Ghrelin, a growth-hormone-releasing acylated peptide, was originally isolated from rat and human stomachs (1, 2). Ghrelin is derived from a prohormone (proghrelin) by posttranslational processing. Obestatin is another product of proghrelin that was recently discovered by Zhang et al. (3). During posttranslational modification the (117) 94-amino acid (pre)proghrelin is cleaved into 28-amino acid ghrelin, 23-amino acid obestatin, and signal peptides. Cells immunoreactive to ghrelin are widely distributed in the gastric mucosa in domestic animals (pig, horse, sheep, cow) and rats (4). In the rat, peripheral or central administration of ghrelin stimulates the secretion of growth hormone (GH) from the pituitary gland, and recent works suggest that ghrelin plays an important role in energy homeostasis, body weight (b. wt.) control, and food intake. Ghrelin is the natural endogenous ligand for the GH secretagogue receptor (GHS-R) (1). Obestatin binds to the orphan G protein-coupled receptor GPR39, which is distinct from the ghrelin receptor (3). In contrast to ghrelin, obestatin does not cross the blood-brain barrier and seems to act rather at the periphery (5). According to Samson et al. (6), obestatin failed to alter basal and stimulated growth hormone secretion in pituitary cell cultures in vitro.

The development of the gastrointestinal (GI) tract and associated organs, the salivary glands, pancreas and liver, is regulated by a complex mechanism involving a great number of hormones, neurotransmitters and neuromodulators (7). Following the discovery of ghrelin it soon became evident that this hormone may play a role in perinatal tissue development. The role of obestatin in GI tract function and development needs further investigation. The aim of this article is to recapitulate the progress in our knowledge made after the discovery of ghrelin regarding its role in controlling the development of gastrointestinal tissues. The article discusses three periods of development, i.e., prenatal, neonatal and post-weaned, to emphasize distinct sources of ghrelin and some discrepancies in its action.

FOETAL DEVELOPMENT

Ghrelin and GHS-R were detected in mouse morula and more advanced embryo stages, blastocyst and hatched blastocyst stage embryos (8). In this study ghrelin expression was detected in endometrium epithelium, and ghrelin mRNA was expressed in the uterus of mice in early pregnancy. Ghrelin was secreted during early pregnancy, and its level in uterine fluid significantly increased in fasting mice as compared with those with free access to food. This finding suggests that ghrelin is produced and secreted from the endometrial epithelium and may regulate in a paracrine and/or autocrine manner the function(s) of the preimplantation embryo during its development (8). This hypothesis is also supported by Tanaka et at. (9), who showed that ghrelin was involved as a paracrine/autocrine regulator of decidualization of human endometrial stromal cells, and tentatively, in the cross-talk between the endometrium and embryo during implantation. These authors described the expression of ghrelin and GHS-R genes in non-pregnant and decidualized endometrium. High levels of ghrelin could inhibit the development of mouse preimplantation embryos through its specific receptor, GHS-R. Addition of ghrelin to mouse embryo culture media inhibited preimplantation embryo development from the two-cell stage embryo to the blastocyst, fully expanded blastocyst, and hatched blastocyst in vitro in a dose-dependent manner. This effect was blocked by the ghrelin antagonist, [D-Lys-3]-GHRP-6. These results suggest that the elevation of plasma ghrelin due to reduction in food intake or malnutrition may inhibit the development of preimplantation embryos. Thus, ghrelin could play a role as a peripheral factor which does not allow continuation of costly metabolic processes (i.e., pregnancy and lactation) under conditions of insufficient nutrient supply (8).

Gualillo et al. (10) detected ghrelin messenger RNA and ghrelin peptide in human and rat placentas. In human placenta, ghrelin mRNA was detected during pregnancy, but the ghrelin peptide appears to be mainly expressed in the first half of pregnancy, as it could not be detected at term. In the rat placenta, expression increased during pregnancy and appeared to be present in its later stages, with a still quite large degree of expression appearing to be present at term. The cited authors suggest that ghrelin disappears as differentiation of placental tissues progresses. One of the most significant findings in their study, however, was the time of pregnancy-related changes in ghrelin expression found in rat samples. This observation strongly suggested that ghrelin is not just a marker of placental tissue, but that it plays a physiological role in placenta function. Whether ghrelin affects placental regulation of peripheral functions or if it is needed for placental development clearly needs further elucidation. According to Guallillo et al. (10) ghrelin may have several functions during intrauterine development, such as local modulation of GH release, and influencing maternal and/or foetal GH secretion from the pituitary. Placenta-derived ghrelin could influence foetal growth and maturation. To support this idea, it was shown that administration of exogenous GH increased placental and foetal growth. On the other hand, GH deficiency is associated with foetal growth retardation. It remains unknown if a change in the expression of the ghrelin gene could led to growth deficiency and/or intrauterine growth retardation. Interestingly, the ghrelin receptor is closely located to the map position of the Brachmann-de-Lange Syndrome, a pre- and postnatal growth deficiency (11). Finally, ghrelin could act by controlling the foetal hypothalamus-pituitary-adrenal axis, since it was shown that GHS administration to adult rodents and humans led to increased circulating levels of ACTH and cortisol (12). No studies are available, however, to confirm these findings in foetuses. Nevertheless, studies in placenta show that in pregnant females, placental expression of ghrelin is distinct from that in the stomach, and may have physiological functions in gestation (10).

Cortelazzi et al., (13) showed that ghrelin was clearly detectable in the umbilical venous blood of the human foetus from 20 weeks to term, and without differences in the sex of examined foetuses. The circulating ghrelin might originate from either the placenta (10), stomach (14) or other tissues such as the pancreas or lung (15) that are known to synthesize large amounts of this hormone during early foetal life (Table 1). The stomach could be the main source of ghrelin in the foetus, just as it is in the adult, but other tissues could also contribute to the foetal pool of ghrelin. Significant synthesis of ghrelin has been demonstrated in the foetal, but not adult, human pancreas (16). Results from a study by Cortelazzi et al. (13) provided evidence for the foetal origin of the peptide, since neither significant umbilical veno-arterial differences in plasma ghrelin concentrations in the term foetus nor a correlation between maternal and foetal plasma levels were observed. Observations that ghrelin seems to be almost absent in the placenta during the last trimester (10) while it persists in the foetal circulation until term, supports the hypothesis on the foetal tissue origin of ghrelin in the umbilical blood. In another study, ghrelin concentrations in the umbilical vein were significantly higher than those in the umbilical artery, which suggest that the placenta is an important source of foetal ghrelin (17). These results indicate that both placental ghrelin and foetal-tissue ghrelin may be important for the foetal pool of ghrelin.

Table 1. Expression of ghrelin in different stages of pre- and postnatal development

Farquhar and co-workers (18) measured ghrelin concentrations in umbilical cord blood in human neonates of different size. Plasma ghrelin was higher in SGA (small for gestational age) neonates as compared with AGA (appropriate for gestational age) and LGA (large for gestational age) neonates, and the difference decreased with advancing gestational age. SGA and LGA were defined as a birth weight below the 10th, and above 90th percentile for gestational age, respectively (18). The ghrelin concentration was also significantly higher in SGA than in AGA foetuses in the study by Kitamura et al. (17). The ghrelin concentration in foetal blood was significantly higher than in cord blood, suggesting that ghrelin may contribute to foetal and neonatal growth (17). Indeed, intrauterine growth restriction (IGR) was found to be associated with a significant increase in the plasma ghrelin level as compared with AGA foetus (13). In conclusion, the high level of ghrelin observed in IGR foetuses and in SGA newborns suggests that ghrelin may play a physiological role in foetal adaptation to intrauterine malnutrition and adverse intrauterine environment. To our knowledge, no studies on the role of ghrelin in the development of the foetal gastrointestinal tissues are available.

The presence of ghrelin immunoreactive cells was found in rat foetal stomachs from day 18 of pregnancy, and their number increased along with stomach development (4). In the study by Lee et al. (19), ghrelin expression in the rat foetal stomach was marginal or undetectable, however, it increased dramatically during the second and third week after birth. The discrepancy in the abundance of ghrelin expression in early stage foetuses may be due to differences in the analytical methods used, nevertheless, the observed tendencies over the time of foetal growth were similar. Ghrelin immunoreactive cells were fairly represented in human foetal stomach, duodenum, pancreas, and lung from week 10 of gestation (16). Ultrastructural studies of P/D1 cells showed that ghrelin cells were found to be among the earliest cells to differentiate in foetal gastric epithelium, earlier than the histamine enterochromaffin-like (ECL), parietal and chief cells of the oxyntic gland. In adult human tissue, in addition to gastrin cells, rare ghrelin immunoreactive cells are present in the upper small intestine and lungs, and no reactive cells are found in the pancreas (16). The expression of GHS-R was also shown in the human foetal pituitary (20). Volante et al. (15) showed that ghrelin is expressed in the neuroendocrine cells of the foetal and infant lung and, to a very limited extent, also in the adult lung (Table 2). Immunoreactive ghrelin was present in single cells or small clusters in the bronchial wall as early as week 7 of gestation and was detected in decreasing amounts in late foetal lungs. In the postnatal period, single endocrine cells scattered in the bronchial mucosa expressed ghrelin during the first 2 years of life. After that the cells become progressively rare and were only occasionally encountered in adult lung parenchyma. A fraction of foetal lung cases also expresses the ghrelin receptor, GHS-R, indicating that ligand-receptor interactions may be actively operating in developing lungs. In the paediatric age group, 2 of 13 and 7 of 13 cases expressed the type 1a and 1b GHS-R, respectively. In seven lungs of adult patients, only four expressed the type 1b receptor, while type 1a was not expressed at all.

Table 2. Tissue expression of the GHS receptor in different stages of the pre- and postnatal development

Obestatin was found in foetal and neonatal rat plasma, pancreas and stomach, though its concentration was lower than that of ghrelin (21). After birth the concentration of obestatin was found to decline up to weaning. Chanoine et al. (21) suggested nonetheless that pancreatic obestatin may contribute to mechanisms of insulin secretion.

DEVELOPMENT OF NEWBORN AND SUCKLING ANIMALS

There are few studies on the role of ghrelin in the development of the gastrointestinal tract of newborn and suckling mammals. As in foetuses, newborn and suckling animals can be affected both by exogenous (i.e., maternal) and endogenous ghrelin. Concerning the source of ghrelin, the GI tract tissues can be affected either from the circulation or from the lumen, since the peptide produced by the stomach mucosa is released both into the blood and gastric lumen (2). Moreover, recent studies revealed substantial concentrations of total and active ghrelin in the colostrum and milk of sows (Wolinski et al., unpublished data) and humans (22), thereby giving a chance to affect the gastrointestinal tract mucosa of the neonate and, if absorbed, the tissues behind the GI tract as well. An 8 h restriction of milk caused a reduction of ghrelin in the neonatal rat stomach tissue simultaneously with increased ghrelin concentrations in the blood, suggesting that ghrelin is secreted from the stomach to blood, and may be involved in stimulating the appetite in order to increase milk intake (4).

Ghrelin expression was found in newborn rats (4, 19, 21) (Table 1), and the amount of ghrelin in the glandular part of the rat stomach increased in an age-dependant manner from neonatal to adult stages (4). In the newborn rat pancreas, total ghrelin and obestatin concentrations decreased progressively from birth to weaning, but acylated ghrelin concentrations increased from the fetal period to day 6 of life. In the blood plasma, however, total ghrelin concentrations were found to decrease abruptly after birth, contrasting with a three-fold increase in the concentration of acylated ghrelin between fetuses and non-suckling neonates (21).

In human neonates the expression of ghrelin and GHS receptor 1a and 1b was also found in lungs (15) (Table 2). The increase in the concentration of ghrelin in the stomach during infancy may be related to increased gastric acid output, GH secretion and milk intake. Yokota et al. (23) demonstrated the existence of octanolyated ghrelin in foetal and neonatal circulation. The ghrelin level in humans was found to increase significantly after birth, peaking during first 2 years of life, and then to decrease until puberty (24). A number of studies have demonstrated a negative correlation between ghrelin levels and some anthropometric indices, such as birth weight, body length or ponderal index in term infants and postnatally (17, 24 - 27), but not in preterm infants. Ng et al. (27) suggest that ghrelin behaved differently from adipocyte-secreted hormones, leptin and resistin, in early gestation as their concentrations were closely associated with body fat content and common anthropometric parameters in preterm infants (27, 28). James et al. (29) reported that low cord ghrelin levels in humans are associated with slower weight gain from birth to 3 months of life.

Daily subcutaneous administrations of ghrelin to pregnant rats from day 15 to 21 of pregnancy led to increased body weights of newborn rats (4), suggesting its possible role in gut development in the postnatal period. Studies in rats (30, 31) and pigs (32; Kotunia et al. - unpublished data) indicated, however, that exogenous ghrelin may restrict gastrointestinal growth in neonates. Administration of ghrelin at a dose of 4, 8, 16 nmol/kg b. wt. intraperitoneally twice a day in suckling rats did not affect their body weight. Treatment with exogenous ghrelin caused a significant and dose-dependant increase in plasma ghrelin as compared with controls. The concentration of plasma ghrelin in suckling rats was also almost two-fold higher as compared with 7-week-old rats receiving the same dose of exogenous ghrelin. Treatment with ghrelin increased serum levels of growth hormone in suckling rats, but the response was lower as compared with 7-week-old rats. Serum levels of insulin-like growth factor-1 (IGF-1) were not affected by administration of ghrelin. Exogenous ghrelin reduced gastric growth in suckling rats as illustrated by a decrease in their gastric mucosa weight, gastric DNA synthesis and DNA content (31). Pancreatic blood flow and plasma glucose concentration, an indicator of insulin release, were not affected by administration of ghrelin, whereas pancreatic DNA synthesis and pancreatic DNA content were significantly decreased by the highest dose of ghrelin. All administered doses of ghrelin produced a significant reduction in the weight of the pancreas and activity of pancreatic amylase (30). The authors suggested that ghrelin's inhibitory effects were probably related to the immaturity of the hypothalamus in suckling as well as in weaned young animals. A number of studies have indicated that the orexigenic effect of ghrelin is related to the activity of neuropeptide Y, agouti-related protein (AGRP) and orexinergic neurons in the hypothalamus. Release of neuropeptide Y plays a major role in this process, and before puberty, hypothalamic neuropeptide Y expression and tissue concentrations are high. Dembinski et al. (30) believe that a high basal hypothalamic level of neuropeptide Y blocks the ghrelin-induced release of this peptide in the hypothalamus in prepubertal animals and that this mechanism is probably responsible for the lack of an orexigenic effect in suckling and weaned rats. Our studies with neonatal pigs fed with milk formula showed that repetitive intragastric administrations of ghrelin (15 µg/kg b. wt. every 8 hours during the first week of life) led to significant reduction in body weight and small intestine length as compared with control piglets (32). In contrast, the wet weight of the stomach was increased. Histometric analysis of the small intestine revealed a significant reduction in the length of villi, thickness of the tunica mucosa and tunica muscularis in the mid- and distal jejunum and ileum as compared with controls. In ghrelin-treated piglets, the crypt depth was significantly increased in the entire small intestine. Lysosomal vacuoles, markers of epithelial immaturity (33, 34), were present in the enterocytes of ghrelin-treated piglets and were larger compared with controls. Plasma ghrelin was not affected by intragastric administrations of the exogenous peptide. These data suggest that luminal ghrelin may retard the development of the small intestine in neonatal pigs.

Intracerebroventricular (ICV) injections of ghrelin and GH-releasing peptide (GHRP)-2, another GHS-R agonist, potently inhibited food intake by neonatal chickens (35, 36). Saito et al. (37) indicated that in chickens, central ghrelin does not interact with NPY as observed in rodents, but instead it inhibits food intake by interacting with endogenous corticotropin-releasing factor (CRF) and its receptor. CRF plays a major role in behavioural responses to stressors and in activation of the hypothalamo-pituitary-adrenal (HPA) axis (38). In mammals, ICV injection of CRF inhibits food intake, elicits anxiety behaviour, and induces glucocorticoid release from the adrenal glands (39, 40). In chicks, ICV injection of CRF also induces anxiety behaviour (hyperactivity) and anorexia (41, 42). In rodents, it is reported that ghrelin stimulates CRF and arginine vasopressin (AVP) release from hypothalamic explants (43). Another study suggests that the orexigenic effects of ghrelin in rats depend on the experimental conditions (44). Ghrelin effects were the most pronounced when injected in the middle of the light phase. In contrast, when injected at onset of darkness, in ad libitum fed rats, ghrelin's effects were less obvious, and in food-deprived rats, ghrelin was unable to significantly increase feeding above the control level. Most likely, these differences in the effectiveness of ghrelin are due to variations in baseline food intake. Ghrelin seems to be the most effective in increasing food intake to above control levels when the baseline food intake is low. These differences, however, could also be influenced by circadian variation in plasma ghrelin. The concentration of plasma ghrelin is known to be increased shortly before a meal and quickly reduced after food intake. The results from this study suggest that ghrelin increases food intake chiefly in young (100-150 g b. wt.), fast-growing rats. Ghrelin may therefore link the high energy needs to the body growth of young individuals. In older rats (300-500 g b. wt.), peripheral ghrelin increased feeding when injected repeatedly over several days.

THE ROLE OF GHRELIN IN WEANING, PERIPUBERTAL AND ADULT ANIMALS

In human, canine, and rodent stomachs, ghrelin is produced by a well-defined cell type that is distinct from other functionally characterized endocrine cells of the oxyntic gland, and rarely occurs in the pyloric gland (2, 16). Immunohistochemical studies in mouse small intestine showed a low number of ghrelin-containing cells in duodenal Brunner's glands, crypts, and villi. Abundant ghrelin-positive cells were found in the rat and human duodenum and proximal jejunum, and lower amounts were found in the distal jejunum, ileum and colon (2, 19). Accordingly, ghrelin mRNA and GHS-R mRNA expression was found in these tissues. The expression of ghrelin was shown in brain, hypothalamus, pituitary, pancrease, lung, kidney, adrenal, thyroid, and immune cells (15, 16, 45 - 48) (Table 1). Expression of GHS-R was described in a wide range of tissues (2, 15, 16, 49 - 54) (Table 2). Controversial results were reported by Gnanapavan et al. (47), who demonstrated the tissue distribution of mRNA of the two subtypes of GHS receptor. Type 1a GHS-R mRNA expression was in the pituitary gland, a lower level of expression was found in a few other tissues (thyroid, pancreas, spleen, myocardial, and adrenal), and was negative in the majority of samples. Interestingly, the expression of unspliced, non-functional type 1b receptor mRNA was widespread, including the pituitary, thyroid, pancreas, spleen, myocardium, adrenals, oesophagus, stomach (antrum, fundus), duodenum, jejunum, ileum, colon, skin, liver, breast, placenta, lung, fundus, lymph node, gall bladder, atrium, lymphocytes, kidney, bladder, prostate, ovary, vein, muscle, and fat. Their findings suggest that some of ghrelin's effects may occur via a receptor different from the cloned type 1a GHS-R.

Ghrelin and weaning

Weaning is associated with drastic dietary and environmental changes for the offspring and brings a new dimension to the development of their gastrointestinal tract tissues. In pig industry practice, weaning is particularly stressful due to regrouping and transportation to a new environment (immunological challenge) as well as the rapid change from a liquid to a solid diet. Following weaning, the gastrointestinal tract of piglets undergoes substantial developmental changes in structure and function resulting in adaptation to new dietary conditions, though little is known about the contribution of ghrelin to these processes. Nevertheless, GI tract development is often disturbed, leading to weaning anorexia during the first 4 days of this process (and thereby a rise in ghrelin production), and inflammation of the gastrointestinal tract (55). A number of post-weaning changes have been identified in intestinal morphology and brush border enzyme activity. In weaned pigs there is a reduction of villous height to 75% of the pre-weaning values (56), an increase in crypt depth (57, 58) coincidently with this effect there is a decrease in the activity of brush-border enzymes (59). These changes are maximal on day 3 post-weaning, and during following days gradual recovery of the small intestine is observed (60). Salfen et al. (61) investigated the effects of exogenous ghrelin on the feed intake, weight gain and feeding behaviour in weaning pigs. In their study, pigs received an infusion of ghrelin at a dose of 2 µg/kg b. wt. three times daily for 5 days. The weaning period resulted in a temporary decrease in body weight in both control and ghrelin-treated animals, however, the ghrelin-treated pigs regained the lost weight more rapidly than the control ones, and after 5 days of ghrelin infusion, weight gain was greater than in controls. In this study there was no significant difference in feed intake between the two groups, however, in behavioural observations, the pigs from the ghrelin group spent more time on eating as compared with control animals. Higher plasma GH, insulin, and cortisol levels were also observed in ghrelin-treated pigs as compared with controls. The results from this study indicate that exogenous ghrelin has a variety of endocrine effects, and in contrast to sucklings, in weaned animals it has a potential to enhance development. Salfen and co-workers (61) suggest that if ghrelin can reduce the duration of weaning anorexia and increase body weight gain, pigs will potentially be able to better resist the postweaning pathological and environmental changes. The effect of ghrelin on morphological changes in the pig GI tract need to be further investigated since this peptide seems to have quite opposite actions on the small intestine. In neonatal pigs it seems to slow down intestinal mucosa development, whereas after this stage, it helps to minimize the negative consequences of weaning.

In studies in weaned rats, ghrelin decreased body weight and tended to reduce food intake, but only at the highest apparently pharmacological dose of 16 nmol/kg b. wt. ip were the effects significant (30). On the other hand, pancreatic weight, DNA content, and amylase activity were significantly increased by all ghrelin doses used in this study, including the doses that can be considered to be within the physiological range. Pancreatic DNA synthesis was significantly increased only by the higher doses. There were no differences in pancreatic blood flow or plasma glucose concentration. The increase in plasma growth hormone concentration after ghrelin at a dose of 8 and 16 nmol/kg b.w. was significantly higher than after 4 nmol/kg b. wt. The concentration of plasma IGF-1 was significantly higher after all applied doses as compared with controls (30).

Ghrelin in adolescent animals

In 7-week-old peripubertal rats, exogenous ghrelin was shown to increase plasma ghrelin in a dose-dependent manner. It also increased food intake and animal body weight (31). The effects were accompanied by a significant increase in gastric mucosa weight, DNA synthesis, and DNA content. Administration of ghrelin (4 and 8 nmol/kg b. wt.) significantly enhanced plasma growth hormone and IGF-1 concentrations, but did not affect plasma glucose. Pancreatic weight, DNA synthesis, DNA content and pancreatic amylase activity were significantly increased by all applied ghrelin doses. Maximal stimulatory effects were, however, observed at the dose of 8 nmol/kg b. wt. (30). Interestingly, the pancreatic blood flow was not affected by exogenous ghrelin, which seems to be an unique feature of this regulatory peptide. Taken together, the rat findings in sucklings and weaners showed ghrelin to have a biphasic effect on gastric growth in rats dependent on animal age, i.e. reduction of gastric growth in suckling animals, and stimulatory action in 7-w-old rats. Puberty was found to be a threshold, which when crossed caused ghrelin to exhibit stable and strong orexigenic effects. Recently, Warzecha and co-workers (31) suggested that the growth-promoting effect of ghrelin in 7-w-old rats seems to depend chiefly on stimulation of food intake and release of IGF-1. This biphasic action of ghrelin was also confirmed in pigs, in which the threshold for stimulatory activity occurs earlier, i.e. in the weaning period. The difference between rats and pigs may be involve species-related differences in the action of ghrelin (for references see the article by T.L. Peeters in this issue). Ghrelin's effect on pancreatic growth in rats was also biphasic, interestingly, ghrelin increased pancreatic growth not only in peripubertal rats, but also in weaned rats. The growth-promoting effects of ghrelin on the pancreas also seem to be related to the anabolic effects of IGF-1.

Ghrelin in plants - a challenge for future research?

Considering the source of ghrelin in peripubertal and adult mammals, it appears that most of this hormone is endogenous. The stomach fundus seems to be the major source of circulating ghrelin since fundectomy resulted in a 30-fold reduction in plasma ghrelin in rats (62). Quite surprisingly, a very recent study by Aydin and co-workers (63) demonstrated strong expression of a peptide identical to human ghrelin in two plants, Prunus x domestica L. and Marus alba using immunohistochemistry and reverse-phase HPLC. In general, fruits had higher levels of ghrelin than vegetative parts. There are no other published studies to our knowledge, though it seems quite probable that in addition to plums and mulberries, other types of fruit may contain the ghrelin peptide as well. The question thus arises whether plant ghrelin can affect the gastrointestinal mucosa following ingestion of fruit. It will be fascinating to see if ingested plant ghrelin can affect GI tract structures just as endogenous ghrelin does. If so, this may open new ways of controlling gastrointestinal tract development using plant preparations.

Acknowledgments: Supported by the Polish Committee for Scientific Research (Solicited Project PBZ-KBN-093/P06/2003).


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