Disparate effects of relaxin and TGF{beta}1: relaxin increases, but TGF{beta}1 inhibits, the relaxin receptor and the production of IGFBP-1 in human endometrial stromal/decidual cells

J. Mazella, M. Tang and L. Tseng1

Department of Obstetrics and Gynecology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8091, USA

1 To whom correspondence should be addressed. e-mail: litseng{at}notes.cc.sunysb.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of this study was to determine the effect of progestin, relaxin (RLX) and transforming growth factor {beta}1 (TGF{beta}1) on the content of relaxin receptor (LGR7) mRNA. The effect of RLX on insulin-like growth factor binding protein-1 (IGFBP-1) production was determined to evaluate the biological function of RLX/receptor in human endometrial cells. METHODS AND RESULTS: The levels of LGR7 mRNA and the effect of hormones were determined by real-time PCR in endometrial cells. LGR7 mRNA was found to be relatively abundant in endometrial glands and decidual cells and much less in endometrial stromal cells. In stromal cells, medroxyprogesterone acetate (MPA), or MPA plus RLX, significantly increased the LGR7 mRNA and RLX alone had little effect. In decidual cells, RLX increased LGR7 mRNA in a dose- and time-dependent fashion. TGF{beta}1 reduced the LGR7 mRNA. In stromal cells, MPA alone caused a slight increase (2–4-fold) of the production rate of IGFBP-1 whereas MPA plus RLX synergistically increased (>40-fold) the IGFBP-1 production. In decidual cells in which the basal production rate was already ~50-fold higher than in stromal cells, RLX alone caused an additional increase (>30-fold) on the production rate. TGF{beta}1 inhibited the IGFBP-1 production. CONCLUSION: The present study showed that in undifferentiated endometrial stromal cells, progestin increases the RLX receptor content to enhance the effect of RLX on the target gene (IGFBP-1). In decidual cells, RLX alone up-regulates its receptor, resulting in a large scale induction of IGFBP-1. TGF{beta}1 has an inhibitory effect on LGR7 and IGFBP-1.

Key words: endometrial decidual and stromal cells/relaxin/relaxin receptor/TGF{beta}/IGFBP-1


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Estrogen and progesterone promote growth and differentiation of endometrial cells during the menstrual cycle (Dallenbach et al., 1987Go). In a fertile cycle, endometrial stromal cells transform into decidual cells under the influence of progestin. Progestin, however, has limited capacity to induce decidualization, namely stromal cells are transformed to predecidual cells which produce a relatively low amount of decidual cell marker proteins, such as insulin-like growth factor binding protein (IGFBP-1) and prolactin (PRL). Formation of decidual cells and extensive induction of these proteins are enhanced by growth factors and cytokines provided that receptors of these factors are present in endometrial cells. One of the probable effects of progestin is to induce local production of growth factors/cytokines and/or their receptors.

Among various types of factors, relaxin (RLX) and transforming growth factor (TGF{beta}) have been considered to be the essential factors for tissue remodelling in endometrium (Lane et al., 1994Go; Tseng, 1994Go; Parekh et al., 2002Go). It has been shown that these two factors are expressed in endometrial tissue (Casslen et al., 1998Go; Palejwala et al., 2002Go; Parekh et al., 2002Go). However, the target genes activated by RLX or TGF{beta} appear to be different in endometrial cells.

The biological effect of RLX on endometrium was first demonstrated four decades ago by Hisaw and Hisaw (1964Go), who showed that administration of RLX in conjunction with estrogen and progesterone resulted in a pregnant-like endometrium in monkey. More recent studies from our laboratory have shown that RLX enhances decidualization based on the ultrastructural analysis of human endometrial stromal cells in long-term culture under the influence of medroxyprogesterone acetate (MPA) and RLX (Lane et al., 1994Go). RLX causes a rapid increase in cAMP accumulation and induces glycodelin production in human endometrial glandular epithelial cells (Chen et al., 1988Go; Tseng et al., 1999Go). In endometrial stromal cells, accumulation of cAMP and target gene activation were noticed after a long period of incubation with progestin and RLX (Tseng et al., 1992Go; Tseng, 1994Go). It is unclear how progestin enhances the RLX action or vice versa. We hypothesized that progestin increases RLX receptor content which determines the function of RLX.

Although binding of RLX to its receptor has been demonstrated in numerous publications, RLX receptor protein was recently cloned and identified as two orphan leucine-rich G-protein-coupled receptors, LGR7 and LGR8 (Hsu et al., 2002Go; Hsu, 2003Go). Subsequently, RLX receptor mRNA and protein expression in human and primate endometrium have been identified in both the epithelial and stromal compartments (Ivell et al., 2003Go; Luna et al., 2004Go). Hormone, however, appears to be ineffective in regulating LGR7 mRNA (Ivell et al., 2003Go). In this study we investigated the effect of hormones on RLX receptor in endometrial cells by real-time PCR which is able to quantify low levels of mRNA and to detect any changes on the level of message affected by the hormone. In addition to endometrial stromal and glandular cells, decidual cells were included in this study.

TGF{beta} has been claimed to play a role in endometrial cell differentiation. It has been shown to enhance tissue remodelling and homeostasis in endometrial cells (Bruner et al., 1995Go; Casslen et al., 1998Go; Ulloa and Tabibzadeh, 2001Go) and inactivation of TGF{beta} has been implicated to endometrial carcinogenesis (Parekh et al., 2002Go). However, it is unclear whether TGF{beta} would regulate those proteins that are abundantly produced in decidual cells, such as IGFBP-1 and PRL. In this study, we have examined the effect of RLX and TGF{beta} to determine whether these two factors have similar or disparate effects on RLX receptor and the induction of the decidual marker protein, IGFBP-1.

Although it is well known that decidual cells secrete a large amount of IGFBP-1, regulation by hormones in decidual cells is unclear. We have investigated the induction patterns of IGBP-1 in decidual and stromal cells, run in parallel.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometrial cell culture
Human endometrial specimens were obtained from women in their reproductive age (35–50 years) and having menses. These patients had undergone hysterectomies for a variety of medical reasons but not related to any abnormality of the endometrium. Decidual tissue pieces were obtained from the decidual layer attached to the fetal membrane at term of gestation. Permission for using these human specimens was approved by the Human Subject Committee of our institution in accordance with US Department of Health regulations.

Stromal and glandular epithelial cells (>95% purity) were isolated from endometrium by digesting the tissue fragments with collagenase as described previously (Zhu et al., 1990Go; Lane et al., 1994Go). Decidual layer attached to the fetal membrane contains a mixture of decidual fibroblasts, newly formed decidual cells, and terminally differentiated decidual cells. Decidual cells were isolated by a method described previously (Tseng, 2003Go). Briefly, the decidual layer was dissected from the fetal membrane, washed extensively with saline, minced into small pieces and digested with 0.25 mg/ml collagenase P (Roche, USA) for 20–30 min to loosen the tissue structure. The remaining tissue fragments were dispersed into cell suspension by 0.05% trypsin for 5 min. Cells were subcultured twice to eliminate the non-adherent cells (which includes terminally differentiated decidual cells, vascular endothelial cells and blood cells). Newly formed decidual cells are able to attach to the surface of a Petri dish. Decidual cells (~95% purity) were identified by positive immunohistochemical staining of vimentin, PRL and IGFBP-1 (Zhu et al., 1990Go; Lane et al., 1994Go; Tseng et al., 1999Go; Palejwala et al., 2002Go). Cells isolated from a single specimen were individually cultured (no mixed culture from different specimens). To study the effect of hormone, culture medium RPMI (Roswell Park Memorial Institute 1640 medium and 2% charcoal–dextran-stripped fetal bovine serum) was supplemented with progestin, MPA, porcine RLX (Sherwood et al., 1993Go) and human recombinant TGF{beta}1 (Sigma, USA), separately or in combination for various periods of time specified in each experiment.

Determination of LGR7 and LGR8 mRNA by real-time PCR
Total RNA was isolated from cells using RNAqueous Kit (Ambion, USA). We used real-time PCR for quantification of LGR7 and LGR8 mRNA. Real-time PCR quantifies the message at the exponential growth phase of PCR product (Figure 1). Optimal primers used in this study are shown in Table I. Primers of LGR7(1) were selected from reported cDNA (Hsu et al., 2000Go) to detect the full length of LGR7 mRNA only and primers of LGR7(1,2) were designed to detect both the full length and a possible splicing variant, missing 34 amino acids (Hsu et al., 2000Go). The two messages can be distinguished by the length of their PCR products, 204 bp for full length and 102 bp for the splicing variant. QuantiTec SYBR Green PCR Kit (204143; Qiagen, USA) was used for real-time PCR to quantify the mRNA. Briefly, 5 µg total RNA was reversed-transcribed (RT) using random primers. Diluted RT products (equivalent 0.25 µg total RNA) were subject to real-time PCR [10 µl SYBR Green Master mix, 1 µl (8 pmol/µl) each forward and reverse primer and 8 µl cDNA]. Standard curves were generated from either plasmids containing the gene of interest or from amplified PCR product.



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Figure 1. LGR7 mRNA in endometrial cells determined by real-time PCR. Fluorescence signal versus number of cycles from samples of stromal and decidual cells (black lines) and glands (grey line). CT: cycle threshold when DNA amplification rises above the background and enters the exponential phase. Inset shows the PCR products of LGR7 mRNA and {beta}-actin run on agarose gel-amplified 40 cycles.

 

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Table I. Primers for real-time PCR and product sizes
 
ELISA assay of IGFBP-1 content in cell culture medium
Immunoreactive IGFBP-1 secreted from endometrial stromal/decidual cells was measured in the culture media by sandwich ELISA (Bell et al., 1991Go; Lane et al., 1994Go). Recombinant IGFBP-1 (Upstate Biotechnology Inc., USA) was used as standard. Antibodies of IGFBP-1 (Mab 6301 and 6303; Medix Biochemica, Finland) were used as capture antibody and detection antibody (conjugated with HRP) respectively. The assay was performed in Maxisorp Nunc immunoplates (Nalgen International, USA). The assay has a lower limit of detection of 0.5 ng/ml and it is linear up to 5 ng/ml. The inter- and intra-assay coefficient of variation is ~8%.

Fibronectin synthesized and secreted from decidual cells determined by [35S]methionine labelling
Fibronectin (FN) synthesis was determined by [35S]methionine labelling and immunoprecipitation (Zhu et al., 1990Go). Cells were incubated with 0.1 mCi/ml [35S]methionine/cysteine (SA >1000 Ci/mmol; NEN Life Science, USA) in methionine free culture media for 3 h. Labelled medium (0.5 ml: one-third of total volume) was incubated with Mab of FN (ICN Biochemicals, USA) and absorbed to protein G agarose beads. After extensive washing, the agarose beads were boiled in the sodium dodecyl sulphate (SDS) loading buffer and analysed by SDS–polyacrylamide gel electrophoresis (PAGE). Authentic FN was run in parallel.

Statistical analyses
LGR7 mRNA content in endometrial stromal, glandular and decidual cells, and induction of LGR7 mRNA by progestin or RLX, were performed in number of specimens specified in each condition. For P-value computation, statistics was performed by Student’s unpaired t-test on the levels of LGR7 mRNA in stromal, glandular and decidual cells and paired t-test on the induction by hormones. P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Relaxin receptor, LGR7 mRNA, is abundant in human endometrial glandular cells and decidual cells
Relaxin receptor mRNA was determined by real-time PCR using primers LGR7(1), LGR7(1,2) and LGR8 (Table I). A strong band (192 bp) was identified using the primers LGR7(1) (Figure 1, inset). PCR products were sequenced and shown to match the corresponding sequences of LGR7 cDNA published by (Hsu et al., 2000Go). A putative splicing variant (missing 34 amino acids, at the N-terminus of the coding region; Hsu et al., 2000Go) was not detected in endometrial cells, since no 102 bp PCR product was found, in agreement with results reported by Ivell et al. (2003Go). We used LGR7(1) primers for data determined by real-time PCR. Figure 1 shows the real-time PCR of samples of stromal, glandular and decidual cells. We found a large degree of variation in the content of LGR7 mRNA in different specimens with no clear correlation with the morphology of the specimen or with patient’s age. Such variations have also been reported by Luna et al. (2004Go). Presumably, it is caused by different hormonal milieu in each individual. LGR7 mRNA content was estimated from cycle threshold (CT, Figure 1) and summarized in Table II. Glandular cells and decidual cells contained significantly higher LGR7 mRNA than in stromal cells (glands versus stroma and decidual cells versus stromal cells, P < 0.001, Student’s unpaired t-test). These variations were detected only in the exponential phase of the PCR reaction (Figure 1) and became less apparent when PCR products were amplified beyond the log-linear range (Figure 1 insets). Endometrial cells also express LGR8 mRNA; however, it was barely detectable with an amount <1/1000 of the LGR7 mRNA and it was not up-regulated by hormone (data not shown).


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Table II. LGR7 mRNA in human endometrial cells and induction by hormones
 
Progestin stimulates LGR7 mRNA in endometrial stromal cells and RLX stimulates LGR7 mRNA in decidual cells
The effects of MPA and RLX on the levels of LGR7 mRNA were studied and summarized in Table II. In endometrial stromal cells, MPA increased the LGR7 mRNA ranging from 2- to 15-fold (mean ± SD 6.4 ± 4.6, n = 7, P < 0.02) and RLX alone had little effect. In glandular epithelial cells, MPA increased the mRNA content ranging from 1.8- to 4-fold (mean ± SD, 2.2 ± 0.9, n = 5). RLX increased the LGR7 mRNA by 2-fold (n = 1). In decidual cells, MPA had a moderate effect (2–3-fold, n = 4). RLX alone increased the LGR7 mRNA 4–12-fold (mean ± SD 7 ± 3, n = 8, P < 0.001). The effect of RLX was dose dependent (Figure 2A). A total of 1 ng/ml RLX was sufficient to stimulate 80% of the maximal induction. Time study showed that ~5-fold increase in mRNA after cells were incubated with RLX for 2 days and continued afterward (Figure 2B). These results indicate that LGR7 is up-regulated by progestin and RLX in a cell type-specific manner. RLX is the major inducer in decidual cells.



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Figure 2. Effect of relaxin (RLX) on the content of LGR7 mRNA in decidual cells. (A) Cells were incubated with various concentrations of RLX (0, 1, 5, 20 and 100 ng/ml) for 4 days. (B) Cells were incubated with 20 ng/ml RLX for 1–7 days. Total RNA was isolated from each sample to determine LGR7 mRNA content by real-time PCR (mean of two determinations).

 
TGF{beta}1 reduces LGR7 mRNA content and inhibits the MPA- and RLX-induced stimulation in endometrial stromal and decidual cells
The effect of TGF{beta}1 on the content of LGR7 mRNA was studied in both stromal and decidual cells (Figure 3). TGF{beta}1 alone reduced LGR7 mRNA content (30–50% of the control values). TGF{beta}1 also inhibited the stimulation induced by MPA or RLX, confirmed in three specimens. To ensure that TGF{beta}1 was functional in this system, we determined the fibronectin synthesis since TGF{beta}1 is known to induce fibronectin (Hocevar et al., 1999Go). We found that TGF{beta}1 increased the biosynthesis of fibronectin in decidual cells (Figure 4), indicating that the inhibitory effect on LGR7 by TGF{beta}1 is specific.



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Figure 3. Effect of hormones on LGR7 mRNA in stromal and decidual cells. Decidual cells were incubated without/with 20 ng/ml relaxin (RLX), 0.1 µmol/l medroxyprogesterone acetate (MPA), 2 ng/ml transforming growth factor (TGF{beta}1) alone or in combination. After 7 days incubation, cells were harvested for RNA isolation. LGR7 mRNA was determination by real-time PCR (mean of two determinations).

 


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Figure 4. Effects of relaxin (RLX), medroxyprogesterone acetate (MPA) and transforming growth factor (TGF{beta}1) on the synthesis/secretion of fibronectin (FN) in decidual cells. Cells were incubated with/without 2 ng/ml TGF{beta}1, 20 ng/ml RLX or 0.1 µmol/l MPA for 4 and 9 days and then incubated with [35S]methionine/cysteine (0.1 mCi/ml) in methionine-free culture media for 3 h. Labelled medium (0.5 ml: one-third of total volume) was incubated with monoclonal antibody of FN and absorbed to protein G agarose beads. After extensive washing, the agarose beads were boiled in the SDS buffer and analysed by SDS–PAGE. Authentic FN (first lane) was run in parallel.

 
Induction pattern of endometrial IGFBP-1 is in parallel with the induction profiles of LGR7 mRNA in endometrial cells
Previous studies have shown that progestin and RLX synergistically induce IGFBP-1. In this study, we provide evidence that the effect of RLX depends on the receptor content and that it is independent of progestin. Endometrial stromal cells and decidual cells were separately cultured with MPA, RLX, TGF{beta}1 or in combination (same experiment shown in Figure 3). In stromal cells, MPA caused a moderate increase in the production of IGFBP-1 (~5-fold) through 7 days incubation. MPA and RLX synergistically increased the production from 0.02 to 0.86 µg/day/1x106 cells (>40-fold). RLX or TGF{beta}1 had little effect (Figure 5A). In decidual cells, the basal production rate of IGFBP-1 was much higher (1–2 µg/day/1x106 cells, >50-fold) than that in stromal cells. RLX further increased the production rate from 2 to ~60 µg/day/1x106 cells (Figure 5B). The effect of MPA was relatively small. TGF{beta}1 inhibited the induction by RLX (Figure 5B). The unique production patterns in decidual cells—induction by RLX, little response to MPA and inhibition by TGF{beta}1—were in parallel with the regulation of LGR7 mRNA by these hormones (Figure 3). Similar results were obtained in three other specimens.



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Figure 5. Effects of hormones on the production of IGFBP-1 in stromal cells (A) and decidual cells (B). Cells were incubated without/with 20 ng/ml relaxin (RLX), 0.1 µmol/l medroxy progesterone acetate (MPA), 2 ng/ml TGF{beta}1 alone or in combination. Culture medium was collected every other day. IGFBP-1 content in culture media was determined by enzyme-linked immunosorbent assay (mean of three determinations).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we showed that in undifferentiated stromal cells, the LGR7 mRNA content is extremely low (2–3 pg/10 µg RNA, equivalent to ~1–5 copies/cell, which is apparently insufficient to activate RLX-induced IGFBP-1 production (Figure 5A). Therefore, the level of LGR7 is critical for the action of RLX. As shown in Table II, LGR7 mRNA is increased by progestin in stromal and also in glandular cells. We conclude that the role of progestin is to induce RLX receptor. In decidual cells, we showed that LGR7 mRNA is up-regulated by its own ligand (Table II and Figure 2) and progestin is no longer required for the induction of IGFBP-1 (Figure 5B), indicating that RLX action is independent of progestin. These observations demonstrate that sequential actions of progestin and then RLX provide an optimal hormonal milieu to induce the process of decidualization and induction of decidual cell protein such as IGFBP-1. Perhaps RLX could be beneficial for the treatment of those individuals with persistent luteal phase defect syndrome but this would require in vivo validation.

The effect of progestin on the induction LGR7 mRNA is likely caused by progestin-induced stromal cell differentiation since the process of induction is slow and it requires ~5 days incubation for a noticeable increase of LGR7 mRNA. Also, the extent of induction in stromal cells is relatively small compared to that in decidual cells. These observations suggest that other unidentified cellular factors are also involved. The up-regulation of LGR7 mRNA by RLX, however, is time- and dose-dependent and it is likely that RLX exerts a direct effect on the transcription of LGR7 mRNA.

In this study, we presented a function of LGR7 (induction of IGFBP-1) rather than detecting the receptor protein in endometrial cells. The extent of induction of IGFBP-1 is proportional to the amount of LGR7 mRNA, i.e. a small induction correlates with the low level of LGR7 mRNA in stromal cells and a large induction correlates with the higher mRNA content in decidual cells. Thus the mRNA content reflects the relative content of LGR7 protein in endometrial cells.

It should be mentioned that other factors, namely, prostaglandin (PGE2), hCG, FSH or LH and cAMP generating agents exhibit similar or redundant functions as RLX to enhance the production of IGFBP-1 and PRL (Tang and Gurpide, 1993Go; Frank et al., 1994Go; Telgmann et al., 1997Go). The effect of RLX appears to be more effective than the others. As shown in the present study, the induction of LGR7 is highly specific. In addition, the stimulation of IGFBP-1 is sustained from sub-microgram in stromal cells to microgram levels in decidual cells with an overall increase >10 000-fold (Figure 5A and B). Maximal production is similar to the accumulation of IGFBP-1 in amniotic fluid during early to mid gestation (Bell et al., 1988Go). Similar results were observed on the production of PRL in endometrial cells, i.e. synergistic effect induced by MPA/RLX in stromal cells, major induction by RLX in decidual cells and inhibition by TGF{beta}1 in both types of endometrial cells (data not shown). These results imply that the productions of endometrial IGFBP-1 and PRL are sustained by RLX in decidual cells during gestation.

As shown in Figures 3 and 5, TGF{beta}1 had an inhibitory effect on the content of RLX receptor and IGFBP-1 production. The degrees of inhibition by TGF{beta}1 shown in Figure 3 are different in two types of endometrial cells. In general, inhibition by TGF{beta}1 is greater in stromal cells than in decidual cells, suggesting that decidual cells may contain factor(s) that neutralize the effect of TGF{beta}1. One such factor could be ‘lefty’, which is known to be highly expressed in deciduas (Brar et al., 2001Go). Further study is required to elucidate the roles of these cytokines in deciduas.

The inhibitory effect of TGF{beta} was unexpected since it is believed that TGF{beta} promotes cell differentiation of numerous mammalian systems by stimulating the production of extracellular matrix proteins, e.g. induction of fibronectin in mammalian cells (Hocevar et al., 1999Go) and in decidual cells (Figure 4). However, endometrial cells cultured with TGF{beta} had no noticeable change in morphology (not shown). On the other hand, stromal cells treated with MPA and RLX exhibited changes in morphology associated with decidualization (Lane et al., 1994Go). The inhibitory effect on LGR7 and IGFBP-1 by TGF{beta}1 suggests that it has little contribution to endometrial cell decidualization. Further study is necessary to elucidate the inhibitory effect of TGF{beta} on RLX-mediated signalling transduction pathways.

In summary, we showed that TGF{beta}1 inhibits the induction of LGR7 mRNA and IGFBP-1. Sequential stimulation by progestin and then RLX is the major driving force for the production of IGFBP-1. RLX exerts a direct effect to induce LGR7 and IGFBP-1 production in decidual cells. Thus, we conclude that RLX receptor is a rate-limiting factor for the biological functions of RLX and it is essential to sustain the production of decidual cell marker protein.


    Acknowledgements
 
We thank the clinicians in the Department of Obstetrics/Gynecology and Reproductive Medicine and Department of Pathology and the Labor and Delivery division at SUNY–Stony Brook for providing us with viable endometrial specimens. Supported by NIH grant HD 19247.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on January 27, 2004; accepted on March 24, 2004.