1 Department of Operative Dentistry, Faculty of Dentistry, Hiroshima University,
1-2-3 Kasumi, Hiroshima 734-8553, Japan
2 Department of Biochemistry, Faculty of Dentistry, Hiroshima University, 1-2-3
Kasumi, Hiroshima 734-8553, Japan
3 Department of Preventive Dentistry, Faculty of Dentistry, Hiroshima
University, 1-2-3 Kasumi, Hiroshima 734-8553, Japan
Author for correspondence (e-mail:
ykato{at}hiroshima-u.ac.jp)
Accepted 27 January 2003
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Summary |
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Key words: Chondrocyte, Concanavalin A, GPI-anchored protein, Membrane-bound transferrin-like protein
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Introduction |
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MTf was originally identified with the use of monoclonal antibodies as a 97
kDa human tumor-associated antigen
(Woodbury et al., 1980;
Dippold et al., 1980
). Its
amino acid sequence shows
40% identity with transferrin and lactoferrin
(Rose et al., 1986
). It binds
to iron (Brown et al., 1982
)
and stimulates iron uptake in the absence of transferrin and transferrin
receptor (Kennard et al.,
1995
). Interestingly, MTf can cross the blood-brain barrier
(Demeule et al., 2002
) and it
accumulates in amyloid plaques of Alzheimer's disease, where iron is also
concentrated (Jefferies et al.,
1996
). Furthermore, it has been suggested that MTf is involved in
the proliferation and differentiation of melanoma cells and eosinophils
(Estin et al., 1989
;
McNagny et al., 1996
).
However, the precise physiological roles of MTf remain unknown.
Previous studies have shown that MTf is a major ConA-binding protein on the
chondrocyte surface (Kawamoto et al.,
1998). Because cartilage contains MTf at a much higher level than
other normal tissues, chondrogenic cells are a good model for studies on MTf.
In the present study, we examined the effect of anti-MTf antibodies on cell
shape and the expression of differentiation-related genes in cultures of
chondrocytes or chondrogenic cells (ATDC5 and C3H10T1/2 cells) in the presence
or absence of ConA. The anti-MTf antibodies markedly suppressed the effect of
ConA on the cell shape and the phenotypic expression in these cultures, or
they mimicked the action of ConA only when the cells synthesized MTf at high
levels. These effects of ConA and the anti-MTf antibodies on MTf-expressing
cells were observed within 24-48 hours. The findings on the ConA-MTf system
obtained in this study will be useful in the understanding of the remarkable
actions of plant lectins on animal cells.
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Materials and Methods |
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Immunoblotting
The proteins extracted from the cultured chondrocytes (10 µg protein per
lane) were resolved by 4-20% SDS-PAGE under nonreducing conditions. After
blotting onto a PVDF membrane (Towbin et
al., 1979) and blocking with 4% nonfat milk in PBS for 2 hours at
room temperature, the membrane was incubated at 4°C with anti-MTf sera
(1:500 dilution in PBS) or anti-MTf-mAb2 (5 µg ml1) for
14 hours and then incubated with 125I-labeled sheep anti-mouse IgG
(Fab')2 fragment in PBS for 2 hours at room temperature.
Chondrocyte culture
The chondrocytes were isolated from the resting cartilage of the ribs of
4-week-old male Japanese White rabbits as described previously
(Shimomura et al., 1975;
Kato and Gospodarowicz, 1985
).
The cells were seeded at a density of 106 cells per 150-mm tissue
culture dish and maintained in 30 ml
-modified Eagle's medium
supplemented with 10% fetal bovine serum, 50 µg ml1
ascorbic acid, 50 U ml1 penicillin, 60 µg
ml1 kanamycin and 250 ng ml1 amphotericin
B (medium A). When the cultures became subconfluent, the cells were harvested
with trypsin and EDTA, and seeded at 5x104 cells per 16-mm
well in 0.5 ml medium A. When the cultures again became subconfluent, the
cells were preincubated in 0.5 ml of a 1:1 mixture of Dulbecco's modified
Eagle's medium and Ham's F-12 medium (Nissui Pharmaceutical, Tokyo, Japan)
supplemented with 0.5% fetal bovine serum (medium B) for 24 hours. The medium
was replaced by 0.5 ml of fresh medium B in the absence or presence of
anti-MTf antibodies, ConA (5 µg ml1) or both, and the
incubation was continued for 24 hours.
ConA affinity chromatography of plasma membrane from
retinoic-acid-exposed cultures
The cell membrane was isolated from retinoic-acid-exposed or -free
chondrocyte cultures in three 150 mm dishes by the method of Mollenhauer et
al. (Mollenhauer et al., 1984)
with some modifications. The plasma membrane fraction was dissolved in 8 ml of
buffer A [10mM Tris/HCl, pH 7.4, 10 µM (p-amidinophenyl)
methanesulfonyl fluoride, 10 µM pepstatin A and 1% sodium deoxycholic acid]
and applied to a ConA-Sepharose column (1 cm x 3 cm) (Amersham,
Piscataway, NJ) equilibrated with buffer A. The ConA-bound glycoprotein was
eluted with buffer A containing 0.5 M methyl-
-mannopyranoside as
described previously (Kawamoto et al.,
1998
).
Northern blotting
Total RNA was prepared from chondrocytes which had been treated with
106 M retinoic acid for 4 days using the
guanidine-thiocyanate method (Smale and
Sasse, 1992). RNA samples (10 µg) were electrophoresed on a 1%
agarose gel containing 2.2 M formaldehyde and transferred to a Hybond-N
membrane (Amersham). The membrane was hybridized with a 32P-labeled
rabbit MTf cDNA probe or a 32P-labeled glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe as described previously
(Kawamoto et al., 1998
). The
membrane was exposed to BioMax X-ray film at 80°C with an
intensifying screen.
Proteoglycan synthesis
The chondrocytes obtained from the rabbit primary cultures were seeded at
104 cells per 6-mm microwell and maintained in medium A. When the
cultures became confluent, the cells were preincubated in 0.1 ml of medium B
for 24 hours. The cells were incubated with ConA (5 µg
ml1), succinyl-ConA (sConA) (10 µg
ml1), dibutyryl cyclic AMP (DBcAMP) (1 mM) or insulin (10
µg ml1) in 0.1 ml of fresh medium B in the absence or
presence of anti-MTf antibodies for 24 hours. [35S]sulfate (5
µCi ml1 final concentration) was added 6 hours before the
end of the incubation (Kato et al.,
1980). Uronic acid was determined by the method of Bitter and Muir
(Bitter and Muir, 1962
).
DNA synthesis
The chondrocytes obtained from the rabbit primary cultures were seeded at
104 cells per 6-mm microwell and maintained in medium A. When the
cultures became confluent, the cells were preincubated in 0.1 ml of
-modified Eagle's medium supplemented with 0.5% fetal bovine serum for
24 hours and then incubated with ConA (5 µg ml1) or sConA
(10 µg ml1) in 0.1 ml of
-modified Eagle's medium
supplemented with 0.5% fetal bovine serum in the absence or presence of
anti-MTf-mAb2 or control mouse IgG for 24 hours. [3H]thymidine (10
µCi ml1) was added 3 hours before the end of the
incubation (Kato et al.,
1983
). The lymphocytes were isolated from the thymus of 4-week-old
rabbits. The cells were seeded at a density of 6x105 cells
per 16-mm well and maintained in 0.2 ml medium A for 72 hours. The cells were
exposed to anti-MTf-mAb2, control mouse IgG and/or ConA for 72 hours.
[3H]Thymidine (3 µCi ml1) was added 6 hours
before the end of the incubation.
Plasmids, transfections and ATDC5 and C3H10T1/2 cells
Rabbit full-length MTf cDNA was inserted into the mammalian expression
vector pDNA3.1/Zeo (Invitrogen, Carlsbad, CA) at the
EcoRI-NotI site to construct pcDNA-MTf. pcDNA3.1/Zeo or
pcDNA-MTf was transfected into ATDC5 and C3H10T1/2 cells (RIKEN, Tsukuba,
Japan) using the SuperFect transfection reagent (Qiagen, Valencia, CA). The
transfected cells were selected with 0.2 mg ml1 Zeocin
(Invitrogen). Individual colonies were isolated, and the expression levels of
MTf were determined by immunoblotting with anti-MTf-mAb2.
ATDC5 cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's
medium and Ham's F-12 medium supplemented with 5% fetal bovine serum, 10 µg
ml1 human transferrin (Boehringer Mannheim, Mannheim,
Germany) and 3x108 M sodium selenite (Sigma, St Louis,
MO) in the presence or absence of 10 µg ml1 bovine
insulin (Sigma) as previously described
(Atsumi et al., 1990). The
inoculum density of the cells was 4x104 cells per 16-mm dish
or 20x104 cells per 10 cm dish. The medium was replaced every
other day.
C3H10T1/2 cells were maintained in medium A. The inoculum density of the cells was 8x104 cells per 35-mm dish or 20x104 cells per 10-cm dish. The medium was replaced every other day. On day 8, the culture medium was changed to Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1) containing 1% fetal bovine serum, insulin (6.25 µg ml1), transferrin (6.25 µg ml1), selenite (6.25 ng ml1), ascorbic acid (50 µg ml1), dexamethasone (1010 M) and transforming growth factor ß1 (TGF-ß1, 5 ng ml1). The medium was replaced every other day.
Number of spherical/polygonal/spindle-like cells
Spherical/polygonal/spindle-like cells and spread cells were counted
separately under a phase-contrast microscope. At least 300 cells were
evaluated and the proportion of spherical/polygonal/spindle-like cells among
total cells was calculated.
RT-PCR and Southern blot analysis
The first-strand cDNA was synthesized with 1 µg ml1
total RNA from ATDC5 cells. PCR was performed using an aliquot of the
first-strand cDNA as a template under standard conditions with Klentaq
Polymerase (Clonetech, Palo Alto, CA) for 18, 23 and 18 cycles for aggrecan,
type II collagen and GAPDH, respectively. These cycles were optimal for
comparison between the amplified products. The PCR products were separated on
1.5% agarose gels and transferred to NytranN membranes (Schleicher &
Schuell, Dassel, Germany). The membranes were hybridized with
32P-labeled mouse aggrecan, 32P-labeled mouse-type II
collagen or 32P-labeled mouse GAPDH cDNA
(Nakamasu et al., 1999).
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Results |
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|
In monolayer cultures in a low-serum medium, the chondrocytes took a
fibroblastic configuration (Fig.
2A) and the addition of ConA induced a cell shape change from
fibroblastic to spherical chondrocytes within 24 hours
(Fig. 2B)
(Yan et al., 1990). However,
pretreatment with retinoic acid for 4 days prevented MTf expression, as
indicated by immunoblotting (Fig.
1D), and the responsiveness of the chondrocytes to ConA
(Fig. 2D), and removal of
retinoic acid for 3 days restored the MTf level
(Fig. 1E) and responsiveness to
ConA (Fig. 2F). These findings
indicate a close relationship between MTf level and responsiveness to
ConA.
|
Inhibition of ConA-induced chondrocyte phenotypic expression by pAb1
and mAb2 and ConA-like actions of pAb4
If MTf is a receptor for ConA, antibodies to MTf should modulate the lectin
action on chondrocytes. To test this hypothesis, we purified MTf from rabbit
chondrocyte plasma membrane using HPLC and lectin-affinity chromatography
(Kawamoto et al., 1998). Using
the purified MTf, we prepared four polyclonal antisera (anti-MTf-pAb1, -pAb2,
-pAb3 and -pAb4) and a monoclonal antibody (anti-MTf-mAb2) that had proven to
be specific for MTf in immunoblot analysis with chondrocyte extracts
(Fig. 3A). Rabbit chondrocytes
were incubated with anti-MTf-pAb1 serum or the control serum in the absence or
presence of ConA for 24 hours. The chondrocytes adopted a fibroblastic
configuration in monolayer cultures at a low serum concentration
(Fig. 3B, control)
(Yan et al., 1990
). The
addition of ConA to the cultures altered the cell shape within 12 hours, with
almost all of the lectin-exposed cells becoming spherical at 24 hours
(Fig. 3B, ConA)
(Yan et al., 1990
). This
effect of ConA was eradicated by the anti-MTf-pAb1 serum
(Fig. 3B, ConA+pAb1).
Anti-MTf-pAb2 and -pAb3 suppressed the action of ConA on the cell shape to a
lesser extent, whereas anti-MTf-pAb4 had little effect on the cell-shape
change (Fig. 3B, ConA+pAb2,
ConA+pAb3 and ConA+pAb4). The anti-MTf-mAb2 suppressed the ConA-induced
cell-shape change dose-dependently (Fig.
3B, ConA+mAb2), and the F(ab')2 fragment of mAb2
also suppressed the cell-shape change (Fig.
3C, ConA+ F(ab')2). The anti-MTf-mAb2 was
prepared from the mouse that produced anti-MTf-pAb1.
|
The anti-MTf-mAb2 suppressed the ConA-stimulation of the incorporation of
[35S]sulfate into glycosaminoglycans synthesized by chondrocytes
(Fig. 4A, left panel). Under
these conditions, the majority of 35S-labeled glycosaminoglycans
were associated with cartilage-characteristic proteoglycan (aggrecan)
(Yan et al., 1990).
|
Previous studies have shown that a divalent succinylated derivative of ConA
(sConA) enhances chondrocyte phenotypic expression without inducing rapid
changes in cell shape, although native tetravalent ConA enhances both rapid
cell-shape change and chondrocyte phenotypic expression (proteoglycan
synthesis) (Yan et al., 1997).
ConA-but not sConA-induces extensive crosslinking of the cell surface
glycoproteins and patch/cap formation (clustering of lectin-binding membrane
proteins) (Gunther et al.,
1973
). In our study, the anti-MTf-mAb2 also suppressed the
sConA-stimulation of proteoglycan synthesis by the chondrocytes
(Fig. 4A, right panel).
Anti-MTf-mAb2 had little effect on the stimulation of proteoglycan
synthesis by either a permeable analogue of cyclic AMP or insulin at the
pharmacological level (Fig.
4B). The anti-MTf-mAb2 did not interfere with the effect of ConA
on DNA synthesis in the chondrocytes (Fig.
4C). In addition, the anti-MTf-mAb2 had little effect on the ConA
stimulation of lymphocyte proliferation
(Fig. 4D), because MTf is
barely expressed in lymphocytes (Kawamoto
et al., 1998; Nakamasu et al.,
1999
).
Interestingly enough, the addition of the anti-MTf-pAb4 serum, as in the case of ConA, induced the expression of the spherical phenotype (Fig. 5A, pAb4). The anti-MTf-pAb2 and -pAb3 sera also induced cell-shape change, although their effect was far less than that of the anti-MTf-pAb4 (Fig. 5A, pAb2 and pAb3). IgG purified from the anti-MTf-pAb4 serum using a Protein-A-affinity gel column also elicited the ConA-like action at 24 hours (Fig. 5A, pAb4 IgG), with the ConA-like action of the anti-MTf-pAb4 being indicated by the increase in incorporation of [35S]sulfate into glycosaminoglycans (Fig. 5B). In cultures exposed to the anti-MTf-pAb4 for 7 days, all of the cells were eventually surrounded by an abundant refractile matrix (Fig. 5C). All of the mice that were immunized with MTf but none of the control mice produced neutralizing and/or mimicking antibodies for the ConA action on chondrocytes.
|
Effects of ConA on chondrocyte phenotypic expression in ATDC5
cultures overexpressing MTf
MTf is upregulated in the mouse embryonic carcinoma-derived ATDC5 cells
during chondrogenic differentiation
(Nakamasu et al., 1999). These
cells initiate chondrogenic differentiation only after the addition of some
growth factor, such as insulin/insulin-like growth factor-I, TGF-ß or
bone morphogenic proteins (Shukunami et
al., 1996
; Atsumi et al.,
1990
; Fujii et al.,
1999
). Using this model, we examined the role of MTf in
ConA-induced chondrogenic differentiation. Rabbit MTf cDNA was expressed under
the control of the CMV promoter in stably transfected ATDC5 cells, and two
MTf-expressing clones were isolated (ATDC5-MTf1 and ATDC5-MTf5);
immunoblotting confirmed the expression of rabbit MTf in these clones
(Fig. 6A). In the absence of
added growth factors, parental ATDC5 cells and empty vector-integrated cells
(Pc1 and Pc2) did not differentiate chondrogenic cells, regardless of the
presence or absence of ConA (Fig.
6B), because of an absence of MTf expression
(Nakamasu et al., 1999
). By
contrast,
30% of the MTf-overexpressing cells became spherical
chondrocytes even in the absence of ConA, and ConA further increased the
number of spherical chondrocytes dose-dependently
(Fig. 6B), with the cell-shape
change being accompanied by an increase in the uronic-acid-containing
macromolecule (proteoglycan) (Fig.
6C). Even in the absence of ConA, the mRNA levels of aggrecan and
collagen type IIB (chondrocyte specific), as well as the collagen type IIA
(prechondrogenic stage characteristic) mRNA level, were much higher in the
MTf-overexpressing cells than in the control cells
(Fig. 6D), indicating the
involvement of MTf in chondrogenic differentiation. Furthermore, sConA
increased these mRNA levels in the MTf-overexpressing cells but not in the
control cells. And, within 48 hours, the anti-MTf-mAb2 or anti-MTf-pAb1
suppressed expression of the spherical phenotype induced by the
MTf-overexpressing ATDC5 cells in response to ConA
(Fig. 6E).
|
Effect of ConA on cell shape in cultures of C3H10T1/2 cells
overexpressing MTf
We isolated the C3H10T1/2 cells (T4) overexpressing rabbit MTf to examine
whether ConA would induce cell shape change in the mouse pluripotent
mesenchymal cell line expressing MTf at high levels. Wild-type C3H10T1/2 and
T4 cells showed MTf mRNA expression at low and high levels, respectively (data
not shown), and immunoblotting confirmed the expression of rabbit MTf at a
high level in T4 cells, and its absence in the wild-type C3H10T1/2 cells
(Fig. 7A). ConA induced cell
shape change from fibroblastic to spherical or spindle-like cells within 48
hours in T4 cells, but not in the wild-type or empty vector-integrated cells
(Fig. 7B,C). Furthermore, the
cell-shape change was suppressed in the presence of the anti-MTf-mAb2
(Fig. 7D,E).
|
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Discussion |
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ConA alters the shape, proliferation and/or differentiation of various
animal cells. MTf is not always expressed in these cells, suggesting that
other ConA receptors are present in non-chondrogenic cells including
lymphocytes. However, it is noteworthy that MTf is expressed at high or
moderate levels in many tumors and fetal tissues
(Brown et al., 1981;
Danielsen and van Deurs, 1995
),
as well as several adult tissues, including the capillaries
(Rothenberger et al., 1996
),
salivary glands (Richardson,
2000
) and eosinophil precursors
(McNagny et al., 1996
). In
these cells, MTf might work, at least partially, as a ConA receptor.
ConA modulated both cell shape and the expression of aggrecan and type II
collagen genes in chondrocytes and ATDC5 cells, via crosslinking of MTf and/or
simple binding to MTf. Crosslinking of MTf by ConA appears to be essential for
rapid cell-shape change but not for the expression of the
differentiation-related genes, because simple binding of sConA induced the
gene expression in chondrocytes (Fig.
4A) but not the rapid cell-shape change
(Yan et al., 1997). Unlike
ConA, sConA cannot induce clustering of cell-surface proteins. The lack of
effect of sConA on cell shape suggests that the ConA-induced cell-shape change
from fibroblastic cells to spherical cells is not directly linked with the
ConA-induced, aggrecan and type II collagen expressions. This conclusion was
unexpected because the cell shape has a great effect on the differentiation of
prechondrogenic cells and dedifferentiated cartilage cells in some
experimental systems (Zanetti and Solursh, 1984;
Brown and Benya, 1988
).
However, in cultures of MTf-overexpressing C3H10T1/2 cells, ConA altered cell
shape but did not enhance the expression of aggrecan or type II collagen (data
not shown), which also suggests that MTf has two distinct roles: modulating
cell shape and stimulating chondrocyte phenotypic expression.
Cell shape is determined by extracellular proteins (e.g. adhesion factors, matrix proteins and proteases) and membrane-bound proteins (e.g. integrins, cytoskeleton-associated proteins, small G proteins and tyrosine kinases), and the crosslinking of MTf might affect the activity of these proteins. Signals for the cell-shape change induced by ConA remain unknown. However, in subconfluent cultures of rabbit chondrocytes, SB203580 [an inhibitor of p38 mitogen-activated protein (MAP) kinase] at 10 µM suppressed the ConA-induced cell-shape changes, whereas U0126 [an inhibitor of MAP kinase kinase (MEK)] or SP600125 [an inhibitor of c-Jun N-terminal kinase (JNK)] had little effect at the same concentrations (data not shown). These findings suggest that p38 MAP kinase is involved in the cell shape change induced by the ConA-MTf system.
MTf is a glycosylphosphatidylinositol (GPI)-anchored protein
(Food et al., 1994) and,
because MTf does not have a cytoplasmic domain, the effect of ConA might be
mediated by the binding of a ConA-MTf complex to a transmembrane receptor. One
GPI-anchored protein [CD14, which binds to lipopolysaccharide (LPS)] forms a
complex with a transmembrane Toll-like receptor-4 to induce inflammatory
responses (Chow et al., 1999
).
The ciliary neurotrophic factor receptor is also a GPI-anchored protein that
binds to a transmembrane gp130 protein, the signaling component of the IL-6
receptor (Davis et al., 1993
).
Similarly, the GPI-anchored glial-cell-derived neurotrophic factor receptor
associates, after ligand binding, with a transmembrane tyrosine kinase
receptor Ret (Jing et al.,
1996
). A more likely mechanism for ConA actions is activation of
signaling molecules in lipid rafts via crosslinking of MTf or simple binding
to MTf. GPI-anchored proteins are found on the outer surface of lipid rafts,
and signalling molecules, such as the Src kinase family and G proteins, are
located in the inner surface of lipid rafts
(Rodgers et al., 1994
;
Mumby, 1997
;
Simons and Ikonen, 1997
).
Binding of specific antibodies to some GPI-anchored proteins (e.g. CD14,
Thy-1, Ly-6 and Qa-2) elicits striking biological reactions, including
tyrosine phosphorylation, increase of cytoplasmic calcium, cell aggregation,
phagocytosis, IL-2 production and/or DNA synthesis in various cells
(Horejsi et al., 1998
).
It is clear that MTf is not a ConA receptor for lymphocyte activations. We
speculate, however, that some GPI-anchored proteins might work as a ConA
receptor in lymphocytes, because ConA and antibodies to some GPI-anchored
proteins have similar effects on lymphocytes, including increases in
cytoplasmic calcium, cell-shape change, DNA synthesis and IL-2 production
(Robinson, 1991;
Horejsi et al., 1998
).
Some GPI-anchored proteins, including Thy-1, have been shown
physiologically to be involved in signaling via immunoreceptors
(Hueber et al., 1997;
Romagnoli and Bron, 1997
). We
showed here that even in the absence of ConA, the overexpression of MTf
moderately altered the shape of ATDC5 cells and enhanced the expression of
cartilage-characteristic genes, which suggests that GPI-anchored MTf plays a
specific physiological role in chondrocyte differentiation.
We are now using ConA to enhance chondrogenic differentiation of human bone marrow mesenchymal cells and to enhance the phenotypic expression of cultured chondrocytes in vitro. The cartilage-like tissue formed in the presence of ConA in vitro might be useful for cell therapy. The results obtained in this study should be useful in promoting the application of ConA to tissue engineering.
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Acknowledgments |
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Footnotes |
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References |
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