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Conditional Overexpression of Bioactive Transforming Growth Factor–ß1 in Neonatal Mouse Lung

A New Model for Bronchopulmonary Dysplasia?

Department of Pediatrics, Albert Einstein College of Medicine and Children's Hospital at Montefiore, Bronx, New York; Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; and Department of Medicine II, Giessen University School of Medicine, Giessen, Germany

Address correspondence to: Jack A. Elias, M.D., Chief, Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, 1 Gilbert Street, S441 TAC, New Haven, CT 06520-8057. E-mail: jack.elias{at}yale.edu

	Abstract

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Research interest in bronchopulmonary dysplasia (BPD) has steadily increased, and numerous potential mediators have been implicated in the development of the disease. Among such mediators is transforming growth factor (TGF)-ß. Unfortunately, commonly utilized murine transgenic models are not optimal to investigate the effects of TGF-ß specifically during the 2–3 wk period of alveolar formation, the developmental stage that corresponds histologically to early alveolar development in humans, and the time frame during which BPD develops. In the current study, we utilized a triple-transgenic construct to overexpress bioactive TGF-ß1 in the neonatal mouse lung during the period of alveolar formation. Lungs were then examined by histologic, Western blot, and immunofluorescent methods. We found that overexpression of bioactive TGF-ß1 in neonatal mouse lungs resulted in structural changes that have been described in BPD. Included in those characteristics are abnormal alveolar structure, cellular composition, and vascular development. Our study indicates that TGF-ß1 overexpression in the neonatal mouse lung results in histologic alterations that have striking similarities to pathologic descriptions of BPD. We encourage the use of conditional transgenic models for the study of BPD, and hypothesize that the TGF-ß system is a central mediator for the histologic alterations described in association with the disease.

Abbreviations: bronchopulmonary dysplasia, BPD • constitutive heat shock protein-70, HSC-70 • mean linear intercept, MLI • Postnatal Day, P • phosphate-buffered saline, PBS • proliferating cell nuclear antigen, PCNA • platelet endothelial cell adhesion molecule, PECAM • smooth muscle actin, SMA • tetracycline operator, tet-operator • tetracycline transactivator silencer, tTS • transgene, TG • transforming growth factor, TGF

	Introduction

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Bronchopulmonary dysplasia (BPD) is a chronic lung disease seen in premature infants exposed to high oxygen concentrations and mechanical ventilation (1, 2). Numerous investigators have described the pathologic characteristics of BPD in both autopsy specimens of patients as well as in various animal models. Included in these characteristics are abnormal alveolar formation with thick, hypercellular septae, abnormal localization and proliferation of –smooth muscle actin (-SMA)–positive cells (a marker for smooth muscle and myofibroblasts), and abnormal capillary development (3–6). Whereas the histologic alterations of BPD have been fairly well described, the molecular mechanisms involved in such changes have remained elusive, partly because existing models for the disease are not optimal. For example, premature baboons, although useful for investigating BPD, require elaborate facilities and well-trained staff to care for these critically ill animals (7, 8). Similarly, mouse models, which can also be very useful, are limited due to the rodent's size and rapid transition ( 2–3 wk) from the saccular stage of lung development to the end of alveolarization (9). This time frame in mice (from a histologic standpoint) corresponds roughly with the first 1–2 yr in premature human infants, many of whose lungs at birth are still in the saccular stage of development (10). Importantly, it is during this stage of lung development (alveolarization) that the histologic alterations of BPD initially appear. Thus, effective utilization of transgenic models for investigation of this disease requires the ability to manipulate genes of interest during this well-defined and somewhat limited period of time.

One example of the importance of time-specific manipulation of gene products to investigate such diseases is the transforming growth factor (TGF)-ß system. TGF-ß, a 25-kD multifunctional protein involved in cellular proliferation and differentiation (11), is crucial for normal lung morphogenesis, and has been implicated in the development of BPD. Previous studies have demonstrated the importance of this system in early lung development, as well as its potential role in the lung's response to stressors, such as hyperoxia (9). Moreover, other investigators have reported increased levels of total and bioactive TGF-ß in bronchoalveolar lavage fluid and tracheal aspirates of premature infants who subsequently developed BPD (12, 13). Unfortunately, as alluded to previously, the effects of TGF-ß, specifically in the neonatal lung, have been difficult to investigate; the currently available models are either lethal in the perinatal period (14) or are technically difficult to adequately preserve lung morphology due to the size of the animals at the age in question (15). To circumvent these issues, we have utilized a triple-transgenic construct to conditionally overexpress bioactive TGF-ß1 in the mouse lung specifically during the period of alveolar formation. Our results indicate that overexpression of bioactive TGF-ß1 in the neonatal mouse lung not only results in a phenotype that differs from that described in adult models, but also parallels the histologic features described in patients with BPD and existing animal models of the disease.

	Materials and Methods

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Triple-Transgenic Model for Conditional Overexpression of Bioactive TGF-ß1 in the Neonatal Mouse Lung
The triple-transgenic construct used for this study, and its capabilities for lung- and time-specific overexpression, are described in detail elsewhere (16–18). Briefly, the triple construct consists of cDNA for bioactive TGF-ß1, reverse tetracycline transactivator, and tetracycline transactivator–silencer (tTS). A tetracycline operator (tet-operator) is inserted immediately upstream of the bioactive TGF-ß1 cDNA, and the entire construct is coupled to a CC10 promoter for lung-specific expression. In the absence of doxycycline, the tTS product binds the tet-operator, preventing transcription of bioactive TGF-ß1. In the presence of doxycycline (which is added to maternal drinking water and is able to cross into the mother's milk), the tTS product dissociates from the tet-operator, leaving the reverse tetracycline transactivator product free to bind and thereby initiate transcription of bioactive TGF-ß1. In litters used for the current investigation, 25–50% of the pups were positive for the transgene (TG). Thus, each litter served as its own control; the doxycycline in the mother's drinking water was subsequently transferred to TG-positive (TG[+]) as well as TG-negative (TG[–]) pups via maternal breast milk. For the current study, doxycycline was added to the mother's drinking water at Postnatal Day (P) 7 and continued until P14.

Processing of Lung Tissue
Animals were killed and lung tissue was processed as described previously (19). Briefly, lungs were inflated through tracheotomies to 20 cm pressure with 4% paraformaldehyde (pH 7.4), and tracheas were ligated. Lungs/hearts were excised en bloc, submersed in 4% paraformaldehyde overnight, and processed for paraffin embedding and sectioning. Lung tissues were then stained with hematoxylin or trichrome for histologic analysis. Other litters were similarly killed, and lungs were excised and frozen in liquid nitrogen.

Measurement of Mean Linear Intercept
Random tissue sections were photographed under 20x magnification and viewed under a field of equally spaced horizontal lines. After removing any line that crossed a large airway or vessel, each septum that intercepted a given line was counted. The total length of all lines counted was divided by the total number of intercepts per field examined to obtain the mean linear intercept (MLI), which is inversely proportional to alveolar surface area (20, 21).

Immunohistochemical Analysis
Lung tissue sections were processed for immunohistochemical analysis as described previously (19). Briefly, sections were deparaffinized in xylene and rehydrated in ethanol and distilled H₂O. Antigen retrieval was performed in a pressure cooker using 6.5 mM sodium citrate (pH 6.0). Sections were incubated with primary antibody at 1:50 dilutions of pancytokeratin (Sigma Biotechnologies, St. Louis, MO), -SMA (Sigma), proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnologies, Santa Cruz, CA), platelet endothelial cell adhesion molecule (PECAM; Santa Cruz Biotechnologies), phospho-Smad2 (p-Smad2; Cell Signaling, Inc., Beverly, MA) in a 0.3% saponin/phosphate-buffered saline (PBS) solution and washed for 4 x 3 min in 0.1% saponin/PBS. Immunofluorescence was detected after incubation with tagged immunofluorescent secondary antibodies (Vector Laboratories, Burlingame, CA) and washing (0.3% saponin/PBS and 0.1% saponin/PBS, respectively). Signal for p-Smad2 was detected using a peroxidase staining kit (R&D Systems, Minneapolis, MN), per the manufacturers instructions.

Western Blot Analysis
Frozen lung tissue was processed as described previously (19). Briefly, frozen tissues were homogenized, sheared, and centrifuged, and supernatants were taken as whole-tissue lysates. Protein concentration was measured using the Bradford assay according to the manufacturers instructions. Equal amounts of protein (10 µg) were separated on 4.5–12% gradient sodium dodecyl sulfate–polyacrylamide gels (NuPage) and transferred to polyvinylidene difluoride membranes. Western blots were performed with antibodies against constitutive heat shock protein (HSC)-70 1:5,000 (Stressgen, Victoria, BC, Canada; Smad2 1:1,000 (Cell Signaling, Inc.); p-Smad2, 1:1,000 (Cell Signaling, Inc.); pancytokeratin, 1:1,000 (Sigma) ; -SMA, 1:1,000 (Sigma); PCNA, 1:1,000 (Santa Cruz Biotechnologies); and PECAM, 1:1000 (Santa Cruz Biotechnologies). Specific bands were visualized after incubation with the respective secondary antibodies by autoradiography using enhanced chemiluminescense according to the manufacturer (SuperSignal; Pierce Chemicals, Rockford, IL).

Densitometric Analysis
Densitometry measurements of western blots from each experimental group were obtained (n 5 for each group), and absolute values were equalized with HSC-70. Results are reported in arbitrary units, comparing each value with that obtained from each respective HSC-70 measurement.

Statistical Analysis
All data are reported as mean values +/– standard deviation and analyzed using the Student's t test. P values < 0.05 were considered to be statistically significant.

	Results

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Effect of Either the TG or Doxycycline on Normal Alveolar Development
To ensure that the presence of the TG or the administration of doxycycline did not affect alveolar development during early postnatal life, we initially examined lung sections at P3 and P14 from three different groups of animals: (i) TG(–), no doxycycline; (ii) TG(–), with doxycycline administered from P7 to P14; and (iii) TG(+), no doxycycline. As demonstrated in Figure 1A, P3 lung sections from all three groups of animals appeared to be in the saccular stage of development. At P14, lung sections from all groups demonstrated well-formed, thin-walled alveoli, characteristic of normal lung architecture at this age. Figure 1B confirms that MLI counts decrease with age, indicating an increase in surface area. Importantly, MLI counts did not differ between the groups at either P3 or P14, indicating that the presence of either the TG or doxycycline alone did not affect early alveolar development. For each group analyzed, at least two separate fields per slide, three slides per animal, and three animals per group were counted.

View larger version (77K): [in this window] [in a new window]   Figure 1. Effect of either the TG or doxycycline on normal alveolar development. In mice, alveolar formation is a postnatal event. We examined lung histology in three groups of animals (open bars, TG(–), no doxycycline; shaded bars, TG(–), doxycycline; hatched bars, TG(+), no doxycycline) to determine the effect of either the TG or doxycycline on alveolar development. (A) At P3, lung sections from all groups of animals appear to be in the saccular stage of development. By P14, all groups demonstrate well-formed, thin-walled alveoli characteristic of normal postnatal development. (B) For all groups, MLI counts decreased with age, indicating an increase in surface area, as expected. MLI counts did not differ between the groups at either P3 or P14, confirming the histologic findings. For each group analyzed, at least two separate fields per slide, three slides per animal, and three animals per group were counted (scale bars = 200 µm).

View larger version (113K): [in this window] [in a new window]   Figure 2. Bioactivity of doxycycline-induced TGF-ß1 in neonatal mouse lung. Expression of bioactive TGF-ß1 in doxycycline-fed, TG(+) animals was confirmed with Western blot analysis on lung homogenates utilizing antibodies specific for Smad2 and p-Smad2. (A) At P14, after litters were maintained on doxycycline from P7 to P14, there was a slight increase in expression of Smad2 from lungs of TG(+) animals when compared with TG(–) control animals, but there was a more dramatic increase in p-Smad2 from TG(+) animals when compared with control animals. (B) These results were confirmed by densitometry (n = 4 animals in each group; shaded bars, TG(–), doxycycline; closed bars, TG(+), doxycycline). (C) In TG(–) animals, immunohistochemical analysis demonstrated that p-Smad2 localized predominately to airway epithelial cells (arrows), with little signal noted in alveolar tissue or vasculature (open arrows). (D) In comparison, TG(+) animals demonstrated intense signal in both airway epithelium (arrows) and cells within abnormal alveolar structures; less intense signal was seen in vascular structures (open arrows; scale bars = 200 µm [low magnification views], 100 µm [high magnification views]; boxes indicate area of enlargement).

Overexpression of Bioactive TGF-ß1 in Early Postnatal Lung Results in Abnormal Lung Alveolar Development
Overexpression of bioactive TGF-ß1 in the neonatal mouse lung resulted in dramatic alteration of lung structure. Figure 3 demonstrates the morphologic changes that occurred in the lungs of TG(+) animals maintained on doxycycline from P7–P14, and compares these changes with three different control groups. As shown in Figure 3A, TG(–) P14 animals that were not maintained on doxycycline demonstrated well-formed alveolar structures with thin-walled septae, as expected. Neither the presence of the TG (Figure 3B) nor doxycycline (Figure 3C) alone seemed to affect lung histology at this age, as detailed previously. In comparison, lungs from TG(+) animals maintained on doxycycline from P7–P14 (Figure 3D) consisted of large respiratory exchange units with thick, hypercellular septae and poor alveolar development. These animals were smaller than all other control groups (4.84 ± 0.33 g versus 7.23 ± 0.62 g for TG[–], no doxycycline; 6.95 ± 0.43 g for TG[–], doxycycline; and 7.09 ± 0.56 g for TG[+], no doxycycline ). Furthermore, TG(+) animals on doxycycline exhibited kyphoscoliosis and signs of respiratory distress with high respiratory rates. In comparison, none of the other groups demonstrated such characteristics.

View larger version (131K): [in this window] [in a new window]   Figure 3. Overexpression of bioactive TGF-ß1 during early alveolar formation. Lung sections from TG(+) animals maintained on doxycycline from P7–P14 were examined and compared with sections from three different control groups. For each group, low- (4x) and high- (20x) magnification views are included (small boxes indicate area of magnification). (A) TG(–), no doxycycline, killed at P14. (B) TG(–), doxycycline administered from P7–P14. (C) TG(+), no doxycycline, killed at P14. (D) TG(+), doxycycline administered from P7–P14. As demonstrated, TG(+) animals maintained on doxycycline showed dramatic alterations in lung structure, including large respiratory exchange units, hypercellular septae, and poor development of alveoli. Scale bars = 500 µm (low-magnification views) and 250 µm (high-magnification views); each photograph is representative of at least four animals from different litters).

View larger version (96K): [in this window] [in a new window]   Figure 4. Parenchymal characteristics of lung tissue after administration of doxycycline from P7–P14. Lung parenchyma at P14, after 7 d of doxycycline administration, was evaluated. (A, B) Extracellular matrix accumulation in lung tissues was assessed with Trichrome staining. In TG(–) and TG(+) animals, fibrosis was localized mostly to the subepithelial space surrounding larger airways and vessels (arrows). Whereas the parenchymal tissues of TG(–) animals demonstrated little staining for collagen, those of TG(+) animals showed some staining within alveolar structures. (C, D) Pancytokeratin signal in TG(–) control animals was intense in airway epithelium (arrows), with less intense signal in alveolar structures. In TG(+) animals, the signal was confined to the airways (arrow) and outer borders of the septae (arrowhead), whereas the interior sections of septae showed little immunoreactivity. (E, F) In TG(–) control animals, intense -SMA–positive signal was limited to airway (arrows) and vascular smooth muscle, with minimal signal noted in alveolar structures. In TG(+) animals, signal was noted in airway smooth muscle (arrows), but also in numerous cells within the septal walls (scale bars = 200 µm; all panels are representative of four separate animals from two different litters).

Proliferation of -SMA–Positive Cells within the Septal Walls in TG(+) Animals
Because the septal walls of TG(+) animals appeared to be hypercellular, we performed Western blot analysis using antibodies against PCNA to determine whether increased cellular proliferation was present. As shown in Figure 5A, lung homogenates from TG(+) animals demonstrated increased PCNA, suggesting increased cellular proliferation. These results were confirmed by densitometry (Figure 5B; n = 5 animals in each group). Because TGF-ß1 is known to cause myofibroblast proliferation in adult models, and because we identified numerous -SMA–positive cells in septal walls as well as increased expression of PCNA from lung homogenates of TG(+) animals, we performed fluorescent double-labeling experiments to determine whether the -SMA–positive cells were the cells undergoing proliferation. As demonstrated in Figure 5C, PCNA colocalized with -SMA in many of the septal cells. Note that airway smooth muscle (arrows) stained positive only for -SMA.

View larger version (59K): [in this window] [in a new window]   Figure 5. Western blot analysis, densitometry and double-labeling experiments for cell proliferation. (A) Western blot analysis performed at P14 (doxycycline from P7–P14) demonstrated that there was an increase in PCNA in whole-lung homogenates of TG(+) animals compared with TG(–) control mice. (B) These results were confirmed by densitometry. Samples were normalized with HSC-70 (n = 5 animals in each group). (C) Fluorescent double-labeling experiments were employed to determine whether the -SMA–positive cells were the cells undergoing proliferation. As shown, numerous cells in the septal walls of TG(+) animals demonstrated colocalization of the antigens. Note that airway smooth muscle (arrows) stained positive only for -SMA, as expected (green = -SMA; red = PCNA; yellow = colocalization). Scale bars = 200 µm; all photos are representative of three animals for each group.

View larger version (87K): [in this window] [in a new window]   Figure 6. Western blot analysis, densitometry and double-labeling experiments for platelet–endothelial cell adhesion molecule. (A) Western blot analysis demonstrated that lung homogenates from TG(+) animals had a lower expression of PECAM when compared with TG(–) control animals. (B) These results were confirmed by densitometry. Samples were normalized with HSC-70 (n = 5 animals for each group). (C) In TG(–) control animals, PECAM signal (green) is intense in large vessels (arrows) and in alveolar structures. Pancytokeratin (red) is mostly localized to airway epithelial cells (arrowheads), with less intense signal in alveoli. In TG(+) animals, PECAM signal (green) is seen mostly in larger vessels (arrows), with only minimal signal scattered throughout the lung parenchyma, particularly from smaller vessels within the abnormal septal walls (green arrow). Pancytokeratin (red) remained in airway epithelium (arrowhead) and, to a lesser extent, alveolar septae (scale bars = 200 µm; all photos are representative of three animals for each group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although a number of transgenic models exist today, few are ideal for investigating the effects of gene products during specific and limited points in time. As mentioned previously, this concept is particularly important for the study of BPD, a disease that arises in the context of a still-developing lung, and within a specific and limited period of time. In the case of TGF-ß, past investigators have employed techniques to overexpress the ligand prenatally, resulting in perinatal death of the animals (14). Thus, the specific effects of TGF-ß during the alveolar stage of development could not be fully assessed. Other investigators have successfully overexpressed TGF-ß in adult rodents utilizing vector inoculation techniques (15). Although such models allow for temporally controlled as well as lung-specific overexpression, the techniques can be difficult to employ in extremely small neonatal mice. Although such investigations have formed the backbone of our current knowledge regarding the effects of TGF-ß in the lung, the use of conditional transgenic models is more suited to investigate a disease such as BPD, which develops during a specific period of time. Utilizing the model described in this article, genes of interest (in this case, TGF-ß) can easily be turned on/off specifically during the period of alveolar formation to more accurately mimic changes that might occur in the premature infant.

Recent pathologic descriptions of BPD suggest that the characteristics of the disease have changed over the era. In the presurfactant years, BPD pathology encompassed airway and parenchymal inflammation and marked fibrosis (26). Today, investigators have focused on three major pulmonary systems affected in BPD: (1) cellular responses to injury, including epithelial, inflammatory, and mesenchymal cells; (2) formation of alveolar structures; and (3) vascularization. In BPD, alveolar development is abnormal, resulting in fewer and larger respiratory exchange units with thick interstitial walls (3, 27). In the current study, we have shown that overexpression of TGF-ß1 in the neonatal lung resulted in similar pathologic findings—large respiratory exchange units with thick, cellular septal walls. In addition, investigators have described abnormal localization and increased proliferation of -SMA–positive cells in terminal airspaces and areas of severe lung injury from patients in various stages of BPD (5, 28). Similarly, the results from our investigation indicate increased proliferation of -SMA–positive cells within the abnormal septal walls. Finally, lung specimens from premature baboons maintained on mechanical ventilation and high oxygen concentrations have revealed not only the alveolar changes noted above, but also abnormal development of pulmonary capillaries (7, 8). As we have shown, overexpression of TGF-ß1 caused diminished expression and abnormal localization of PECAM (a marker for endothelial cells, including capillaries) in the neonatal mouse lung.

To our knowledge, this is the first study that describes the changes induced by overexpression of TGF-ß1 in the neonatal mouse lung using a tetracycline-operated transgenic mouse. However, since the initial preparation of this article, Dr. Gauldie's and Dr. Warburton's groups have collaborated to publish a similar investigation using adenoviral vector inoculation techniques to overexpress bioactive TGF-ß1 in neonatal rat lungs (29). Although our study confirms their findings and adds valuable information regarding the vascular changes that occur in developing lung in response to TGF-ß1, there are noticeable differences between the models. For example, the changes in lung histology that we observed appear more rapidly and seem to be more severe, as assessed by the health of the animals within 1 wk of TGF-ß1 overexpression. One possibility for this observation could be that the current model induces a more homogeneous expression pattern affecting the entire lung, whereas the adenoviral vector model may result in "patchy" overexpression due to varying efficacies of gene transfer. Alternatively, the genetic differences between the mouse and rat may confer varying susceptibilities to the effects of TGF-ß1. Although neither investigation addresses these issues fully, both models will be useful tools for future experiments involving overexpression of TGF-ß1 during the neonatal period.

Despite the similarities between our findings and those described in infants with BPD and in existing animal models, it is still not clear whether the same sequence of events occurs in premature infants who develop the disease. Furthermore, we recognize that BPD is likely to be a multifactorial disease. Whereas stimuli, such as hyperoxia, mechanical ventilation, and infection, may all contribute to the development of disease, our study, combined with observations from separate investigations, suggests that TGF-ß1 may play a role in, and possibly mediate, the pathologic changes that ultimately occur in this condition. For example, stressors known to contribute to the development of BPD, such as hyperoxia, result in increased expression of TGF-ß1 ligand and other signal-transducing components of the system (30–32), whereas latent TGF-ß1 can be directly activated by reactive oxygen species in vitro (33). Similarly, ventilator-induced lung injury has been shown to upregulate the expression of TGF-ß mRNA in rat lungs (34), and more recent studies have shown that vß6 integrin, which increases in expression with epithelial injury, can bind and activate latent TGF-ß1 (35, 36). Thus, our data provide a potential link between known stimuli for the development of BPD–hyperoxia and mechanical ventilation/epithelial injury and the resulting pathologic changes. The use of conditional knockouts in well-established animal models of BPD (i.e., hyperoxia), or other technologies designed to inhibit the TGF-ß signal transduction pathway, will help to strengthen our findings and possibly uncover novel therapeutic targets for the treatment or prevention of disease. In the present study, we have demonstrated that conditional transgenic models are useful in studying BPD. More widespread use of such technology will enhance our ability to dissect the molecular mechanisms underlying the disease and normal lung development.

	Acknowledgments

 
This study was supported by National Institutes of Health (NIH) grant 5P01HD-32573 to G.G.H., NIH grant HL-64242, HL-56389, HL-61904 to J.A.E., and by the Juvenile Diabetes Foundation International grant #10-2000-71 to O.E.

	Footnotes

 
A.G.V. and C.G.L. contributed equally to this project.

Conflict of Interest Statement: A.G.V. has no declared conflicts of interest; C.G.L. has no declared conflicts of interest; S.J.C. has no declared conflicts of interest; O.E. has no declared conflicts of interest; Y.C. has no declared conflicts of interest; G.G.H. has no declared conflicts of interest; and J.A.E. has no declared conflicts of interest.

Received in original form March 15, 2004

Received in final form August 17, 2004

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