Ludwig Institute for Cancer Research, Melbourne Branch, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
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ABSTRACT |
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The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.
immunoglobulin superfamily; intestinal epithelium
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INTRODUCTION |
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MOUSE MONOCLONAL ANTIBODY A33 (MAb A33) was elicited in mice injected with the human pancreatic carcinoma-derived cell line ASPC1 and found to react with a cell surface protein with a highly restricted pattern of expression. Immunohistochemical analysis of frozen sections of a range of normal human tissues revealed strong and almost exclusive expression of human A33 (hA33) antigen by intestinal epithelial cells, with a suggestion of weak staining in the salivary gland (12). In an analysis of thirty different tumor types, 95% of primary and secondary colorectal carcinomas and >50% of gastric carcinomas were found to express the hA33 antigen (12). Only a few instances of nongastrointestinal A33 antigen expression, including areas of intestinal differentiation in certain testicular and germ cell tumors, were detected (12). In view of this highly restricted pattern of expression, the hA33 antigen has attracted attention as a promising target for immunotherapeutic approaches to the treatment of metastatic colorectal carcinoma. Indeed, phase I/II studies have shown highly selective targeting of radiolabeled MAb A33 to tumors in patients with widely disseminated disease (38-40). Recently, to permit prolonged and repeated treatment regimes, a humanized version of A33 was produced and is currently undergoing phase I biodistribution studies.
The hA33 antigen was recently cloned and characterized (18). cDNAs were obtained from a cDNA library constructed from RNA extracted from a colorectal carcinoma-derived cell line, LIM1215 (18). The nucleotide sequence predicted a type 1 transmembrane protein of 298 amino acids (aa), comprising an extracellular domain with an NH2 terminal variable (V)-type Ig-like domain followed by one of the constant 2 (C2)-type (44), a single-span transmembrane domain, and a highly acidic cytoplasmic domain of 62 aa.
Here we describe the cDNA cloning of the mouse A33 (mA33) antigen, deduce its amino acid sequence, predict its molecular structure, and describe its pattern of expression and subcellular localization during development and in adulthood. On the basis of aa sequence identity and conservation of key structural residues, mA33 and hA33 antigens were found to be closely related to several recently described transmembrane proteins. These included the marker of cortical thymocytes in Xenopus (CTX) (8), its putative chicken ortholog ChT1 (22), mouse and human homologs of CTX (CTM/CTH) (7), and the receptor for group B Coxsackie viruses and adenoviruses types 2 and 5 (CAR) (1, 2, 35). These proteins appear to be founder members of a novel subfamily of molecules within the Ig superfamily (IgSF) that is conserved throughout vertebrates. Our results further suggest that the A33 antigen may provide a definitive marker of the basolateral surfaces of intestinal epithelial cells. This distribution is likely to be of significance to the currently unknown function of the A33 antigen.
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MATERIALS AND METHODS |
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Cell culture. The LIM1863 cell line and embryonic stem (ES) cells were grown as described elsewhere (17, 44). Mouse F9 embryonal carcinoma cells were grown in tissue culture flasks coated with 0.1% gelatin in RPMI medium containing 10% FCS.
Cloning of mouse A33 antigen cDNA.
Sequence similarity searching of available expressed sequence tag (EST)
databases with the full-length hA33 antigen cDNA sequence identified a
249-bp EST (GenBank accession no. D28657) from undifferentiated F9
cells (28) that was 74% identical to hA33 antigen cDNA
(nucleotides 551-792). In the likelihood that this EST represented
part of the mouse ortholog of hA33 antigen, two primers based on the
EST (sense 5'-TGACAAAGAAATACATC-3' and antisense 5'-TCTGGCTTGGAGGGTGG-3') were used to amplify, using a touchdown PCR protocol (10), a 217-bp product from a cDNA library
derived from RNA extracted from purified adult mouse colonic epithelial cells (20). This product was gel purified, radiolabeled
with [-32P]dATP and [
-32P]dCTP
(Geneworks, Adelaide, Australia) using the Megaprime DNA labeling kit
(Amersham Pharmacia Biotech, Uppsala, Sweden), and used to screen
1 × 106 clones of the adult mouse colonic epithelial
cell cDNA library. The cDNA inserts of 20 positive clones were excised
and subcloned into pBluescript (KS+).
5' RACE PCR. To extend the 5' nucleotide sequence, 5' rapid amplification of cDNA ends (RACE) PCR was performed using the Marathon kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions and using total RNA (14 µg) extracted from purified mouse intestinal epithelial cells (41) as the template. PCR amplification of the resultant double-stranded cDNA using the AP1 sense primer (provided with the kit) paired with a mA33 antigen-specific antisense primer (5'-GTGTGTGTCCAGGAACAGAAACGCCATGGT-3') yielded a single 1.65-kb product. The product was subcloned into the TopoTA vector (Invitrogen, Leek, The Netherlands), and the 5' sequences of two individual clones were determined on both strands.
DNA sequencing. DNA sequencing reactions were performed using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit, followed by analysis on an ABI-373 automated DNA sequencer, according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). DNA sequence comparisons were performed using the SeqManII program (DNASTAR, Madison, WI).
Northern blot analysis.
Total RNA was prepared from various tissues of ICR mice according to
the method of Chomczynski and Sacchi (6). Samples (20 µg) were electrophoresed in 1% agarose-0.4 M formaldehyde gels and
transferred to nylon membranes (Hybond N; Amersham). Membranes were
hybridized overnight at 42°C with a 2.1-kb mA33 antigen cDNA probe
labeled with [-32P]dATP (Geneworks) using the
Megaprime DNA labeling kit. All images were generated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Polyclonal antisera production. A peptide containing the most COOH terminal 17 aa of the mA33 antigen, with an additional lysine residue at the NH2 terminus to facilitate conjugation (KHEDRWSSGRSTPDQPFQ), was custom synthesized (Chiron Mimotopes, Clayton, Australia) and conjugated to keyhole limpet hemocyanin. Rabbits were primed with 1.2 mg of peptide in complete Freund's adjuvant (Sigma Chemical, St. Louis, MO) and boosted with 500 µg of peptide in incomplete Freund's adjuvant every 4 wk. Ten milliliters of blood was obtained 12 days after the final boost, and the serum was collected. The IgG fraction was purified from both the antiserum and preimmune serum using protein A-Sepharose chromatography (Amersham).
Western blot analysis.
Membrane proteins were extracted from purified intestinal epithelial
cells (41), homogenized whole mouse bladder, and cultured mouse cells by incubation in lysis buffer [1% Triton X-100 (TX-100), 20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 15 mM
sodium pyrophosphate, and 1× complete protease inhibitor cocktail
(Roche Diagnostics Australia, Castle Hill, Australia)] at 4°C for 30 min. Lysates were centrifuged, and the protein concentration of the
supernatant was determined using the BCA protein assay (Pierce Chemical, Rockford, IL). Protein extract (15 µg) was subjected to
SDS-PAGE in 10% gels in the presence or absence of
-mercaptoethanol, transferred to polyvinylidene difluoride membranes
(Immobilon P; Millipore, Bedford, MA) and blocked overnight at 4°C in
1× PBS-3% BSA-0.05% Tween 20. Membranes were then incubated
overnight at 4°C with either preimmune IgG or anti-mA33 antigen IgG
(both at 1.2 µg/ml) in 1× PBS-0.1% BSA-0.05% Tween 20. These were
washed 3 times and incubated at 25°C for 60 min in horseradish
peroxidase (HRP)-conjugated goat anti-rabbit IgG at a 1:10,000 dilution
(Bio-Rad, Hercules, CA) in 1× PBS-0.1% BSA-0.05% Tween 20. Membranes
were washed 3 times, and HRP activity was determined using enhanced chemiluminescence (Amersham).
Whole mount immunohistochemistry. E4.5 blastocysts were flushed from the uteri of pregnant ICR mice generated by timed matings. Blastocysts were fixed in freshly prepared 4% paraformaldehyde-PBS for 15 min at 4°C, washed in PBS, and permeabilized with 2 × 15 min washes in 1% TX-100-PBS. The blastocysts were then blocked in 2% BSA-1% TX-100-PBS for 15 min at room temperature before incubation with preimmune IgG or anti-mA33 antigen IgG (5 µg/ml) in 0.2% BSA-1× PBS overnight at 4°C. The blastocysts were then washed for 4 × 30 min in 1% TX-100-PBS before incubation in Alexa Fluor 546-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR) overnight. A final series of washes in 1% TX-100-PBS was used before imaging the fluorescent signals in a confocal microscope (1024ES; Bio-Rad, Hemel Hempstead, UK). Segments of adult small intestine and colon from ICR mice were processed for whole mount immunohistochemistry in a similar way but with longer incubation times. Fixation (4% paraformaldehyde) was performed overnight at 4°C; permeabilization and washing steps (1% TX-100-PBS) comprised 4 × 60 min washes, and incubation in primary anti-mA33 antigen IgG or preimmune IgG was for 60 h. All processing steps were performed under conditions of gentle agitation.
Immunohistochemistry and in situ hybridization.
Mouse tissues and embryos were fixed in 4% paraformaldehyde for 8 h, embedded in paraffin, and sectioned (4 µm) onto
3-aminopropyltriethoxy-silane-coated slides. Initial difficulties in
obtaining positive immunohistochemical staining of paraffin-embedded
sections were overcome by using an antigen retrieval step that involved
microwaving the slides on high power for two periods of 5 min in 50 mM
Tris · HCl (pH 9.5) (33). Histological sections of
adult tissue were blocked in 0.2% BSA-1× PBS for 15 min and incubated
in preimmune IgG, anti-mA33 antigen IgG (both at 6 µg/ml), or
anti-mA33 antigen IgG (6 µg/ml) plus an excess of peptide (5 µg/ml)
in 0.2% BSA-1× PBS for 1.5 h. Sagittal sections of embryos were
incubated with anti-mA33 antigen IgG (25 µg/ml) or anti-mA33 antigen
IgG (25 µg/ml) plus peptide (5 µg/ml). Sections were rinsed twice
in 1× PBS, incubated with HRP-conjugated swine anti-rabbit IgG (DAKO, Carpinteria, CA) for 30 min, rinsed again, incubated with the chromogen
3,3'-diaminobenzidine (DAKO), and counterstained with hematoxylin. In
situ hybridization histochemistry was carried out as described
elsewhere with minor modifications (25). Sagittal sections
described above were hybridized with a 437-bp antisense mA33 antigen
cRNA probe, generated by in vitro transcription using [-35S]UTP (1,250 Ci/mmol; Geneworks). Specificity of
hybridization was confirmed by hybridizing adjacent tissue sections
with either a radiolabeled sense mA33 antigen (437 bp) cRNA probe or a
radiolabeled antisense probe for mouse insulin-like growth factor
binding protein-4.
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RESULTS |
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Cloning of mouse A33 antigen cDNAs. For cloning of cDNAs encoding the mouse ortholog of the hA33 antigen, we used a cDNA library constructed from poly(A)+ RNA extracted from purified normal adult mouse colonic epithelial cells (20) that had been used previously to clone cDNAs encoding other intestine-enriched proteins, for example, Cdx1 (20). A specific probe was identified as a result of sequence similarity searching of all available databases with the full-length hA33 antigen cDNA sequence. This identified a 249-bp EST generated from a low-abundance transcript from the mouse teratocarcinoma-derived cell line F9 (28) that was 74% identical to hA33 antigen cDNA in the corresponding region (nucleotides 551-792). Translation of the F9 EST predicted a protein sequence that shared 55% aa identity with hA33 antigen in the region of overlap. In the likelihood that this EST represented part of the mouse ortholog of the human A33 antigen, two oligonucleotide primers based on the EST were synthesized and used to amplify a 217-bp product from the normal adult mouse colonic epithelial cDNA library described above. The PCR product was sequenced, found to share 95% nucleotide identity with the F9 EST, and used to screen the normal adult mouse colonic epithelial cDNA library. Of the 20 putative mA33 antigen cDNA clones obtained, the longest were ~2.1 kb in size. The sequences of two clones were determined completely on both strands, and two more clones were sequenced partially. All four clones encoded the same molecule.
The nucleotide sequence of mA33 antigen cDNA and the deduced protein sequence are depicted in Fig. 1.1 Compared with the full-length hA33 antigen cDNA, the mouse cDNA clones lacked all of the 5' untranslated region (UTR) and the first two nucleotides of the codon for initiation of translation (ATG). The remainder of the 5' sequence was determined by 5' RACE PCR, which yielded a fragment containing the entire coding region, including the putative initiation methionine and 104 bp of the 5' UTR. The mouse 3' UTR was 254 bp shorter than the human 3' UTR, with a polyadenylation signal starting at position 2270 followed 17 nucleotides downstream by a run of adenines. The overall nucleotide sequence identity between human and mouse A33 antigen cDNAs was 62% (77% identity in the coding region). The deduced mA33 antigen protein sequence shared 66% identity (77% similarity) with the product encoded by hA33 antigen cDNA (18). This degree of sequence identity compares favorably with interspecies variation observed between other IgSF orthologs, for example, mouse and human CD2 (54%) and mouse and human CD22 (62%) (32, 24).
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The A33 antigen is related to CTX/ChT1, CAR, CTM/CTH, and JAM. The domain structure of mA33 antigen shows striking similarity to that of the T lymphocyte cell adhesion molecule CD2, for which the crystal structure of the extracellular domain is available (3). However, the primary sequences of their V-type and C2-type domains are only 12% identical, and more marked sequence identity was noted with four recently cloned IgSF members named CTX (7, 8, 11) (25% identity in the V-type domain; 33% identity in the C2-type domain), ChT1 (22) (23% identity in the V-type domain, 26% identity in the C2-type domain), hCAR and mCAR (1, 2, 35) (23% identity in the V-type domain; 29% identity in the C2-type domain), CTM and CTH (7) (21% in the V-type domain, 31% in the C2-type domain), and junctional adhesion molecule (JAM) (26) (22% identity in the V-type domain; 24% identity in the C2-type domain). Meanwhile, the molecules share only weak sequence similarity in their intracellular domains (data not shown).
The sequences of the extracellular and transmembrane domains of the related molecules were aligned (Fig. 2) and compared with human CD2, which allowed us to predict the sequences likely to contribute to each of theLocalization of mA33 antigen in adult mouse tissues.
The expression of mA33 antigen mRNA was examined in multiple mouse
tissues by Northern blot analysis (Fig.
3A). Of the 23 tissues
studied, only small intestine and colon were strongly positive,
producing a single intense band corresponding to a transcript of ~2.4
kb. Mouse stomach and bladder produced weak signals (~10% the
intensity of that obtained from intestine). This is in contrast to the
pattern of expression reported in humans (12), in which expression of hA33 antigen was absent in 13 samples of normal human
stomach and several samples of normal and neoplastic human bladder.
However, 14 of 24 gastric carcinomas expressed hA33 antigen in >80%
of cells (12). All other human and mouse tissues were negative except for very weak staining in human salivary gland, which
was not seen in mice. Differences in expression pattern between mouse
and human have also been observed for other members of the CTX family
(2, 35). For instance, mCAR was strongly expressed in
liver, lung, and kidney, whereas hCAR expression appeared to be minimal
or absent in these tissues. Northern blot analysis of mA33 antigen mRNA
levels throughout the rostrocaudal axis of the intestine revealed
uniformly high expression from duodenum to distal colon, with a
significantly lower level of expression in stomach epithelium (Fig.
3B).
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Mouse A33 antigen expression is regulated during embryonic
development.
Murine A33 antigen expression was not detected in embryos between E5.5
and E13.5 (H. E. Abud, unpublished observations) through immunohistochemistry. Intestinal mA33 antigen expression was first detected in the hindgut endoderm at E14.5 with cytoplasmic as well as
cell surface expression (Fig. 6E). Interestingly,
immunoreactivity along the rostrocaudal axis of the intestine was
polarized at this stage, with the distal end of the intestine being
strongly positive for mA33 antigen expression and the proximal end
being only barely positive. A signal was not observed in the midgut at
this stage of development (data not shown) nor in any other tissue in
the developing embryo (Fig.
7B). Notably, the
initial appearance of mA33 antigen expression was therefore in the
opposite direction to the proximal-to-distal wave of morphogenesis and cytodifferentiation that mediates substantial remodeling of the intestine between E15 and E19 of mouse development (14).
By E15.5, strong mA33 antigen expression was found throughout the intestine and was restricted to the plasma membranes of endoderm cells
(Fig. 6F). At this stage, the endoderm had yet to
convert from a multilayered stratified structure to a simple columnar epithelium (36). By E16.5, the endoderm had thinned to a
columnar epithelium, which maintained strong membranous expression of
mA33 antigen (data not shown). Northern blot analysis confirmed that mA33 antigen mRNA was highly expressed at E16.5 in both small intestine
and colon (Fig. 7A). The mA33 antigen mRNA levels increased gradually until adulthood with no dramatic change in expression after
birth and weaning (24 days postnatal). In contrast, expression in
bladder appeared to be induced between 1 and 4 days after birth but was
always present at much lower levels than in intestine (Fig.
7A). At E18.5, in situ hybridization histochemistry of
sections of whole embryo torsos detected mA33 antigen mRNA specifically in the intestine (Fig. 7B), indicating that the highly
restricted pattern of expression observed in the adult was essentially
established at this stage of development.
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DISCUSSION |
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The characteristics of mA33 antigen expression appear to provide us with a definitive marker for all intestinal epithelial cells irrespective of their cell lineage and proliferative/differentiation status. Although a related molecule, JAM, was localized to tight junctions in endothelial cells and intestinal epithelial cells (26), mA33 antigen appears to be distributed specifically to the basolateral membranes. The subcellular localization of mA33 antigen combined with the structure of its extracellular domain, which is highly reminiscent of CD2, strongly suggest that mA33 antigen may be involved in cell-cell adhesion within the gastrointestinal tract. Here the A33 antigen could be involved in cell-cell adhesion between adjacent epithelial cells or between epithelial cells and the resident population of intraepithelial T lymphocytes (21). The presence or absence of mA33 antigen at tight junctions and other epithelial cell-cell adhesion structures such as desmosomes and adherens junctions (4, 15) must be addressed in the future with the use of electron microscopy.
The exquisite, almost intestine-specific, pattern of mA33 antigen
expression strongly suggests that the regulatory elements of the gene
encoding mA33 antigen would be perfectly suited for directing
expression of gene products implicated in the regulation of epithelial
cell growth and differentiation and colorectal tumorigenesis to the
intestinal epithelium in transgenic mice. Other promoters have been
investigated for these purposes with varying degrees of success
(16, 19, 42, 45). Two of the more promising ones, the
promoters of the rat liver fatty acid-binding protein gene
(Fabpl) (34) and the mouse villin gene
(29), were recently described. An artificial Fabpl
promoter, containing four extra copies of a heptad repeat inserted
in its normal position at nucleotide 132, directed strong transgene
expression to colonic, cecal, and distal small intestinal epithelium
and minimal expression to kidney epithelium (34). Unlike
expression of the endogenous gene, expression of the transgene extended
to the base of the crypt in the large and small intestine. Similarly, a
relatively large region of the mouse villin gene (
3.5 kb to +5.5 kb)
was sufficient to drive expression of a
-galactosidase reporter gene in a manner that essentially recapitulated endogenous villin gene expression in transgenic mice (29). The transgene was
strongly expressed by differentiated colonic and small intestinal
epithelial cells, with reduced activity in the lower crypt region where
some
-galactosidase-negative cells were found.
One important consideration for this type of animal model is whether transgene expression can be induced in intestinal epithelial stem cells or not. The ability to deliver expression to stem cells would provide a particularly powerful system since stem cell anchorage and self renewal would ensure continual expression of the transgene within the proliferative cell compartment (30). At present, it is impossible to identify intestinal stem cells accurately and determine their patterns of gene expression. Our data, although not absolutely conclusive, demonstrated that mA33 antigen-negative cells were conspicuously absent in the region around the base of crypts and raise the intriguing possibility that stem cells express mA33 antigen. Accordingly, we propose that transgenic mice based on the mA33 antigen gene promoter may provide significant new opportunities to investigate the genetic basis of colorectal cancer.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert James for the adult mouse colonic epithelial cell cDNA library, Dr. Guo-Fen Tu for DNA sequencing, Dr. Stacey Gabriel, Dianne Grail, and Melissa Inglese for collection of mouse tissues, Valerie Feakes for expert tissue sectioning and immunohistochemistry, and Janna Stickland for photography.
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FOOTNOTES |
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C. N. Johnstone and N. C. Tebbutt were supported by an Australian Postgraduate Award and a National Health and Medical Research Council (NH&MRC) Medical Postgraduate Research Scholarship, respectively. This study was funded in part by a project grant from the NH&MRC (Australia).
Present address for R. H. Whitehead: G. I. Cancer Program, Vanderbilt Medical Center, Nashville, TN 37232-2583.
Address for reprint requests and other correspondence: J. K. Heath, Ludwig Institute for Cancer Research, Post Office Royal Melbourne Hospital, Parkville, Victoria 3050, Australia (E-mail: joan.heath{at}ludwig.edu.au).
1 The GenBank accession number for the novel murine A33 antigen sequence described in this study is AF24765. Other relevant GenBank accession numbers are hA33 antigen cDNA (U79725), CTX cDNA (U43330), ChT1 cDNA (AF061023), mCAR cDNA (Y10320), hCAR cDNA (U90716), CTM cDNA (AF061024), CTH cDNA (AF061022), JAM cDNA (U89915), and hCD2 cDNA (M16445).
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. §1734 solely to indicate this fact.
Received 28 December 1999; accepted in final form 30 March 2000.
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