Departments of 1Pediatrics and 4Medicine, Division of Gastroenterology and Nutrition, Mattel Children's Hospital, David Geffen School of Medicine at University of California Los Angeles, Los Angeles 90095; 2Department of Biology, California State University Northridge, Northridge, California 91330; and 3Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
Submitted 20 March 2003 ; accepted in final form 13 January 2004
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ABSTRACT |
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passive immunity; development; ontogeny; immunoglobulin
The receptor that transfers maternally derived IgG to the systemic circulation of suckling animal is the Fc receptor of the neonate (FcRn) (28). FcRn is expressed by syncytiotrophoblast of the placenta, where it mediates the transplacental transfer of IgG (14). In the small intestine of suckling mammals, FcRn is expressed by enterocytes located along the entire length of the crypt-to-villus axis and is responsible for the uptake of luminal IgG across the epithelial layer (1). In rodents, FcRn mRNA levels are most abundant in the duodenum, and steadily decline along the horizontal axis of the small bowel (10, 18). In humans, FcRn has been detected in intestinal epithelial cells, where it may play a role in sampling luminal antigens and possibly mediate the transfer of IgG during breast feeding (27).
FcRn is a heterodimer that is noncovalently bound to 2-microglobulin and is related to the family of nonclassic major histocompatibility complex (MHC) class Ib proteins (23). FcRn transports IgG, in a process termed transcytosis, from apical (intestinal lumen) to basolateral membranes (systemic circulation) (2). At the apical membrane, IgG-FcRn complexes form at an acidic pH and are endocytosed in clathrin-coated vesicles, which subsequently sort to intermediate compartments before trafficking to the basolateral membrane. At the basolateral membrane, the complex dissociates at the neutral pH, and IgG is released into the systemic circulation (25). Overall, FcRn serves an essential role in controlling the transfer of IgG from the maternal to the fetal/newborn circulation of all mammals.
In rodents and other mammals, the systemic humoral IgG immune system matures shortly after the cessation of breast feeding, and steady-state FcRn mRNA levels in the small bowel decline dramatically in weaned animals to nearly undetectable levels (10, 17, 18). The FcRn mRNA transcript has also been detected at much lower quantities in liver and vascular endothelial cells compared with levels seen in the small bowel of suckling animals (2). In vascular endothelial cells, FcRn has been shown to represent the "Brambell receptor" and control the extremely low rate of IgG catabolism (3, 11). Interestingly, hypogammaglobulinemia attributable to rapid IgG catabolism has been described in myotonic dystrophy and in a rare autosomal recessive disorder (OMIM 241600 [OMIM] ) (31, 32). Therefore, in humans, the FcRn protein controls serum IgG levels and may sample luminal antigens for presentation to the mucosal adaptive immune system.
Insufficient information is currently available regarding the regulation of the FcRn expression. Corticosteroids, thyroxine, EGF, and insulin have all been shown to decrease IgG transfer via the intestinal enterocyte of suckling animals (30). We have reported that the administration of either corticosteroids or thyroxine to suckling pups abolishes both IgG absorption and FcRn mRNA expression in a dose- and time-dependent manner (16). The reduction of FcRn expression in endothelial cells may explain the benefit of steroids and intravenous immunoglobulins in the management of various autoimmune disorders (16, 33).
Whereas the sequences of the human and murine FcRn genes have been reported, the elements that are critical in directing FcRn promoter activity have not been identified (12, 22). In a recent analysis of the human promoter, several elements were identified that form complexes with nuclear extracts; however, the study failed to adequately define the important elements that drive FcRn gene expression (22). To further characterize the mechanism of how the FcRn gene is regulated, we cloned the upstream region of the rat gene and identified the cis-acting elements that control its basal expression.
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MATERIALS AND METHODS |
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Approximately 106 recombinant phage clones from a rat (Sprague Dawley) genomic library (Stratagene) were plated and blotted in duplicate on nitrocellulose filters. The filters were screened with a PCR-derived cDNA corresponding to nucleotides 500 to 1000 by standard methods (19, 28). Liquid phage stocks were produced of the phage clones that remained positive after tertiary screening, and we performed genomic mapping by digesting DNA with partial or double restriction enzymes that included BamHI, EcoRI, HindIII, and SmaI (New England Biolaboratories). DNA was fractionated on a 0.4% agarose gel, transferred to nitrocellulose filters, and hybridized to [-P32]dATP (New England Nuclear Research Products) end-labeled oligonucleotides including T3, T7, and regions internal to the FcRn cDNA.
Intron/Exon Mapping and DNA Sequence Analysis
Genomic DNA containing each of the exons was digested, subcloned into Bluescript SKI (), and purified by the standard Qiagen method. Each exon, the intron/exon boundary and 2.9 Kb of the 5' upstream region, was sequenced by the dideoxy method with [-35S]dATP (New England Nuclear Research Products) and Sequenase 2 (United States Biochemical).
RNase Protection and Northern Blot Analysis
RNase protection assay was performed according to previously described methods (10). Specifically, total RNA was isolated by standard guanidine-phenol extraction methods and hybridized to radiolabeled -sense riboprobe. After overnight incubation in a specific buffer, the mixture will be digested with RNaseA/T1 mixture for 30 min and electrophoresed on a polyacrylamide gel. Northern blot analysis was performed with 20 µg of total RNA isolated as previously described from Caco-2, HT-29, IEC-6, and endothelial SV40 cells (29). The human (Caco-2 and HT-29) and rodent (IEC-6, endothelial SV40) RNA samples were hybridized to the human or rat cDNA probe, respectively.
DNA Cloning and Substitution Mutagenesis
The FcRn #1 phage clone was digested with EcoRI, and the 3.3-Kb fragment was subcloned into a similarly digested pBluescript vector. The vector was then digested with BamHI, and a 2.9-Kb fragment was subcloned into the BglII site of the reporter clone pGL3-basic (Promega). The orientation was confirmed by sequencing, and the pGL3-basic 2.9-Kb FcRn clone was used to generate nested deletion clones whose size was confirmed by sequencing. Six sense, 450/430 (5'-gca gat ctT CCT GAC AAG ATC TTG GGT TG), 300/283 (5'-gca gat ctG AGA GTG GGC TGC AGC AGG), 216/198 (5'-gca gat ctT GCT CAG AAT GAG TAA ACA C), 157/139 (5'-gca gat ctT GCT CAG AAT GAG TAA ACA C), 101/95 (5'-gca gat ctC CCC AGG AGG CTT CCA GA), and 58/40 (5'-gca gat ctT GCT TAA GAG CTC GTG GGG T) and an antisense +113/+80 (5'-ata agc ttC CCT CTT CCT CAC AGA AGC C) oligonucleotides were developed to amplify various shorter-length products from rat genomic DNA. The fragments were digested with HindIII/BglII (restriction site indicated in lowercase lettering) and subcloned into the pGL3-enhancer luciferase reporter vector (Promega). Clones rFcRn450/+135/E-Luc, rFcRn300/+135/E-Luc, rFcRn216/+135/E-Luc, rFcRn157/+135/E-Luc, rFcRn101/+135/E-Luc, and rFcRn58/+135/E-Luc were sequenced to define their size and authenticity.
To define the nucleotides within the core promoter that were critical to maintain the gene's basal level of expression, 20 clones containing 10-bp mutations were produced by standard scanning mutagenesis. The location of each mutation is shown in Fig. 1. Mutagenizing oligonucleotides that contain 10-bp central transversion mutations (A-C, G-T) were designed and used to perform PCR with the antisense (+113/+80) oligonucleotide (Table 1). The PCR products were gel purified and with the addition of a sense (157/139) oligonucleotide, a second PCR reaction was performed to generate clones with 10-bp mutations. The full-length fragments were cut with BglII/HindIII, gel purified, and subcloned into the pGL3-E vector. All clones were sequenced to verify the presence of only the desired mutation.
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Cell Transfection Procedure
Caco-2, 3T3, Chinese hamster ovary (CHO), HeLa, and m-ICcl2 (kindly provided by Alain Vandewalle) cells were transfected with a calcium phosphate reagents as previously described (21). IEC-6 cells were electroporated with a BioRad RF module. Each pulse had 10 µg of luciferase construct and 1 µg of renilla vector for 4 x 106 cells. This mixture was then pulsed at 0% modulation, 0.2 kV, 50 Hz, for 2 ms, with 1-s intervals for five bursts. Cells were incubated for 48 h, rinsed with PBS, and lysed in SDS containing buffer (Analytical Luminescence Laboratory), and extracts were stored at 80°C. Both luciferase and renilla were measured as previously described (29).
Drosophila SL2 cells were maintained in Schneider's insect medium supplemented with 10% fetal bovine serum and antibiotics and were grown at 25°C without CO2. Transfection of SL-2 cells was performed as previously described (13). Specifically, 200 ng of rFcRn157/+135/Gal were transfected with various amounts of Sp1 or Sp3 expression plasmids containing the Drosophila actin 5 promoter (pPacSp1 and pPacUSp3 were generously provided by Drs. R. Tjian and G. Suske, respectively).
-Gal activity was expressed as relative light units per milligram of protein. The protein concentration was determined by bicinchoninic acid protein assay (Pierce).
DNase I Footprint Analysis
DNase I footprint analysis was performed to define the approximate region of protein-DNA interactions as previously described (20). The rFcRn216/+135/E-Luc vector was cut with HindIII, kinased with [-P32]dATP (6,000 Ci/mol) and then cut with BglII. Samples were electrophoresed on a 5% polyacrylamide gel, purified, and diluted to 6,000 counts/min (cpm)/µl. Footprints were performed with Caco-2 nuclear extracts that were dialyzed with 0.1 M KCl. Either nuclear extracts (6 µg) or BSA was mixed with probe (6,000 cpm), and serial dilution of DNase I was added to the reaction buffer [in mM: 40 CaCl2, 10 HEPES (pH 7.9), 0.1 EDTA, 0.05 KCl, 28 2-
-mercaptoethanol, and 0.1 BSA, with 10% glycerol]. Samples were purified with phenol and chloroform, precipitated with ethanol, and run on a 6% polyacrylamide gel for 100 min at 50 W. The locations of the protein/DNA binding sites were determined by running a DNA ladder sequenced by the Maxam-Gilbert method.
EMSA and Preparation of Nuclear Extract
Nuclear extracts were obtained from both Caco-2 cells by standard methods as previously described, and recombinant Sp1 was also used for a limited number of studies (Promega) (21). EMSA was performed using a series of double-stranded oligonucleotides, which were labeled with Klenow fragments. The identities of each oligonucleotide used in this study are included in results. Approximately 2 x 104 cpm of probe and 2.55 µg nuclear extracts were used for each reaction. Standard competition studies were run using excess unlabeled oligonucleotide duplexes. Antisera against Sp1, Sp2, and Sp3 were purchased from Santa Cruz Biotechnology and were used to perform supershift studies.
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RESULTS |
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We performed Northern blot analysis for the FcRn mRNA transcript in several cell lines including Caco-2, HT-29, IEC-6, and a rodent endothelial (SVEC) cell line. As shown in Fig. 2B, the FcRn transcript was readily abundant in each of the cell lines tested, and these data were interpreted to suggest that these cells lines would be good candidates to perform functional analysis of the FcRn promoter.
DNase I Footprint Analysis of Core Promoter Identifies Several DNA-Protein Complexes
To determine the approximate location of the cis-elements that bind to nuclear extracts, Caco-2 extracts were used to perform DNase I footprint analysis of the core promoter region. These data demonstrate that as many as two distinct footprints (26 to 61 and 90 to 121) and five hypersensitivity sites were identified within the gene's immediate promoter region in the presence of Caco-2 nuclear extracts (Fig. 2C).
Transient Transfection of Clones Containing Various Lengths of the Upstream Region of the Rat FcRn Gene in Intestinal Cell Lines Identified the Core Promoter
To begin assessing the role of the various regions of the 5'-upstream portion of the FcRn gene, chimeric-luciferase reporter clones were transiently transfected into both Caco-2 and IEC-6 cells. Transfection of the full-length (rFcRn2940/+14) and sequentially shorter reporter constructs resulted in reporter activity in both cell lines that ranged from 100- to 40-fold higher than the empty pGL3-basic vector (data not shown). Comparable data were obtained with several clones that contain a shorter length of the 5' region of the FcRn promoter region subcloned upstream of the reporter vector pGL3-enhancer (Fig. 3A). Reporter activity declined abruptly with clones shorter than FcRn157/+135, suggesting that the core promoter region of the FcRn gene is located within nucleotides 157 and +135.
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To assess the cell specificity of the core promoter region of the FcRn gene, we transiently transfected several intestinal (Caco-2, IEC-6, and m-ICcl2) and nonintestinal (3T3, CHO, and HeLa) cell lines. Maximum expression was obtained in the three intestinal cell lines, with the most abundant expression occurring in Caco-2 cells (Fig. 3B). Whereas 3T3 cells supported the expression of the reporter, promoter activity in both CHO and HeLa cells was only marginal. These data suggest that the core promoter region (nt 157 and +135) of the FcRn gene is sufficient to drive abundant expression in primarily cells of intestinal origin.
Site-Directed Mutagenesis of the Core Promoter Revealed Two Critical Regions that Control Promoter Activity
To assess the critical regions within the core promoter (157 to +135) that are responsible for basal expression, a series of 20 clones was developed that contained 10 base pair mutations (Table 1 and Fig. 1). When these clones were transfected into either Caco-2 or IEC-6 cells, mutations in two specific regions were shown to dramatically influence promoter activity (Fig. 4). In Caco-2 cells, promoter activity of the wild-type 157/+135 clone was more than 120-fold greater than the empty pGL3-enhancer vector. Clones with mutations in the M8M11 regions had promoter activities that were 25% of the wild-type rFcRn157/+135 clone. In contrast, promoter activities of the clones containing mutations in M15M17 region were only 30% of the empty pGL3-enhancer vector. Similar albeit less dramatic results were obtained with IEC-6 cells.
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Sp-A, Sp-B, and Sp-C sites. To further evaluate the proteins responsible for controlling the basal promoter activity of the FcRn gene, we developed a series of double-stranded oligonucleotides to the M8M13 region of the promoter. EMSA performed with the Sp-A oligonucleotide that spans from 47 to 22 forms two specific complexes that supershift with antiserum to various members of the Sp family of transcriptional factors including Sp1, Sp2, and Sp3 (Fig. 6, lanes 19). Specifically, the slower migrating complex is formed by binding of Sp1, Sp2, and other unidentified nuclear proteins, whereas the faster complex was entirely supershifted with an anti-Sp3 antibody. The 47/22 probe was also capable of specifically binding recombinant Sp1 (Fig. 6, lanes 10 and 11), and competition with a group of duplex oligonucleotides containing mutations in regions that span 47 to 22 confirmed that the complexes form by interacting with regions located between 28 to 39 (data not shown). This Sp1 site was named Sp-A, and it corresponds to a classic Sp1 consensus sequence (Figs. 5 and 6).
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To further assess the characteristics of the Sp-D and Sp-E complexes, we performed EMSA-competition experiments with the unlabeled Sp-A, Sp-B, and Sp-C oligonucleotides. Competition of the Sp-D-E probe with either Sp-A or Sp-B entirely competed for binding of the Sp family of proteins while failing to compete for the slower migrating complex (Fig. 9, lanes 3133). Moreover, competition with the Sp-C and Sp-d-E oligonucleotides only partially competed for the Sp family of complexes, whereas the Sp-D-e oligonucleotides failed to compete entirely (Fig. 9, lanes 3436). In contrast, removal of the Sp-D site (using probe Sp-d-E) interfered with the ability of even the high-affinity Sp-A site to compete for the Sp-complexes, although still retaining the ability to compete with the unlabeled Sp-d-E oligonucleotide (Fig. 9, lanes 2530). Moreover, the slower migrating complex was completed by excess of either the Sp-d-E or Sp-D-e oligonucleotides but not by Sp-A, Sp-B, or Sp-C (Fig. 9, lanes 1924).
The identities of these complexes were assessed by supershift assay using antiserum specific for Sp1, Sp2, and Sp3. Labeling of the Sp-D-E oligonucleotides formed complexes that were supershifted with each of the three Sp antibodies confirming the identity of each complex (Fig. 10, lanes 19). Similar results were obtained when the Sp-E element was assessed using the Sp-d-E oligonucleotide (Fig. 10, lanes 1927). However, none of the antibodies was capable of interacting with the slower migrating protein that interacts with the Sp-D site as shown with the Sp-D-e oligonucleotides (Fig. 10, lanes 1018). Overall, these data were interpreted to suggest that the Sp-D element binds an unidentified protein complex that is neither Sp1, Sp2, nor Sp3 and that this complex facilitates the binding of Sp-proteins to the Sp-E element.
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Drosophila SL2 Cells were Used to Confirm the Role of Sp1 and Sp3 in Driving Activity of FcRn's Minimal Promoter
To further assess the role of the Sp family of proteins in inducing FcRn's promoter activity, the rFcRn157/+135/-Gal clone was cotransfected with various amounts of Sp1 (pPacSp1) and Sp3 (pPacUSp3) expression vectors in Sp-deficient Drosophila SL2 cells. The data demonstrate that increasing concentrations of Sp1 augments FcRn promoter activity more than fourfold, whereas Sp3 increases activity ninefold (Fig. 11, A and B).
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DISCUSSION |
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A previous analysis of the human FcRn gene suggested the role of GC elements in the control of promoter activity; however, selective mutations that assessed the function of these sites were not performed (22). In the current study, we took an unbiased approach and assessed the entire minimal promoter region for potentially active cis-elements by performing scanning mutagenesis. The immediate upstream region of the rat promoter contains several GC elements that were mutated in the M8M11 clones, and transcriptional activities of these clones were significantly reduced in both Caco-2 and IEC-6 cells (Fig. 4). These mutations disrupt binding of members of the Sp family of proteins to the Sp-A (40/29) and -B (59/50) sites and decrease activity to 25% of the 157/+135 wild-type clone (Fig. 4). EMSA and supershift assays confirmed that both sites bind to members of the Sp family of proteins, including Sp1, Sp2, and Sp3 (Figs. 6 and 7) (24). Specifically, the slower migrating complex was composed of Sp1, Sp2, and other unidentified complexes, whereas the faster migrating complex represents Sp3.
Although Sp1 is considered a ubiquitously expressed transcription factor, protein levels vary dramatically in different organs and cell lines (26). Therefore, members of the Sp family of proteins play an important role in the regulation of many tissue and developmentally specific genes (21). Moreover, the various members of the Sp-multigene family function as both transcriptional activators and repressors and may also interact directly with other transcriptional factors to control gene expression (6, 13, 26). Phosphorylation of the DNA-binding domain of Sp1 and other zinc finger transcriptional factors has recently been shown to inactivate protein-DNA interactions and to inhibit transactivation (5). Therefore, it is plausible that the multiple GC elements identified in the immediate promoter region of the FcRn gene may influence both the developmental and tissue expression of this gene.
Two additional Sp sites were also identified just downstream of Sp-A, which were labeled Sp-B (59/50) and Sp-C (78/69). In this region, the Sp-A element has the highest affinity to members of the Sp family of proteins, whereas the Sp-C site has lower affinity while also interacting with a yet unidentified complex. These findings are consistent with the transfection studies that showed disruption of the Sp-A site inhibited promoter activity to 25% of wild type, whereas mutation of the Sp-C site had luciferase levels that were 50% of wild type (Fig. 4). The slowest migrating complex identified with the Sp-A-B-c probe in Fig. 8 most likely represents multimerzation of the Sp family of proteins binding to the Sp-A and -B elements. The three Sp elements (Sp-A, Sp-B, and Sp-C) are on the sense strand and separated by 14 nucleotides (from the center of each element). The adjacent Sp sites should be capable of simultaneously binding Sp proteins because spatial constraints are minimized if the distance between the center of adjacent elements is greater than 10 nucleotides (4).
We also determined that the Sp-E site forms a complex with the Sp family of proteins, whereas the Sp-D element forms a complex with a slower migrating protein that appears to facilitate binding of Sp-proteins to the Sp-E element (Figs. 8 and 9). Interestingly, mutagenesis of these sites inhibited basal promoter activity more than 200-fold. Our interpretation of this data would be that disruption of either site (Sp-D and Sp-E) may either remove very potent positive transacting factors or, alternatively, could augment the binding of factors that suppress promoter activity. The current studies failed to decipher between these two possibilities. Whereas we confirmed that the Sp-D complex is neither Sp1, Sp2, nor Sp3, the data suggest that in its absence, the binding affinity of the Sp-E site for the Sp proteins declines considerably (Fig. 9). Moreover, the Sp complexes that form with the Sp-d-E probe could not be competed with an excess of even the high-affinity Sp-A and Sp-B elements, suggesting that a protein other than Sp1, Sp2, and Sp3 could be binding to Sp-E in the absence of the Sp-D complex (Fig. 9). The identity of the Sp-D complex remains unclear, and whether its removal augments the binding of other transcription factors to the Sp-E site has not been addressed in the current study.
Nevertheless, the transfection data do suggest that the absence of either the Sp-D or Sp-E sites does dramatically alter the activity of the core promoter region. Moreover, we hypothesize that the promoter activity of the FcRn gene may be controlled by either changes in the zinc concentrations, abundance of various Sp-proteins, or alteration of the phosphorylation state of zinc finger proteins (5). Each of these manipulations will influence binding of these various Sp proteins to the gene's core promoter and alter activity. The physiological relevance of these sites in controlling the developmental repression expression of the FcRn gene in the intestine of weaned mice is currently under investigation.
Overall, this study represents the first detailed analysis of the elements that control the expression of the FcRn gene in any species. We have identified six specific elements located in the immediate upstream of the rat FcRn promoter that interact with members of the Sp family of proteins (Fig. 5). The Sp proteins that interact with the Sp-A, -B, or -C sites moderately augment basal expression of the gene core promoter. In contrast, the complexes that form with the Sp-D and -E sites appear to dramatically repress basal expression of the gene. Nevertheless, the role that these elements have in controlling the expression of the FcRn gene in the developing animals has yet to be identified.
Whereas many genes that are expressed by small intestinal enterocytes are developmentally regulated at about the time of weaning, the FcRn and lactase-phlorizin hydrolase genes are the only well-characterized genes that decline in postweaned animals (9, 15). Whereas lactase and FcRn expression are most abundant in the proximal intestine of suckling rodents, the FcRn transcript declines at weaning to undetectable levels throughout the intestine. In contrast, lactase expression in the proximal bowel of postweaned mice declines 50% compared with their preweaned littermates, whereas expression levels in the distal intestine decline to undetectable levels (10). Therefore, FcRn appears to be rather unique among the various developmentally regulated genes of the small intestine, and further analysis of the mechanism that underlies its transcriptional control may lead to further insights into understanding the process of enterocyte development and differentiation.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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