KR100552496B1 - Specific expression of NDRG2 gene in dendritic cells and detection of dendritic cells using the gene and protein expression pattern - Google Patents

Abstract

The present invention relates to a NDRG2 gene specifically expressing dendritic cells and a method for detecting dendritic cells using the same, and more particularly, specific to dendritic cells induced differentiation in human peripheral blood and umbilical cord blood and even hematological cancer cells. NDRG2 (N-myc downstream regulated), which can be used to obtain differentiation-induced dendritic cells, their purity and characterization during clinical use of dendritic cells in intractable diseases and chemotherapy. gene 2) relates to a gene and a method for detecting dendritic cells using the same.

Dendritic cells, NDRG2 (N-myc downstream regulated gene 2), Northern blotting, polyclonal antibodies, immunohistochemical staining

Description

Specific expression of NDRG2 gene in dendritic cells and detection of dendritic cells using the gene and protein expression pattern}             

Figure 1a is expressed in dendritic cells induced differentiation for 7 days in culture conditions containing recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant IL-4 (Interleukin 4) in monocytes isolated from human peripheral blood The resultant antigen was combined with an antibody labeled with fluorescence and analyzed using a flow cytometer.

Figure 1b is the result of observing the morphology of the cells of the differentiated dendritic cells using an electron microscope (SEM).

2 is a result of comparing the expression of the NDRG2 gene with the differentiation time of dendritic cells and the expression of the NDRG2 gene in different types of immune cell lines by Northern blotting.

Figure 3a is differentiated for 12 days with recombinant GM-CSF, recombinant IL-4, recombinant SCF (Stem cell factor) and recombinant TNF-α (Tumor necrosis factor-α) in stem cells isolated from human umbilical cord blood (CD34 ⁺ ) The antigen of the dendritic cells was analyzed and confirmed by flow cytometry.

Figure 3b is a result of comparing the degree of expression of NDRG2 gene and optical micrographs of the differentiation process of dendritic cells differentiated from human stem cells compared with dendritic cells differentiated from monocytes.

Figure 4a is to differentiate the MUTZ-3 hematologic cancer cell line into dendritic cells for 4 days in a medium containing recombinant GM-CSF, recombinant IL-4 and recombinant TNF-α, and then analyzed the antigen expressed on the cell surface by flow cytometry This is the result of comparative analysis of cell morphology with optical microscope.

Figure 4b was compared and confirmed the expression of NDRG2 gene in the dendritic cells differentiated from human peripheral blood monocytes and MUTZ-3 cell line, dendritic cells differentiated from MUTZ-3 cell line by Northern blotting, the degree of gene expression according to differentiation time Observed.

5 is a result of comparing and observing expression of NDRG2 gene after culturing HL-60, THP-1 and U937 cell lines similar to monocytes under the same conditions of differentiating dendritic cells from primary monocytes derived from blood. .

Figure 6a is a result of observing the differentiation of dendritic cells using a flow cytometer after treatment of differentiation of dexamethasone (Dexamethasone) and vitamin D3 from monocytes to dendritic cells, which is known to inhibit the differentiation of dendritic cells.

Figure 6b is the result of observation by Northern blotting whether the expression of the gene of NDRG2 is also inhibited in dendritic cells suppressed differentiation.

7A shows the results of Northern blotting confirming the expression of NDRG2 gene in dendritic cells differentiated from U937 cell line transfected with NDRG2 gene, cell line transfected with vector only, U937 cell line, and peripheral blood monocytes.

Figure 7b is the result of confirming the expression of NDRG2 protein in dendritic cells differentiated in MUTZ-3 cell line and NDRG2 gene transduced by Western blotting using NDRG2 polyclonal antibody.

FIG. 8 shows the results of immunohistochemical staining using NDRG2 polyclonal antibody to specifically express NDRG2 protein in dendritic cells of human lymph node tissue. FIG.

The present invention relates to a NDRG2 gene specifically expressing dendritic cells and a method for detecting dendritic cells using the same, and more particularly, specific to dendritic cells induced differentiation in human peripheral blood and umbilical cord blood and blood cancer cells. NDRG2 (N-myc downstream regulated), which can be used to obtain differentiation-induced dendritic cells, their purity and characterization during clinical use of dendritic cells in intractable diseases and chemotherapy. gene 2) relates to a gene and a method for detecting dendritic cells using the same.

Dendritic cells are antigen-presenting cells (APCs) that are essential for antigen presentation and immune cell function. The dendritic cells are also unique immune cells capable of inducing a primary immune response that can stimulate naive T cells that have never been exposed to antigen. Therefore, studies on the role of dendritic cells in infectious disease and tumor immunity are actively conducted, and in the case of refractory disease without special measures for prevention and treatment, specific immune responses using dendritic cells are applied. In order to develop an induction vaccine, many researchers are trying to elucidate the biological function of the cells through the study of the characteristics of the dendritic cells and the expression of genes specifically expressed. In the future, dendritic cells are expected to be widely used as vaccines that induce immune responses against specific antigens and as cells that can be used to act as modulators to easily modulate immune responses.

As a result of ongoing clinical studies, it has been reported that the application of dendritic cells as a cell vaccine or an immune vaccine is effective in treating tumors. However, there are difficulties in obtaining a large amount of dendritic cells and a problem in which markers specific to these cells are not found yet. In addition, there is a difficulty in clinical applications because the characteristics of dendritic cells are not fully understood. Therefore, if the study of dendritic cell characteristics is in-depth, the search for specific expression of genes or proteins, and the study of its function makes an effective method, dendritic in the treatment and prevention of various diseases in clinical Cells will be available.

On the other hand, NDRG2 (N-myc downstream regulated gene 2) gene found in the present invention as reported so far, mainly as a gene that is expressed in a variety of tissue cells, such as brain, heart, muscle, or kidney cells, There have been no reports of expression in immune cells [Qu et al ., 2002; Mol Cell Biochem. 229: 35-44, Kokame et al ., 1996; J Biol Chem. 271: 29659-29665, Ulrix et al. 1999; FEBS Lett. 455: 23-26.

NDRG gene has been reported four different genes, they have a high homology (Homology), and it can be seen that the expression form is changed according to the development and growth of the individual. NDRG1 is expressed in relatively all tissues and cells, whereas NDRG2 and NDRG3 are expressed in brain, heart, muscle and kidney cells, and NDRG4 is limited in brain and heart cells.

Thus, although these genes are expected to function differently, there are no reports on their exact function yet.

On the other hand, in the method of detecting dendritic cells, there are conventional methods for determining the presence of dendritic cells mainly based on the expression level of surface antigens, but these methods do not use antigens specific to dendritic cells, The problem of having to determine the relative purity by comparing the expression of is raised. In addition, the use of the expression of these surface antigens in identifying dendritic cells in vivo has not provided an absolute criterion.

Therefore, the present inventors have studied the dendritic cells that activate the immune system to combat intractable disease and cancer, the results showed that the N-myc downstream regulated gene 2 (NDRG2) gene in human peripheral blood monocytes, umbilical cord blood stem cells or cancer cells It was confirmed that the expression is specific only to differentiated dendritic cells, there is no expression in other kinds of immune cells, to obtain a cell line transduced with the NDRG2 gene, and to prepare the NDRG2 recombinant protein to confirm the expression of the NDRG2 protein After immunization in mice, polyclonal antibodies were obtained to confirm the expression of NDRG2 protein in the transduced cell line, and the present invention was confirmed by confirming the possibility of studying the characteristics of dendritic cells using the cell line and the NDRG2 antibody. It was completed.

Accordingly, an object of the present invention is to provide the genes and gene products as markers of dendritic cells by confirming the expression of NDRG2 gene, which is specifically expressed only in dendritic cells, unlike other immune cells.

Another object of the present invention is to provide a method for easily detecting dendritic cells with only the NDRG2 gene, unlike conventional dendritic cell detection methods.

The present invention is characterized by a NDRG2 (N-myc downstream regulated gene 2) gene specifically expressed in dendritic cells differentiated from human peripheral blood monocytes, umbilical cord blood stem cells or cancer cells, and a method for detecting dendritic cells using the same. .

Referring to the present invention in more detail as follows.

As a method for differentiating human dendritic cells in a laboratory, two methods are widely used depending on the kind of precursor used for differentiation. Differentiation from monocytes isolated from human peripheral blood and stem cells from umbilical cord blood. In the present invention, first, dendritic cells which differentiated monocytes isolated from human peripheral blood for 7 days were used, and antigens such as CD1a, CD54, CD80, CD83, CD86 and HLA-DR were well developed on the cell surface by flow cytometry. It was confirmed that there is [Fig. 1a and 1b]. As a result, it was confirmed that the dendritic cells used in the present invention are not significantly different from those of previously reported dendritic cells. On the other hand, CD34-positive stem cells are isolated from cord blood using MACS (Magnetic activated cell sorting), which utilizes antibodies specific for CD34 antigen in cord blood, and cultured for 12 days in the presence of cytokines to differentiate into dendritic cells. After making, using the flow cytometer in the same manner as described above it was confirmed that the dendritic cells similar to those reported so far [Fig. 3a].

The present invention searches for a specific gene possessed by dendritic cells by comparing and analyzing genes expressed in dendritic cells and genes expressed in monocytes and stem cells as precursors thereof. The NDRG2 gene (GenBank Accession No .: NM_016250) was confirmed to be specific. The cDNA of the NDRG2 gene was obtained by reverse transcription and then amplified by a PCR method and cloned using a pEZ vector as a cloning vector. The NDRG2 gene is in Chromosome 14, with an mRNA size of 2 Kb and 1074 nucleotides making about 40 KDa of protein.

The NDRG2 clone thus obtained was used to synthesize a probe with ³² P-CTP labeled with radiation, and then compared with expression patterns in other immune cells by Northern blotting. The expression of NDRG2 was observed in U937, a lymphoid cell line, THP-1 and HL-60 cell lines, and B-cell lines Wil2NS, BJAB, T-cell line Molt4, natural killer cell line NK92, and monocytes isolated from human peripheral blood. There was no gene expression in dendritic cells induced differentiation in monocytes [Fig. 2].

In the experiment of inducing differentiation of dendritic cells in CD34 positive stem cells, expression of NDRG2 gene appeared as differentiation progressed and it was confirmed that its expression was increased [FIG. 3B].

Recently, research has shown that MUTZ-3 cell line, a type of blood cancer cell, can differentiate into dendritic cells. Therefore, MUTZ-3 cell lines were differentiated into dendritic cells according to the reported method [FIG. 4A], and the expression of the NDRG2 gene was also confirmed in these [FIG. 4B].

To confirm that the expression of this gene is specific to dendritic cell differentiation rather than the effect of the added cytokine, cytokines with the same conditions were applied to the U937, THP-1 and HL-60 cell lines, a type of blood cancer cell line. When the cells were added and cultured to induce differentiation and confirmed that the NDRG2 gene was expressed, these cell lines did not differentiate into dendritic cells, and thus, expression of NDRG2 was not observed [FIG. 5].

Dexamethasone and vitamin D3, which inhibit the differentiation of dendritic cells, were added to the medium used for differentiation of dendritic cells and then induced to differentiate into dendritic cells. Reduction of the expression of highly expressed CD1a, CD80, and HLA-DR antigens was confirmed by flow cytometry to confirm that the differentiation of dendritic cells was inhibited [Fig. 6a], Northern blotting that the expression of the NDRG2 gene in these is reduced It was confirmed as [Fig. 6b].

On the other hand, cloned the NDRG2 gene into the pcDNA3.1 vector and transfected the U937 cell line with electrophoration to obtain a clone, and Northern blotting confirmed that the gene was well transduced in the clone. Could be [FIG. 7A].

In addition, genes were cloned into the pET28a vector to make NDRG2 antibodies, and then transformed into BL21 Escherichia coli to synthesize NDRG2 recombinant proteins. 30 μg of the NDRG2 recombinant protein and the same amount of Complete Freund adjuvant were mixed well until emulsification and injected into the abdominal cavity of mice. After four booster injections at 10-day intervals, mouse serum was isolated to obtain an NDRG2 polyclonal antibody. As a result, the expression of NDRG2 in the U937 cell line transduced with the NDRG2 gene was confirmed in the Western blotting experiment using the obtained NDRG2 polyclonal antibody [FIG. 7B]. In addition, the expression of this protein, which was weakly expressed in the MUTZ-3 leukemia cell line, induced differentiation into the dendritic cell form (MUTZ-3-DC), it was confirmed that the expression increased significantly (Fig. 7b).

Finally, the immunohistochemical staining method using the NDRG2 antibody was used to determine whether the protein produced by this gene was specifically expressed in dendritic cells in human lymph nodes. As a result, it was confirmed that NDRG2 protein is specifically expressed in dendritic cells in the paracortical area where T lymphocytes and iDCs (interdigitating dendritic cells) are mainly present in the lymph nodes, whereas B lymphocytes are mainly present. In the germinal center, which is known to be present, follicular dendritic cells (FDC), which are well known to have a different origin from iDCs, are present in which the expression of this protein is very weak or not at all [FIG. 8]. Therefore, NDRG2 could be confirmed to be specifically expressed in dendritic cells including iDCs.

Therefore, the present invention is expected to be useful to study the degree of differentiation and characteristics of dendritic cells by using the NDRG2 gene and the product of the gene is specifically expressed in dendritic cells.

In particular, a gene specifically expressed in the dendritic cells in a method for detecting existing dendritic cells by hybridizing the RNA obtained from the cells or their fragments with the gene specifically expressed in the dendritic cells to determine whether to hybridize By confirming that the NDRG2 gene is very useful, a dendritic detection method using the same is included in the present invention.

The present invention also includes a method of detecting dendritic cells by confirming that NDRG2 protein is specifically expressed in dendritic cells using NDRG2 polyclonal antibody.

It is expected to be useful for the treatment of intractable disease or cancer by producing a cell vaccine or an immune vaccine using the dendritic cells thus detected.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1: Differentiation of Dendritic Cells

Human blood was isolated from Peripheral blood mononuclear cells by Density centrifugation using Histoqueque 1077, and the cells were attached to a T-75 plastic incubator. After about 3 hours, the cells attached to the plastic incubator were incubated for 7 days in RPMI 1640 medium containing 400 ng / ml recombinant GM-CSF, 20 ng / ml recombinant IL-4, and 10% FBS. Cells were obtained [FIGS. 1A and 1B].

Stem cells were isolated from cord blood using MACS for dendritic cell differentiation using stem cells isolated from human cord blood (CD34 ⁺ ), and 400 ng / ml recombinant GM-CSF and 2.5 ng / ml recombinant TNF-α for 5 days. , Dendritic cell differentiation was induced by culturing in RPMI 1640 medium containing 20 ng / ml recombinant SCF, 10% FBS and then incubating for 7 days in medium in which SCF was replaced with 20 ng / ml IL-4 [FIG. 3A and FIG. 3b].

Example 2: Antigen Screening of Dendritic Cells Using Flow Cytometry

In order to search for the characteristics of the dendritic cells differentiated in Example 1, the antigen expressed on the cell surface was combined with an antibody labeled with fluorescence and analyzed by flow cytometry.

First, 1 to 5 × 10 ⁵ cells were washed twice with PBS, followed by CD80, CD83, and CD86 monoclonal conjugated CD1a, CD54, HLA-DR monoclonal antibody (Phycoerythrin) with FITC (Fluorescein isothiocyanate) The antibody was reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed twice with PBS, and FITC or PE fluorescence was measured using 530 nm and 575 nm filters to detect the amount of antigen expressed on the cell surface.

Example 3: RNA Separation and Northern Blotting of NDRG2 Gene in Immune Cell Lines and Dendritic Cells

Acid cell lines HL-60, U-937, THP-1, Wil2-NS, BJAB, Molt-4, NK- by acid guanidinium thiocyanate-phenol-chloroform extraction 92 and total RNA was isolated from peripheral blood, monocytes and dendritic cells. First, the cultured cells (2-10 × 10 ⁶ ) were washed with PBS, and the cells were lysed with 4M guanidine isothiocyanate containing 100 mM 2-mercaptoethanol and 2M sodium acetate, followed by phenol and chloroform-iso. After reacting with amyl alcohol for 1 hour at 4 ° C., centrifuged for 30 minutes, 600 μl of the supernatant was recovered, the same amount of isopropanol was added, the reaction was carried out at −20 ° C. for at least 1 hour, and further centrifuged to obtain RNA pellets. The obtained RNA was dissolved in water containing 0.1% DEPC after drying, and the concentration and purity of the RNA were measured using a UV spectrophotometer.

The obtained RNA was subjected to electrophoresis on a 1.2% denatured agarose gel to confirm RNA bands of 18S and 28S, and then transferred to a nylon membrane to obtain a blot. The base sequence was 5'-ATGGCGGAGCTGCAGG-3 '( Chain polymerization of NDRG2 in 35 cycles (95 ° C. 1 minute, 57 ° C. 1 minute, 72 ° C. 1 minute) using primers SEQ ID NO: 1) and 3'-TCAACAGGAGACCTCCAT-5 '(SEQ ID NO: 2) Northern blot expression of NDRG2 gene in each cell by labeling ³² P radiation using a 1073 bp NDRG2 gene obtained through the N-B as a probe, hybridizing to an RNA blot and exposing it to an X-ray film for 2 days. It was confirmed as [Fig. 2].

Example 4: Differentiation of Dendritic Cells Using MUZT-3 Cells and Northern Blotting of NDRG2 Gene

Induce differentiation into dendritic cells using a method published in the Blood Journal in 2002 by Masterson et al. (Blood 100; 2, 701-703, 2002) to differentiate a cell line called MUTZ-3, a type of blood cancer cell, into dendritic cells. It was.

First, a 5 × 10 ⁵ cells / ml MUTZ-3 cell line was prepared with 400 ng / ml recombinant GM-CSF and 10 ng / ml recombinant IL-4, 2.5 ng / ml recombinant TNF-α, and α-MEM containing 20% FBS. Differentiation was induced into dendritic cells by incubation for 4 days in the medium. Searching for the antigen expressed on the surface of the MUTZ-3 cell line induced differentiation into dendritic cells was detected the expression level of the antigen in the same manner as the flow cytometry method described in Example 2 [Fig. 4a].

In addition, the total RNA of dendritic cells differentiated from MUTZ-3 and MUTZ-3 and dendritic cells differentiated from monocytes was isolated in the same manner as described in Example 3, and then the expression of NDRG2 gene was searched by Northern blotting. 4B.

Example 5 Inhibition of Dendritic Cell Differentiation and Northern Blotting of NDRG2 Gene Using Dexamethasone and Vitamin D3

In order to inhibit dendritic cell differentiation, the method described in Example 1 was used to differentiate dendritic cells from monocytes to dendritic cells, followed by incubation for 10 days with 10 ⁻⁸ M of dexamethasone or vitamin D3, and then using the flow cytometry method. It was confirmed that there is an inhibition in the surface antigen expression of [Fig. 6a].

In addition, the total RNA was isolated from dendritic cells inhibited by differentiation by dexamethasone and vitamin D3 in the same manner as in Example 3, and then the expression level of the NDRG2 gene was confirmed by northern blotting. 5'-GCCATGTACGTTGCTATCCAGGCTG-3 '(SEQ ID NO: 3) and 3'-AGCCGTGGCCATCTCTTGCTCGAAG-5' for the use of β-actin as a control.

Using (SEQ ID NO: 4) as a primer, 300 bp β-actin gene obtained through RT-PCR of 30 cycles (95 ° C 1 minute, 54 ° C 1 minute, 72 ° C 1 minute) was used as a probe. Northern blotting in the same manner (FIG. 6b).

Example 6: Gene Delivery and Clone Selection by Electro-stimulation

In the TOPO vector cloned with the NDRG2 gene, the NDRG2 gene was cut out with restriction enzymes XhoI and BamHI and then conjugated to the pcDNA3.1 vector. 1.3 × 10 ⁷ cells were suspended in 300 μl RPMI 1640 medium containing pcDNA3.1 vector conjugated with 10 μg of NDRG2 gene. After standing at room temperature for 15 minutes, the electric shock at 300 volt, 960 ㎌, and after 15 minutes at room temperature mixed with RPMI 1640 medium containing 10% FBS and maintained in 37 ℃ CO ₂ incubator. After 24 hours, clones were selected by dispensing in 96-well plates to 0.5 cells / well in medium containing G418 to a final concentration of 1 mg / ml. Northern blots were compared to dendritic cells to select clones transfected with the NDRG2 gene. The mRNA of the NDRG2 gene has a size of 2 Kb, and the 1074 nucleic acid (Neucleotide) that makes the protein is combined with several proteins for the transcription of the protein and an untranslated portion that helps stabilize the mRNA. region) and 926 nucleic acids. However, in the case of NDRG2 cloned into pcDNA3.1 vector, only 1074 nucleic acid sites that make proteins are contained in the vector, and when translated into mRNA, 250 nucleic acids, which are untranslated portions of the vector, are transcribed and mRNA The size of is about 1.3 Kb. Thus, Northern blotting results in differences in the size of NDRG2 mRNA in dendritic cells and the size of NDRG2 mRNA in transduced cells. However, the size of the NDRG2 protein they make did not change (Fig. 7b).

Example 7: Expression of recombinant NDRG2 protein in E. coli cells, isolation of protein and production of mouse polyclonal antibody using the same

The NDRG2 gene cloned in the pEZ vector was cut using restriction enzymes BamH I and Xho I, conjugated to the pET28a vector, an E. coli expression vector, and transformed into BL21 Escherichia coli to obtain an NDRG2 recombinant protein.

To obtain immunized mice for the development of NDRG2 antibodies, 30 μg recombinant NDRG2 protein and the same amount of Complete Freund Adjuvant were mixed well until emulsified and injected intraperitoneally of 4-6 week old Balb / c mice. . After a total of four booster injections at 10-day intervals, mouse serum was isolated to obtain NDRG2 polyclonal antibodies. As a result, the expression of NDRG2 in the U937 cell line transduced with the NDRG2 gene was confirmed in the Western blotting experiment using the obtained NDRG2 polyclonal antibody [FIG. 7B].

Example 8: Specific expression of NDRG2 protein in dendritic cells present in human lymph nodes by immunochemical staining with polyclonal antibodies

Human lymph nodes were fixed with formalin in 10% buffer and paraffin blocks. This was cut into 3 μm thick sections and placed on slides covered with 3-amino-propyltriethoxysilane. In a 10 mM sodium citrate buffer (pH 6.0) (Sodiumm nitrate buffer) was heated up to 4 minutes in an autoclave to allow paraffin removal and antigen recovery. Mouse NDRG2 polyclonal antibody was diluted 1: 3200 using background reducing diluent (Dako, Carpinteria, CA). Immunostaining was performed using the EnVision Kit (Dako). After preincubation with blocking serum for 15 minutes, NDRG2 polyclonal antibody was added as a primary antibody, incubated for 30 minutes in a wet chamber, and washed with PBS. The slides were incubated with peroxidase reagent (Dako) in the EnVision Kit for 30 minutes and then the slides were continuously incubated with DAB (3,3-diaminobenzidine) chromogen for 5 minutes. The slides were then counter-stained with Meyer's hematoxylin and framed. Careful washing was performed by changing PBS several times between each of these processes. Staining without the addition of a primary antibody was used as a negative control.

As a result, it was confirmed that NDRG2 protein is specifically expressed in dendritic cells in the paracortical area where T lymphocytes and iDCs (interdigitating dendritic cells) are mainly present in the lymph nodes, whereas B lymphocytes are mainly present. In the germinal center, which is known to be present, follicular dendritic cells (FDC), which are well known to have a different origin from iDCs, are present in which the expression of this protein is very weak or not at all [FIG. 8]. Therefore, it was confirmed that NDRG2 is specifically expressed in dendritic cells including iDCs.

As described above, the NDRG2 gene according to the present invention is specifically expressed only in human dendritic cells to isolate, culture, purify dendritic cells and study dendritic cell function, and furthermore, in the cell therapy process using dendritic cells. It can be utilized.

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Claims (4)

delete delete In the method of detecting dendritic cells by hybridizing the RNA obtained from the cells or their fragments with the genes specifically expressed in the dendritic cells to determine whether the hybridization, The method for detecting dendritic cells, characterized in that the gene specifically expressed in the dendritic cells is NDRG2 (N-myc downstream regulated gene 2) gene. In the method of detecting dendritic cells by confirming the expression of a protein using an antibody, Dendritic cell detection method characterized by confirming the expression of NDRG2 protein using the NDRG2 (N-myc downstream regulated gene 2) polyclonal antibody as the antibody.

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