Full text of DOI:10.1007/s004180000131

Histochem Cell Biol (2000) 113:279–286
Digital Object Identifier (DOI) 10.1007/s004180000131
O R I G I N A L PA P E R
Siru Haila · Ulpu Saarialho-Kere
Marja-Liisa Karjalainen-Lindsberg · Hannes Lohi
Kristiina Airola · Christer Holmberg
Johanna Hästbacka · Juha Kere · Pia Höglund
The congenital chloride diarrhea gene is expressed
in seminal vesicle, sweat gland, inflammatory colon epithelium,
and in some dysplastic colon cells
Accepted: 24 January 2000 / Published online: 3 March 2000
© Springer-Verlag 2000
Abstract Congenital chloride diarrhea (CLD) is an au-
tosomal recessive disorder of intestinal electrolyte trans-
Introduction
portation caused by mutations in the anion transporter
protein encoded by the down-regulated in adenoma
(DRA), or CLD, gene. In this study, in situ hybridization
and immunohistochemistry were performed to investi-
gate the expression of CLD in extraintestinal normal epi-
thelia and in intestinal inflammatory and neoplastic epi-
thelia. The expression of the closely related anion trans-
porter diastrophic dysplasia sulfate transporter, DTDST,
was also examined and compared with that of CLD in
colon. The only extraintestinal tissues showing CLD ex-
pression were eccrine sweat glands and seminal vesicles.
In inflammatory bowel disease and ischemic colitis, ex-
pression of CLD mRNA in colon epithelium was similar
to histologically normal colon epithelium, but the protein
was found deeper in crypts, including proliferative epi-
thelial cells. In intestinal tumors, the expression pattern
of CLD was dependent on the differentiation status of
the tissue studied: epithelial polyps with no or minor
dysplasia showed abundant expression, whereas adeno-
carcinomas were negative. The DTDST gene was abun-
dantly expressed in the upper crypt epithelium of colonic
mucosa.
Congenital chloride diarrhea (CLD) is an autosomal reces-
sive disorder of intestinal electrolyte transportation (Norio
et al. 1971). A defect in ileal and colonic absorption of Cl–
in exchange for HCO3– results in a lifelong acidic diarrhea
with a high chloride content (Holmberg et al. 1977; Holm-
berg 1986). CLD is caused by mutations in the down-reg-
ulated in adenoma (DRA) gene (Höglund et al. 1996)
which was originally cloned as a candidate tumor suppres-
sor gene due to its downregulation in colon adenomas and
adenocarcinomas (Schweinfest et al. 1993). The CLD pro-
tein is highly homologous to two other known human
members of the sulfate transporter gene family, namely
the human diastrophic dysplasia gene (DTDST) and the
human Pendred syndrome gene (Hästbacka et al. 1994;
Everett et al. 1997). Mutations in the human DTDST gene
cause disorders of skeletal development such as diastroph-
ic dysplasia (Hästbacka et al. 1994), atelosteogenesis type
II (Hästbacka et al. 1996), and achondrogenesis type IB
(Superti-Furga et al. 1996). By Northern analysis it is ex-
pressed in a wide variety of human tissues, including co-
lon (Hästbacka et al. 1994), whereas studies on rat dtdst
have suggested a more restricted expression profile limit-
ed mainly to cartilage and intestine (Satoh et al. 1998).
CLD mRNA has been shown to be expressed in the
mature surface epithelium, especially in brush border
cells of normal ileum and colon (Byeon et al. 1996;
Höglund et al. 1996) whereas no expression studies on
DTDST have been published. These proteins share com-
mon features such as suggested transmembrane topology
and function as anion transporters. The CLD protein has
been shown to act as a Na+-independent Cl–/OH– or
Cl–/HCO3– exchanger (Melvin et al. 1999; Moseley et al.
1999), but it transports also other anions such as sulfate
and oxalate (Silberg et al. 1995). The DTDST protein
transports sulfate (Hästbacka et al. 1994; Satoh et al.
1998) which has a major role in the pathogenesis of car-
tilage growth defects.
S. Haila ( ) · H. Lohi · J. Hästbacka · J. Kere · P. Höglund
✉
Department of Medical Genetics, Haartman Institute,
P.O.Box 21 (Haartmaninkatu 3), 00014 University of Helsinki,
Finland
e-mail: Siru.Haila@helsinki.fi
Tel.: +358-9-1911, Fax: +358-9-19126677
U. Saarialho-Kere · K. Airola
Department of Dermatology,
00029 Helsinki University Central Hospital, Finland
M.-L. Karjalainen-Lindsberg
Department of Pathology, Haartman Institute,
00014 University of Helsinki, Finland
C. Holmberg
Hospital for Children and Adolescents,
00029 Helsinki University Central Hospital, Finland

280
Fig. 1 Western blot detection
of the congenital chloride
diarrhea (CLD) protein. The
band of approximately 75 kDa
is specifically recognized by
anti-CLD serum (1), while
preimmune serum of the same
rabbit detects no specific bands
from homogenized colon
epithelium (2). Molecular size
markers are indicated in kilo-
daltons
In this study, we have characterized epithelial tissues
kD
1
2
and specific cell types that express CLD in vivo, which
is an important step in elucidating physiological func-
tions of the protein and pathophysiology of chloride di-
arrhea as a disease entity. The effect of inflammation on
CLD expression was examined in specimens of several
types of colitis. Since the CLD gene has been suggested
to play a role in neoplastic transformation or tumor pro-
gression in colon (Schweinfest et al. 1993; Antalis et al.
1998), a set of normal tissues, benign neoplasms, and
adenomas with dysplasia, as well as invasive carcino-
mas, were studied to obtain a better understanding of
this process. Since by Northern analysis, the rat dtdst
has been shown to be expressed mainly in cartilage and
intestine, human DTDST was included in this study to
elucidate its possible expression and distribution in hu-
man colon.
211
124
73
43
31
Immunohistochemistry
Antiserum was raised in rabbits against the synthetic peptide
FNPSQEKDGKIDFT corresponding to amino acids 2375–2416
of the published cDNA sequence (GenBank number: L02785).
Peptide synthesis and antibody production were purchased
from Research Genetics (Huntsville, Ala., USA). The specificity
of the antibody was demonstrated by Western blotting (Fig. 1)
using homogenized human colon epithelium. After centrifugation
at 12000 g for 10 min, the supernatant was collected and diluted
1:2 in Laemmli sample buffer (Pharmacia, Uppsala, Sweden) con-
taining 5% β-mercaptoethanol. Denatured proteins were separated
on a 4–20% polyacrylamide gradient gel and the gel was blotted
onto Hybond C-extra (Amersham) membrane using standard pro-
tocols. The primary antibody serum was diluted 1:500. Biotin con-
jugated anti-rabbit IgG (Boehringer Mannheim, Mannheim, Ger-
many) 1:2000 in 0.1% Tween 20/PBS containing 5% non-fat milk
was used as the secondary antibody and was then detected using
streptavidin-POD (Boehringer Mannheim, Mannheim, Germany)
1:5000 in 0.1% Tween 20/PBS containing 5% non-fat milk. The
protein bands were visualized by chemiluminescence according to
standard protocols.
Serial sections to those used for in situ hybridization were used
for immunohistochemistry. The peroxidase–antiperoxidase tech-
nique was performed using an automatic immunostaining device
(Ventana Medical Systems, Tucson, Ariz., USA) and Ventana kits.
For pretreatment, the deparaffinized slides were boiled in a micro-
wave oven for 15 min in 10 mM citrate buffer (pH 7.0). Anti-CLD
serum was diluted 1:100–1:200 and diaminobenzidine was used as
the chromogenic substrate. Preimmune serum was used as a nega-
tive control in parallel sections.
Materials and methods
Tissues
Formalin-fixed, paraffin-embedded archival specimens from adult
patients were obtained from the Department of Pathology, Haart-
man Institute, University of Helsinki. The following specimens
were examined: Crohn’s (colon; n=5), ulcerative (colon; n=6), and
ischemic colitis (colon; n=4); hyperplastic polyps (n=5), villous
(n=3) and tubular (n=3) adenomas, colonic adenocarcinomas
(n=5); mastopathy (n=5), lactating mammary gland (n=2), endo-
metrium at different menstrual phases (n=10), dermatofibroma
(n=1), scalp skin with alopecia (n=1), and prostate hyperplasia
(n=1). Samples from normal ventricle (n=4), duodenum (n=5), je-
junum (n=3), ileum (n=6), appendix (n=2), colon (n=4), sigma
(n=1), rectum (n=1), anal canal (n=2), liver (n=4), pancreas (n=3),
parotid gland (n=3), skin (n=4), heart (n=1), lung (n=3), kidney
(n=3), prostate (n=9), seminal vesicle (n=3), and placenta (n=4)
were studied as well.
In situ hybridization
A 622-bp fragment corresponding to positions 1650–2271 of the
published human CLD/DRA cDNA (GenBank number: L02785)
was generated by polymerase chain reaction (PCR). This fragment
was designed with a T7 RNA polymerase promoter at the 3′ end
and an SP6 RNA polymerase promoter at the 5′ end. Both sense
and antisense probes were transcribed from this PCR product
using the riboprobe in vitro transcription system (Promega,
Madison, Wis., USA). Antisense and sense RNA probes were
labeled with alpha-35S-UTP and purified probes were used at
4×105 cpm/µl of hybridization solution. The DTDST probe was
constructed in a similar way corresponding to the nucleotide posi-
tions 1440–2061 of the DTDST sequence (GenBank number:
U14528), corresponding to the homology region of the CLD
probe.
To assess proliferative activity of epithelial cells, Ki-67 stain-
ing was performed. For pretreatment, deparaffinized sections were
boiled in 10 mM citrate buffer for 3×5 min. After primary anti-
body reaction with rabbit anti-human Ki-67 antigen (Dako, Glos-
trup, Denmark), the slides were processed with StreptABCom-
plex/HRP Duet kit (Dako) utilizing the biotinylated secondary an-
tibody followed by a complex of streptavidin and biotinylated per-
oxidase. Aminoethylcarbazole was used as a chromogenic sub-
strate and slides were counterstained with Mayer’s hematoxylin.
As previously described (Prosser et al. 1989; Höglund et al.
1996), deparaffinized 5-µm tissue sections were digested with
0.6–1.0 µg/ml proteinase K for 30 min at 37°C and treated with
0.25% acetic anhydride in 0.1 M triethanolamine buffer for 10 min
at room temperature. Hybridization was carried out overnight at
52°C. After hybridization, the slides were washed under stringent
conditions, including RNase A, and exposed to LM-1 emulsion
(Amersham, Aylesbury, UK) for 21–60 days at 4°C. The slides
were developed and counterstained with hematoxylin and eosin.
Normal colon samples known to be positive were used as controls
in each experiment and a sense RNA probe was used as a negative
control (Höglund et al. 1996).
Results
Extraintestinal normal tissues
Our previous Northern analysis revealed strong expres-
sion of CLD mRNA in colon and prostate (Höglund
et al. 1996). In this study both stromal and epithelial tis-
sues of the prostate itself were constantly negative for

281
A
C
B
D
E
Fig. 2A–E CLD is expressed in seminal vesicles and eccrine
sweat glands. In seminal vesicle, strong signal for CLD mRNA
(A; darkfield image) and protein (B) are seen in luminal border
of ductal epithelium. Similarly, the luminal side of secretory por-
tion of the eccrine sweat gland demonstrates intensive CLD im-
munoreactivity (C,D) while no signal is detected in sections pro-
cessed with preimmune rabbit serum (E). Original magnification:
A,B ×100; C ×400; D,E ×200
epithelium showed intense CLD expression both at the
apical side of nucleus in the cytoplasm (Fig. 3A–C) and
focally in the brush border (Fig. 3C) of the epithelial
cells.
Hyperplastic polyps, generally considered to lack ma-
lignant potential, demonstrated cytoplasmic expression
of the CLD protein identical with the normal epithelium
(Fig. 3D), but no brush border staining could be detected
due to a total lack of brush border in this set of samples.
CLD mRNA and protein, while signal was found in in- Hyperplastic polyps showed similar distribution of pro-
tra- and extraprostatic seminal vesicles (Fig. 2A,B). liferating epithelial cells by Ki-67 immunostaining when
Strong immunoreactivity was observed especially on mi- compared to normal colonic mucosa (data not shown).
crovilli of the secretory columnar cells of the ductal epi-
thelium (Fig. 2B).
Among other epithelial tissues studied only eccrine Inflammatory bowel tissues
sweat glands showed CLD protein expression. Abundant
immunoreactivity was detected on the luminal side and To assess possible alterations in CLD expression in re-
in intercellular canaliculi of epithelial cells in the coiled sponse to inflammation, altogether 15 samples of ulcer-
secretory part of the eccrine sweat gland (Fig. 2C–E). ative, Crohn’s, and ischemic colitis were studied by in
Ventricle, duodenum, liver, pancreas, parotid gland, situ hybridization and immunohistochemistry. No signif-
small salivary glands, lung, heart muscle, prostate, endo- icant change in the expression of CLD mRNA and cyto-
metrium, placenta, mammary gland, and kidney were re- plasmic immunoreactivity for the CLD protein was de-
petitively negative for both CLD mRNA and protein.
tected in the colon mucosal surface in any samples stud-
ied. Even cases with active inflammation (Fig. 3E–G)
and the gut epithelium bordering ulcerations (Fig. 3H)
demonstrated normal cytoplasmic immunoreactivity. In
Crohn’s and ischemic colitis also the brush border stain-
Normal colon and hyperplastic polyps
We have previously shown that in normal colon CLD ing was similar to normal control (Fig. 3I), but was miss-
mRNA is detected in the surface epithelium, mainly in ing in samples with ulcerative colitis.
the absorptive epithelial and goblet cells, whereas the
In serial sections of inflammatory intestinal samples,
signal was absent at the bottom of the crypts (Höglund Ki-67 immunostaining showed constant redistribution of
et al. 1996). By immunohistochemistry, normal colon the proliferative cell compartment to the upper crypt re-

282
A
C
D
B
F
E
G
I
H
Fig. 3A–I Expression of the CLD protein in normal, hyperplastic, well as immunoreactivity for CLD protein (F) are detected in a se-
and inflammatory colon. A,C Immunostaining for CLD protein is verely inflamed ulcerative colitis specimen in nearby sections. Ar-
detected in the apical surface of absorptive enterocytes in histolog- rows mark corresponding structures. G Higher magnification of
ically normal colon tissue. B No immunoreactivity is seen on sec- the same specimen. H Expression is present even in epithelium
tions processed with preimmune rabbit serum. D Hyperplastic pol- bordering ulcerations. I Brush border staining could also be de-
yps showed cytoplasmic expression of the CLD protein similar to tected focally, here shown in Crohn’s colitis. Original magnifica-
the normal epithelium. E,F Strong signal for CLD mRNA (E) as tion: A,B,E,F ×100; C,H ×400; D,G ×200; I ×500

283
Fig. 4A,B In inflammatory
bowel disease the expression of
CLD is found also in the areas
with proliferative activity.
Colocalization of Ki-67 (A)
and CLD (B) in the lower crypt
regions in nearby sections of
Crohn’s colitis specimens.
Arrows mark corresponding
structures. Original magnifica-
tion: A,B ×100
A
B
Fig. 5A–D CLD is expressed
in some dysplastic colon cells.
A–C Tubular (A) and villous
(B) adenomas with minor dys-
plastic features demonstrated
only slightly decreased apical
cytoplasmic immunoreactivity,
while in C the expression is
more clearly downregulated in
adenomas with moderate dys-
plasia. D No CLD-specific
immunoreactivity is detected in
an adenocarcinoma. Arrow
marks the border line between
adenocarcinoma and normal
epithelium. Original magnifica-
tion: A–C ×100; D ×200
B
D
A
C
gions. Colocalization of the Ki-67 and CLD signal in the showed slightly reduced cytoplasmic immunoreactivity
lower crypt regions suggested an ability of proliferating (Fig. 5A,B), but with moderate dysplasia the cytoplasmic
cells to express CLD (Fig. 4A,B).
immunoreactivity was clearly reduced (Fig. 5C). Total
lack of expression was observed in adenomas with se-
vere dysplasia and in adenocarcinomas (Fig. 5D).
Proliferative epithelial lesions of the colorectum
Multiple colorectal proliferative lesions and carcinomas Diastrophic dysplasia sulfate transporter
were studied to elucidate whether CLD contributes to
cellular mechanisms leading to neoplastic transformation Intense colonic expression of the closely related human
or tumor progression. Proliferative lesions of the colo- DTDST gene was detected in the epithelial cells of
rectum revealed expression of CLD that was associated the upper one-third of the normal crypts (Fig. 6A,B,D).
with the state of differentiation of the tumor: highly At this region the CLD signal was normally present
differentiated but proliferative lesions showed expres- (Fig. 6C). Expression of DTDST decreased through
sion similar to normal epithelium, whereas poorly differ- the opening of the crypt and was absent both in the lumi-
entiated invasive tumors lacked expression. Both tubular nal surface epithelium and in the bases of the crypts
and villous adenomas with minor dysplastic features (Fig. 6B).

284
A
C
B
D
Fig. 6A–D The diastrophic dysplasia sulfate transporter (DTDST)
gene is abundantly expressed in colonic upper crypt epithelium.
A Brightfield view of a section hybridized with DTDST antisense
probe. B In a darkfield image of the same section, DTDST mRNA
is expressed in the epithelial cells of the upper one-third of the
normal crypts where in darkfield image of parallel section (C)
CLD expression was present. D No signal was detected in dark-
field view of a serial section with the DTDST sense negative con-
trol probe. Original magnification: A–D ×100
Interestingly, CLD expression was found in the semi-
nal vesicle epithelium and not in prostate as our earlier
Northern analysis suggested. It seems now obvious that
the prostate sample in our commercial Northern filter
also contained intraprostatic seminal vesicular tissue that
might be difficult to separate from glandular posterior
part of prostate. The seminal vesicle fluid contains a
–
high concentration of HCO3 (Okamura et al. 1985),
which is suggested to be secreted by an active epithelial
transport system (Mann and Lutwak-Mann 1981). CLD
might well be involved in this process since its intestinal
Discussion
–
manifestation includes a defect in Cl–/HCO3 exchange
We have investigated the pattern of expression of the (Holmberg et al. 1975). At ejaculation, stored sperm mix
human CLD gene in various normal glandular tissues, with the alkalic seminal vesicle fluid and the motility of
as well as in histologically normal, inflammatory, hy- the primarily quiescent sperm is rapidly activated. De-
perplastic, and neoplastic intestinal tissues using in situ fects in the neutralization process result in the acidity of
hybridization and immunohistochemistry. We also stud- the luminal fluid, causing hypomotility of the sperm and
ied the colonic expression pattern of the closely related infertility/subfertility (Breton et al. 1996). This hypothe-
anion transporter, DTDST. In normal colon, most abun- sis is supported by the finding that low levels of bicar-
dant CLD mRNA and protein expression were demon- bonate have been associated with hypomotile spermato-
strated in the mature colon surface epithelium, especial- zoa (Okamura et al. 1986). It is important to re-evaluate
ly at the apical side of the nucleus in the cytoplasm and the clinical significance of the seminal vesicular expres-
in the brush border of absorptive enterocytes. Brush sion of the CLD gene in male CLD patients.
border staining corresponds well with the mature, func-
Considering the complexity of sweat secretion physi-
tional form of the protein at the membrane. In contrast, ology, the finding of CLD protein expression in the se-
the role and function of the abundant cytoplasmic pro- cretory sweat gland epithelium is not surprising even
tein remains to be clarified. Most likely it reflects a though no abnormalities concerning sweat secretion are
newly synthesized form of the CLD protein which is un- known in CLD patients. The mechanisms of electrolyte
der terminal processing, or retarded form in an intracel- transportation across cellular borders and to the sweat
lular compartment, such as endoplasmic reticulum or gland lumen are not well established but complicated co-
Golgi apparatus, and finally targeted to the plasma operation of multiple electrolyte transporters has been
membrane.
proposed. The main role has been suggested to belong to

285
Na-K-2Cl cotransporters (Sato et al. 1989) that have
In adenomatous polyps with mild to moderate dyspla-
been recently shown to be present and regulated in simi- sia, the cytoplasmic signal was reduced but clearly pres-
an eccrine clear cells (Toyomoto et al. 1997). At least ent in all samples. However, there were no regions with
–
during cholinergic sweat secretion Cl–-HCO3 and Na-H brush border immunoreactivity normally found in the
exchangers have also been implicated in the control of apical membrane. In more advanced dysplasias and
sweat composition (Sato et al. 1989). Localization of the adenocarcinomas also the cytoplasmic staining was grad-
CLD protein to the coiled secretory part of the eccrine ually lost, whereas the non-neoplastic epithelium adja-
sweat gland suggests it to be one of the mediators of cent to tumors expressed CLD normally. We propose that
electrolyte transportation involved in primary sweat for- there is a gradual loss of expression that is most likely
mation.
associated with the decreased differentiation status of
In inflammatory bowel disease (IBD), the disturbed those tissues. Our results show that CLD is intensely ex-
balance between proinflammatory and anti-inflammatory pressed in normal and benign colon surface epithelium
cytokines has been suggested to play an important role in and that its downregulation is a common phenomenon,
maintaining chronic inflammation (Casini-Raggi et al. but a later event in neoplastic evolution of colonic muco-
1995; Noguchi et al. 1998). Cytokines have also been sa than previously thought. The significance of this find-
shown to alter epithelial ion transport and permeability ing remains open; however the proposed tumor-suppres-
(McKay and Baird 1999), although little is known about sor capability of CLD appears unlikely. More likely, loss
the regulation of enterocytic protein expression by in- of expression reflects loss of terminal differentiation in
flammatory mediators. Previously, inflammation and colon surface epithelium.
IL-1 stimulation have been reported to reduce the ex-
The human DTDST gene was strongly expressed in
pression of the CLD gene (Yang et al. 1998). Here we colon with distribution to the top one-third of the crypt
show that colonic mucosa with IBD, compared with nor- and lacking signal at the surface level. This localization
mal colonic surface mucosa, show no significant change was partially overlapping with that of CLD. Clinical ab-
in the expression of the CLD gene or cytoplasmic form normalities known in diastrophic dysplasia are restricted
of the CLD protein. In contrast, the brush border immu- to cartilage and bone and no intestinal manifestations
nostaining was present in samples with Crohn’s and have been reported. In vitro functional studies have dem-
ischemic colitis, but could not be detected in ulcerative onstrated that both CLD and DTDST mediate the trans-
colitis samples. These findings suggest that reduced port of sulfate (Silberg et al. 1995; Satoh et al. 1998) that
CLD expression reported earlier is not a constant phe- is inhibited by extracellular chloride. In physiological
nomenon in inflammatory intestinal tissue.
circumstances, CLD transports chloride preferentially
Increased proliferative activity in IBD has already (Moseley et al. 1999), whereas the loss of sulfate trans-
been previously shown in terms of expansion of the pro- port in cartilage has been shown to be the major source
liferative zone of the colonic crypt up to the lower two- of cartilage pathology in diastrophic dysplasia. The
thirds of the crypt (Serafini et al. 1981). In our set of physiological role of DTDST in human colon and regu-
samples with IBD CLD is expressed also in these areas lation of its expression require further studies. However,
suggesting that the expression of CLD is not limited to the clinical phenotype of CLD suggests that DTDST is
non-proliferating cells, although in normal tissues, no unlikely to be able to compensate CLD transport func-
CLD expression is usually observed in the cells of the tion in human intestine.
proliferative cell compartment.
Acknowledgements The skillful technical assistance of Ms Alli
Tallqvist and Ms Merja Nissinen is gratefully acknowledged. This
study was supported by the Helsinki University Research Fund,
the Academy of Finland, The Finnish Medical Foundation, the Re-
search and Science Foundation of Farmos, Sigrid Juselius Founda-
tion, and the Finnish Pediatric Foundation, Ulla Hjelt Fund.
Originally, a role of CLD/DRA in colon carcinogenesis
was proposed due to its downregulation in adenomas and
adenocarcinomas (Schweinfest et al. 1993). Further stud-
ies have suggested the downregulation to correlate with
tumor progression and to be particularly significant in the
earliest phases of neoplastic evolution (Antalis et al.
1998). Little clinical support to this hypothesis was given
by an epidemiological survey studying incidence of can-
cer among CLD patients with a non-functional V317del
germline mutation and their parents (obligatory carriers).
The total cancer incidence was unchanged, although
a slightly elevated risk of colon cancer was found
(Hemminki et al. 1998). However, since patients homozy-
gous for this mutation thrive well without prominent can-
cer predisposition, a major role of CLD in the initiation of
cancer is unlikely. These findings led us to study system-
atically the expression of CLD in a set of different colonic
tumors, including all stages of colon tumor progression, to
clarify whether the downregulation of the CLD gene is as-
sociated with neoplastic transformation and progression.
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