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Stem Cells 2005;23:230-239 www.StemCells.com
© 2005 AlphaMed Press

Early Fetal Liver Readily Repopulates B Lymphopoiesis in Adult Bone Marrow

Ko-Tung Chang, Ludek Sefc, Oskar Psenák, Martin Vokurka, Emanuel Necas

Institute of Pathological Physiology, First Medical Faculty, Charles University, Prague, Czech Republic

Key Words. Fetal liver • Hematopoietic stem cell • B lymphopoiesis • Gene expression

Correspondence: Ko-Tung Chang, Ph.D., Institute of Pathological Physiology, First Medical Faculty, Charles University, U Nemocnice 5, 128 53 Prague, Czech Republic. Telephone: 420-2-2496-5934; Fax: 420-2-2491-2834; e-mail: kotungc{at}bcm.tmc.edu


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fetal liver (FL) becomes a major organ of hematopoiesis at mouse embryonic day (E) 11 and E12, when definitive hematopoietic stem cells, originating from the aorta-gonads-mesonephros region, colonize the hepatic tissue. Unipotent B-cell progenitors are very rare in FL by day 12, whereas erythropoiesis prevails. We have studied hematopoiesis in FL from different gestational ages, with special emphasis on B lymphopoiesis. The mRNA levels of selected liver-specific genes, hematopoietic lineage-specific genes, and genes for selected cytokines/hormones as well as for their receptors were evaluated by real-time polymerase chain reaction in FL from E12.5, E14.5, and E17.5, adult liver and adult bone marrow (BM). The level of B lineage–related gene expression in FL was very low at E12.5. There was also a significantly lower fraction of B220+ and CD19+ B cells in E12.5 FL compared with E17.5 FL. To analyze whether these differences reflect different stem cell potentials occurring during FL development, 106 or 5 x 106 of FL cells collected from embryos at E12.5 or E17.5 and those from adult BM were transplanted into sublethally irradiated (3- or 6-Gy) congenic mice. Short-term and long-term repopulation of B and T cells and granulocyte/macrophage lineages from donor FL or adult BM cells were evaluated in competition to adult hematopoiesis of sublethally irradiated recipients. In short-term repopulation, the transplantation of E12.5 FL cells resulted in a lower blood chimerism compared with that of E17.5 FL cells. However, the proportion of B lymphopoiesis exerted by E12.5 FL cells was not different from that of E17.5 FL or adult BM. This study demonstrates that E12.5 FL contains hematopoietic stem cells with fully developed B-cell repopulating capacity and that the developmental period of fetal hematopoiesis between E12.5 and E17.5 is not an obligatory phase for the adult B lymphopoiesis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hematopoiesis is a time- and a site-dependent event during ontogeny of vertebrates [1, 2]. During gastrulation in mice, ventral mesodermal cells segregate to the two sites where blood cells form independently. The first wave of hematopoietic activity, known as primitive hematopoiesis, appears in the ventral blood islands of the yolk sac (YS) at embryonic day (E) 7.5 [3]. This transient extraembryonic hematopoiesis gives rise to primitive nucleated erythrocytes between E7 and E11 to cope with the oxygen demand of a rapidly growing embryo. Adult-type definitive hematopoiesis begins as the second wave of hematopoiesis in the dorsolateral plate of aorta-gonads-mesonephros (AGM) region at E10.5 [4, 5]. This intraembryonic hematopoiesis thereafter shifts to the fetal liver (FL) at E11 and E12, where production of all hematopoietic cells is initiated. Finally, from near birth until the end of life, hematopoiesis resides in the bone marrow (BM), spleen, thymus, and lymphatic tissues.

Multipotent progenitors are detected readily by in vitro culture of YS cells. However, long-term repopulating hematopoietic stem cell (LTR-HSC) activity is not present in the YS before E11 [5–7], because cells from the YS are unable to reconstitute the entire hematopoietic system in lethally irradiated adult mice for more than several months [8]. LTR-HSCs emerge in the AGM region just before the establishment of the hematopoietic liver activity and subsequently colonize the hepatic tissue. FL becomes a main hematopoietic organ during the fetal period, and HSCs expand dramatically there between E12 and E16 [9]. However, the microenvironment of FL undergoes continuous changes during this time, and the metabolic function of hepatocytes gradually becomes the major function of the liver [10, 11].

Definitive erythropoiesis and fetal T lymphopoiesis peak in the FL at day 12, whereas B lineage–restricted progenitors are still rare to be detected [12, 13]. Although B-lymphoid activity can be found beginning at E8 or E9 in the YS by fetal thymic organ culture [14], B-lineage precursors were detected in FL from E11 and pre-B cells in E13 FL [15]. A population of fully B-committed progenitors that can be differentiated in vitro into B-lineage cells emerges in the FL at E10 or E11 [16]. This shows that the B-cell differentiation program is not delayed during hematopoietic ontogeny. However, it does not address the question of whether the B-lineage progenitors/ stem cells from early FLs are ready to function in the environment of adult BM. The question is pertinent, because LTR-HSC activity of the cells derived from E11.5 AGM is enhanced when cultivated in the presence of FL nonhematopoietic cells [17]. These reports prompted us to study whether HSCs of the AGM origin developing additionally in the FL may be specifically imprinted by the interaction with the FL microenvironment to convert into fully functional adult hematopoietic tissue. To test the tentative instructive role of FL regarding B-lymphopoiesis ontogeny, we studied the gene expression associated with normal hematopoiesis in FL from E12.5, E14.5, and E17.5. We also compared the short- and long-term repopulating potential of FL HSCs from E12.5 and E17.5 by measuring competitive reconstitution of B- and T-lymphoid and granulocyte/macrophage lineages from unfractioned FL cells in sublethally irradiated adult mice.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
C57BL/6 mice (B6-Ly5.2 and congenic B6-Ly5.1 mice) were maintained in a clean conventional animal facility. B6-Ly5.2 male and female mice from 6 weeks to 4 months of age were maintained on a constant light-dark cycle before mating. To set up mating, females were examined in the afternoon, and those in estrus were placed in cages with males (two females with one male). The morning after mating, the females were checked for the presence of a copulation plug in vagina, and this day was designated as day 0.5 postconception. Eight- to 12-week-old B6-Ly5.1 male mice were used as recipients in the transplantation experiments.

Cells
Embryos and extraembryonic membranes were dissected free of maternal tissue and Reichert’s membrane under a dissecting microscope. FLs were obtained from embryos at E12.5, E14.5, and E17.5. Adult liver was obtained from the euthanized pregnant mice. Adult BM was collected from both femur and tibia pairs of normal male mice. The cell suspensions were prepared in phosphate-buffered saline (PBS) solution containing 0.5% albumin by repeated flushing through needles of 18 to 27 gauges. The cells were passed through a nylon mesh with a pore size of 70 µm (Falcon 2350, Becton, Dickinson Labware, Franklin Lake, NJ). Viability of cells was verified by trypan blue dye exclusion.

Real-Time Reverse Transcription–Polymerase Chain Reaction
Total RNA was extracted from E12.5, E14.5, and E17.5 FL and adult liver by the single-step RNA extraction protocol (RNAzol, Tel-test, Friendswood, TX). Synthesis and normalization of cDNA were performed using first-strand cDNA synthesis (Fermentas, Hanover, MD) as described [18]. All RNA samples were treated with DNase I (amplification grade, from Gibco/BRL, Paisley, U.K.) before cDNA synthesis to eliminate any contaminating genomic DNA. cDNA samples were amplified by Light Cycler (Roche Diagnostic GmbH, Mannheim, Germany) using Light Cycler-FastStart DNA-Master SYBR green. Briefly, each polymerase chain reaction (PCR) reaction was prepared in a capillary tube that contained 1 µl of diluted cDNA, 1 µl of FastStart mixture buffer, 1 µl of primer pair (5 µM), 0.8 µl of MgCl2 (25 mM), and 6.2 µl of deionized water up to 10 µl of total volume, and the following PCR conditions were used: initial incubation at 95°C for 8 minutes, 45 cycles of denaturation at 95°C for 0 seconds, annealing at 60°C for 5 seconds, elongation at 72°C for 27 seconds, and a final elongation step of 10 minutes. The specificity of the PCR product was confirmed by 1% agarose gel electrophoresis stained with ethidium bromide and by melting curve (MC) analysis. The level of gene expression was measured at the crossing point (CP) against ß-actin. CP represents the number of PCR cycles at which the fluorescence levels of all samples are identical. Both MC and CP were performed by the Light Cycler software version 3. Specific primer pairs, provided in Table 1Go, were designed by OLIGO 4.0 software (National Bioscience, Chester, NY). Primers were tested before their use in PCR to exclude dimer and undesired product formation.


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  		Table 1. A list of mRNAs examined by real-time polymerase chain reaction in fetal liver, adult liver, and adult bone marrow.
 
We used cDNA for real-time PCR directly after reverse transcription without dilution. ß-Actin cDNA was used as an internal reference standard. For each sample (e.g., E12.5 FL), we ran the reaction for both the target gene and the reference gene in separate capillary tubes and both in duplicates. Equal fluorescent intensity was generally reached in fewer PCR cycles for the reference gene relative to the target gene. We used the absolute difference in the number of PCR cycles between the target cDNA and the reference ß-actin cDNA to compare our samples collected at different times of FL development or from different tissues. We have not compared expression of different genes because of a lack of evidence that efficiency of the PCR amplification process was equal for different genes.

Reconstitution of Sublethally Irradiated Mice
Eight- to twelve-week-old B6-Ly5.1 male mice were sublethally irradiated with a single dose of 3 or 6 Gy from a 60Co source. Between 1 and 2 hours later, mice receiving 3-Gy irradiation were injected intravenously with 106 B6-Ly5.2 FL cells, which were resuspended previously in 200 µl of PBS with 0.5% albumin. Mice receiving 6-Gy irradiation were injected intravenously with 106 or 5 x 106 B6-Ly5.2 FL cells or 5 x 106 B6-Ly5.2 male BM cells from normal donors. The donors of BM were thus sex matched with the recipients, but the FL cells’ mixture was from sex-undetermined embryos. The presence of donor-derived cells was followed in the peripheral blood for up to 16 weeks.

Analysis of the Recipients
At 2, 4, 8, 12, and 16 weeks after transplantation, peripheral blood from the recipients was obtained from the retro-bulbar plexus. Three aliquots (~50 µl) from each blood sample were added to separate tubes filled with 3 ml lysis buffer (0.15 M NH4Cl, 0.035 M NaCl, and 0.1 mM EDTA), and red blood cells were lysed for 5 minutes. Cells were washed twice, resuspended in PBS, and stained with phycoerythrin (PE)–conjugated anti-Ly5.1 and fluorescence isothiocyanate-conjugated anti-Ly5.2. They were simultaneously stained with biotinylated anti-B220 or with a mixture of biotinylated anti-Gr-1 and anti-Mac-1 or with biotinylated anti-CD3 antibodies. This was followed by addition of streptavidin-PE-Cy5. All antibodies and reagents were purchased from PharMingen (San Diego). Multicolor analysis was performed on a FACS Calibur (Becton, Dickinson, San Jose, CA). Donor-derived cells were determined by gating for donor Ly5.2+ as well as host Ly5.1– cells to omit Ly5.1+Ly5.2+ artificial doublets from the analysis.

Statistics

Gene Expression Studies   Six independent experiments were done to determine mRNA levels in the FL (a pooled sample from four to five fetuses from a single pregnant mouse in each experiment), adult liver (a pooled sample from two to three pregnant mice), and adult BM (a pooled sample from two to three normal mice). Real-time PCR reactions were performed in duplicates, and mean value of the cycle difference from that of ß-actin was used in the calculations. One-way analysis of variance (ANOVA) together with Tukey test [19], using the mean values and standard deviations from the six repeated experiments, was used to evaluate significance of differences from the adult BM samples.

Flow Cytometry Analysis   Three independent experiments were done, and p values were calculated by ANOVA together with Tukey test.

Transplantation Experiments   Groups of eight irradiated mice recipients given the same treatment were used to calculate the mean values and standard deviations of the percentage of donor and recipient cells. These values were used to calculate the p values using the two-tailed Student’s t-test.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mRNA Levels in Early-, Mid-, and Late-Gestational FL and in Adult Liver Compared with Adult BM (Table 2Go)

Liver Developmental Genes   The mRNA levels of albumin, transferrin receptor 2, and X-box binding protein-1 increased during liver development. They were significantly different from adult BM in the case of albumin and transferrin receptor 2.


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  		Table 2. Comparison of the levels of mRNA for selected genes in fetal liver from E12.5, E14.5, and E17.5 and in adult liver with that of adult BM taken as 1.0
 

Lineage-Specific Genes   The transcription factor Pax5 and pre-B lineage antigen CD19 mRNAs levels were low in FL from the early and middle gestational ages as well as in adult liver but were slightly higher in E17.5 FL than in adult BM.

The expression of {alpha}-globin gene was high throughout the whole FL development. Surprisingly, the mRNA level in the adult liver was shown to be comparable to that in BM. It might have been caused by contamination with reticulocytes from blood contained in the liver, which was not perfused before sample collection.

Integrin   Very late antigen 4 (VLA-4) mRNA tended to be higher in FL at days 14.5 and days 17.5 of gestation compared with adult BM, but the difference was not significant.

Cytokines and Their Receptors   SCF and c-kit
The expression of both kinds of mRNA had a tendency to be higher in FL from all stages studied compared with adult BM.

SDF-1 (CXCL12) and CXCR4
The highest expression of both stromal-derived factor-1(SDF-1) and CXCR4 was in FL from E17.5, but SDF-1 mRNA was also high in adult liver. CXCR4 mRNA expression was significantly lower in the early and middle stages of FL compared with that of E17.5 FL (p < .05).

Erythropoietin and Erythropoietin Receptor
Erythropoietin (Epo) mRNA was only marginally expressed in BM, whereas all stages of FL significantly expressed the Epo gene. Epo receptor (EpoR) mRNA was higher in FL compared with BM.

Oncostatin M and Oncostatin M Receptor
Oncostatin M mRNA was highly expressed in all stages of FL. Oncostatin M receptor mRNA peaked in adult liver.

Vascular Endothelial Growth Factor and KDR/flk-1
The vascular endothelial growth factor (VEGF) mRNA, as well as the mRNA for its receptor (KDR/flk-1), was more expressed in FL, as well as in adult liver, compared with adult BM.

Flt3 Ligand
Flt3 ligand mRNA was significantly more expressed in all FL and adult liver samples compared with adult BM.

Interleukin-7 Receptor {alpha}
The receptor mRNA was expressed in middle and late FL comparably to its expression in adult BM.

Flow Cytometry Analysis of B-Cell Lineage Antigenic Surface Markers B220 and CD19 in Early and Late Gestational FL and Adult BM
Flow cytometry analysis of unfractioned FL from E12.5 and E17.5 and adult BM was performed using antibodies against the pan-B-cell markers (Fig. 1Go). The mean percentages of B220+ cells in FL from E12.5 and E17.5 and in adult BM were 1.42%, 5.72%, and 13.64%, and those of CD19+ cells were 0.89%, 5.69%, and 14.04%, respectively. All of these differences were highly significant (p < .001).



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  		Figure 1. Flow cytometry analysis of B-cell antigenic surface markers B220 and CD19 in FL at days 12.5 and 17.5 of gestation and in adult BM. A representative result from three independent experiments is shown. FL cells were obtained, on average, from six embryos of the same mother. Adult BM was obtained from both femur and tibia pairs of a normal mouse. Relative proportions of B-lineage cells: B220+ (A) and CD19+ (B). Abbreviations: BM, bone marrow; FL, fetal liver.

 
Comparison of Short- and Long-Term Repopulating Capacity of Progenitor/Stem Cells from Early- and Late-Gestation FL and from Adult BM
Eight mice irradiated with 6 Gy were transplanted with 5 x 106 of FL cells from E12.5 or E17.5 or with 5 x 106 cells from adult BM. The percentage of donor cells at different times after the transplantations is presented in Figure 2AGo. At up to 16 weeks, the chimerism resulting from transplantation of E12.5 FL cells was significantly lower than that achieved by transplantation of FL cells from E17.5. This difference was much more pronounced during the first 2 months after transplantation, corresponding with short-term repopulation. The proportions of B- and T-lymphoid cells in total donor-derived nucleated cells were not different among recipients of either E12.5 or E17.5 FL cells or adult BM cells (Figs. 2B–2DGo).



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  		Figure 2. Competitive repopulating capacity of E12.5 and E17.5 FL cells against adult BM. Five million cells collected from FL at day 12.5 or 17.5 of gestation and from adult BM were transplanted into sublethally irradiated recipients (6 Gy). (A): Total contribution to peripheral blood nucleated cells from donor cells. (B–D): Percentage of B and T lymphocytes and granulocytes/macrophages in donor-derived nucleated cells (total donor nucleated cells = 100%). n = 8; means ± standard error of the mean are shown. Significant difference between E12.5 and E17.5 of FL: **p < .001 and *p < .05, respectively (two-tailed Student’s t-test). Abbreviations: BM, bone marrow; FL, fetal liver.

 
Inhibition of Donor-Derived Granulocyte/ Macrophage Formation in Recipients with a Low Level of Engraftment
A total of 106 FL cells from day 17.5 was transplanted into mice that received either 3 or 6 Gy of irradiation. There was a very low engraftment in 3-Gy irradiated mice (<1% of donor cells in the peripheral blood) until 4 weeks. This, however, increased to 7.77 ± 1.52% after 12 weeks. In 6-Gy irradiated mice, the engraftments were 62.9 ± 2.8% after 4 weeks and 85.0 ± 1.2% after 12 weeks. The percentage of granulocytes/macrophages in total donor-derived nucleated cells was significantly lower (p < .0001) in 3-Gy irradiated mice compared with 6-Gy irradiated mice after 12 weeks (Fig. 3Go). On the other hand, there was no apparent difference in B and T cells. A cytospin preparation of cells from the nonstained fraction (B220–, CD3–, and Gr-1–/Mac-1–) contained 81% of lymphocytes, 17% of epithelial-like cells, and 1% of both macrophages and eosinophilic granulocytes.



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  		Figure 3. Inhibition of donor-derived granulocyte/macrophage formation in recipients with a low level of engraftment. A total of 106 cells collected from fetal liver at day 17.5 of gestation was transplanted into 3- or 6-Gy irradiated recipients (n = 8). Data show the percentage (means ± standard error of the mean) of B and T lymphocytes and granulocytes/macrophages in donor-derived peripheral blood after 12 weeks. There was a significant difference of granulocytes/macrophages fraction derived from transplanted cells (6.06 ± 1.6% versus 22.83 ± 1.8%, *p < .0001; two-tailed Student’s t-test).

 

   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is evidence for the presence of stem cells, multipotent progenitors, or lineage-restricted progenitors in the murine FL [20]. A large proportion of progenitors at day 12 of gestation are T-cell lineage-restricted progenitors, but their frequency significantly decreases with gestational age. In contrast, B-cell lineage-restricted progenitors are very rare by day 12, but they rapidly increase thereafter [21].

To further characterize FL hematopoiesis in relation to gestational age, with special emphasis on B-cell lymphopoiesis, we examined the expression of several genes in FL collected at early, middle, or late gestational age regarding the period of FL development. Furthermore, we compared the capacity of FL cells from early and late gestational ages to engraft in the adult hematopoietic tissue in competition with hematopoietic progenitor/stem cells from adult BM of sublethally irradiated recipients. Because the incidence of repopulating units (a repopulating activity corresponding to that of 1 x 105 BM cells [22]) per 105 FL cells does not change in FL between E12.5 and 16.5 [9], the results in our study were not biased by a different proportion of progenitor/stem cells among other FL cells by using unfractioned FL cells.

Our gene expression studies demonstrated a continuous maturation of the liver tissue toward its metabolic function, as well as presence of erythropoiesis in FL in all examined developmental stages. This was supported by significant expression of the Epo gene. The Epo mRNA was also detected in adult BM, in concert with the previous demonstration that Epo mRNA and Epo were present in hematopoietic cells [23].

In contrast, the genes highly specific for B lymphopoiesis were only marginally expressed in FL from E12.5 (Pax 5, CD19, CXCR4, interleukin [IL]-7 receptor a, and, relatively, VLA-4), and those of Pax 5, CD19, and CXCR4 were still low in FL from E14.5 (Fig. 4Go and Table 2Go). There were also low numbers of FL cells carrying the pan-B-lineage antigenic markers detected by CD19 and B220 monoclonal antibodies in the E12.5 FL (Fig. 1Go). FL expressed genes for several hematopoietic and angiogenic cytokines and their receptors. Some of these genes were still highly expressed in the adult liver (SDF-1, flt3 ligand, oncostatin M, and VEGF).



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  		Figure 4. mRNA expression associated with B-cell lymphopoiesis in E12.5 FL, E14.5 FL, E17.5 FL, adult liver, and adult BM. The mean values of cycle differences from ß-actin in real-time polymerase chain reactions are shown with their standard deviations calculated from six independent experiments. For each mRNA, its level is also expressed as percentage of that from the adult BM. Note that fewer numbers of cycles reflect a higher amount of mRNA. Abbreviations: BM, bone marrow; FL, fetal liver.

 
Pax-5 (BSAP) transcription factor inactivates myeloid signaling by repressing the M-CSF-R gene in pro-B cells [24, 25] as well as in mature B cells [26]. Pax-5 can also activate pre-B-cell receptor signaling by promoting VH-DHJH rearrangements at the immunoglobulin H locus [27] through an IL-7R–mediated signal [28] and by inducing the expression of B-cell linker protein (BLNK) [29] as well as CD19 [30, 31] and immunoglobulin {alpha} [31].

The B-lymphoid phenotype was thus only marginally developed in FL from E12.5. On the other hand, various markers of B lymphopoiesis were significantly expressed in FL from E17.5 at both the mRNA and the protein level. The expression was comparable or higher to that in adult BM. Therefore, we anticipated a higher efficiency of E17.5 FL cells in reconstitution of B lymphopoiesis in irradiated mice compared with FL from E12.5 and with adult BM. However, there was no apparent difference in contribution of transplanted FL cells originating from E12.5 and E17.5 to B lymphopoiesis in adult sublethally irradiated recipients. Only the total level of engraftment of donor cells was lower in the recipients of FL cells from E12.5 compared with those receiving FL cells from E17.5 or adult BM. Our results thus demonstrate that progenitor/stem cells present in E12.5 FL, including those for B lymphopoiesis, are ready to home and function in the environment of adult hematopoiesis, and they do not need to be imprinted for this during the period of their significant expansion in the FL occurring between E12.5 and E16.5 [9].

Although our results did not reveal any significant differences between hematopoietic stem cells from early and late FL compared with adult BM, they do not exclude presence of differences like those described by Kincade et al. [32, 33], who demonstrated that progenitor cells belonging to the B lymphopoiesis and derived from FL cells are deficient in functional estrogen receptors [32] and also differ in other respects [33] from those of the adult BM.

Our results also demonstrate that significantly different grafts may result in a same outcome after transplantation into recipients that received identical treatment (Fig. 2Go). On the other hand, they additionally demonstrate that an outcome of transplantation of identical cells may be different when recipients obtained different treatments (Fig. 3Go). Therefore, the final outcome of grafting hematopoietic tissue seems to be principally determined by the environment and special requirements of the host. This conclusion is in keeping with the results of Rivera et al. [34], who reported that host progenitor/stem cells repopulated the lymphoid lineage when the BM graft originated from genetically deficient mice incapable of generating B and T cells, whereas the remaining cell lines were repopulated by donor cells.

We conclude from our results that the potential of murine FL from E12.5 stage is as high as that of FL from E17.5 regarding long-term repopulation of the adult hematopoiesis, including B lymphopoiesis, despite that B lymphopoiesis still has very little phenotypical expression in FL from E12.5.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by research grant MSM 111100003 from the Ministry of Education of the Czech Republic. We thank D. Dyrová and D. Singerová for excellent technical assistance and Z. Volkánová and J. Jobbiková for the animal maintenance. We also thank Drs. Max Cooper, Paul W. Kincade, and Josef T. Prchal for their critical review of the manuscript.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on March 25, 2004; accepted for publication on October 18, 2004.




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