Colonic mucin release in response to immobilization stress is mast
cell dependent
Ignazio
Castagliuolo1,
Barry K.
Wershil2,3,
Katia
Karalis1,4,
Asiya
Pasha1,
Sigfus T.
Nikulasson5, and
Charalabos
Pothoulakis1
Divisions of 1 Gastroenterology
and 2 Experimental Pathology, Beth
Israel Deaconess Medical Center,
3 The Combined Program in
Pediatric Gastroenterology and Nutrition, and
4 Division of Endocrinology,
Children's Hospital, Harvard Medical School, Boston 02215; and
5 Department of Pathology,
Boston University School of Medicine, Boston, Massachusetts
02118
 |
ABSTRACT |
We recently reported that immobilization
stress increased colonic motility, mucin, and prostaglandin
E2
(PGE2) release and mucosal mast
cell degranulation in rat colon [Proc. Natl.
Acad. Sci. USA 93: 12611-12615, 1996;
Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34):
G884-G892, 1996]. To directly assess the contribution of
mast cells, we compared colonic responses to stress in mast cell-deficient
KitW/KitW
v
and normal (+/+) mice. Mucin and
PGE2 release were measured in colonic explants cultured from
KitW/KitW
v
and (+/+) mice 30 min after immobilization stress. We found that stress
stimulated colonic mucin release (1.8-fold), goblet cell depletion
(3-fold), and PGE2 (2.3-fold)
release in (+/+) but not mast cell-deficient
KitW/KitW
v
mice. However, mast cell-deficient mice that had their mast cell population reconstituted by injection of bone marrow-derived mast cells
from (+/+) mice had colonic responses to stress similar to those of
normal (+/+) mice. In contrast, colonic transit changes in response to
stress, estimated by fecal output, were similar between
KitW/KitW
v
and normal (+/+) mice. We conclude that mast cells regulate colonic mucin and PGE2 release but not
colonic transit changes in response to immobilization stress.
mast cell-deficient mice; prostaglandin
E2; colonic motility; hypothalamic-pituitary-adrenal axis; central nervous
system
 |
INTRODUCTION |
RECENT EVIDENCE SUPPORTS substantial "cross talk"
between the central nervous system and the endocrine and immune systems in control of intestinal function. Several laboratories reported stress-mediated stimulation of ion secretion (16, 27), intestinal permeability (27), and increased colonic motility (19, 39) in
experimental animal models of acute, nontraumatic stress such as
immobilization. We have recently shown that immobilization stress in
rats causes colonic mucin secretion from goblet cells and stimulated
release of PGE2 from colonic
explants (6, 7). We also found that immobilization stress in rats
caused activation of colonic mucosal mast cells (6, 7) and that
pretreatment of rats with the mast cell "stabilizer" lodoxamide
reduced colonic mucin secretion and
PGE2 release in response to
immobilization stress (6). These results provided indirect evidence
that mast cells are involved in stress-mediated secretion of colonic
mucin and PGE2.
To directly assess whether mast cells contribute to stress-induced
colonic responses, we investigated the effects of immobilization stress
on colonic mucin and PGE2 release,
colonic goblet cell depletion, and fecal pellet output in normal and
mast cell-deficient mice. These mice have been used previously to
delineate the participation of mast cells in intestinal secretory
responses to antigenic and electrical field stimulation (22) as well as
to the neuropeptide substance P (36). In our studies, we compared
colonic mucin and PGE2 secretion
and colonic motility changes in response to immobilization stress in
normal and mast cell-deficient
KitW/KitW
v
mice. Stress-mediated colonic responses were also examined in mast
cell-deficient
KitW/KitW
v
mice that had undergone selective reconstitution of their mast cell
populations by the systemic injection of bone marrow-cultured mast
cells derived from congenic normal mice. Because immobilization stress
activates the hypothalamic-pituitary-adrenal (HPA) axis, leading to
increased corticotropin-releasing factor (35), ACTH, and finally
corticosterone release (1, 34), we also evaluated the corticosterone
levels in these mice under basal and stress conditions. We found that
despite corticosterone levels similar to their congenic normal
controls, mast cell-deficient mice exhibited reduced colonic mucin and
PGE2 secretion in response to
restraint stress.
 |
MATERIALS AND METHODS |
Immobilization stress model.
Adult male mast cell-deficient
WBB6F1-KitW/KitW
v
mice (referred to as
KitW/KitW
v)
(Charles River Breeding Laboratories, Wilmington, MA) and male congenic
normal WBB6F1-(+/+) mice [referred to as (+/+)] were used
in all experiments. Mice were housed individually under controlled conditions on a 12:12-h light-dark cycle and provided with food and
water ad libitum. Mice were handled daily for 7 days before stress
experiments. Experiments were performed between 10:00 and 11:00 AM to
minimize the influence of the circadian rhythm. Immobilization stress
was applied by placing the mice in a restraint cage (Harvard Apparatus,
Cambridge, MA); control mice walked freely. After 30 min, mice were
killed with a bolus of pentobarbital sodium (120 mg/kg ip), their
abdomens were opened, and colons were removed and cut longitudinally.
Sections (1 × 1 mm) were cut and cultured for measurements of
mucin and PGE2 as described below.
Colonic motility was estimated by counting the number of fecal pellets expelled during the immobilization period (19). This study was approved
by the Beth Israel Deaconess Medical Center Institutional Animal Care
and Use Committee.
Measurement of colonic mucin release.
Colonic mucosal explants were cultured in 35-mm tissue culture dishes
(Fisher, Springfield, NJ) in 1.5 ml Trowell's medium (GIBCO BRL,
Gaithersburg, MD) containing 10 µCi/ml
[3H]glucosamine (New
England Nuclear, Boston, MA) and 1% penicillin-streptomycin as
previously described (6, 7). After incubation (37°C for 18 h) in a
5% CO2-95%
O2 atmosphere, the medium was
aspirated and TCA and phosphotungstic acid (TCA/PTA; Sigma, St. Louis,
MO) were added to a final concentration of 10% TCA and 1% PTA. The mixture was kept overnight at 4°C and then centrifuged (1,600 g for 30 min at 4°C). The pellets
were resuspended in ice-cold TCA/PTA and centrifuged several times
until unincorporated
[3H]glucosamine was
completely removed. Pellets were then dissolved in 1 ml of 0.3 M NaOH
and neutralized with 0.1 ml of 0.6 M acetic acid, and radioactivity was
measured. In some samples, to validate that
[3H]glucosamine was
incorporated into mucin, the TCA/PTA precipitate was examined by
density-gradient ultracentrifugation as described previously (6). In
keeping with our previous findings (6), our results here also showed
that the majority (>75%) of the
[3H]glucosamine
migrated in fractions of density >1.45 g/ml, consistent with the
density of mucin (data not shown).
In some experiments, colonic mucosal explants from nonimmobilized mast
cell-deficient or control (+/+) mice were incubated in medium
containing 10 µCi/ml
[3H]glucosamine with
10
7 M of either the known
mucin secretagogues PGE2 (28) and
forskolin (9) or medium alone (control). After incubation at 37°C
for 6 h, mucin glycoprotein release was measured as described above.
Mucin secretion was also examined histologically by counting the number
of goblet cells depleted of mucin, as previously described by us (6,
7). Briefly, colonic samples were fixed in Formalin, embedded in
paraffin, and stained with hematoxylin and eosin and Alcian blue. The
number of goblet cells containing mucus was quantified in each sample
in an area including 10 parallel colonic crypts. We only quantified the
number of surface goblet cells, because our previous results showed
that restraint stress caused mucus discharge from surface but not crypt
goblet cells (6). Coded sections were examined by a gastrointestinal
pathologist (S. T. Nikulasson) in a blinded fashion, and results are
expressed as the number of goblet cells containing mucin per 100 colonic surface epithelial cells.
Colonic PGE2 release.
Colonic explants (3 sections per dish) were incubated (37°C for 30 min) in 1 ml of modified Krebs buffer (15). After 30 min, the medium
was replaced with fresh medium, and explants were incubated for 2 h at
37°C. PGE2 was measured in
aliquots of supernatant by an immunoenzymatic assay (PerSeptive
Diagnostics) as previously described (6, 7). Results are expressed as
picograms PGE2 per milligram of
tissue wet weight.
Selective mast cell reconstitution of mast cell-deficient
KitW/KitW
v.
Mast cell-deficient
KitW/KitW
v
mice exhibit several abnormalities as a result of the mutations in the
W locus, including macrocytic anemia, absence of melanocytes,
sterility, diminished numbers of interstitial cells of Cajal (ICC), and
age-dependent changes in intestinal intraepithelial lymphocytes, as
well as profound mast cell deficiency (11, 14, 24). However, the
expression of immunologic or inflammatory reactions that do not involve
mast cells is generally similar in mast cell-deficient and congenic normal mice (10). The W locus encodes a member of the tyrosine kinase
receptor type III family termed c-kit.
Thus cells expressing c-kit are
affected in these mice, including mast cells. Nakano et al. (20) have
shown that mast cell populations can be selectively reconstituted in
KitW/KitW
v
mice by the adoptive transfer of immature mast cells derived from the
bone marrow cells of congenic normal (+/+) mice. Briefly, femoral bone
marrow cells from (+/+) mice were cultured for 4 wk in WEHI 3 medium
until >99% of total cells assessed by neutral red staining were mast
cells.
KitW/KitW
v
mice were injected with Dulbecco's modified Eagle's medium (0.2 ml
iv) containing 2 × 106 bone
marrow-derived mast cells or an equal volume of medium alone. Ten weeks
later, the hematocrit was measured in all mice to establish that mice
were still anemic, and then mice were used for immobilization stress
experiments. Histological examination of colonic tissues obtained at
the end of the immobilization experiments confirmed reconstitution of
colonic mast cells. In these experiments, a full-thickness section of
mouse colon was processed into Epon-embedded, Giemsa-stained sections
(40). The sections were examined by a single observer, who was blinded
as to the identity of the specimens.
Effect of immobilization stress on blood corticosterone levels of
normal (+/+)
and mast cell-deficient
KitW/KitW
v
mice.
Blood samples were collected in heparinized capillary tubes from the
retroorbital plexus from five mice of each genotype before and after 30 min of immobilization stress. Corticosterone levels (expressed as
µg/dl) in unextracted plasma samples were measured by a commercially
available radioimmunoassay kit (ICN Biomedicals, Costa Mesa, CA).
Statistical analyses.
Statistical analyses were performed with the use of SigmaStat version
1.00 (Jandel Scientific Software, San Rafael, CA). ANOVA was used for
intergroup comparisons. All data are expressed as means ± SE, and
probabilities are regarded as significant at 95% confidence level
(P < 0.05), using Student's
t-test.
 |
RESULTS |
Mast cell-deficient
KitW/KitW
v
mice exhibit normal colonic mucin glycoprotein release in response to
mucin secretagogues.
We compared mucin glycoprotein secretion in colonic explants of mast
cell-deficient and control (+/+) mice in response to the known mucin
secretagogues PGE2 and forskolin.
Incubation of colonic explants of control (+/+) mice with
PGE2 and forskolin showed
increased mucin glycoprotein release compared with mucin release from
explants exposed to medium alone (Table 1).
Colonic explants from mast cell-deficient
KitW/KitW
v
mice also showed increased mucin release in response to
PGE2 and forskolin (Table 1).
Furthermore, the levels of colonic mucin after administration of both
secretagogues were statistically indistinguishable between (+/+) and
KitW/KitW
v
mice (Table 1). These data demonstrate that there is no
difference in colonic mucin release between mast cell-deficient and
normal mice in their ability to respond to direct stimulation with
mucin secretagogues in vitro.
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Table 1.
Effect of secretagogues on mucin glycoprotein release from colonic
explants of mast cell-deficient KitW/KitW v
and congenic normal (+/+) mice
|
|
Mast cell-deficient
KitW/KitW
v
mice exhibit reduced colonic mucin release in response to
immobilization stress.
The levels of colonic mucin release measured either biochemically or by
counting the number of goblet cells containing mucin were similar in
nonimmobilized
KitW/KitW
v
and normal (+/+) mice (Fig.
1A;
Table 2). We next compared mucin release in
colonic explants of normal mice and mast cell-deficient mice in
response to 30 min of immobilization stress. As shown in Fig.
1A, colonic explants from stressed
(+/+) mice released significantly more
[3H]glucosamine-labeled
glycoproteins in vitro vs. nonimmobilized (+/+) mice
(P < 0.05). Histological examination
of colonic tissues also showed a significant reduction in the number of
superficial goblet cells containing mucus in stressed vs. nonstressed
(+/+) mice (Fig. 2; Table 2).
In contrast, 30-min immobilization stress had no significant effect on
colonic mucin glycoprotein release in mast cell-deficient
KitW/KitW
v
mice (Fig. 1A). Furthermore, there
was no significant reduction in the number of superficial goblet cells
containing mucus in immobilized vs. nonimmobilized
KitW/KitW
v
mice (Fig. 2; Table 2).

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Fig. 1.
Mast cell-deficient
KitW/KitW v
exhibit reduced colonic mucin and
PGE2 release in response to
immobilization stress. Mice were immobilized as described in
MATERIALS AND METHODS; control mice
moved freely in their cages. After 30 min, mice were killed, colons
were removed, and colonic explants were cultured.
A: for mucin measurements explants
were cultured in medium containing
[3H]glucosamine and
incubated at 37°C for 18 h. Mucin release was measured by
incorporation of
[3H]glucosamine into
TCA-phosphotungstic acid (TCA/PTA) precipitates of culture
supernatants. B: for
PGE2 measurements, colonic
explants were placed in culture dishes and incubated (37°C for 2 h)
in modified Krebs buffer as described previously.
PGE2 levels were measured in
aliquots of supernatant by an immunoenzymatic assay as described
previously. Bars are means ± SE of 5-13 different
experiments, each with duplicate determinations.
* P < 0.05 vs. all other
groups.
|
|
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Table 2.
Effect of immobilization stress on goblet cell depletion in mast
cell-deficient KitW/KitW v
and mast cell-reconstituted
KitW/KitW v mice
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Fig. 2.
Effect of immobilization stress on goblet cell depletion in mast
cell-deficient
KitW/KitW v
and mast cell-reconstituted
KitW/KitW v
mice. Mice were immobilized as described in MATERIALS
AND METHODS; control rats moved freely in their cages.
After 30 min, rats were killed and full-thickness samples of colon were
fixed in Formalin and stained with hematoxylin and eosin and Alcian
blue. Colons from a nonimmobilized normal (+/+) mouse
(A), a mast cell-deficient mouse
(C), and a mast cell-deficient mouse
that had been selectively reconstituted with bone marrow-cultured mast
cells obtained from congenic (+/+) mice
(E) show normal mucosal
architecture, with many goblet cells containing mucin in the crypts and
the superficial epithelium. Colon obtained from an immobilized normal
(+/+) mouse (B) shows disappearance
of superficial mucin-containing goblet cells, in contrast to colon
obtained from an immobilized mast cell-deficient mouse showing normal
colonic architecture (D). Colon from
an immobilized mast cell-reconstituted mast cell-deficient mouse
(F) shows goblet cell degranulation
comparable to colon of an immobilized normal (+/+) mouse
(B). Original magnification,
×140.
|
|
Mast cell-deficient
KitW/KitW
v
mice exhibit reduced colonic PGE2 release in
response to immobilization stress.
There was no significant difference in basal colonic
PGE2 levels of nonimmobilized
KitW/KitW
v
and (+/+) mice (Fig. 1B). As
observed in rats (6, 7), immobilization for 30 min resulted in a
2.3-fold increase in PGE2 release
in (+/+) mice compared with nonimmobilized (+/+) mice
(P < 0.05, Fig.
1B). However, immobilization of
KitW/KitW
v
mice did not increase colonic PGE2
release (Fig. 1B).
Effect of stress on colonic transit in mast cell-deficient
KitW/KitW
v
mice.
As shown in Fig. 3, immobilization stress
for 30 min resulted in an 18.5-fold increase in fecal pellet output in
normal (+/+) mice compared with nonimmobilized mice
(P < 0.01). Immobilization of mast
cell-deficient mice also caused a 15.2-fold increase in fecal pellet
output compared with nonstressed mast cell-deficient mice
(P < 0.01). Furthermore, there was
no significant difference in the number of fecal pellets after
restraint stress between (+/+) and
KitW/KitW
v
mice (Fig. 3).

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Fig. 3.
Mast cell-deficient
KitW/KitW v
mice exhibit normal fecal pellet output in response to immobilization
stress. After 30-min immobilization, mice were killed, and the number
of fecal pellets produced during immobilization was quantified. Bars
are means ± SE of 7-12 animals per group.
** P < 0.01 vs. respective
controls.
|
|
Mast cell reconstitution of
KitW/KitW
v
mice normalizes stress-induced colonic responses.
To further elucidate the contribution of mast cells in colonic
responses to immobilization stress, we selectively reconstituted mast
cells in
KitW/KitW
v
mice. As previously reported (20), this procedure only corrects the
mast cell deficiency and does not affect the other abnormalities that
result from mutations at the W locus. Quantitative analysis showed that
the numbers of mast cells in the mucosa, submucosa, and muscularis
propria of the colon of mast cell-reconstituted KitW/KitW
v
mice 10-12 wk after injection of (+/+)-derived mast cells was similar to those of normal (+/+) mice
(n = 5 per group, data not shown).
Colonic mucin and PGE2 release and
colonic goblet cell depletion elicited by immobilization stress were
not statistically different between mast cell-reconstituted
KitW/KitW
v
mice and age-matched normal (+/+) mice (Fig.
4; Table 2).

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Fig. 4.
Mast cell reconstituted
KitW/KitW v
mice exhibit normal colonic mucin (A) and
PGE2 (B) release in
response to immobilization stress. Age-matched normal (+/+) mice and
mast cell-deficient mice that had been selectively reconstituted with
bone marrow-derived mast cells obtained from congenic (+/+) mice were
immobilized as described in MATERIALS AND
METHODS; control mice moved freely in their cages.
After 30 min, animals were killed, colons were removed, and colonic
mucin and PGE2 release were
measured as described in Fig. 1 legend. Bars are means ± SE of
4-5 experiments, each with duplicate determinations.
* P < 0.05 vs. respective
controls.
|
|
Mast cell-deficient
KitW/KitW
v
mice exhibit normal stress-induced corticosterone levels in response to
immobilization stress.
We examined the possibility that altered stress-induced corticosterone
release in mast cell-deficient mice may account for their reduced
colonic response to stress. We found that after 30-min restraint
KitW/KitW
v
and (+/+) mice achieved similar plasma corticosterone levels (53.8 ± 6.1 and 56.9 ± 6.2 µg corticosterone/dl of plasma,
respectively; n = 5 per group). Also,
basal corticosterone plasma levels in the two groups were statistically
indistinguishable [12.2 ± 3.4 and 9.5 ± 3.2 µg
corticosterone/dl of plasma in
KitW/KitW
v
and (+/+) mice, respectively; n = 5 per group].
 |
DISCUSSION |
We report here that genetically mast cell-deficient
KitW/KitW
v
mice have diminished colonic mucin release from goblet cells and colonic PGE2 release, but not
fecal pellet output, in response to acute restraint stress. We also
show that mast cell reconstitution of
KitW/KitW
v
mice completely normalized stress-induced mucin and
PGE2 release. These findings
provide the first direct evidence that release of colonic mucin and
PGE2 in response to immobilization
stress is mast cell dependent. These results are consistent with our previous studies, which provided indirect evidence that colonic mast
cells participate in colonic mucin secretion and
PGE2 release after immobilization
stress (6).
Mast cells have been implicated in the pathogenesis of several
gastrointestinal conditions, including acute (5, 23) and chronic
colonic inflammation (see Ref. 37 for review) and functional bowel
disorders (17, 38). Earlier studies indicated that events in the
central nervous system may activate intestinal mast cells (18, 25) and
that mucosal mast cells reside in close anatomic proximity to
intestinal nerves (30, 31). Several studies have also shown that stress
activates mast cells in various organs. For example, Theoharides et al.
(32) reported that immobilization stress induces degranulation of mast
cells in the dura matter of rats, and Spanos et al. (29) showed
activation of bladder mast cells in response to restraint stress. Along
the same lines, immobilization and cold stress in rats caused
proliferation and degranulation of mast cells in the testis (33),
isolation stress increased hypothalamic histamine content (2), and
Pavlovian conditioning in rats caused activation of mucosal mast cells
(18). We have previously reported that immobilization stress in rats stimulated colonic mucin and PGE2
release and caused degranulation of mucosal mast cells (6).
Interestingly, in a preliminary report, Santos et al. (26) showed that
exposing human volunteers to 30-min cold stress caused activation of
jejunal mast cells, as evidenced by increased luminal levels of the
mast cell mediators tryptase, histamine, and
PGD2.
Although the results presented here indicate that mast cells play a
significant role in the observed colonic responses to stress, the
pathways that cause mast cell activation during stress remain to be
elucidated. Previous results from our laboratory suggested an
interaction between nerves and mast cells in mediating goblet cell
mucin secretion in response to restraint stress (6). For example,
pretreatment of rats with hexamethonium, atropine, or bretylium not
only inhibited stress-induced mucosal mast cell activation but also
reduced colonic mucin and PGE2
release caused by restraint stress (6), indicating an interaction
between parasympathetic and sympathetic nerves and mast cells in the
mediation of colonic goblet cell secretion. Neurotensin (NT), a peptide that can be released from intestinal and nonintestinal sources (4), may
also interact with mast cells in the colon and participate in mast cell
activation during stress. Castagliuolo et al. (7) showed that
pretreatment of rats with the nonpeptide NT receptor antagonist
SR-48,692 inhibited colonic mucin and
PGE2 release as well as colonic
mast cell activation caused by restraint stress, in agreement with
studies indicating a functional interaction between NT and mast cells
(3).
Similar to (+/+) mice, mast cell-deficient mice have increased colonic
motility in response to restraint stress (Fig. 3), indicating that mast
cells are not involved in stress-mediated motility changes. This is
particularly interesting since mast cell-deficient
KitW/KitW
v
mice have a diminished number of ICC and abnormal intestinal pacemaker
function (14). Thus our finding would suggest that ICC are not involved
in stress-related motility changes. Our results are quite consistent
with our previous data showing that pretreatment of rats with the mast
cell stabilizer lodoxamide inhibited colonic mucin and
PGE2 release but did not affect
colonic motility in stressed rats (6). Thus immobilization stress
induces mucin secretion, which involves mast cell participation,
whereas colonic motility changes are mediated through a mast
cell-independent pathway. Previous studies suggested that colonic
motility changes in response to immobilization stress most likely
involve parasympathetic nerves and the neuropeptide substance P (6).
Mast cell-deficient mice had decreased colonic responses during stress,
although their basal and stress-induced corticosterone levels were
similar to those of normal (+/+) mice. This indicates that their HPA
axis response to immobilization stress is not impaired and suggests
that mast cells are not required for stress-induced HPA activation,
although they mediate stress-induced colonic responses. Our findings
are different from previous studies in rats, showing a requirement of
adrenal mast cells for ACTH-induced corticosterone release (12, 13).
Differences in the species (mice vs. rats) and/or
methodological approaches (in vivo vs. in vitro) used in our study and
the studies of Hinson et al. (12, 13) may account for these
discrepancies.
In summary, our results directly demonstrate participation of mast
cells in colonic goblet cell discharge and
PGE2 secretion caused by restraint
of mice. These findings provide direct evidence for a link between mast
cells and the intestinal epithelium in the pathogenesis of
stress-related responses. Our findings could be of importance for
understanding the pathophysiology of irritable bowel syndrome, in which
intestinal mast cell activation (17, 21, 38) and mucus discharge (8)
have been reported.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47343 (C. Pothoulakis), DK-33506, and DK-46818 (B. K. Wershil). I. Castagliuolo was supported by a Research Fellowship from the Crohn's and Colitis Foundation of
America, Inc.
 |
FOOTNOTES |
Address for reprint requests: C. Pothoulakis, Division of
Gastroenterology, Beth Israel Hospital, 330 Brookline Ave., Boston, MA
02115.
Received 23 September 1997; accepted in final form 27 February
1998.
 |
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