Division of Biomedical Sciences, University of California, Riverside, California 92521
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ABSTRACT |
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The purpose of the current study was to determine whether maternal prolactin (PRL) had any effects on bone formation in the developing rat pup. Because the most prevalent forms of PRL in rats are unmodified and phosphorylated PRL, both recombinant PRL and a molecular mimic of phosphorylated PRL (PP-PRL) were administered to pregnant animals. Blood samples from the dams showed normal estrogen and progesterone and no effect of extra PRL on parathyroid hormone (PTH), calcium, or alkaline phosphatase (AP). In newborn pups, however, there was a 30% decrease in blood AP in both PRL-treated groups, whereas PTH and calcium levels were not different from controls. When primary rat osteoblasts were exposed to both PRLs, AP activity was reduced, with PP-PRL being the more potent form of the hormone. Histological examination of pup bone formation showed reduced calvarial bone and reduced endochondral ossification in pups exposed to PP-PRL. These results are the first to show a direct inhibitory effect of PRL on osteoblast function.
phosphorylated prolactin; intramembranous bone formation; endochondral bone formation
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INTRODUCTION |
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IT HAS LONG BEEN APPRECIATED that both physiological and pathological hyperprolactinemia is associated with alterations in bone physiology. Thus 1) tumor-induced hyperprolactinemia in people (16, 21, 30, 31) and rats (2) is associated with osteopenia; 2) elevation of prolactin (PRL) by the administration of neuroleptic drugs (13) also results in osteopenia (3); 3) prolonged lactation results in significant bone loss (9, 23, 26, 35); and 4) aging is correlated with increased PRL (36) and bone loss (28) and, in postmenopausal women, the degree of osteopenia can be predicted by PRL levels (22).
However, because hyperprolactinemia in the adult frequently results in hypogonadism (29), most authors have concluded that prolactin's effects on bone are likely to be indirect (1, 17). More recently, however, PCR analyses of human osteosarcoma cell lines (4) and primary cultures of mouse osteoblasts (7) have rekindled interest in the idea that PRL may also have direct effects on bone metabolism by demonstrating the presence of PRL receptors in these cells. Although most studies have focused on the potential role of PRL in the remodeling of the adult skeleton, our aim was to analyze potential effects of PRL on initial bone development. The perinatal period is a time of substantial bone formation, as evidenced by the threefold higher levels of alkaline phosphatase (AP) in newborn vs. adult animals (6) and is therefore an unusually sensitive period in which to study potential influences on bone metabolism.
In most studies, PRL is treated as if it were a single hormone. With increasing evidence of different biological activities of different forms of PRL (10, 14, 25, 32, 38), however, it is important to begin to consider the potentially different activities of each form in each tissue. In the rat, the two PRL forms, which together account for 90-99% of the material released from the pituitary, are unmodified and phosphorylated PRL (18, 19). The relative proportion of unmodified and phosphorylated PRL released from the pituitary varies with physiological state (19), including the duration of gestation (18).
In this study, we have analyzed the effects of elevations in unmodified and phosphorylated PRL by use of recombinant versions of both. In the case of unmodified PRL, the recombinant version was human methionyl wild-type PRL (5). In the case of phosphorylated PRL, a molecular mimic was used. In this mimic, the serine, phosphorylated inside intact pituitary secretory granules (37), was replaced by an aspartate residue. Because this molecular mimic of phosphorylated PRL contains no phosphate, it cannot be dephosphorylated during in vitro or in vivo experiments. The molecular mimic is human methionyl S179D PRL and has been shown to accurately mimic naturally phosphorylated PRL in vitro (5, 38). Also, as shown by analysis of signaling molecules, the human methionyl S179D PRL competes with human methionyl PRL for signaling through the rat PRL receptor (8). We have dubbed this S179D molecular mimic "pseudophosphorylated" PRL (PP-PRL).
Administration of PP-PRL during pregnancy resulted in decreased calvarial bone and decreased endochondral ossification in the pups at the time of birth, without effect on maternal gonadal steroids or measures of maternal bone formation or turnover rate. Administration of the wild-type PRL had no histologically obvious effect on the amount of bone. When direct effects of each PRL were tested on osteoblasts in primary culture, both PRLs reduced AP activity, but PP-PRL was more potent. Together, these results support a direct role for PRL in the regulation of bone formation.
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MATERIALS AND METHODS |
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Immunocytochemistry.
Neonatal rats were killed, and calvariae were removed and fixed for
3 h in cold fixative [2% paraformaldehyde, 0.01 M periodate, 0.75 M lysine, and 6% sucrose in phosphate buffer-PLP
(27)]. Tissues were then cryoprotected by overnight
incubation in 15% sucrose. The tissues were mounted with
tissue-freezing medium, and 6-µm frozen sections were cut on a Zeiss
cryotome (Heidelberg, Germany). Sections were washed with wash buffer
(20% goat serum, 0.2% BSA in 0.01 M PBS), and nonspecific binding was
blocked by incubation in goat serum. Sections were incubated with U6
anti-prolactin receptor monoclonal antibody (kindly provided by Paul
Kelly, Institut National de la Santé et de la Recherche
Médicale, Faculté de Médecine Necker, Paris, France),
diluted in wash buffer to 5 µg/ml, for 2 h. Separate sections
were incubated with nonimmune mouse IgG-1 diluted to the same
concentration in wash buffer, as a control. A secondary antibody-only
control was also performed. After three washes, sections were incubated
with fluorescein goat anti-mouse IgG (Molecular Probes, Eugene, OR)
diluted to 20 µg/ml in wash buffer for 1 h. Sections were washed
three times in wash buffer and twice in PBS before they were mounted
with Prolong antifade (Molecular Probes) and examined on the
fluorescence microscope. Both traditional microscopy and scanning
confocal fluorescence microscopy were used, and illustrations were
chosen from the most informative images. For osteoblasts, this was
mainly from the traditional microscope, because phase contrast could
confirm the identity of the cells. For chondrocytes, identity is clear
from their position in the cartilagenous matrix, and the confocal image better illustrates cell membrane fluorescence. All immunocytochemical localizations were performed a minimum of 3 times.
Protein expression. Wild-type recombinant PRL and S179D PP-PRL were produced and characterized as previously described (5). Both proteins were expressed and purified in parallel and were expressed at similar levels. Each preparation was tested for its biological activity by Nb2 bioassay (5, 34). Nb2 cells are T lymphoma cells isolated from the lymph node of an estrogen-treated rat that proliferate in response to PRL (34). Proteins were concentrated to 1 mg/ml of saline using Amicon Centripreps (Amicon, Danvers, MA).
Animals. Sprague-Dawley female rats were maintained in constant light-dark conditions (12:12-h light-dark cycle) and were fed ad libitum. After mating and observation of a vaginal plug, rats were randomly assigned, 10 to each of 3 different groups: a control group, a group treated with wild-type recombinant PRL, and a group treated with PP-PRL. Alzet minipumps (Alza, Palo Alto, CA, model 2004), containing the appropriate protein, were implanted at day 0.5 on the morning after observation of the plug. The pumps delivered 6 µg/24 h throughout pregnancy. One-half of the dams were killed on day 19.5 of gestation and one-half after delivery. On days 6.5 and 11.5 of gestation, blood was obtained from the tail and, on day 19.5, from the trunk. To reduce the potential influence of stress, no individual animal was bled more than once from the tail. After delivery, pups were weighed and killed by metafane overdose, and representative pups from each litter were fixed by immersion in PLP fixative. Others were decapitated after being killed for the collection of trunk blood. Trunk blood from 5-6 pups was pooled for each litter. All procedures were approved by the University of California Riverside Committee on Laboratory Animal Care and were in accordance with National Institutes of Health guidelines.
Determination of circulating levels of recombinant PRLs. To eliminate any possible distortion of RIA values caused by cross-reactivity of antisera with rat lactogens, circulating levels of administered recombinant proteins were determined by immunoprecipitation of biosynthetically 35S-labeled recombinant proteins (15). Use of excess antibody ensured maximal precipitation from serum samples. Greater than 85% of counts present in samples taken on days 1-16 remained in immunoprecipitable PRL. Knowing the specific activity of the radiolabeled PRL allowed us to determine that administration of 6 µg/24 h resulted in circulating levels of 50 ng/ml by day 4 of pump implantation; levels remained constant thereafter. Steady-state levels after pump implantation were the same for each form of PRL. Clearance for the 1st h after a bolus injection was determined in anesthetized animals (45 mg/kg pentobarbital sodium) treated with bromocryptine (500 µg/kg to reduce endogenous PRL release) before cannulation. Radiolabeled PRLs were injected as 750 ng in 300 µl of saline containing 0.05% bovine serum albumin.
AP, calcium, parathyroid hormone, and progesterone in blood. Sigma kit 104-LL was used for measurements of blood AP. This assay uses p-nitrophenyl phosphate as substrate and measures all types of AP activity. Parathyroid hormone (PTH) levels were measured using a PTH RIA/IRMA kit from Nichols Diagnostic Institute (San Juan Capistrano, CA). Progesterone and estrogen levels were measured by RIA with kits from Diagnostic Products (Los Angeles, CA). Calcium levels were measured by atomic absorption spectrophotometry on a Perkin-Elmer 400.
All samples presented in a given figure were measured in the same assay, thereby limiting errors to intra-assay variation. Measurements for dams' parameters were performed on plasma or serum, whereas for pups' parameters, measurements were performed on plasma. Heparin was used as the anticoagulant. All assays were validated for use on heparinized plasma samples.Histological staining. Fixed tissue from neonatal rats from the three treatment groups was dissected, dehydrated in an ethanol series, and embedded with the same orientation in Paraplast. Serial 6-µm sections were cut until at least one-third of the way across the calvaria or halfway across the digit. Equivalent sections were stained with hematoxylin and eosin or Von Kossa's stain or Alizarin red. Multiple sections per pup, two pups per dam, and four dams per group were examined. For measurements, calavarial width at three places per section was ascertained.
Primary bone cell culture.
Calvariae were removed from nontreated neonatal rats and digested with
collagenase [2.4 mg/ml, (200 units/mg) and 10 µg/ml N-p-tosyl-L-lysine
chloromethylketone trypsin inhibitor in HEPES buffer containing 0.1%
BSA and 0.5% glucose] for 2 h at 37°C. Supernatant from the
first 15 min of digestion was discarded to enrich the eventual
preparation with osteoblasts. The subsequent supernatant, containing
released osteoblasts, was then transferred to a new tube. Cells were
washed and plated in DMEM with 10% fetal bovine serum. Cells were
maintained in log phase and were grown at 37°C in a humidified
atmosphere of 5% CO2. Cells were used for experiments at
passages 2 or 3.
RT-PCR. Total cellular RNA was extracted with TRI-reagent (Molecular Research Center, Cincinnati, OH). First strand complementary DNA was synthesized from 5 µg total RNA from osteoblasts and Nb2 cells and 1 µg RNA from rat pituitary by use of reverse transcriptase (Promega, Madison, WI) for 1 h at 37°C after random priming. Twenty microliters of the product were diluted to 50 µl, and 2 µl were used in the PCR reaction. Five micromoles final concentration of each primer (5'-CCCACCCACCATAACTGATG and 3'-TCCAGCAGATGGGTATCAAATC for PRL receptor and, in a separate reaction for rat PRL, 5'-CCTCCTGCTGATGATGTCAAACC and 3'-GGGAGTTTCTGGGCTTGTTCCT) were added to the 50-µl final volume PCR buffer with 2 U Amplitaq Gold (Perkin-Elmer, Branchburg, NJ) per reaction and 1.5 and 3 mM MgCl2 for PRL and PRL receptor, respectively. Thirty-six cycles of amplification were performed with the following profile: 1 min at 95°C, 1 min at 63°C, and 2 min at 72°C, followed by extension for 10 min at 72°C. Amplification products were resolved on a 1.5% agarose gel.
Cell proliferation assay. Osteoblasts were plated in 96-well plates at 4,000 cells/well (such that they would reach confluence at or near the beginning of treatment) or 1,000 cells/well (so as to be proliferating during treatment) in 100 µl of DMEM with 10% horse serum. Horse serum was used because horse PRL cannot activate rat receptors (34). Cells were allowed to attach overnight, and on the next day, 100 µl of twofold-concentrated PRL in the same medium were added to the wells. After 72 h, 20 µl/well 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) solution (Promega, Madison, WI) were added, and the color was allowed to develop for 2 h. Absorption was read on an ELISA reader using a 492-nm filter. MTS color development was shown to be linear for primary osteoblasts between 1,000 and 14,000 cells/well.
Cell AP assay. For AP activity, cells were treated as described for the proliferation assay and were then washed and lysed after 72 h of treatment. Lysis was in 200 µl 0.1% Triton X-100 in 25 mM carbonate buffer, pH 10.3. One hundred microliters of cell extract were then added to 200 µl of 250 mM carbonate buffer, pH 10.3, containing 1.5 mM MgCl2 and 15 mM p-nitrophenyl phosphate. Color development was quantified by absorption at 405 nm.
Statistical analyses. Statistical significance was assessed by using Duncan's modified t-test for multiple comparisons against a single control group or by one-way analysis of variance.
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RESULTS |
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PRL receptors on cells of bone and cartilage.
Although previous investigators have demonstrated the presence of PRL
receptor mRNA and protein in primary osteoblasts (7), potential contamination of such cultures by other cell types made it
important that PRL receptors be localized to morphologically recognizable osteoblasts in intact bone. Figure
1 shows immunocytochemical evidence of
the presence of PRL receptors on osteoblasts of normal rat skull bones.
No osteoclast staining was observed. A comparison of the specific
antibody- and nonspecific antibody-treated sections clearly shows
specific osteoblastic staining in the former. Osteoblasts adhere to the
surface of the developing bone and have a substantial cytoplasm. Bone
matrix reflects fluorescent emission, and hence, bone to which the
positively stained osteoblasts adhere has a higher background
fluorescence than that in the two controls. This is eliminated by the
use of confocal fluorescence microscopy, as is illustrated in Fig.
1B. Figure 1B also illustrates a more punctate
staining of the receptors.
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Effects of PRL administration on pup bone development.
Administration of PP-PRL to dams throughout pregnancy resulted in
reduced pup calvarial bone. Calvariae of the control group were
52.12 ± 1.72 µm thick, those in the wild-type PRL-exposed group
were 53.87 ± 1.72 µm thick, and those from the PP-PRL group were 24.88 ± 1.28 µm thick. (PP-PRL different from the other
two groups with P value < 0.0001.) Also evident in the
PP-PRL-exposed group was reduced endochondral ossification (Fig.
3, C and F). For
the latter, the cartilagenous precursor was formed essentially normally, and hypertrophy of the diaphyseal chondrocytes was evident. There was, however, no evidence of the formation of a bony collar or
diaphyseal endochondral ossification, both of which were evident in the
controls. By contrast, administration of wild-type PRL had no obvious
effect (Fig. 3, B and E). Sections stained with Von Kossa's silver stain or Alizarin red to demonstrate calcified matrix showed no differences in the degree of calcification of the
matrix present (data not shown).
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Clearance of wild-type PRL and PP-PRL.
As illustrated in Fig. 4, both PRLs were
cleared from the circulation at very similar rates. Their differential
efficacies are therefore not due to different half-times in the
circulation.
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Maternal parameters during pregnancy.
Administration of either wild-type PRL or PP-PRL had no effect on
progesterone levels on any of the 3 days measured during treatment
(Table 1). Also unaltered were PTH
levels, serum calcium, AP, or estrogen at day 19.5 of
pregnancy (Fig. 5), i.e., after 19 days
of treatment with this level of additional PRL.
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Pup parameters. Pups in all three groups were the same weight at birth (6.6 ± 0.1 g control; 6.5 ± 0.1 g wild-type PRL; 6.7 ± 0.1 g PP-PRL), consistent with the formation of equivalent cartilagenous skeletons.
Plasma concentrations of PTH were very variable in the newborn pups, and there was no significant difference among groups (Fig. 6A). Plasma calcium levels, which were about one-third those seen in the dams, also showed no statistically significant difference from controls (Fig. 6B).
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Primary osteoblast cultures. Because blood levels of calcium and AP can be aggregates of effects on several tissues, we investigated direct effects of the two forms of PRL on osteoblasts in primary culture.
To determine that expression of the PRL receptor was maintained when rat osteoblasts were placed in primary culture, we performed RT-PCR for the message. The primers chosen did not distinguish between the long and short forms of the receptor. As can be seen in Fig. 7, cells at passage 3 express message for the PRL receptor, but not for PRL. RNA from Nb2 cells and pituitary was used as the positive control for receptor and RNA from pituitary as the positive control for PRL. Lack of osteoblast expression of PRL makes it more likely that osteoblast PRL receptors respond to circulating rather than locally produced PRL. Expression of the PRL receptor protein was confirmed by immunocytochemistry (data not shown).
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DISCUSSION |
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Preliminary experiments had established that administration at a rate of 6 µg/24 h of either PRL form to pregnant animals would have no effect on progesterone or estrogen output by the maternal ovary, and this was confirmed in the present study. During the current experiments, therefore, we could be certain that pup bone development would not be influenced by altered levels of maternal steroid hormones. Also unaltered by this protocol were maternal levels of PTH, elevations of which would increase bone turnover rate (11); calcium, an aggregate marker of intestinal uptake; kidney reabsorption and deposition/resorption in bone, the first two of which are reportedly influenced by PRL (2, 24); and AP, a marker of bone formation (33). Thus we had good evidence that changes in fetal bone development were not secondary to changes in maternal bone metabolism or the availability of calcium. The duration of rodent pregnancy is really too short a period in which to study effects on maternal bone, so our results do not rule out the possibility that longer exposure to the administered PRLs at this level would have had an effect on the maternal skeleton.
Pup blood AP levels are about threefold those in adult animals, indicative of the high rate of bone formation ongoing in the perinatal period (Ref. 6 and results herein). A 30% decrease in pup AP levels after in utero exposure to administered PRL therefore represents a major and important change, suggesting reduced bone formation. Histological analysis at the time of birth, however, showed obvious decreased bone formation only in the pups exposed to PP-PRL. To ensure that the decrease in AP was in fact an effect on osteoblast AP, a series of studies using osteoblasts in primary culture was undertaken. These studies showed both forms of PRL to decrease AP activity in cultures with a high cell density. This effect occurred in the dose range used in the intact animals and is consistent with the lower blood AP observed in both sets of pups at the time of birth. In cultures with a lower cell density more closely approximating the beginnings of bone formation, only the PP-PRL inhibited AP activity. Thus it seems reasonable to suggest that the lowered amount of bone observed in the animals exposed to PP-PRL was a consequence of an early effect on bone formation, in other words, an effect during initial osteoblast differentiation in calvarial islands or the formation of the long bone bony collar. At the time of birth, both PRLs were likely inhibiting bone formation, but for the wild-type hormone, this had not been going on long enough to see any clear histological evidence of same.
Contrary to our expectations, this direct effect of PRL on osteoblasts was on cell-associated AP activity and not on osteoblast proliferation. Elevated PRL, therefore, seems to influence bone cell differentiation, because osteoblasts express high levels of AP during osteoid formation (33).
Despite the presence of numerous PRL receptors on chondrocytes, elevation of PRL by administration of either form had no very distinct effect on the growth of cartilage. The pups in each group weighed the same, and all stages of interstitial growth and so-called chondrocyte hypertrophy occurred normally in the developing long bones, although there was some evidence of hyperplasia. The defect in endochondral bone formation was observed only when osteoblasts would normally be functional. The common theme in both types of bone formation, therefore, seems to be an inhibition of osteoblast function by elevated PRL. The role of the PRL receptor in chondrocytes remains to be established, but cartilage formation and growth do not seem to be adversely affected by elevated PRL.
Other investigators have recently suggested that osteoblasts are a target for PRL. This suggestion was based on the presence of PRL receptor mRNA and protein in primary osteoblast cultures and effects on bone metabolism in PRL receptor knockout mice (7). No direct effect of PRL on osteoblast function was demonstrated, however, and interpretation of the results in the knockout animal model is severely complicated by disturbances in the levels of gonadal steroids, calcium, and PTH (7). Thus defects in bone formation may be secondary to other changes. Unexpectedly, with regard to calvariae, the fetal/newborn phenotype is similar in terms of bone mass in the PRL receptor knockout mice and our rat pups exposed to PP-PRL. This could be simply a coincidence related to disturbed bone metabolism resulting from two different mechanisms. Alternatively, it could be a function of a biphasic response to PRL. In other words, some PRL is necessary for osteoblast function, but elevated levels inhibit osteoblast function. If this were the explanation, however, one might have expected to see evidence of a biphasic response in our in vitro studies, and none was observed.
The fetal calvariae in the PRL receptor knockout mice were also undercalcified, as demonstrated using Von Kossa's stain (7). We could detect no reduced calcification of the amount of osteoid produced in the PP-PRL-exposed pups by use of the same approach. It is possible, therefore, that alterations in mineralization in this knockout animal are secondary to the documented disturbances in serum calcium and PTH (7).
Analyses of the PRL knockout animal, by contrast, report no similar calvarial phenotype to the PRL receptor knockout (20). Others have suggested that this may be because placental lactogens, which also activate the PRL receptor, can substitute during fetal bone development (7) or are, in fact, the normal ligand for fetal bone PRL receptors. However, given the negative effect of PRL receptor activation demonstrated in our in vitro studies, it does not seem likely to us that placental lactogens, which reach microgram-per-milliliter concentrations in late pregnancy, would be the normal ligand for fetal bone PRL receptors.
Regardless of what the normal ligand for fetal bone PRL receptors is, our study has demonstrated an adverse effect of elevated maternal PRL on pup bone development, an effect that can be duplicated in the same dose range in vitro. This study therefore reports the first clearly direct effect of PRL on osteoblast function that can be correlated with a reduction in bone formation in vivo.
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ACKNOWLEDGEMENTS |
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The authors thank Sally Scott for expert animal husbandry, Nancy Price for preparation of the manuscript, and June Bishop and Anthony Norman for advice about blood calcium measurements. The authors acknowledge the pituitary hormone program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Child Health and Human Development, and United States Department of Agriculture and Dr. A. F. Parlow for generous provision of the standard human PRL used in this study.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant ES-06410 to R. A. Luben and by a grant from Sensus Corporation, Austin, Texas and a generous gift from Bill and Charis Bennett to A. M. Walker.
Address for reprint requests and other correspondence: A. M. Walker, Division of Biomedical Sciences, Univ. of California, Riverside, CA 92521-0121 (E-mail: ameae.walker{at}ucr.edu).
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. Section 1734 solely to indicate this fact.
Received 28 February 2000; accepted in final form 14 July 2000.
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