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1 Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, United Kingdom; and 2 Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114-2696
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-After injury
and loss of epithelial cells, intestinal barrier function is
reestablished by migration of viable epithelial cells from the wound
edge (restitution). Myofibroblasts are located close to the basal
surface of epithelial cells. This study aimed to investigate the role
of human colonic subepithelial myofibroblasts in epithelial
restitution. Primary cultures of subepithelial myofibroblasts were
established. Monolayers of the epithelial cell lines IEC-6 and T84 were
"wounded" in a standard manner to create an in vitro model of
restitution. Migration of epithelial cells across the wound edge was
assessed following culture in myofibroblast-conditioned medium.
Myofibroblast expression of transforming growth factor (TGF)-
isoforms was examined using RT-PCR, and TGF-
isoform bioactivity was assessed using Mv 1 Lu bioassay.
Myofibroblast-conditioned medium, via a TGF--dependent pathway,
significantly enhanced migration of epithelial cells across the wound
edge and significantly inhibited cell proliferation in wounded
monolayers. Messenger RNA for TGF-1, -2, and -3 was detected
in the myofibroblasts, and Mv 1 Lu bioassay showed the presence of
predominantly bioactive TGF-3. This study shows that human colonic
subepithelial myofibroblasts secrete predominantly bioactive TGF-3
and enhance restitution in wounded epithelial monolayers
via a TGF--dependent pathway.
transforming growth factor-; wound repair
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE GASTROINTESTINAL TRACT performs the critical functions of extraction of nutrients, minerals, and electrolytes but excludes luminal microorganisms and toxic molecules. These essential functions are mediated by a monolayer of epithelial cells that lines the intestinal lumen. The epithelial monolayer provides a barrier to luminal toxic molecules and microorganisms via intercellular tight junctions. This barrier is disrupted following injury and loss of epithelial cells, directly exposing the lamina propria cells to luminal contents. In the normal intestinal mucosa, epithelial continuity and barrier function are rapidly reestablished following the loss of injured epithelial cells. This occurs via a process designated restitution, in which viable epithelial cells migrate from the wound edge to reestablish epithelial continuity (15, 16, 21, 29, 31, 37, 43, 47). This process can be complete within minutes to hours, depending on the extent of epithelial injury. Cell proliferation over the subsequent 24-48 h allows the replacement of the lost epithelial cells. There is increasing appreciation for the fact that epithelial restitution in vivo involves complex interaction between epithelial cells, the underlying basement membrane, as well as cells within the lamina propria matrix. The cells in the lamina propria may interact with the epithelial cells via pores in the basement membrane (26).
Intestinal subepithelial myofibroblasts are present immediately subjacent to the basement membrane and close to the basal surface of epithelial cells. Ultrastructural studies have shown that these cells share characteristics of both fibroblasts (7, 14, 19) and smooth muscle cells and have therefore been designated myofibroblasts (18, 20, 36, 38). Their location below the basement membrane suggests that these cells may play an important role in the regulation of a number of epithelial cell functions (45). We have recently established an ex vivo model that allows, for the first time, pure populations of subepithelial myofibroblasts from adult human intestinal mucosa to be available (24). The isolated myofibroblasts retain a representative and differentiated phenotype despite prolonged culture and passage (24). Myofibroblasts have been shown to have an important role in wound healing and fibrosis (10), but there have been no studies to date investigating the role of human intestinal myofibroblasts in the initial wound healing process of restitution.
In this study, we have investigated the role of isolated normal primary
adult human colonic subepithelial myofibroblasts in the regulation of
epithelial restitution. We demonstrate that the myofibroblasts enhance
epithelial cell migration via secreted bioactive transforming growth
factor (TGF)- and that the predominant bioactive isoform is
TGF-3.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture. The nontransformed rat small intestinal epithelial cell line IEC-6 was obtained from the European Collection of Animal Cell Cultures (ECACC; Porton Down, UK) and studied at passages 26-31. The cells were maintained in DMEM (GIBCO BRL, Gaithersburg, MD) supplemented with 5% FCS (GIBCO), 2 mM glutamine (Sigma Chemical, St. Louis, MO), 100 U/ml penicillin (Britannia Pharmaceuticals, Surrey, UK), 0.1 mg/ml streptomycin (Evans Medical, Surrey, UK), and insulin (final concentration 4 µg/ml; Sigma).
The human colon cancer cell line T84 was obtained from the ECACC and studied at passages 70-75. The cells were maintained in DMEM-Ham's F-12 medium (GIBCO) supplemented with 10% FCS (GIBCO), 2 mM glutamine (Sigma), 100 U/ml penicillin (Britannia Pharmaceuticals), and 0.1 mg/ml streptomycin (Evans Medical). Human colonic subepithelial myofibroblasts were isolated from normal colonic mucosal samples obtained from six colonic resection specimens. The normal colonic mucosal samples were obtained >5 cm from the tumor, and myofibroblasts were isolated as previously described (24). In brief, the mucosal samples were completely denuded of epithelial cells by three 30-min periods of incubation (at 37°C) in 1 mmol/l EDTA (Sigma). The deepithelialized mucosal samples were subsequently cultured (at 37°C in 5% CO2) in RPMI 1640 (GIBCO) containing 10% FCS. The cells in suspension were removed after every 24- to 72-h culture period, and the denuded tissue was maintained in culture for up to 6 wk. Established colonies of myofibroblasts were cultured in DMEM supplemented with 10% FCS and 1% nonessential amino acids (GIBCO), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). The cells were passaged using 0.1% (wt/vol) trypsin-0.2% (vol/wt) EDTA in a 1:2 to 1:3 split ratio. Studies were carried out at passages 2-7 of myofibroblasts isolated from six resection specimens. For myofibroblast-conditioned medium, subconfluent and confluent monolayers of cells were washed with DMEM and subsequently cultured in 0.1% FCS-DMEM for 24 h. The conditioned medium was stored at
70°C until use in studies on wounded epithelial monolayers and TGF- bioassay (see below).
Wounding assays.
Wound assays were performed in multiples of six, using a previously
described method (27) with modification. Confluent monolayers of IEC-6
and T84 cells in six-well tissue culture plates (Nunc, GIBCO) were
wounded under microscopic vision using a razor blade and a Gilson
"p2" pipette tip. Cells were washed three times with fresh
serum-deprived medium (0.1% FCS-DMEM), and the wounded monolayers were
further cultured in fresh serum-deprived medium in the presence or
absence of recombinant human TGF-1 (5 ng/ml; R&D Systems, Minneapolis, MN) or myofibroblast-conditioned medium.
1, -2, and -3
(1 µg/ml; R&D Systems).
As previously described, migration of IEC-6 cells was assessed in a
blinded fashion by the determination of the mean number of cells found
across the wound border in a standardized wound area (12), and
migration of T84 cells was assessed in a blinded fashion by
determination of the reduction in wound width over 24 h (34). Wound
areas were standardized by taking photographs at 100-fold magnification
using an Olympus CK-2 inverted microscope and an Olympus OM-1 camera.
Experiments were performed in quadruplicate, and data are presented as
means ± SD.
Cell proliferation in wounded epithelial monolayers. "Wounded" monolayers of IEC-6 cells in six-well plates were incubated as described in wounding assays. After culture for 20 h, [3H]thymidine (Amersham International, Buckinghamshire, UK) was added to each well (1 µCi/well). After a further incubation of 4 h, the cells were subsequently fixed with methanol-acetic acid (vol/vol, 3:1) at room temperature for 1 h, washed twice with 80% methanol, and lysed with 1 M NaOH. Uptake of [3H]thymidine was determined using an LKB (Wallac, Milton Keynes, UK) beta counter.
Proliferation was also assessed by determination of bromodeoxyuridine (BrdU) uptake, as reflected in the mean number of BrdU-positive cells observed per low-power field as previously described (4). Briefly, BrdU (final concentration of 10 µmol/l; Sigma) was added to the wounded monolayer. BrdU uptake was assessed after culture for 24 h in either serum-deprived medium or myofibroblast-conditioned medium by addition of mouse IgG anti-BrdU antibody (final concentration of 200 mU/ml; Sigma) and detected using a universal Vectastain ABC detection kit (Vector Laboratories, Peterborough, UK).TGF- bioassay.
The presence of bioactive TGF- in myofibroblast-conditioned medium
was determined using a specific bioassay, which is based on the ability
of TGF- to inhibit proliferation of the mink lung epithelial cell
line Mv 1 Lu (ECACC) (25). Latent TGF- present in the
myofibroblast-conditioned medium was activated by the addition of
concentrated HCl to pH 2 and left to stand at room temperature for 60 min, followed by neutralization with NaOH and HEPES (to a final
concentration of 16 mmol/l).
1 were added and incubated for
20 h at 37°C (95% O2, 5%
CO2). After 20 h,
[3H]thymidine (1 µCi/well) was added, and the incubation was continued for an
additional 4 h. The cells were then fixed with methanol-acetic acid
(3:1), washed twice with 80% methanol, and lysed with 1 M NaOH. Uptake
of [3H]thymidine was
determined as before. From the standard curve obtained, the
concentration of total and biologically active TGF- present in the
myofibroblast-conditioned medium could be calculated.
The contribution of each TGF- isoform to total bioactivity was
assessed using Mv 1 Lu bioassay performed in the presence of TGF-
isoform-specific monoclonal antibodies (1 µg/ml; R&D Systems).
Conditioned medium from confluent and wounded IEC-6 and T84 monolayers
was also assessed for TGF- bioactivity.
RNA isolation and reverse transcription.
RNA was isolated from myofibroblasts using RNAzolB (Biogenesis, Poole,
UK). Random hexamer primer (Pharmacia Biotech, Brussels, Belgium) was
mixed with 10 µg RNA (final volume of 37.5 µl) and heated to
70°C for 10 min and allowed to cool on ice. Reverse transcription
to cDNA was performed by adding the following and incubating at
37°C for 60 min: 5 µl of 10× PCR buffer [0.5 M Tris (pH 8.3), 0.75 M KCl, and 30 mM
MgCl2 (Stratagene, La Jolla,
CA)], 1.5 µl of 5 mM 2'-deoxyribonucleotide
5'-triphosphate mix (containing dATP, dCTP, dGTP, and dTTP each
at 25 mM; Ultrapure dNTP set, Pharmacia), 1 µl of Moloney murine
leukemia virus RT (200 U/µl; GIBCO), and 5 µl of 0.1 M DTT.
Subsequent enzyme deactivation was performed by heating to 90°C for
5 min, and the cDNA was stored at 20°C.
Polymerase chain reaction.
The following reaction mixture was added to 5 µl of the cDNA product:
5 µl of enzyme buffer [0.5 mM KCl, 0.1 M
Tris · HCl (pH 9.0), and 1% Triton X-100 (Promega,
Madison, WI)], 2 µl of 5 mM dNTPs, 0.5 µl of
Taq DNA polymerase (5 U/µl;
Promega), and sterile water to make a final solution of 50 µl. The
following primer pairs were used (to a final concentration of 5 µM)
based on published nucleotide sequences: 5'-CAG AAA TAC AGC AAC
AAT TCC TGG-3' (sense) and 5'-TTG CAG TGT GTT ATC CGT GCT
GTC-3' (antisense) to amplify 187-bp human TGF-1 product (8);
5'-TCC AAA GAT TTA ACA TCT CCA ACC-3' (sense) and
5'-TCC CAC TGT TTT TTT TCC TAG TGG-3' (antisense) to
amplify 310-bp human TGF-2 product (23); 5'-ACA TTT CTT TCT
TGC TGG-3' (sense) and 5'-GGG GAA GAA CCC ATA ATG-3'
(antisense) to amplify 685-bp human TGF-3 product (9); and
5'-GGT GAA GGT CGG AGT CAA CGG A-3' (sense) and
5'-GAG GGA TCT CGC TCC TGG AAG A-3' (antisense) to amplify
240-bp human glyceraldehyde-6-phosphate dehydrogenase product.
1, -2, and -3 was confirmed by hybridization of
the PCR product using specific digoxigenin-labeled internal
oligonucleotide probes and subsequent detection using alkaline
phosphatase-labeled rabbit anti-digoxigenin antibody (28) and by DNA
sequence analysis. The following digoxigenin-labeled probes were used:
5'-GTT GTG CGG CAG TGG-3' (to identify TGF-1 product),
5'-GAG CAG AAG GCG AAT-3' (to identify TGF-2 product), and 5'-GGG AGA AGG AAG GGC-3' (to identify TGF-3 product).
Statistical analysis. Results are expressed as means ± SD. Statistical analyses were performed using one-way ANOVA and Student's t-test.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Myofibroblast-conditioned medium enhances epithelial migration in
wounded IEC-6 and T84 monolayers.
The results illustrated in Table 1 and
Figs. 1 and
2 show that conditioned medium of
myofibroblast cultures derived from mucosal samples of six different
colons consistently enhanced repair of wounded IEC-6 and T84
monolayers. As expected (4, 12) and shown in Table 1, rTGF-1 also
enhanced wound repair.
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1 (data not shown). In wounded monolayers cultured in
control medium, scattered BrdU-labeled epithelial cells were seen in
the remaining monolayer, as previously described (5). In wounded
monolayers cultured in myofibroblast-conditioned medium or rTGF-1,
there was a significant reduction in the number of BrdU-labeled cells
(data not shown).
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Myofibroblasts enhance epithelial migration via secreted
TGF-.
The similarity with rTGF-1 in the effect on cell migration and
proliferation in wounded epithelial monolayers suggested that the
myofibroblast-conditioned medium contained bioactive TGF-. This was
confirmed by inhibition of myofibroblast-conditioned medium-mediated
epithelial migration by polyclonal neutralizing antibody to TGF-
(Fig. 1A). The
myofibroblast-mediated enhanced restitution in wounded T84 cells was
also shown to be partially dependent on bioactive TGF- (Fig.
1B). Addition of neutralizing antibody alone to wounded IEC-6 monolayers (cultured in control medium)
did not significantly inhibit cell migration (data not shown).
in myofibroblast-conditioned medium
was confirmed using the Mv 1 Lu bioassay (Table
3). After acidification, there was only a
small increase in TGF- bioactivity detected in the conditioned
medium, implying that the majority of TGF- secreted by the
myofibroblasts is in the biologically active form. The predominance of
bioactive TGF- in myofibroblast-conditioned medium was confirmed by
lack of a difference between acid-treated and untreated samples in the
enhancement of restitution in wounded IEC-6 monolayers (Fig. 2).
TGF- bioactivity present in conditioned medium of IEC-6 and T84
epithelial monolayers was negligible in comparison to that present in
myofibroblast-conditioned medium. After acid treatment, the TGF-
concentration of IEC-6- and T84-conditioned medium was 60 and 6 pg/ml,
respectively (TGF- concentration range in myofibroblast-conditioned
medium was 600-1,500 pg/ml). There was no significant difference
in total TGF- bioactivity in conditioned medium collected from
confluent vs. subconfluent myofibroblast monolayers or from wounded vs.
confluent IEC-6 and T84 monolayers (data not shown).
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isoform to total bioactivity was
assessed by the use of monoclonal isoform-specific neutralizing antibodies. As shown in Fig. 3, TGF-3
was the predominant bioactive isoform secreted by the myofibroblasts.
Neutralizing antibodies to TGF-1 and -2 reduced the overall
percentage of TGF- bioactivity by 16.2 ± 6.7% and 3.2 ± 3.8%, respectively. In contrast, anti-TGF-3 antibody reduced total
bioactivity by 81.0 ± 17.6%.
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1,
-2, and -3 are expressed by myofibroblasts (Fig.
4). These PCR products were confirmed to be
those of the individual isoforms of TGF- by hybridization using
digoxigenin-labeled internal oligonucleotide probes and by DNA
sequencing as described in MATERIALS AND
METHODS.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the intestine, the surface monolayer of epithelial cells plays a
critical role in the maintenance of mucosal integrity, providing an
essential barrier to penetration by luminal microorganisms and their
products. Injury to the epithelial cells results in a loss of monolayer
continuity and barrier function and allows penetration by luminal
contents into the lamina propria. It is therefore of importance to the
host to repair any such breach in the barrier to avoid or limit an
inflammatory response. Epithelial continuity and barrier function is
normally rapidly restored by migration of viable cells adjacent to the
wound edge to cover the mucosal defect, a process termed restitution
(15, 16, 21, 29, 31, 37, 43, 47). This process is independent of
proliferation and begins within minutes of the initial injury. Proliferation of epithelial cells, required to make up for the lost
cells, occurs over 24-48 h after the injury (16). Previous studies
of epithelial cell restitution have shown that a large number of
peptide growth factors (interleukin-1, interferon-
, epidermal
growth factor, TGF-
, hepatocyte growth factor, basic fibroblast
growth factor, intestinal trefoil factor) can influence this process
(11-13, 32). However, TGF- plays a central role in mediating
the effects of many of these peptide growth factors (12).
It is recognized that the process of restitution in vivo involves
complex interactions between epithelial cells, cells within the lamina
propria, and components of the extracellular matrix. Previous studies
have shown that components of the extracellular matrix can regulate
epithelial restitution (2, 3, 17); however, there is little information
on the role of individual cell types within the lamina propria in the
regulation of epithelial restitution. In this study, we have examined
the role of primary adult human colonic subepithelial myofibroblasts in
epithelial cell migration. Cultures of subepithelial myofibroblasts
were established as recently described (24). During culture of mucosal samples denuded of epithelial cells, myofibroblasts migrate out of the
lamina propria via pores in the basement membrane. These cells
subsequently become adherent to the culture dish and proliferate to
form monolayers of myofibroblasts. We have previously shown that,
despite prolonged culture and passage, the myofibroblasts retain a
representative and differentiated phenotype (24). Myofibroblast cultures used in this study were established as previously reported, and their phenotypes were confirmed by demonstration of the expression of -smooth muscle actin and vimentin (data not shown). The use of
the IEC-6 and T84 cell lines to study restitution in vitro is well
recognized. Extensive studies have shown the similarity between the
untransformed small intestinal rat epithelial IEC-6 cell line and the
normal rat crypt intestinal epithelial cell (35). These cell lines
allow the study of epithelial cell responses without the ambiguity of
contamination inherent to studies with preparations of primary
epithelial cells.
The studies in this report show that myofibroblasts secrete a factor or
factors that enhance cell migration in wounded epithelial monolayers while they paradoxically inhibit epithelial cell
proliferation, suggesting that this effect was mediated by TGF-.
This was confirmed by the addition of neutralizing antibody to TGF-,
which abrogated the enhanced epithelial cell migration seen in response
to the myofibroblast-conditioned medium. This response was more
pronounced in the nontransformed IEC-6 cell line compared with the
transformed T84 cell line. This could be explained by the relative
resistance of transformed cell lines to the effect of TGF-, one of
the postulated mechanisms for the development of colonic carcinoma.
Previous reports have shown that cells can produce latent TGF- as
well as plasminlike protease activity required to effect bioactivation
through liberation of the mature TGF- dimer (39, 40, 44). We have
shown that myofibroblasts themselves express mRNA transcripts for all
three human TGF- isoforms (TGF-1, -2, and -3). The secreted
TGF- is biologically active, suggesting that the myofibroblasts also
produce plasminlike protease activity. This finding differs from that
of TGF- secretion by platelets (33) or other cell types (6, 22, 46).
In previous studies, TGF- mRNA in the normal colon has been shown to
be expressed predominantly in the lamina propria closest to the surface
epithelium (1). From our current studies, we postulate that TGF-
derived from myofibroblasts may play an important role in epithelial
restitution in vivo. The myofibroblasts lie close to the epithelium,
and the secreted bioactive TGF- could reach the epithelial cells
both by passive diffusion and via pores in the basement membrane (26). It is also possible that there are other myofibroblast-derived factors
that enhance epithelial restitution by inducing secretion of TGF- by
the epithelial cells (4, 5). In addition, the subepithelial network of
myofibroblasts may facilitate repair of the injured intestinal
epithelium by contraction, thereby reducing the denuded surface area to
be reepithelialized (30).
It is recognized that myofibroblasts also participate in the formation
of the extracellular matrix and granulation tissue leading to fibrosis
during repair (10), processes that appear to be influenced to a large
extent by the TGF- superfamily. This superfamily is one of the most
complex groups of cytokines, with widespread effects on many aspects of
growth, development, and wound healing. The three mammalian isoforms of
TGF- (TGF-1, -2, and -3) have been localized in healing
wounds, and there is evidence that manipulation of the ratios of the
TGF- isoforms may influence the latter stages of the repair process,
namely scarring and fibrosis (41). Thus recent studies maintain that either neutralization of TGF-1 and -2 or exogenous addition of
TGF-3 reduces scarring in cutaneous rat wounds (42). In our study,
we have shown for the first time that primary cultures of normal human
colonic subepithelial myofibroblasts release predominantly bioactive
TGF-3. Such predominant expression of bioactive TGF-3 in vivo
would imply a capacity for wound healing without fibrosis. Myofibroblasts may therefore play a pivotal role in regulating several
stages of wound repair from restitution to end-stage fibrosis.
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ACKNOWLEDGEMENTS |
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This work was supported by the Digestive Disorders Foundation (UK) and The National Association for Colitis and Crohn's Disease (UK).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: Y. R. Mahida, Division of Gastroenterology, Univ. Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Received 25 September 1998; accepted in final form 4 February 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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