1 Faculté de médecine, Institut National de la Santé et de la Recherche Médicale U38, 2 Faculté de pharmacie, Unité Propre de Recherche de l'Enseignement Supérieur Associée au Centre National de la Recherche Scientifique 6032, and 3 Centre de microscopie et d'analyse d'images, Faculté de médecine, Université de la Méditerranée, 13385 Marseille, France
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ABSTRACT |
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Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 ± 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.
cell size; cAMP; thyrotropin control; iodide control
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INTRODUCTION |
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CELL VOLUME
DEPENDS ON the equilibrium between the intracellular ion
concentration and the transport of electrolytes, particularly inorganic
ions (Na+, K+, and Cl) and water.
As a steady state of cell volume is essential to maintaining the
metabolic functions of cells, any variations in ion and water transport
that are liable to modify the cell volume will result in metabolic
dysfunctions (18). Various regulatory mechanisms serve to
prevent any excessively large changes in cell volume. The authors of
several studies have described the effects of cell volume variations
induced by changes in cell osmolarity triggered by anisotonic media and
the resulting regulatory mechanisms (24). Furthermore,
several effectors, including hormones and neurotransmitters, can also
induce cell swelling or shrinkage via their effects on ion transport
and the state of cellular hydration. Hormonal effects on cell volume
have been described in detail in various tissues, especially in
hepatocytes, where insulin leads to cell swelling whereas glucagon
results in cell shrinkage (15). Moreover, in hyperthyroid
states, thyroid hormones increase the size of cardiomyocytes
(33) and induce renal hypertrophy (23). Some
hormonal effects on cell volume are mediated by cAMP. However, depending on the target cells involved, an increase in the cAMP pool
can lead to either a decrease in the cell volume, as observed in
hepatocytes, MDCK cells, and pancreatic epithelial cells, or an
increase, as is found to occur in sweat gland cells (24). Therefore, as suggested by Haussinger and Lang (17), the
cell volume variations occurring in response to hormonal stimuli can be
said to act like another second messenger in the hormonal processes.
In the thyroid gland, epithelial cell metabolism is modulated
by thyrotropin (TSH) and iodide. Like other epithelial cells, thyroid
cells transport Na+ in the apical-to-basal direction, as
well as transporting K+ and Cl in the
opposite direction. This transport of electrolytes is regulated by TSH
(28, 29, 30, 38)
via the cAMP pathway (3), like most of the
effects of TSH (12), especially the expression of the
Na+/I
symporter (32). Variations
in thyroid gland volume can be observed in some pathological states;
the goiter resulting from hypertrophy and hyperplasia of the thyroid
gland are often associated with a decrease in the serum iodide level
(14) or overstimulation by excessive TSH effects
(9). Experiments on rat gland have shown that a
correlation exists between hypertrophy of the thyroid cells and the
regulation of follicular volume by iodide (7, 31). The increase in thyroid cell volume observed in human
pathologies and animal models is accompanied by a corresponding
decrease in the colloidal space that is probably associated with fluid
transport. On the other hand, atrophic thyroid glands are also known to
occur when the effects of TSH on thyroid cells are either reduced or completely lacking. This pathological state is generally
characterized by a decrease in thyroid cell volume.
No data have been published so far, however, on the respective and/or correlated roles of TSH and iodide in the regulation of thyroid cell volume and the associated fluid transport nor are any quantitative data available to date on thyroid cell volume. The aims of the present study were, therefore, 1) to determine the volumes (total cell volume and cytosolic volume) of thyroid cells in the steady state and 2) to investigate the respective and combined effects of TSH and iodide on those volumes and on fluid transport. For this purpose, we used porcine thyroid cells cultured on collagen-coated filters in which all of the thyroid functions, even thyroid hormone synthesis (16), were kept. With this cell culture system, it was possible to study the effects of TSH and iodide separately after adding them on the basolateral side, as well as to measure the volumes of interest in living thyroid cells and in the colloid-like space. It was established here that TSH and iodide have opposite regulatory effects on thyroid cell volumes and on apical-to-basal net fluid transport. These antagonistic effects may be partly due to the fact that TSH and iodide affect the production of cAMP in opposite ways. These findings are discussed in relation to the data available on the regulation of ion exchanges and fluid transport in thyroid cells, as well as those on pathological states caused by TSH or iodide deficits.
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MATERIALS AND METHODS |
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Cell culture. Thyroid epithelial cells were isolated from porcine glands and cultured on porous filters coated with collagen, as previously described (5). The isolated cells, suspended (2.5 × 106/ml) in phenol red-free DMEM containing 1 g/l glucose (GIBCO BRL, Cergy Pontoise, France), were seeded into the apical compartment of chambers. The same medium was introduced into the basal compartment outside the insert. The culture was carried out at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. From day 4 onward, 10% newborn calf serum (Biomedia, Boussens, France) was added to the basal compartment whenever the media were changed. On day 6, the tightness of the monolayer to serum protein was checked in the apical media by performing a colorimetric protein assay, and only tight monolayers were kept (5). From day 6 onward, the basal media were supplemented, or not, with 100 µU/ml b-TSH (Calbiochem, San Diego, CA). From day 8 onward, potassium iodide was added daily, or not, to the basal media at 0.5 µM final concentration, and the apical media were replaced by B-DMEM (a saline medium DMEM devoid of amino acids and vitamins). Moreover, on the same day, an analog of cAMP, 8-chloro-cAMP (Roche Diagnostics, Meylan, France), was added at 0.5 mM final concentration in some experiments instead of TSH. Then, the basal media were subsequently changed every 3 days, maintaining the same TSH or cAMP concentration. From days 8 to 14, apical media were not changed.
Measurement of total cell volume by confocal laser-scanning microscopy. On day 14, apical media were removed, and cells were washed with B-DMEM and loaded with 20 µM octadecylrhodamine B (chloride form, Molecular Probe, Eugene, OR) in B-DMEM for 20 min at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. Then cells were washed three times with PBS. The filter was placed between lamina that were separated by spacers and mounted on the microscope stage.
A Leica system based on an inverted Leitz microscope (DMIRBE, Leica, Heidelberg, Germany) was used to perform confocal laser-scanning microscopy. Living cells labeled with octadecylrhodamine B were observed with a 40X apochromat lens (NA 1.0). Red fluorescence of octadecylrhodamine B, excited at 514 nm with an argon-krypton ion laser, was collected with a 590-nm long-pass filter. Serial optical slices through the cell thickness were obtained. Images (512 × 512 pixels) with a 0.49- × 0.49- × 0.9-µm voxel in size were collected and imported into a PC workstation equipped with MATROX image analysis cards (image series 640, Dozval, Canada). A homemade computer program was drawn up from 3-D reconstitution data (19) with the SAMBA software program (Unilog, Grenoble, France) and used to pile up n consecutive optical slices and then evaluate the total cell volume of each cell (in pl). The same computer program was also used to count the number of cells per culture filter.Determination of the 3-O-methyl-D-glucose space. On day 14, cells were used to measure their cytosolic volume with the nonmetabolizable 3-O-methyl-D-glucose (3-OMDG). This was done when both the intracellular and the extracellular 3-OMDG (Sigma, St. Louis, MO) concentrations had reached equilibrium as previously described (21). Briefly, the apical and basal media were changed, the basal medium was adjusted with 1 mM 3-OMDG (final concentration), and the cells were incubated with 5 µCi/ml 3-O-methyl-D-[1-3H]glucose (5 Ci/mmol, Amersham France, Les Ulyss, France) for 30 min at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. By this stage, the equilibrium between the intracellular and extracellular concentration of 3-OMDG was reached for all of the culture conditions used. Cells were promptly washed three times with ice-cold PBS containing 0.6 mM phloretin (Sigma), a sugar transport inhibitor, to prevent the extrusion of 3-OMDG. Cells were then solubilized in a 1% SDS solution, and aliquots were counted in a liquid scintillation counter (Tricarb 2100TR, Packard Instruments, Meriden, CT). The 3-OMDG space was deduced from the radioactivity remaining in the cells (dpm/cell) and from the volumic radioactivity (dpm/µl) of the extracellular compartment (25).
Protein and DNA assays. Proteins were evaluated by micro-bicinchoninic acid protein assay (Pierce, Rockford, IL).
Quantitative determination of DNA was performed by measuring DNA fluorescence in the presence of Hoechtst 33258 (Roche Diagnostics, Meylan, France) by use of the procedure previously described by Teixeira et al. (37). Samples and standards were prepared in the same way, and the fluorescence was measured in 96-well plates with a PC-controlled fluorometer (Fluostar +, Salzburg, Austria) with the Biolyse 1.7 program.Determination of net fluid transport. From the beginning of the culture, the volumes of apical (1 ml) and basal (2.3 ml) media were adjusted so as to preserve the hydrostatic equilibrium. On day 8, the apical (1 ml) and basal (2.3 ml) media were changed, and the apical volumes were measured carefully up to day 14. It was verified that the volume loss in the apical media was recovered in the basal medium; thus evaporation was found to be negligible. The net fluid transport was determined from the slopes of the linear regression curves and was expressed as microliters per hour per 106 cells.
Statistical analyses. The data presented here are expressed as means ± SE for n values. Variations between assays and control were shown to be significant using the Student's t-test with a 1% statistical significance limit. The data presented for the net fluid transport were calculated from the slopes of the linear regression curves ± SD determined with the ORIGIN Software program (Microcal Software, Northampton, MA).
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RESULTS |
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In all experiments, porcine thyroid cells were cultured for 14 days as monolayers on permeable filters coated with collagen. This
experimental culture system leaves the main features of thyroid cells
intact, especially their functional bipolarity. In addition, it gives
separate access to the apical and basal compartments and makes it
possible to culture the thyroid cells without any hormones or other
effectors. In contrast, functional, well oriented follicles can be
obtained only in the presence of TSH or cAMP analog (27).
Moreover, when cell monolayers are cultured on plastic supports, TSH
and iodide are in contact only with the apical membranes, and this does
not correspond to physiological conditions. Conversely, under the
bicameral culture conditions used here, the TSH and iodide added to the
basal compartment come into contact with the basolateral membranes on
which the TSH receptor and the Na+/I
symporter are present. In this study, cells cultured with TSH and
iodide were used as the control cells, because these conditions are
similar to the in vivo ones. Three other conditions were then obtained
by removing the iodide, the TSH, or both, which might mimic what occurs
in some pathological states.
TSH and iodide regulate total volume of thyroid cells.
To estimate the total cell volume, thyroid cells were loaded with
octadecylrhodamine B, a fluorescent probe generally used to label cell
membranes by intercalating it between the lipid bilayers. Optical
slices were performed on the z axis with a confocal laser-scanning microscope. Median optical slices of control cells showed that each cell of the monolayer was labeled with
octadecylrhodamine B (Fig.
1A). The absence of
TSH and/or iodide did not give rise to any differences in the shape of
the cells (Fig. 1, B, C, and D).
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TSH and iodide control the cytosolic volume of thyroid cells.
We measured the cytosolic portion of thyroid cells, which is the
soluble part of the cytoplasm and represents 50-55% of cell volume in most cell types. The remaining volume includes the nucleus and the intracellular organelles, and their variations under each culture conditions may be evaluated in part by the determination of
protein content. The cytosolic portion has been shown to be the space
occupied by the nonmetabolizable sugar, 3-OMDG, in the cases of
leucocytes (13) and glioma cells (20). The
3-OMDG probably did not enter into the nucleus and cytoplasmic
organelles (13). Thus, to evaluate the cytosolic volume,
experiments were first performed either with the usual media (5.6 mM
glucose) or with glucose-depleted media. There was no difference
between these two conditions as far as the 3-OMDG equilibrium time and
the intracellular space occupied by the 3-OMDG were concerned (data not
shown), as was previously observed in glial cells (25).
Media containing 5.6 mM glucose were chosen because they correspond to
the physiological concentrations used in our culture media. The effects
of TSH and iodide on the 3-OMDG space of thyroid cells were studied
under each of the culture conditions (Table
2). In the absence of iodide or TSH, the
3-OMDG space increased, whereas it decreased clearly when the two
effectors were absent. The proportion of the space occupied by 3-OMDG
in relation to the total cell volume was calculated (Table 2). In the
control cells, this proportion was ~54%, which shows that, in our
cells, the 3-OMDG probably accounted for the cytosolic volume of the
cell. This proportion decreased in the absence of iodide but increased
in the absence of TSH (Table 2). TSH and iodide, therefore, had
antagonistic effects on the proportional size of the cytosolic fraction
in thyroid cells.
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TSH and iodide regulate apical-to-basal fluid transport in thyroid
cells.
The apical volumes decreased differently depending on the culture
conditions; these decreases in the apical volumes corresponded to a net
fluid transport in the apical-to-basal direction. We carefully measured
the apical media in the culture chambers under each of the culture
conditions for 6 days. In the control cells, the volume of apical
medium decreased from 1 ml to 356 ± 19 µl. In the case of cells
cultured in the absence of iodide, the volume of apical medium was
lower (37%) than that with the control cells, whereas in the absence
of TSH, the volume of the apical medium was greater either with (37%)
or without (46%) iodide than that in the control cells. Such
changes in the volume of the apical medium were also observed when
8-chloro-cAMP was added to the basal medium instead of TSH (results not
shown), as was previously reported (5). After 5 days, the
volumes of apical media became stabilized, which shows that the effects
of TSH and iodide on the net fluid transport reached equilibrium. We
determined the fluid flux rate
(µl · h
1 · 106
cells
1) corresponding to the slopes obtained by
performing linear regression analysis on the time-dependent apical
volumes (Fig. 5). In the absence of
iodide, this rate increased by 27%, whereas in the absence of TSH, it
decreased by 27% with iodide and by 37% without iodide. These results
show that the absence of iodide increases net fluid transport, whereas
the absence of TSH decreases it. These findings demonstrate that TSH
and iodide have antagonistic effects on apical-to-basal net fluid
transport.
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DISCUSSION |
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Porcine thyroid cells cultured as monolayers on permeable collagen-coated filters were used to study cell volume regulation by TSH and iodide, the two main effectors of thyroid cells. In cells obtained in this way, both the epithelial phenotypes and the metabolic functions are preserved compared with in vivo thyroid cells. This culture system was suitable for this study, because it makes it possible to observe the effects of TSH deprivation on the physiology of thyroid cells and gives access to the apical medium (the colloid-like space). It was used here to measure the volume of living cells by performing confocal laser-scanning microscopy and computer-assisted 3-D reconstitution. We had first used calcein as a marker to label the total intracellular space. Unfortunately, calcein was very rapidly extruded from the TSH-stimulated cells. It has been reported that calcein was also actively extruded from other cell types by transporters that carry organic anions such as multidrug resistance-associated protein (MRP), and this ABC transporter was expressed in thyroid follicular cells (35). Thus calcein was not suitable for measuring the total volume of thyroid cells. We therefore used octadecylrhodamine B, which labels plasma membranes, to determine the total volume of living cells.
In this study, we quantified TSH and iodide regulation of thyroid cell
volumes. This is the first time that the values of the total volume of
living thyroid cells have been determined under a variety of
stimulation conditions. The results clearly show that, under our
culture conditions, only the cell height varied. The values of the cell
heights obtained on cells cultured with TSH and without iodide were
similar to those recorded in vivo on rat thyroid follicular cells under
iodide deficiency (31): 16.5 vs. 20 µm, respectively.
Those authors observed a decrease in cell height when rats were refed
with iodide and therefore were in a state corresponding to our culture
conditions with TSH and iodide. The absence of TSH decreased the cell
volume in the same way whether or not iodide was present. When TSH was
replaced by a cAMP analog, similar variations in cell volume were
observed. TSH and iodide were also found to regulate the cytosolic
volumes and the total amount of proteins without affecting the number of cells. In the absence of iodide, the absolute cytosolic volume increased, but its proportion vs. the total cell volume decreased, showing an increase in the protein content when TSH stimulation was
applied alone, partly by increasing the endoplasmic reticulum and the
intracellular organelles (10, 31). In the
absence of TSH but with iodide, the decrease in protein content was
associated with an increase in the absolute cytosolic volume and its
proportion vs. the total cell volume. Now it is well known that, in the
presence of TSH, iodide decreases TSH-stimulated protein synthesis via the effects of 2-iodoaldehydes, which decrease adenylylcyclase activity
(2). However, we also observed a decrease in the protein level in the presence of iodide and the absence of TSH, which was found
to be necessary for the Na+/I symporter to be
activated and, consequently, for the formation of the 2-iodoaldehydes
to occur. It is possible that, under our culture conditions, iodide
influx may have occurred only when traces of TSH were present in the
serum of the basal medium, and this hypothesis still remains to be
investigated. Taking our data as a whole, two states were found to be
relatively "well-balanced", that occurring when the cells were
regulated either by both TSH and iodide (control cells) or without TSH
and without iodide; this last state was characterized by values of the
parameters amounting to ~80% of those recorded in the control cells.
The other two states, namely that without iodide or that without TSH, which may correspond to pathological conditions, can be said to be
"unbalanced" states, because the proportions of the cytosolic volumes and protein content were inverted, particularly in the absence
of TSH. All these findings suggest that iodide and TSH control the
total cell and cytosolic volumes, as well as the protein content, by
exerting antagonistic effects.
In comparison with the control cells, the apical medium decreased in the absence of iodide and increased in the absence of TSH, which shows that the net fluid transport was also regulated in opposite ways by iodide and TSH. The changes in apical volume occurred during the first few days of TSH and iodide action, probably before reaching equilibrium. Our results on the effects of TSH are in good agreement with previous data showing that both TSH and the cAMP analog increased the height of the domes that developed when cell monolayers were cultured on a plastic support (3). This dome formation process was also dependent on fluid transport in the apical-to-basal direction. Although the cultured thyroid epithelium is tight (29), a slight fluid transport at the paracellular level is not excluded. Moreover, transcytosis, which was previously observed to be weak (1% during 48 h) in TSH-stimulated cells (5), could represent only a minor part of the apical-to-basal fluid transport.
The changes in cell volume occurring in response to hormones and/or
other effectors are correlated with the regulation of water transport
and closely associated ion exchanges through both the apical and the
basolateral membranes of epithelial cells (15, 17). More specifically, Na+, K+,
and Cl transport are involved in the changes in cell
volume (24). In the thyroid gland, the regulation of cell
volume as well as colloidal space effected by TSH and iodide
may also be mediated via Na+, K+, and
Cl
exchanges; TSH via cAMP increases basolateral
Na+-K+-ATPase activity (28,
30) and apical Na+ channel conductance
(3, 29), resulting in the extrusion of
Na+ ions from the cells and the uptake of K+
ions on the basolateral side. TSH also increases the secretion of
Cl
ions, mainly by controlling the basolateral
Na+-K+-2Cl
symporter
(4, 38) and apical Cl
channels
(4). At the apical membrane, Cl
and
Na+ exchanges may also be regulated by the cystic fibrosis
transmembrane conductance regulator (CFTR) via cAMP, as previously
described in airway and colonic epithelial cells (26,
34), because it has been established that CFTR is present
in thyroid cells (11). The movement of Na+ in
the apical-to-basal direction was probably associated with the
basal-to-apical secretion of Cl
as well as with the
transepithelial apical-to-basolateral fluid transport (4,
38). Moreover, the fluid transport may also depend on the
expression of water channels called aquaporin-2, as observed in kidney
epithelial cells, where this protein is regulated by vasopressin via
cAMP (22). Hormonal control of these ionic and water
transport processes has been found to be cAMP dependent in several
types of epithelial cells. In our thyroid cells, any changes in the
intracellular cAMP pool triggered by TSH or iodide may therefore affect
the total cell volume, the proportion of cytosol, and the protein
level, as well as the size of the colloid-like space. Consequently, the
opposite effects of TSH and iodide on the parameters studied here may
be mediated via control of adenylylcyclase activity.
Antagonistic effects of TSH and iodide on thyroid cell and colloid volumes have also been observed in in vivo studies on both induced animal pathologies and human pathologies. Eliminating iodide from the diet of rats resulted in the development of goiter caused not only by the absence of iodide but also by an increase in the serum TSH level. This goiter was accompanied by a decrease in the colloidal space, which may have reflected an increase in the net fluid transport. Restoring iodide reduced the hypertrophy of the cells and increased the colloidal volume, probably by decreasing the net fluid transport (7, 31). In human pathologies, numerous authors have reported that goiter size can be reduced by application of an iodide treatment, which results in changes in the size of cells and follicles (14).
When our cells were cultured without TSH, the total cell volume decreased, showing that the absence of TSH had an atrophic effect on the thyroid cells, whereas the volume of the apical media increased. In human pathological states, this atrophic effect may also be due to the TSH effects being either decreased or abolished; these can occur for two major reasons, namely a dysfunction of the hypothalamic-pituitary axis (8) or a dysfunction of the TSH receptor induced either by gene mutations (1) or by the presence of autoantibodies blocking this receptor (36). The absence of either iodide or TSH in our cultured cells can therefore be said to have mimicked the state of imbalance present in pathological thyroid conditions.
In conclusion, the present results show that TSH and iodide regulate thyroid cell volumes and fluid transport in opposite ways, probably via up- and downregulation of adenylylcyclase activity, respectively. However, interrelations exist between these antagonistic effects; by upregulating the iodide uptake and transport, TSH controls its own antagonist, which in turn slows down the effect of TSH. TSH therefore exerts self-control via the iodide regulation process. In vivo, these regulatory mechanisms might maintain a steady state of the intracellular concentration of ions and other molecules through the control of the cell volumes as well as of the fluid transport.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. P. Carayon for interest in this study and the Institut Federatif de Recherche. Jean Roche is thanked for the use of the confocal microscope. D. Cauvi is the recipient of a grant from the Ministère de l'Education Nationale de la Recherche et de la Technologie.
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FOOTNOTES |
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This study was supported by INSERM (U38), CNRS (SDI 401038, UPRESA 6032) and the Université de la Méditerranée.
Address for reprint requests and other correspondence: O. Chabaud, INSERM U38, Faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille, cedex 05, France (E-mail: Odile.Chabaud{at}medecine.univ-mrs.fr).
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.
Received 2 December 1999; accepted in final form 6 April 2000.
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