Growth Hormone-Dependent Effects of Human Serum on the in Vitro Growth Characteristics of Human Skin Fibroblasts MARGARET H. MACGILLIVRAY, CLAUDIA HASTINGS, AND JUDITH A. BROWN Department of Pediatrics, Children's Hospital Divisions of Endocrinology and Human Genetics, State University of New York at Buffalo Medical School, Buffalo, New York 14222 pituitary children during successful treatment with growth hormone exhibit normal monolayer confluency. These observations are compatible with the explanation that serum from GH-deficient children lacks a GH-dependent component which is essential for the normal in vitro growth of human skin fibroblasts. (/ Clin Endocrinol Metab 40: 62, 1975)
ABSTRACT. Skin fibroblasts derived from either a normal child or from untreated hypopituitary children become progressively more rounded and finally detach and float free when cultured in medium containing serum from growth hormone (GH)-deficient patients. In contrast, human fibroblasts cultured in medium supplemented with serum obtained from hypo-
I
NVESTIGATIONS have shown that segments of costal cartilage from hypophysectomized rats exhibit an increased uptake of radiolabeled sulfate when incubated in medium containing serum from normal rats or human subjects (1-4). In contrast, incorporation of S35 is less when the cartilage is cultured in medium supplemented with serum from hypopituitary rats or patients (1-4). After initiation of in vivo GH replacement therapy, serum obtained from hypopituitary rats or humans enhances the uptake of radioactive sulfate into hypophysectomized rat cartilage (1,2). These effects have not been consistently demonstrated when GH is added to cartilage explants in vitro (1). Additional studies have demonstrated that the active serum component, originally named sulfation factor, has a broad spectrum of other biological effects including stimulation of DNA, RNA and protein synthesis (5-8). On the basis of these findings, it has been postulated that control of cellular growth is mediated by a serum conponent whose synthesis is regulated by growth hormone.
The general term, somatomedin, has been proposed as a more appropriate name for this growth hormone-dependent serum growth factor (9). The observations concerning the differential effects of normal and hypopituitary rat serum on the in vitro metabolism of cartilage prompted the present investigations into the effects of serum from normal subjects and GH-deficient patients on cultured human fibroblasts. In this communication, the morphological changes which occurred when human skin fibroblasts were grown in medium supplemented with serum from hypopituitary dwarfs prior to and during GH treatment are described. Materials and Methods Skin biopsies were obtained from a normal boy (age 10) and from 2 untreated GH-deficient patients (female, age 10, and male, age 5). Blood was drawn from several fasting normal adult subjects and from 6 euthyroid, hypopituitary dwarfs both before and during the period of GH treatment. The normal serum specimens (NHS) were pooled, whereas the samples from GH deficient patients were used individually. GH deficiency in the patients was confirmed by plasma GH concentration of less than 5 ng/ml following insulin -hypoglycemia and L-arginine stimulation tests (10). All diagnostic studies were performed in the Clinical Research Center. During the treatment period, human
Received May 21, 1973. Reprint address: M. H. MacGillivray, M.D., Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, New York 14222. 62
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS GH, provided by the National Pituitary Agency, was administered intramuscularly three times weekly using a dose of 0.1 U/kg. Fibroblasts were initially established in monolayer culture in T25 cm2 Falcon flasks containing F10 medium [Grand Island Biological Company (GIBCO), Grand Island, New York] supplemented with 20% fetal calf serum (v/v), penicillin (100 U/ml) and streptomycin (100 /tig/ml). The cells were subcultured at weekly intervals after washing twice with Hank's Balanced Salt Solution (HBSS) and
63
incubating for 3-5 min at 37 C in 0.25% trypsin solution (GIBCO). Prior to use, the fibroblasts were initially transferred to medium supplemented with 10% fetal calf serum and finally, to medium supplemented with 10% normal human serum. For the assays, approximately 1 x 106 cells in 5 ml of medium plus 10% test serum were transferred to T15 cm2 flasks and incubated at 37 C in an atmosphere of 5% carbon dioxide in 95% air. The experimental design consisted of culturing fibroblasts for at least two passages in
FEG. 1. Skin fibroblasts from normal individuals cultured in medium containing normal human serum.
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medium supplemented with one of the following types of serum: (a) 10% normal serum, (b) 10% autologous or homologous hypopituitary patient serum before GH treatment, (c) 10% autologous or homologous hypopituitary patient serum during GH treatment. In additional studies, fibroblasts previously cultured for two passages in medium supplemented with 10% serum obtained from untreated GH-deficient patients were gently centrifuged, washed three times and replated into fresh medium contain-
JCE & M • 1975 Vol 40 • No 1
ing either. 10% normal human serum or serum from treated GH-deficient patients. The direct effects of human GH (10 fxg and 100 /u,g/ml) were evaluated by adding these two concentrations to fibroblasts cultured in 10% normal or hypopituitary serum. Morphological changes were recorded using a Unitron Inverted Photo-microscope with high contrast copy film (Kodak, Rochester, New York). Photos were taken at 100 x magnification and enlarged 20 x for the illustrations.
FIG. 2. Normal fibroblasts in homologous hypopituitary serum obtained during GH treatment. Photomicrograph taken 24 hr after second passage.
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS Results Skin fibroblasts from the control subject exhibited normal monolayer confluency and cellular morphology when cultured for numerous passages in medium containing normal human serum (Fig. 1). Fibroblasts from the GH-deficient patients when grown in normal serum were indistinguishable from the control fibroblasts.
65
Similar growth patterns were observed when fibroblasts obtained from the control subject or untreated hypopituitary patients were cultured in medium containing serum from hypopituitary patients undergoing successful treatment with GH (Fig. 2). In contrast, control and hypopituitary patient fibroblasts, during the second passage, exhibited strikingly abnormal cellular structure and disruption of the monolayer
FIG. 3a. Normal fibroblasts in untreated hypopituitary serum. Photomicrograph taken at 48 hr after second passage.
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JCE & M • 1975 Vol 40 • No 1
FIG. 3b. Hypopituitary fibroblasts in untreated hypopituitary serum at 72 hr after second passage.
when grown in medium containing serum from untreated patients. Within 24 to 48 hr, the cells became rounded, detached from the growing surface and floated free in the medium (Figs. 3a, 3b). The floating fibroblasts, whether derived from the control subject or the GH deficient patients, when replated into medium containing normal serum or serum from hypopituitary patients during GH treatment, re-established normal monolayer confluency (Figs. 4a, 4b). In fact, these cells retained their ability to
form a confluent monolayer even after being allowed to float free for as long as 8 days in medium supplemented with pre-GH treatment hypopituitary serum. In the 6 hypopituitary patients studied to date, the differences in fibroblast morphology observed during culture in pre- and post-treatment serum were documented in two or three separate evaluations per serum. Addition of GH (10 /ig/ml) to fibroblasts cultured in normal serum or untreated
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS
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FIG. 4a. Clumps of fibroblasts, previously floating in untreated hypopituitary serum, begin to exhibit new monolayer growth within 24 hr after passage into normal human serum.
hypopituitary serum had no detectable ef- pronounced morphologic changes in their fect on the growth characteristics other growth characteristics when cultured in than those seen when only serum was medium containing serum from untreated used. However, GH at a concentration of hypopituitary patients. During the initial 100 /u,g/ml proved to be toxic to the exposure to serum from untreated hypomonolayer and caused disruption and lift- pituitary dwarfs, the fibroblasts appeared ing of the cells. less healthy with marked granulation and vesiculation of the cytoplasm after the fifth Discussion and sixth day of culture. Subsequent pasIn these studies, human skin fibroblasts sage of these cells into fresh medium derived from either a normal child or from containing untreated hypopituitary serum untreated hypopituitary children showed resulted in scant monolayer growth during
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HASTINGS, AND BROWN
the first 2 days followed by striking alterations in monolayer morphology between days 2 to 4. The fibroblasts became progressively more rounded andfinallybecame detached and floated free in the medium. In contrast, human skin fibroblasts cultured in medium supplemented with serum obtained from hypopituitary children during successful treatment with human GH exhibited normal monolayer
JCE & M • 1975 Vol 40 • No 1
confluency throughout the two passages. The gross morphologic appearance of these fibroblasts did not differ from cells maintained in medium supplemented with either normal human or fetal calf serum. Although fibroblasts exposed to untreated hypopituitary serum appeared "necrotic" because of their tightly packed, rounded appearance, they were capable of re-establishing monolayer confluency
FIG. 4b. Clumps of fibroblasts, previously floating in untreated hypopituitary serum exhibit confluent monolayer growth within 5 days after passage into normal human serum.
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS when replated into fresh medium containing either normal serum or serum from GH-treated patients. The abnormalities in skin fibroblast morphology were not observed until the second passage of the cells into medium containing serum from untreated hypopituitary dwarfs. A possible explanation for this delay may be that one passage in hypopituitary serum is required to dissipate the growth-promoting stimulus in fibroblasts previously cultured in normal human or fetal calf serum. The evidence in support of this hypothesis is derived from studies which showed that cartilage segments from normal rats had to be incubated from a period of time in medium before the hormonal stimulation of growth subsided (2). Data presented here indicate that serum from GH deficient children lacks a components), possibly somatomedin, which is essential for the normal in vitro growth of human skin fibroblasts. This component(s), like somatomedin, is detectable in serum following growth hormone replacement therapy in hypopituitary patients. In a related study utilizing HeLa cells, Salmon and Hosse (11) reported stimulation of cell growth by a serum factor with "sulfaction factor" activity. Although the precise nature of the serum component is not identified at present, the fibroblast bioassay has potential use as a sensitive morphologic index for evaluating the growth-promoting properties of sera from children with a variety of growth disorders.
69
Acknowledgments This investigation was supported by grants from the Department of Health, Education and Welfare, Maternal and Child Health Service (Project 417), by General Research Support Grant RR05493 from the General Research Support Branch, Division of Research Facilities and Resources and by the Department of Health, Education and Welfare, Public Health Service, NIH 1 PO1 HD 06321-01. All clinical diagnostic studies were supported by a grant from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health and by the National Pituitary Agency.
References 1. Salmon, W. D., and W. H. Daughaday, J Lab Clin Med 49: 825, 1957. 2. Daughaday, W. H., W. D. Salmon, and F. Alexander,/ Clin Endocrinol Metab 19: 743, 1959. 3. Almqvist, S., Ada Endocrinol (Kbh) 35: 381, 1960. 4. , D. Ikkos, and R. Luft, Acta Endocrinol (Kbh) 36: 577, 1961. 5. Salmon, W. D., and M. R. DuVall, Endocrinology 86: 721, 1970. , Endocrinology 87: 1168, 1970. 6. , and 7. Daughaday, W. H., and C. Reeder, / Lab Clin Med 68: 357, 1966. 8. Van Wyk, J. J., K. Hall, J. L. Van den Brande, R. P. Weaver, K. Uthne, R. L. Hintz, J. H. Harrison, and P. Mathewson, In Pecile, A., and E. Mueller, (eds.), Proc Second Int Symp Growth Hormone, 1972, p. 155. 9. Daughaday, W. H., K. Hall, M. S. Raben, W. D. Salmon, J. D. Van den Brande, and J. J. Van Wyk, Nature (Lond) 236: 107, 1972. 10. Frohman, L. A., T. Aceto, Jr., and M. H. MacGillivrayj Clin Endocrinol Metab 27: 1409, 1967. 11. Salmon, W. D., and B. R. Hosse, Proc Soc Exp Biol Med 136: 805, 1971. 12. Daughaday, W. H., J. N. Heins, L. Srivastava, and C. Hammer,/ Lab Clin Med 72: 803, 1968.
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ABSTRACT. Skin fibroblasts derived from either a normal child or from untreated hypopituitary children become progressively more rounded and finally detach and float free when cultured in medium containing serum from growth hormone (GH)-deficient patients. In contrast, human fibroblasts cultured in medium supplemented with serum obtained from hypo-
I
NVESTIGATIONS have shown that segments of costal cartilage from hypophysectomized rats exhibit an increased uptake of radiolabeled sulfate when incubated in medium containing serum from normal rats or human subjects (1-4). In contrast, incorporation of S35 is less when the cartilage is cultured in medium supplemented with serum from hypopituitary rats or patients (1-4). After initiation of in vivo GH replacement therapy, serum obtained from hypopituitary rats or humans enhances the uptake of radioactive sulfate into hypophysectomized rat cartilage (1,2). These effects have not been consistently demonstrated when GH is added to cartilage explants in vitro (1). Additional studies have demonstrated that the active serum component, originally named sulfation factor, has a broad spectrum of other biological effects including stimulation of DNA, RNA and protein synthesis (5-8). On the basis of these findings, it has been postulated that control of cellular growth is mediated by a serum conponent whose synthesis is regulated by growth hormone.
The general term, somatomedin, has been proposed as a more appropriate name for this growth hormone-dependent serum growth factor (9). The observations concerning the differential effects of normal and hypopituitary rat serum on the in vitro metabolism of cartilage prompted the present investigations into the effects of serum from normal subjects and GH-deficient patients on cultured human fibroblasts. In this communication, the morphological changes which occurred when human skin fibroblasts were grown in medium supplemented with serum from hypopituitary dwarfs prior to and during GH treatment are described. Materials and Methods Skin biopsies were obtained from a normal boy (age 10) and from 2 untreated GH-deficient patients (female, age 10, and male, age 5). Blood was drawn from several fasting normal adult subjects and from 6 euthyroid, hypopituitary dwarfs both before and during the period of GH treatment. The normal serum specimens (NHS) were pooled, whereas the samples from GH deficient patients were used individually. GH deficiency in the patients was confirmed by plasma GH concentration of less than 5 ng/ml following insulin -hypoglycemia and L-arginine stimulation tests (10). All diagnostic studies were performed in the Clinical Research Center. During the treatment period, human
Received May 21, 1973. Reprint address: M. H. MacGillivray, M.D., Children's Hospital of Buffalo, 219 Bryant Street, Buffalo, New York 14222. 62
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS GH, provided by the National Pituitary Agency, was administered intramuscularly three times weekly using a dose of 0.1 U/kg. Fibroblasts were initially established in monolayer culture in T25 cm2 Falcon flasks containing F10 medium [Grand Island Biological Company (GIBCO), Grand Island, New York] supplemented with 20% fetal calf serum (v/v), penicillin (100 U/ml) and streptomycin (100 /tig/ml). The cells were subcultured at weekly intervals after washing twice with Hank's Balanced Salt Solution (HBSS) and
63
incubating for 3-5 min at 37 C in 0.25% trypsin solution (GIBCO). Prior to use, the fibroblasts were initially transferred to medium supplemented with 10% fetal calf serum and finally, to medium supplemented with 10% normal human serum. For the assays, approximately 1 x 106 cells in 5 ml of medium plus 10% test serum were transferred to T15 cm2 flasks and incubated at 37 C in an atmosphere of 5% carbon dioxide in 95% air. The experimental design consisted of culturing fibroblasts for at least two passages in
FEG. 1. Skin fibroblasts from normal individuals cultured in medium containing normal human serum.
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MACGILLIVRAY,
HASTINGS, AND BROWN
medium supplemented with one of the following types of serum: (a) 10% normal serum, (b) 10% autologous or homologous hypopituitary patient serum before GH treatment, (c) 10% autologous or homologous hypopituitary patient serum during GH treatment. In additional studies, fibroblasts previously cultured for two passages in medium supplemented with 10% serum obtained from untreated GH-deficient patients were gently centrifuged, washed three times and replated into fresh medium contain-
JCE & M • 1975 Vol 40 • No 1
ing either. 10% normal human serum or serum from treated GH-deficient patients. The direct effects of human GH (10 fxg and 100 /u,g/ml) were evaluated by adding these two concentrations to fibroblasts cultured in 10% normal or hypopituitary serum. Morphological changes were recorded using a Unitron Inverted Photo-microscope with high contrast copy film (Kodak, Rochester, New York). Photos were taken at 100 x magnification and enlarged 20 x for the illustrations.
FIG. 2. Normal fibroblasts in homologous hypopituitary serum obtained during GH treatment. Photomicrograph taken 24 hr after second passage.
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS Results Skin fibroblasts from the control subject exhibited normal monolayer confluency and cellular morphology when cultured for numerous passages in medium containing normal human serum (Fig. 1). Fibroblasts from the GH-deficient patients when grown in normal serum were indistinguishable from the control fibroblasts.
65
Similar growth patterns were observed when fibroblasts obtained from the control subject or untreated hypopituitary patients were cultured in medium containing serum from hypopituitary patients undergoing successful treatment with GH (Fig. 2). In contrast, control and hypopituitary patient fibroblasts, during the second passage, exhibited strikingly abnormal cellular structure and disruption of the monolayer
FIG. 3a. Normal fibroblasts in untreated hypopituitary serum. Photomicrograph taken at 48 hr after second passage.
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JCE & M • 1975 Vol 40 • No 1
FIG. 3b. Hypopituitary fibroblasts in untreated hypopituitary serum at 72 hr after second passage.
when grown in medium containing serum from untreated patients. Within 24 to 48 hr, the cells became rounded, detached from the growing surface and floated free in the medium (Figs. 3a, 3b). The floating fibroblasts, whether derived from the control subject or the GH deficient patients, when replated into medium containing normal serum or serum from hypopituitary patients during GH treatment, re-established normal monolayer confluency (Figs. 4a, 4b). In fact, these cells retained their ability to
form a confluent monolayer even after being allowed to float free for as long as 8 days in medium supplemented with pre-GH treatment hypopituitary serum. In the 6 hypopituitary patients studied to date, the differences in fibroblast morphology observed during culture in pre- and post-treatment serum were documented in two or three separate evaluations per serum. Addition of GH (10 /ig/ml) to fibroblasts cultured in normal serum or untreated
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS
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FIG. 4a. Clumps of fibroblasts, previously floating in untreated hypopituitary serum, begin to exhibit new monolayer growth within 24 hr after passage into normal human serum.
hypopituitary serum had no detectable ef- pronounced morphologic changes in their fect on the growth characteristics other growth characteristics when cultured in than those seen when only serum was medium containing serum from untreated used. However, GH at a concentration of hypopituitary patients. During the initial 100 /u,g/ml proved to be toxic to the exposure to serum from untreated hypomonolayer and caused disruption and lift- pituitary dwarfs, the fibroblasts appeared ing of the cells. less healthy with marked granulation and vesiculation of the cytoplasm after the fifth Discussion and sixth day of culture. Subsequent pasIn these studies, human skin fibroblasts sage of these cells into fresh medium derived from either a normal child or from containing untreated hypopituitary serum untreated hypopituitary children showed resulted in scant monolayer growth during
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HASTINGS, AND BROWN
the first 2 days followed by striking alterations in monolayer morphology between days 2 to 4. The fibroblasts became progressively more rounded andfinallybecame detached and floated free in the medium. In contrast, human skin fibroblasts cultured in medium supplemented with serum obtained from hypopituitary children during successful treatment with human GH exhibited normal monolayer
JCE & M • 1975 Vol 40 • No 1
confluency throughout the two passages. The gross morphologic appearance of these fibroblasts did not differ from cells maintained in medium supplemented with either normal human or fetal calf serum. Although fibroblasts exposed to untreated hypopituitary serum appeared "necrotic" because of their tightly packed, rounded appearance, they were capable of re-establishing monolayer confluency
FIG. 4b. Clumps of fibroblasts, previously floating in untreated hypopituitary serum exhibit confluent monolayer growth within 5 days after passage into normal human serum.
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GH-DEPENDENT EFFECTS OF SERUM ON FIBROBLASTS when replated into fresh medium containing either normal serum or serum from GH-treated patients. The abnormalities in skin fibroblast morphology were not observed until the second passage of the cells into medium containing serum from untreated hypopituitary dwarfs. A possible explanation for this delay may be that one passage in hypopituitary serum is required to dissipate the growth-promoting stimulus in fibroblasts previously cultured in normal human or fetal calf serum. The evidence in support of this hypothesis is derived from studies which showed that cartilage segments from normal rats had to be incubated from a period of time in medium before the hormonal stimulation of growth subsided (2). Data presented here indicate that serum from GH deficient children lacks a components), possibly somatomedin, which is essential for the normal in vitro growth of human skin fibroblasts. This component(s), like somatomedin, is detectable in serum following growth hormone replacement therapy in hypopituitary patients. In a related study utilizing HeLa cells, Salmon and Hosse (11) reported stimulation of cell growth by a serum factor with "sulfaction factor" activity. Although the precise nature of the serum component is not identified at present, the fibroblast bioassay has potential use as a sensitive morphologic index for evaluating the growth-promoting properties of sera from children with a variety of growth disorders.
69
Acknowledgments This investigation was supported by grants from the Department of Health, Education and Welfare, Maternal and Child Health Service (Project 417), by General Research Support Grant RR05493 from the General Research Support Branch, Division of Research Facilities and Resources and by the Department of Health, Education and Welfare, Public Health Service, NIH 1 PO1 HD 06321-01. All clinical diagnostic studies were supported by a grant from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health and by the National Pituitary Agency.
References 1. Salmon, W. D., and W. H. Daughaday, J Lab Clin Med 49: 825, 1957. 2. Daughaday, W. H., W. D. Salmon, and F. Alexander,/ Clin Endocrinol Metab 19: 743, 1959. 3. Almqvist, S., Ada Endocrinol (Kbh) 35: 381, 1960. 4. , D. Ikkos, and R. Luft, Acta Endocrinol (Kbh) 36: 577, 1961. 5. Salmon, W. D., and M. R. DuVall, Endocrinology 86: 721, 1970. , Endocrinology 87: 1168, 1970. 6. , and 7. Daughaday, W. H., and C. Reeder, / Lab Clin Med 68: 357, 1966. 8. Van Wyk, J. J., K. Hall, J. L. Van den Brande, R. P. Weaver, K. Uthne, R. L. Hintz, J. H. Harrison, and P. Mathewson, In Pecile, A., and E. Mueller, (eds.), Proc Second Int Symp Growth Hormone, 1972, p. 155. 9. Daughaday, W. H., K. Hall, M. S. Raben, W. D. Salmon, J. D. Van den Brande, and J. J. Van Wyk, Nature (Lond) 236: 107, 1972. 10. Frohman, L. A., T. Aceto, Jr., and M. H. MacGillivrayj Clin Endocrinol Metab 27: 1409, 1967. 11. Salmon, W. D., and B. R. Hosse, Proc Soc Exp Biol Med 136: 805, 1971. 12. Daughaday, W. H., J. N. Heins, L. Srivastava, and C. Hammer,/ Lab Clin Med 72: 803, 1968.
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