Molecular Human Reproduction, Vol. 6, No. 10, 943-950,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Localization of E-cadherin in villous, extravillous and vascular trophoblasts during intrauterine, ectopic and molar pregnancy
1 Institute of Pathology, Odense University Hospital, 2 Institute of Human Genetics, Aarhus University Hospital, 3 Department of Obstetrics and Gynaecology, Odense University Hospital, and 4 Department of Immunology and Microbiology, Odense University, Odense University Hospital, DK-5000 Odense C, Denmark
Abstract
Previous reports have described down-regulation of E-cadherin in trophoblasts differentiating to an invasive phenotype. This study shows the localization of E-cadherin in a prospective design with stereological sampling of fetal and maternal first, second and third trimester tissue. E-cadherin was observed in villous cytotrophoblasts, and in non-proliferating, intermediate trophoblasts (IT) within cell columns and islands in intrauterine, ectopic and partial molar placentas. Highly proliferating IT with cytological atypia in complete molar placentas were also E-cadherin-positive. E-cadherin was present in trophoblasts throughout the anchoring cell columns. Trophoblasts undergoing epithelial mesenchymal transformation (EMT) detaching from the distal cell columns and deeper located single extravillous interstitial trophoblasts (EVT) showed E-cadherin-negative breaches in the cell membrane. Prior to the late second trimester, the relative number of E-cadherin-positive single EMT and EVT differed from the total number of cytokeratin-positive trophoblasts. Intraluminal, endovascular and perivascular trophoblasts adjacent to the maternal vessels were also E-cadherin-positive, but a highly varying pattern was observed at different ages of gestation. Our results indicate a temporary shift in E-cadherin expression in extravillous trophoblasts possessing a migrating and invasive potential. Functional E-cadherin may be restored as trophoblasts aggregate in the decidua and the vessel wall after completion of migration.
E-cadherin/hydatidiform mole/invasion/placentation/trophoblasts
Introduction
E-cadherin is supposed to be essential for embryonal development and blastocystic implantation. E-cadherin is a Ca2+-dependent cell–cell adhesion molecule expressed in epithelial cells. The intact form of E-cadherin is a 120–130 kDa integral membrane glycoprotein linked to the cytoskeleton via intracellular ligands termed catenins 
, ß and 
respectively, (Dalseg et al., 1993
; Behrens, 1994; MacCalman et al., 1998) and the complex is essential for cell adhesion. In normal cells, the presence of E-cadherin is thought to stabilize epithelial architecture by restraining invasion thereby regulating the differentiation of benign, non-invasive phenotypes (MacCalman et al., 1998). Consequently, E-cadherin appears to be down-regulated following de-differentiation in metastatic malignant phenotypes (Siitonen et al., 1996; Charpin et al., 1997; Mori et al., 1998; Bankfalvi et al., 1999).
E-cadherin has mainly been characterized in anchoring placental villi and is considered to be absent or gradually down-regulated following extravillous differentiation in vitro and in vivo (Babawale et al., 1996; Zhou et al., 1997a,b). Zhou et al. describe a substantial down-regulation of the protein in distal trophoblasts of anchoring villi as well as in interstitial and vascular trophoblasts in normal pregnancies between the first and second trimester (Zhou et al., 1997a). In contrast, placental bed biopsies from patients with preeclampsia have shown an apparent up-regulation of E-cadherin in interstitial trophoblasts and vascular trophoblasts colonizing maternal blood vessels (Zhou et al., 1997b). The localization of E-cadherin has furthermore been described in a small series of placental site trophoblastic tumours, epithelioid trophoblastic tumours and choriocarcinomas demonstrating dispersed E-cadherin expression patterns (Shih and Kurman, 1997, 1998).
Whether normal, pre-eclamptic or molar trophoblasts share biological features with tumour cells is poorly elucidated. It has been suggested that trophoblast invasion of maternal tissue partly imitates tumorous growth, but occurs in a strictly controlled fashion (Pijnenborg, 1994). An uncontrolled trophoblastic invasion more frequently accompanys diploid, androgenic complete hydatidiform molar pregnancies (CM) when compared with triploid, partial hydatidiform moles (PM) (Genest et al., 1991; Silverberg and Kurman, 1992; Paradinas and Fisher, 1995; Shih and Kurman, 1997). Another example of uncontrolled trophoblastic invasion is that of benign ectopic pregnancies (EP) (Randall et al., 1987; Floridon et al., 1996, 1999). These pathological pregnancies can each proceed to persistent trophoblastic disease (PTD) which is the clinical consequence in 15% of CM, 1% of PM (Paradinas and Fisher, 1995) and 5% of EP (Floridon and Thomsen, 1994) respectively. Accordingly, a broad spectrum of proteins, their receptors, proteases and cell-cycle related markers of proliferation have been studied to predict PTD (Genest et al., 1991; Rice et al., 1991; Persaud et al., 1993; Suresh et al., 1993; Cheung et al., 1994; Greenfield, 1995; Jefferset al., 1996; Montes et al., 1996; Horn and Bilek, 1997; Floridon et al., 1999), but only serial assessments of serum human chorionic gonadotrophin (HCG) are currently accepted as markers for the development of PTD (Rice et al., 1991; Greenfield, 1995).
The aim of the present study was to analyse trophoblastic E-cadherin localization in vivo in a prospective design with stereological tissue sampling (Gundersen et al., 1988a,b) from patients with normal, molar and ectopic pregnancies. The main objective was a systematic evaluation of E-cadherin in the villous and extravillous trophoblastic subpopulations within the basal plate, placental bed and maternal vessels. The significance of E-cadherin for development of post-molar PTD was furthermore evaluated by semi-quantification of E-cadherin expression.
Materials and methods
Study design
Prospectively collected formalin-fixed paraffin-embedded and cryostat sections of placental tissue were analysed by a monoclonal anti-human E-cadherin antibody. The study design included a pilot study where five different epitope retrieval protocols were tested on two multi-tissue blocks comprising 23 placental, 27 normal and 14 malignant tissues.
The localization of E-cadherin was evaluated in the different fetal and maternal cellular subpopulations. The histopathological analysis was performed by systematical analysis of different locations separately, i.e. the villous part of the placenta, the villi anchoring to the basal plate and the placental bed including the maternal vessels. E-cadherin expression within sections were classified as positive or negative regardless of the staining intensity.
The study included a semi-quantitative analysis of E-cadherin in placental tissue from normal and molar specimens with development of post-molar PTD. The analysis was performed by two individual pathologists. The estimation of the percentage of immunoreactive cells positive for E-cadherin was assessed in cell islands of intermediate trophoblasts from normal pregnancies and the corresponding cell islands of molar trophoblasts. The observations were related to the gestational age of the pregnancy. Semi-quantification of E-cadherin was scored into three groups according to the percentage of positive cells: group 1 <33%; group 2 33–67%; and group 3 >67%. Computer-assisted analysis was performed using the C.A.S.T – Grid system (Olympus Denmark). A counting frame containing the representative area in the villous part of the placenta was marked, and within this frame 10 random fields of vision containing at least 500 cells, i.e. the intermediate trophoblasts within cell islands was scored in 2-D. The magnification used for scoring was x20 objective lens.
Tissue preparation
Tissue specimens from 32 normal intrauterine pregnancies (gestational age 6–40 weeks) and 92 complete and partial moles (gestational age 6–22 weeks) were collected by stereological random sampling (Gundersen et al., 1988a,b). The intrauterine molar and normal placental tissue in the patients was firstly evacuated. Secondly, a deep scraping was done until `muscle sound' to achieve maternal tissue from the basal plate and placental bed encompassing decidua and myometrium with interstitial trophoblastic invasion. Afterwards, 10–15 tissue blocks were chosen in a haphazard way and fixed in formalin overnight at random orientation. The tissue samples from the 92 complete and partial moles have previously been analysed to document the molar chromosomes and parental origin (Sunde et al., 1993, 1996).
Tissue specimens from 50 unruptured ectopic pregnancies (gestational age 6–11 weeks) were surgically removed including the entire Fallopian tube and the pregnancy. To avoid distortion, the Fallopian tube was carefully extended and mounted with needles on polystyrene before fixation overnight in formalin. After fixation, consecutive cross-sections of the entire Fallopian tube were performed.
Endometrial tissue from the intrauterine evacuate in 20 of the 50 patients with an unruptured ectopic pregnancy was also analysed. In addition, 10 specimens with morphologically verified endometrial tissue in the proliferative phase and 10 specimens in the secretory phase from 20 supplementary non-pregnant, premenopausal women were included in the study. The hormonal status of these patients, i.e. serum progesterone concentrations, was not analysed.
Antibodies and immunohistochemistry
The monoclonal anti-human E-cadherin antibody (clone HECD-1; R&D Systems, Abingdon, UK) was tested prior to application. The antibody specifically recognizes an epitope in the extracellular domain of E-cadherin with the molecular weight of 120 kDa. The HECD-1 antibody is previously characterized biochemically and lacks cross-reactivity to other members of the cadherin family (Shimoyama et al., 1989, 1999; Takeichi, 1991).
The following parameters were considered: (i) identical staining pattern of formalin-fixed paraffin embedded and frozen sections; (ii) a specificity in accordance with the literature when analysing multi-tissue blocks; (iii) minimal unspecific reaction on multi-tissue blocks; (iv) reaction in relation to formaldehyde fixation time (6, 24, 48 h and 1 week) and (v) epitope retrieval protocol, i.e. no retrieval, protease type 14 (0.05% in Tris-buffered saline, pH 7.4) for 12–18 min at 37°C, pepsin (0.4% in 0.01 mol/l HCl) for 20 min at 37°C and microwave heating at 600 W (Grabau et al., 1998) in 10 mmol/l citrate buffer at pH 6.0 or 10 mmol/l Tris 0.5 mmol/l EGTA at pH 9.0 (TEG buffer) for 25 min.
Before immunostaining, 4 µm sections of formalin-fixed paraffin-embedded tissues were mounted onto ChemMate capillary gap slides (Dako, Glostrup, Denmark), dried in a slide oven at 60°C for 1 h, deparaffinized with xylene, and rehydrated with ethanol to distilled water. The staining procedures were performed on an automated immunostainer (TechMate 1000; Dako) using the biotin–streptavidin detection system (ChemMate-HRP/DAB; Dako). The primary antibody was diluted in ChemMate diluent, and incubation performed overnight at 4°C. All following procedures were carried out at room temperature in accordance with the ChemMate protocol. Each TechMate holder included a positive and negative control slide, i.e. a multi-tissue block containing normal and malignant tissue incubated with the primary antibody and a multi-tissue block where the primary antibody was substituted with TechMate diluent.
The results of this analysis revealed that the optimal procedure was epitope retrieval in microwave heating/TEG buffer with the anti-human E-cadherin antibody (clone HECD-1) diluted 1:1600 as the primary antibody. Using this procedure there was no effect on the signal after prolonged fixation of up to 1 week. Epithelial cells and proliferating cells were identified in consecutive parallel sections with monoclonal antibodies to cytokeratin (CAM 5.2; Beckton Dickinson, Orangeburg, NY, USA) or against Ki67 (Immunotech, Marseilles, France) respectively.
The number of patients in the present study were 214. In total, 1588 formalin-fixed paraffin-embedded sections stained with haematoxylin and eosin, 338 with anti E-cadherin, 338 with anti cytokeratin and 338 with Ki-67 were evaluated. In addition, frozen tissue from two early and one mature placenta were used as controls for the antibody.
Results
The localization of E-cadherin in the fetal and maternal cellular subpopulations in intrauterine, ectopic and molar pregnancies are summarized in Table I.
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Localization of E-cadherin in the villous placenta
Membrane-associated E-cadherin staining was seen in the cytotrophoblastic (CT) monolayer covering the chorionic villous core. The polarized proliferating CT (Ki67-positive) at the proximal tip of cell columns and the distal, non-proliferating (Ki67-negative) intermediate trophoblast (IT) of normal intrauterine (Figure 1a
), ectopic and partial villous placental tissue were also E-cadherin-positive. The corresponding highly proliferating (Ki67-positive), molar IT within the polypoid cell columns or cell islands were likewise E-cadherin-positive (Figure b, c and d). In four of the complete molar cases, E-cadherin staining was negative in all IT, but only one of these cases developed postmolar PTD. In partial moles, the cytotrophoblasts of the stromal inclusions were E-cadherin-positive (Figure 1e). Otherwise, villous trophoblastic E-cadherin expression did not differ between normal, ectopic and partial or complete molar pregnancies. However, the total number of villous cell islands comprising IT were markedly reduced beyond the early second trimester in the normal and partial molar placentae whereas the total number of villous cell islands comprising IT in the complete molar placentae remained almost unchanged up to 22 weeks gestation. The multinucleated syncytiotrophoblasts (ST) lining the intervillous space as well as ST within molar cell columns and islands were E-cadherin-negative (Figure 1a, b and e).
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Localization of E-cadherin in anchoring villi, basal plate, placental bed and endometrium
The trophoblasts throughout anchoring cell columns were E-cadherin-positive (Figure 2a
). The most distal trophoblasts of anchoring villi presented a phenotypical epithelia–mesenchymal transformation (EMT). E-cadherin staining was diminished in single cell EMT trophoblasts detached from the distal row of the anchoring columns (Figure 2a). Within first and early second trimester basal plate samples, E-cadherin staining of single cell extravillous interstitial trophoblasts appeared focally discontinuous along the cell membrane with E-cadherinnegative breaches (Figure 2a). By contrast, when extravillous interstitial trophoblasts were aggregating, E-cadherin staining was positive throughout the cell membrane (Figure 2b and c). Placental bed trophoblast giant cells were E-cadherin-negative (Figure 2b). The trophoblastic colonization of the maternal basal plate, placental bed and vessels were much more extensive in ectopic pregnancies (Figure 2b) than in normal and molar pregnancies.
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The E-cadherin staining pattern of single cell extravillous interstitial trophoblasts differed markedly before early second trimester placentae when compared with later gestations, i.e. late second and third trimester placentae. The relative number of E-cadherin-positive single cell extravillous interstitial trophoblasts in basal plate or placental bed specimens was lower prior to the early second trimester (results not shown) when compared with the total number of cytokeratin-positive trophoblasts. In contrast, extravillous E-cadherin-positive trophoblasts were mainly grouped in large aggregates in the late second and third trimester normal and molar specimens. However, there were no third trimester tissue specimens comprising deep placental bed with myometrium, since no pregnant hysterectomies were performed during the study. In the superficial part of basal plate third trimester placentae, aggregating E-cadherin-positive extravillous trophoblasts were consistently observed within areas of maternal cells and fibrinoid deposits in the maternal–fetal junctional zone (Figure 2c
). Furthermore, the number of E-cadherin-positive trophoblasts was equal to the total number of cytokeratin-positive cells in consecutive parallel sections (results not shown). E-cadherin staining was positive in all endometrial glandular cells (results not shown). E-cadherin expression did not deviate throughout the menstrual cycle when related to proliferative or secretory phases (results not shown). Decidual cells were E-cadherin-negative and there was no decidual-like reaction of the maternal stromal cells in the Fallopian wall of the highly colonized ectopic pregnancies.
Localization of E-cadherin in trophoblasts of the maternal vessels
First trimester intraluminal trophoblasts attached to endothelial cells (Figure 3a) as well as peri- and endovascular trophoblasts were E-cadherin-positive (Figure 3b and 3c) when trophoblasts were aggregated. The vessel associated E-cadherin-positive trophoblasts of first trimester specimens consisted with cytokeratin-positive cells of consecutive parallel sections.
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In the Fallopian tube, all stages of invasion, i.e. submucosal, intramural and subserosal trophoblastic spread was observed. The submucosal central vessels of the Fallopian wall were much more extensively colonized with trophoblasts (Figure 3c
) than basal plate and placental bed spiral arteries of first trimester intrauterine placentae. Trophoblasts that were invading the peripheral vessels beneath the circumferential muscular layer in the tubal wall were E-cadherin-positive (results not shown).
When vascular trophoblasts presented as single cells or were embedded in fibrinoid deposit within the vessel wall, E-cadherin staining was similar to that of single cell extravillous interstitial trophoblasts with E-cadherin-negative breaches of the cell membrane (Figure 3d). The relative number of E-cadherin-positive vascular trophoblasts in the basal plate and superficial placental bed demonstrated a highly varying pattern in different second trimester specimens and/or even within the same section. In several sections, the number of E-cadherin-positive trophoblasts was significantly lower (Figure 3e) than the total number of cytokeratin-positive trophoblasts (Figure 3f). Other second trimester cases, or different locations within same sections, demonstrated an apparent down-regulation of E-cadherin (Figure 3d) or a staining pattern similar to that of first trimester pregnancies (Figure 3b). Trophoblasts in the deep placental bed invading the myometrial vessel was apparent in most of the evacuated specimens from first and second trimester normal and molar pregnancies and showed a similar expression pattern. In contrast, the vascular trophoblasts of third trimester vessels within the placental septae of the superficial basal plate were E-cadherin-positive (Figure 3g) in accordance with the cytokeratin-positive vascular trophoblasts of consecutive parallel sections (results not shown).
Semi-quantitative analysis
The results of the semi-quantitative analysis are shown in Table II. In 11 patients (12%) of the 92 cases (49 of complete and 43 of partial mole) of molar pregnancy a subsequent clinical need for re-evacuation or additional chemotherapy was diagnosed. The specimens from these 11 patients were classified as cases with post-molar development of PTD.
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Initially, the inter-observer variability showed low concensus between the two pathologists. This high inter-observer variance was due to disagreement in scoring E-cadherin-positive molar intermediate trophoblasts. This was primarily biased by discrepancies of an apparent decreased staining intensity in the loosely attached molar intermediate trophoblasts. In a second review, after disputing E-cadherin staining reaction bias, the pathologists agreed in scoring E-cadherin in 88 of the 92 molar cases. Nevertheless, when performing the non-parametric
2-test the result was not significant. Consequently, semi-quantitative evaluation of E-cadherin was not useful to predict development of post-molar PTD. Discussion
E-cadherin is supposed to be essential for murine embryogenesis and placental development (Larue et al., 1994; Riethmacher et al., 1995). Previous in-vivo immunofluorescence studies have suggested a down-regulation of E-cadherin accompanying human trophoblast differentiation within anchoring cell columns from first to second trimester normal pregnancies (Zhou et al., 1997a,b, 1998). Absence of E-cadherin in trophoblasts colonizing second trimester maternal vessels in normal pregnancies was furthermore described. In contrast, an apparent up-regulation of E-cadherin in trophoblasts in placental bed biopsies from patients with pre-eclampsia has been stated, and a corresponding shallow interstitial and vascular invasion is claimed to be the result in this condition (Zhou et al., 1997b). Shallow invasion in pre-eclampsia is nonetheless a controversial finding (Pijnenborg, 1998). Co-cultures with trophoblastic and decidual tissue have shown a down-regulation of E-cadherin following epithelial mesenchymal transformation (EMT), and a loss of the protein after further differentiation to extravillous interstitial trophoblasts (Babawale et al., 1996). However, these findings were obtained by different anti E-cadherin antibodies than the presented E-cadherin (HECD-1) antibody which lacks cross-reactivity to other members of the cadherin family (Shimoyama et al., 1989, 1999; Takeichi, 1991).
E-cadherin was in the present study seen in trophoblasts throughout the anchoring cell columns of first and second trimester placentae without an apparent down-regulation. Epithelial mesenchymal trophoblasts detaching distally from cell columns and deeper located single extravillous interstitial trophoblasts (EVT) showed E-cadherin-negative breaches in the cell membrane, but E-cadherin was not completely absent as previously reported (Babawale et al., 1996). However, the relative number of E-cadherin-positive single EMT and EVT differed from the total number of cytokeratin-positive trophoblasts prior to the second trimester. Furthermore, we observed a highly varying E-cadherin staining pattern in vascular trophoblasts of the basal plate and placental bed compared with Zhou et al. (1997a,b). Basal plate intraluminal, endovascular and perivascular trophoblasts as well as trophoblasts related to myometrial vessels were E-cadherin-positive, but the relative number of E-cadherin-positive trophoblasts also differed at different ages of gestations. Thus, a major difference between the present study and previous reports (Zhou et al., 1997a,b) are our observations of E-cadherin-positive trophoblasts throughout gestation and in trophoblasts related to myometrial vessels. These discrepancies may be explained by the sampling procedure and/or the number of patients and tissue specimens presented compared with the smaller series reported by Zhou et al. (1997a,b). In a review (Pijnenborg, 1998), it was concluded that recent placental bed literature has resulted in over-interpretation of trophoblastic observations due to inadequate sampling of basal plate biopsies or specimens primarily from delivered placentas. The studies by Zhou et al. (1997a,b) may also be influenced by a challenged morphological evaluation when using immunofluorescence. Accordingly, we agree with Pijnenborg in this point of view, and the results presented emphasize the importance of adequate sampling procedures comprising several specimens. Our study does not readily confirm previous reports on trophoblastic E-cadherin down-regulation with the acquisition of a more invasive phenotype even though we also observed a temporary shift in E-cadherin expression in extravillous trophoblasts possessing a migrating and invasive potential. Whether experimental cell culture systems as reported by Babawale et al. (1996) reflect the behaviour of extravillous trophoblasts in vivo remains open and the behaviour of deeply invasive aggregations of trophoblasts outside the myometrial vessels is poorly elucidated (Kaufmann and Castellucci, 1997). Studies using co-cultures between trophoblasts and the myometrium and/or vascular system may throw further light on this enigma.
Experimental studies using knock-out mice have demonstrated that E-cadherin is essential for embryonic development (Larue et al., 1994; Riethmacher et al., 1995). E-cadherin homozygous negative (–/–) mouse embryos were unable to form functional trophectoderm and died around the time of implantation whereas both heterozygous embryos (+/–) and wild-type embryos (+/+) developed normally. Immunohistochemical analysis of embryos revealed membrane-associated E-cadherin in the embryos, but only up to the morula stage. This finding is considered to be due to a maternal supply of E-cadherin mRNA from the oocyte. Both mouse and human placentas are haemochorial and the biology of placentation may be related. However, the invasive steps in primates and rodents differ with respect to the mode of trophoblastic attachment and infiltration of the decidua while extravillous interstitial and myometrial invasion is a unique feature of the human placenta (Salamonsen, 1999).
Whether molar trophoblasts are more invasive than normal trophoblasts or if they merely represent de-differentiated phenotypes is not readily understood. The intermediate trophoblasts are the subcellular phenotype supposed to be most essential for invasion of the decidua, myometrium and maternal vessels (Shih and Kurman, 1997). The present study demonstrates E-cadherin in both normal, non-proliferating villous trophoblasts as well as in complete molar, highly proliferating villous and extravillous intermediate trophoblasts with cytologic atypia. Accordingly, no substantial differences were observed between cell–cell adhesion in normal and molar trophoblasts possessing a different phenotype. Nor was semi-quantitative evaluation of E-cadherin useful in predicting postmolar PTD. However, a general problem in trying to identify a prognostic marker for PTD is possibly that the information remains in the molar tissue left behind after the primary evacuation. It has recently been demonstrated that the amounts of a circulating 80 kDa degradation product of E-cadherin (sE-cadherin) were useful as a clinical marker for tumour progression in patients with malignant diseases (Katayama et al., 1994; Griffiths et al., 1996; Velikova et al., 1998). Consequently, serum concentrations of the circulating form of E-cadherin in normal and molar pregnancies after the primary surgical treatment may add further information concerning development of post-molar PTD.
In conclusion, the present study describes the in-vivo subcellular localization of E-cadherin in a prospective study design with stereological sampling of fetal and maternal tissue samples from normal, ectopic and molar pregnancies. The results presented indicate a temporary shift in extravillous and vascular trophoblastic E-cadherin expression in first trimester pregnancies, at which time the trophoblasts possess a migrating and invasive potential. Consequently, functional E-cadherin may be restored as trophoblasts aggregate in the decidua and the vessel wall after completion of migration.
Acknowledgments
This work was supported by a grant from A.J.Andersen Foundation, Meta and Håkon Baggers Foundation (The Danish Cancer Society) and the Institute of Clinical Research, Odense University Hospital, Denmark.
Notes
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Odense University Hospital, Odense University Hospital, DK-5000 Odense C, Denmark. E-mail: floridon{at}yahoo.com 
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Submitted on January 10, 2000; accepted on July 10, 2000.
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