old) were purchased from Japan SLC. (Tokyo, Japan). The Animal Care and Use Committee of the Keio University School of Medicine approved all animal procedures.

Construction of a SOX6 DNA plasmid and transfection

We obtained the full length of the open reading frame (ORF) in mouse SOX6 gene (GenBank entry: D61689.1) with the sites of EcoRI on the 5′ end and SalI on the 3′ end from mouse testis cDNA and cloned in the pGEM-T Easy Vector (Promega) as described previously.10 After sequencing, the full length ORF of mouse SOX6 gene from SOX6-pGEM-T Easy Vector was inserted into the pcDNA3 (Invitrogen, Carlsbad, CA; pcDNA3-SOX6). As a control, pcDNA3 plasmid was used as the empty vector (pcDNA3). All recombinant constructs were confirmed by sequence analysis. Large scale preparation of plasmid DNA was carried out using a Giga kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. The pcDNA3-SOX6 was transfected with LipofectAMINE (Invitrogen) into 293T cells (293T-SOX6). After 48 hr incubation at 37°C, the transfected cells were collected and lysed by RIPA buffer (Cell Signaling).

Western blot analysis of SOX6 in tumor cell lines

SOX6 expression in tumor cells was tested by Western blot analysis. SOX6 protein exists in the nuclei of mammalian cells.12 To isolate nuclear extracts, GL261, EL4 and 293T cells transfected with the full-length murine SOX6 cDNA were homogenized in 0.25 M sucrose and centrifuged for 10 min. Pellets were resuspended in 0.25 M sucrose supplemented with a protease inhibitor cocktail (Sigma Aldrich). Twenty micrograms of total protein was mixed with an equal volume of a sodium dodecyl sulfate (SDS)-sample buffer composed of 2% SDS, 10% β-mercaptoethanol, 10% glycerol, 1 mM EDTA, 40 mM Tris and 240 mM glycine at pH 8.5 and boiled for 3 min. The same amount of total protein was then loaded on each lane, and the proteins were transferred onto a nitrocellulose membrane (Hybond C+; Amersham Pharmacia) by electroblotting. After blocking with 5% skim milk for 2 hr, the sheets were incubated with a rabbit anti-human SOX6 polyclonal antibody (10 μg/ml, CHEMICON International), followed by 1:2,000-diluted peroxidase-conjugated mouse anti-goat immunoglobulin G (IgG) (Cappel, Aurora, Ohio). The proteins were visualized with the help of an ECL Western blot detection system (Amersham Biosciences, Buckinghamshire, UK).

Preparation of DNA-coated gold particles and gene transfer

The Helios gene gun system (Bio-Rad Laboratories, Hercules, CA) was used for intradermal gene delivery. Bullets containing 1.25 μg of DNA/shot were generated according to the manufacturer's protocols. Briefly, 50 μg DNA was precipitated on 30 mg of 1.0 μm gold particles in the presence of 100 μl of 0.05 M spermidine using 100 μl of 1 M CaCl2/preparation. The gold was washed thrice in fresh absolute ethanol and was resuspended in 2.4 ml of 0.1 mg/ml polyvinylpyrrolidone in 100% ethanol. The gold was then loaded into the tubing using the tubing preparation station (Bio-Rad), and the gold-loaded tubing was cut into 1.27-cm (i.e., 0.5-inch) pieces to load into the cartridges. The loaded DNA/gold complexes were transferred using the gene gun into the hair-shaved abdomen skin of mice with a helium gas pulse at 350 p.s.i.

51Cr release assays and interferon-γ ELISA assay

Female C57BL/6 mice (n = 6) were given 1.25 μg pcDNA3-SOX6 or pcDNA3 via a gene gun. These mice were boosted 4 times with the same regimen as the first vaccination at days 7, 14, 21 and 28. Seven days after the last booster, splenocytes from pcDNA3-SOX6-immunized mice (Sp-SOX6) or splenocytes from pcDNA3-immunized mice (Sp-vector) were used as effector cells in a cytolytic analysis. Four hour 51Cr release assays were performed as previously described.11 In brief, target cells (GL261 and EL-4) were incubated with 50 μCi of Na251CrO4 (51Cr) for 60 min. Target cells (1 × 104) were then mixed with effector cells (Sp-SOX6 and Sp-vector) for 4 hr at the indicated effector-to-target ratios. The amount of 51Cr release was determined by γ counting, and the percentage of specific lysis was calculated using triplicate samples as follows: [(experimental cpm-spontaneous cpm)/(maximum cpm-spontaneous cpm)] × 100. The production of interferon (IFN)-γ by the CTLs was also tested with an ELISA. In brief, an Sp-SOX6 or Sp-vector was cocultured with GL261 cells for 5 days. Then, the cultured splenocytes were further incubated with GL261 or EL4 cells in a 96-well plate for 24 hr. The culture supernatant (100 μl) was collected and the amount of IFN-γ was measured using a standard ELISA (Endogen, Woburn, MA).

Histology

Immunized mice were perfused, and postfixed in 4% paraformaldehyde (Sigma). The main organs expressing SOX6 in the embryo, including brain tissue (hippocampus, subventricular zone, cerebral cortex and cerebellum) and cartilage tissue in the trachea were removed from immunized mice, in which glioma-specific CTL responses were induced. Eight-micrometer paraffin sections were deparaffinized, rehydrated and routinely stained with hematoxylin and eosin for histological examination. Histological analysis was performed by independent pathologists from the Sapporo General Pathology Laboratory. (Sapporo, Japan).

Tumor protection and therapy assay

For in vivo tumor prevention experiments, female C57BL/6 mice (n = 5) were immunized 4 times at seven-day intervals with 1.25 μg pcDNA3-SOX6, or pcDNA3 via a gene gun, with nonimmunized mice as controls. Seven days after the booster, mice were anesthesized before all intracranial procedures and placed in a stereotaxic fixation device (Stoelting, Wood Dale, IL). A burr hole was drilled in the skull 2 mm lateral to the bregma. The needle of a Hamilton syringe (Hamilton, Darmstadt, Germany) was introduced to a depth of 3 mm. GL261 cells (5 × 104) resuspended in a volume of 2 μL of PBS were injected into the right striatum as described elsewhere with a minor modification.13 The survival time after the tumor cells inoculation was recorded.

For in vivo therapeutic experiments, female C57BL/6 mice (n = 5) were injected with 5 × 104 GL261 cells/mouse and then immunized with DNA vaccine by gene gun on days 4, 11, 18 and 25 after the inoculation of GL261 cells. The mice were fed until their death and the survival time was recorded to draw a survival curve for evaluating the therapeutic effect of the DNA vaccines.

In vivo depletion of T-cell subsets

T-cell subsets were depleted in vivo as described elsewhere.14, 15 Briefly, mice (n = 10) were vaccinated four times at 7-day intervals with pcDNA3-SOX6, or pcDNA3 via a gene gun, and challenged with 5 × 104 GL261 cells/mouse on day 7 after the fourth immunization. Mice were injected intraperitoneally with 100 μg of either the anti-CD4 antibody (Ab), the anti-CD8 Ab, both the anti-CD4 Ab and the anti-CD8 Ab or the isotype control Ab (rat IgG, ZYMED Laboratories, San Francisco, CA) 6 days after the last immunization and then twice per week for 3 weeks. Depletion was terminated on day 21 after tumor cell injection. The mice were fed until their death and the survival time was recorded to determine the subset of lymphocytes that are important for rejection of GL261.

Statistical analysis

Data are presented as means and standard deviation. Statistical differences between two groups were evaluated using the unpaired Student's t-test. The survival time was calculated by the Kaplan–Meier method. The cure rate of in vivo therapeutic experiments was also evaluated by χ2 for independence test. These statistical analyses were performed using SPSS version 14.0 (SPSS, Chicago, IL) and Statcel 2 (OMS Publishing, Saitama, Japan). p-values less than 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Preferential expression of SOX6 protein in murine glioma cells

We previously confirmed that the anti-SOX6 polyclonal antibody specifically recognizes SOX6.10 Western blot analysis using this antibody revealed that the SOX6 protein was expressed in GL261, but not in EL4 (Fig. 1).

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Figure 1. SOX6 expression in mouse glioma. Western blot analysis with the anti-SOX6 polyclonal antibody of 293T cells transfected with murine SOX6 cDNA (293T cell-SOX6), murine glioma cell line (GL261) and murine T-cell lymphoma cell line (EL4). Murine SOX6 was detected as an 87-kDa band.

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Induction of CTLs against SOX6 by the vaccination with SOX6 plasmid DNA

To determine whether the immune response generated by gene gun-mediated vaccination with a pcDNA3 plasmid DNA encoding full-length murine SOX6 (pcDNA3-SOX6) is tumor-specific, splenocytes isolated from immunized mice were used in an IFN-γ release assay and a 51Cr release assay. Splenocytes from pcDNA3-SOX6-immunized mice (Sp-SOX6) preferentially recognized GL261 glioma cells in vitro when measured using IFN-γ release (Fig. 2a). Moreover, the 51Cr release assay revealed that Sp-SOX6 lysed GL261 but not EL-4, which did not express SOX6 (Fig. 2). These results indicate that CTL responses generated in pcDNA3-SOX6-immunized mice are specific for SOX6-expressing glioma cells.

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Figure 2. Induction of SOX6-specific cytotoxic T lymphocytes by SOX6 DNA vaccination. (a) ELISA analysis of IFN-γ release from splenocytes culture. Splenocytes from pcDNA3-SOX6-immunized mice (Sp-SOX6) or splenocytes from pcDNA3-immunized mice (Sp-vector) were incubated with GL261 (black column) or EL4 (gray column) for 24 hr, and the culture supernatants were analyzed with an ELISA. Asterisks indicate p-values less than 0.05. (b) The average of specific lysis of Sp-SOX6 (▪) and Sp-vector (□) against GL261 (n = 12) analyzed by cytotoxic activity assay. Specific lysis of GL261 in Sp-SOX6 increase significantly compared to Sp-vector when the effector/target ratio is 40:1 (p < 0.05) and 80:1 (p < 0.05). Asterisks indicate p-values less than 0.05.

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Histological analysis of organs from mice which acquired a tumor-specific CTL response

SOX6 has been suggested to regulate embryonic development and determine cell fate. Mouse Sox6 is expressed in the CNS during embryogenesis, but not in the adult brain.16 Sox6, Sox5 and Sox9 are expressed simultaneously and at high levels from early stages of chondrogenesis in all cartilaginous sites in mouse embryos.12 We previously showed that human SOX6 was expressed in fetal brain and gliomas, but not in the adult brain and various normal tissues except for the testes,10, 17 suggesting a low risk of normal tissue damage as a result of immune responses to the SOX6 protein. To evaluate the risk of autoaggression by immunization against SOX6 protein, the tissues of immunized mice, which had acquired a tumor-specific CTL response, were histologically examined. The brain and cartilage tissue in the trachea of 2 mice were intensively examined because SOX6 was mainly expressed in these tissues during embryogenesis. Brain tissues, including the hippocampus, subventricular zone, cerebral cortex and cerebellum, showed normal structure and cellularity in both mice examined, and no pathological changes caused by immune response, such as lymphocyte infiltration or tissue destruction and repair, were observed (Figs. 3a and 3d). In cartilage tissue in the trachea of 1 out of 2 pcDNA3-SOX6-immunized mice, slight degeneration was observed, but a similar change in trachea of pcDNA3-immunized control mice was also noted (Fig. 3e). These results suggest that the CTLs specific for SOX6-expressing glioma cells do not recognize normal autologous-cells that have expressed SOX6 during embryogenesis at physiological levels.

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Figure 3. Histological analysis of organs from pcDNA-SOX6-immunized mice (left panel) and pcDNA-immunized mice (right panel). Hematoxylin and eosin staining of the cerebral cortex (a, original magnification, ×16), subventricular zone of gray matter (b, original magnification, ×80), hippocampus (c, original magnification, ×32), cerebellum (d, original magnification, ×32), and tracheal cartilage tissue (e, original magnification, ×200). Arrows in e indicate slight degeneration of tracheal cartilage tissue.

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Induction of the antitumor activity

To investigate the protective antitumor immunity, we immunized mice with pcDNA3-SOX6 and pcDNA3 with a gene gun before being challenged intracranially with GL261 tumor cells, which typically form solid and lethal tumor lesions in the brain of the syngeneic C57BL/6 mouse (Fig. 4a). As shown in Figure 4b, the survival of the mice treated with pcDNA3-SOX6 was significantly longer than that of pcDNA3-immunized mice and nonimmunized mice. The therapeutic efficacy of the DNA vaccine encoding SOX6 was then tested in glioma-bearing mice. The mice were treated starting at day 4 after the injection of GL261 glioma cells. As shown in Figure 4c, the survival of the mice treated with pcDNA3-SOX6 (survival rate on day 59; 60%) tended to be longer, comparing to that of pcDNA3-immunized mice (survival rate on day 59; 0%), although this difference was not statistically significant by the log-rank test (p = 0.052). The rate of complete response in mice treated with pcDNA3-SOX6 was significantly higher than that in mice treated with pcDNA3 (p < 0.05, by χ2 for independence test), when complete response was defined as survival of an animal for more than 80 days after tumor inoculation. These results suggest that a DNA vaccine with SOX6 could induce a strong antitumor response in a mouse glioma model.

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Figure 4. Vaccination with pcDNA-SOX6 induces antitumor activity in glioma-bearing mice. (a) H&E staining of the tumor in the brain from a control, nonimmunized mouse. Paraffin embedded tissue sections were prepared from the brain of C57BL/6 mice bearing day 45 GL261 glioma in the right frontal lobe. Scale bar = 200 μm. (b) The survival assay in the tumor prevention experiments. C57BL/6 mice (n = 5) were immunized four times with pcDNA3-SOX6 or pcDNA3 at seven-day intervals. Seven days after the booster, GL261 cells (5 × 104) were injected into the brain. The survival of the mice treated with pcDNA3-SOX6 (▪) was significantly longer than that of pcDNA3-immunized mice (□) (p < 0.05, by log-rank test), and than that of nonimmunized mice (▴) (p < 0.05). (c) The survival assay in the tumor therapeutic experiments. C57BL/6 mice (n = 5) were injected with 5 × 104 GL261 cells (day 0) and then immunized with the DNA vaccines on days 4, 11, 18 and 25. The survival of the mice treated with pcDNA3-SOX6 (▪) tended to be longer than that of pcDNA3-immunized mice (□) (p = 0.052, by log-rank test).

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Role of T-cell subsets in pcDNA3-SOX6-induced antitumour activity

To determine the subset of lymphocytes that are important for the rejection of GL261, we depleted CD8 positive (CD8+) and/or CD4 positive (CD4+) T-cell subsets in vivo one day before tumor challenge. When depletions were performed after immunization with pcDNA3-SOX6, the survival of mice treated with rat IgG was significantly prolonged, compared to anti-CD4 Ab, anti-CD8 Ab, or both anti-CD4 Ab and anti-CD8 Ab (Fig. 5). These data suggest that both CD8+ T cells and CD4+ T cells are involved in the antitumor effect induced by SOX6 DNA vaccine against brain tumors.

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Figure 5. Abrogation of antitumor activity by in vivo depletion of the T-cell subsets after immunization with the pcDNA3-SOX6 vaccine. Immunized mice were also injected intraperitoneally with anti-CD4 Ab, anti-CD8 Ab, both anti-CD4 Ab and anti-CD8 Ab, or isotype control rat IgG. The survival of mice treated with rat IgG (▪, n = 10), was significantly longer than that of mice treated with anti-CD4 Ab (□, n = 10, p < 0.01, by log-rank test), anti-CD8 Ab (•, n = 10, p < 0.01), or both anti-CD4 Ab and anti-CD8 Ab (○, n = 10, P < 0.01).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

DNA vaccination has become an attractive immunization strategy against tumors, because it has the ability to induce both cellular and humoral immune responses. However, a major problem of DNA vaccination is its limited potency to induce immune responses.18 Indeed, CTL responses from pcDNA3-SOX6-immunized mice were specific for SOX6-expressing glioma cells, but the killing activity in vitro was not strong. Therefore, a SOX6-DNA vaccine may be enhanced by coadministration of immune-potentiating cytokines,19–21 and/or chemokines which recruit professional host antigen-presenting cells.22

Although the DNA vaccination approach seems to be safe and promising, DNA vaccination with oncogenes still harbor the risk of transformation for the cells that receive and express the oncogenes.23 One of the important problems in immunotherapy with CTLs induced against overexpressed self-proteins such as a SOX6 protein is to avoid any damage to normal tissues expressing such self-proteins. Mouse Sox6 has been reported to be expressed in the CNS during embryogenesis16 and be expressed in all cartilaginous sites in mouse embryos,12 but not in the adult tissues. Recently, SOX6 has been reported to be sufficient to induce astrocytic differentiation when it was artificially overexpressed in adult neural stem cells.24 We also previously reported the possibility that SOX6 may be expressed in bipotential or multipotential cells capable of neuronal and glial differentiation, but not in fully differentiated cells.25 Therefore, we evaluated the risk of autoaggression by immunization against SOX6 protein in the tissues of immunized mice, but CTLs induced against SOX6 protein did not cause damage to normal tissues that had expressed SOX6 during embryogenesis. Moreover, these CTL responses did not affect normal neural stem/progenitor cells located in the hippocampus and subventricular zone of the adult brain. These results suggest that the CTLs specific for SOX6-expressing cells do not recognize normal self-cells in which there is some possibility of SOX6 expression.

To analyze the immune mechanisms elicited by the SOX6-DNA vaccine, we depleted CD8+ or CD4+ T-cell subsets in a group of mice in vivo after immunization. The depletion of CD8+ or CD4+ T-cells during the effector phase impaired the capacity of pcDNA3-SOX6-immunized mice to reject GL261, suggesting that both CD8+ T-cells and CD4+ T-cells were responsible for the antitumor activity by the SOX6-DNA vaccine. It has been reported that removal of CD4+ T-cells during the induction of the response, but not during the effector phase, abolished any capacity to reject the tumor challenge.26, 27 In contrast, the antitumor activity of the SOX6-DNA vaccine was abrogated by the depletion of CD4+ T-cells in the effector phase. CD4+ T lymphocytes can steer and amplify the immune response through the secretion of cytokines and the expression of surface molecules such as costimulatory molecules.28, 29 Therefore, CD4+ T cells are critical for both the complete elimination of tumors and the maintenance of a long term protective antitumor memory response in vivo.30, 31 Our results also suggest the important roles of CD4+ T cells in the antitumor immunity.

SEREX-defined antigens, such as SOX6, can be recognized by CD4+ helper T cells. Moreover, we recently identified CTL epitopes derived from the human SOX6 protein (unpublished data). These facts raise the possibility that the SOX6 protein has the ability to induce both cellular and humoral immune responses. Taken together, the findings in the present study may provide a useful target for DNA vaccine for the treatment of gliomas. Enhancement of antitumor activity by further optimization may be essential to accomplish the ultimate goal of developing therapeutic vaccines for glioma patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Tomoko Muraki (Neuroimmunology Research Group, Keio University School of Medicine) for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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