Neuropeptides (2002) 36(2±3), 96±102 Special Issue on Transgenics and Knockouts with Mutations in Genes for Neuropeptides and their Receptors. ß 2002 Elsevier Science Ltd. All rights reserved. doi: 10.1054/npep.2002.0906, available online at http://www.idealibrary.com on
Lessons from CRH knockout mice M. Venihaki, J. Majzoub Division of Endocrinology, Children's Hospital, 300 Longwood Avenue, Boston, MA, 02115
Summary Corticotropin-releasing hormone (CRH), the major regulator of hypothalamic±pituitary±adrenal (HPA) axis, has a wide spectrum of actions within the central nervous system and the periphery. The development and use of Crh knockout mice (Crh / ) has been an important tool for addressing the physiologic and pathologic roles of CRH. This review describes the generation and characterization of Crh-deficient mice as well as the use of these mice to study the role of CRH in maternal and fetal HPA axes development and in the regulation of the adult HPA axis and behavior. The review concludes with information about recently discovered CRH-related peptides and their possible roles in some of the functions thought initially to be mediated by CRH. ß 2002 Elsevier Science Ltd. All rights reserved.
INTRODUCTION The ability to generate and use mice with genetic mutations or complete deletions of speci®c genes through homologous recombination in embryonic stem (ES) cells has been an important tool to study the in vivo role and functions of many genes and their link to human diseases (Majzoub and Muglia, 1996). However, because such mice are de®cient in the speci®c gene product during their entire fetal and postnatal life, compensatory mechanisms likely develop in response to the de®ciency which may further alter the phenotype of these mice. Thus, conclusions regarding normal physiology or the pathology of knockout mice must be drawn cautiously. Nevertheless, this technique has become a powerful means with which to study gene function in vivo. CRH, a 41-amino acid neuropeptide, is one of the main hypothalamic components of the HPA axis. It is synthesized in the paraventricular nucleus of the hypothalamus (PVH), released into the hypophysial portal blood system and carried to the anterior pituitary gland. At this site, CRH acting via CRH type 1 receptor (Chalmers et al., 1996) stimulates the synthesis of mRNA encoding proopiomelanocortin (POMC, the precursor of ACTH) and the release of ACTH from pituitary corticotrophs (Rivier et al., 1982; Bruhn et al., 1984).
Received 2 May 2002 Accepted 8 May 2002 Correspondence to: Joseph Majzoub, M.D. Division of Endocrinology, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115. Tel.: (617) 355-6421; Fax: (617) 734-0062; E-mail: [email protected]
CRH mRNA and peptide have also been found in many extrahypothalamic brain regions as well as in peripheral tissues (Bruhn et al., 1987; Charlton et al., 1987; Sasaki et al., 1988; Emanuel et al., 1989; Karalis et al., 1991; Muglia et al., 1994). In addition to its endocrine effects, CRH is thought to have a wide spectrum of behavioral, autonomic, and immune functions as suggested by studies in animals and humans using CRH, CRH antagonists or other agents. In this review we discuss the generation and survival of CRH de®cient (Crh / ) mice, the consequences of CRH de®ciency on fetal and maternal HPA axes, the regulation of the adult hypothalamic-pituitaryadrenal (HPA) axis, and the responses to physiological and behavioral stressors. We conclude with information about recently discovered CRH-related peptides and their possible roles in some of the functions thought initially to be mediated by CRH.
GENERATION AND RESCUE OF Crh-DEFICIENT MICE CRH de®cient (Crh / ) mice were generated by targeted gene disruption in ES cells (Muglia et al., 1994; Muglia et al., 1995). Cloned genomic DNA corresponding to the CRH locus was isolated from a mouse Balb/c genomic DNA library. The gene-targeting vector used was constructed such as to replace the entire pre-proCRH coding region with a neomycin resistance cassette. Introduction of the targeting vector into ES cells produced clones having replaced one copy of the endogenous CRH gene with the neo gene. Injection of these targeted ES cells into wild type blastocysts resulted in generation of chimeras capable of transmitting the mutated allele to their
Lessons from the CRH knockout mice 97
offspring (Muglia et al., 1995), yielding mice heterozygous for CRH de®ciency. Heterozygotes were then bred to each other to create mice homozygous for CRH de®ciency. CRH de®ciency was con®rmed with reverse-transcription polymerase chain reaction analysis of whole brain RNA from wild type (Crh / ) and Crh / mice using speci®c primers for CRH mRNA. Crh / mice were born from heterozygous matings at the anticipated Mendelian ratio indicating normal viability (Muglia et al., 1995). Furthermore, Crh-de®cient mice were indistinguishable from their Crh / littermates in all parameters tested such as size, activity, and general behavior (Muglia et al., 1995). They were fertile and had normal longevity despite their very low basal glucocorticoid levels (Muglia et al., 1995). Matings between homozygous Crh-de®cient mice resulted in fetuses which all died on the ®rst day of life with cyanosis and labored breathing. Neonatal demise was due to pulmonary insuf®ciency, which was reversed by supplementation of the pregnant females with corticosterone in their drinking water (Muglia et al., 1995; Venihaki et al., 2000). Detailed histologic analysis of the lungs from pups derived from homozygous Crh-de®cient matings revealed that Crh / fetuses showed an overall failure in morphological maturation of the lungs, with hypercellularity con®rmed by wet weight, dry weight and DNA content (Muglia et al., 1995; Muglia et al., 1999). This hypercellularity was likely the result of continued cell proliferation during gestation since Crh-de®cient fetuses had increased proliferation cell nuclear antigen (PCNA) staining in the lungs (Muglia et al., 1999). Furthermore, Crh / fetuses had delayed appearance of CC10, a speci®c marker for Clara cells, and increased expression of the pulmonary neuroendocrine cell marker PGP9.5, not only in neuroendocrine cells but also in the majority of the epithelial cells (Muglia et al., 1999). Although the pulmonary surfactant system was also affected in Crh / mice, it did not appear to play a major role in their perinatal demise (Muglia et al., 1999). The defects found in Crh / mice suggest that combined maternal and fetal glucocorticoid insuf®ciency causes an overall delay in pulmonary maturation since both isolated maternal glucocorticoid de®ciency, as in Crh / offspring of Crh / mothers, and isolated fetal glucocorticoid de®ciency, as in Crh / offspring of Crh / mothers, allow for normal fetal lung development.
HYPOTHALAMIC-PITUITARYADRENAL (HPA) AXIS REGULATION IN Crh-DEFICIENT MICE Fetal and Maternal HPA axes Regulation The HPA axis is one of the ®rst endocrine systems to develop during fetal life. Recent studies demonstrated ß 2002 Elsevier Science Ltd. All rights reserved.
that both the expression and the secretion of POMC peptides in rat anterior pituitary start as early as e13.5 (e12.5 in mouse), and a similar pattern of expression has been reported in other species (Elkabes et al., 1989; Japon et al., 1994; Ma et al., 1994; Carr et al., 1995). The onset of POMC expression becomes apparent at a stage when brain development is far from being complete and thus, the activation of POMC gene expression is probably independent of the formation of hypothalamic brain structures and hypothalamic CRH expression. Indeed, CRH expression in the hypothalamus is ®rst detected on e17 in rats, and on e13.5 in mice (Grino et al., 1989; Keegan et al., 1994). In support of this hypothesis, Crh / fetal mice appear to have normal pituitary histology and POMC mRNA expression (Venihaki et al., 2000). However, adrenal size, StAR mRNA expression (a rate-limiting enzyme in the biosynthesis of glucocorticoid), and blood corticosterone levels are signi®cantly lower in Crh / fetuses compared to Crh / fetuses (Venihaki et al., 2000). These data suggest that although fetal CRH is not required for the development of corticotrophs and the onset of the expression of pituitary POMC mRNA, it is necessary for the normal development and function of the fetal adrenal gland. The absence of fetal CRH causes poor adrenal growth, diminished corticosterone secretion, and impaired pulmonary development (Venihaki et al., 2000). However, Crh / neonates born from Crh / mothers can survive after birth despite their low morning corticosterone levels similar to those measured in Crh / fetuses born from Crh / mothers. This ®nding can be explained by the fact that circadian evening rise in blood corticosterone which is preserved during pregnancy in rodents (Montano et al., 1991) and is present in Crh / mothers (but absent in Crh / mothers) results to a signi®cant rise (over 10-fold) in the evening blood corticosterone levels in Crh / fetuses carried by Crh / mothers compared to the levels measured in Crh / fetuses from Crh / mothers. In rodents during the last days of pregnancy, maternal levels of plasma corticosterone rise over 20-fold compared to the levels measured in nonpregnant normal females (Cohen and Guillon, 1985; Montano et al., 1991; Venihaki et al., 2000). A moderate rise in corticosterone is even observed in Crh / mothers carrying only Crh / fetuses, since plasma corticosterone levels are 10-fold elevated compared with levels in nonpregnant Crh / females (Venihaki et al., 2000). This ®nding suggests that in the mouse, CRH is not absolutely required for maternal corticosterone secretion during pregnancy. The glucocorticoid levels are even higher in Crh / females carrying Crh / fetuses indicating that the increase in maternal glucocorticoid secretion during the last days of pregnancy is the combined result of both maternal adrenal secretion and the transfer Neuropeptides (2002) 36(2^3), 96^102
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of corticosterone from fetus to mother through the placenta.
secretion. These data indicate an additional, required role for CRH in ACTH secretion.
Adult HPA axis regulation
Role of CRH in circadian rhythmicity
The normal function of the adult HPA axis in rodents and humans is under the control of several neuropeptides, with the major ones being CRH and vasopressin (Giguere and Labrie, 1982; Giguere et al., 1982; Aguilera et al., 1983; Affolter and Reisine, 1985; Whitnall, 1993; Aoki et al., 1997). Hypothalamic CRH binds to CRH receptor 1 located on corticotrophs and stimulates POMC mRNA synthesis and the secretion of ACTH from these cells. ACTH in turn stimulates the release of glucocorticoid from the adrenal gland. Vasopressin released by parvocellular neurons of the PVH and acting via V1b receptors on corticotrophs has been suggested to participate in the regulation of ACTH secretion, although the relative roles of CRH and vasopressin are not clear. The Crh / mouse model has been a useful tool to investigate the importance of CRH in the regulation of POMC synthesis and the release of ACTH. Crh / mice, despite their low glucocorticoid levels and elevated hypothalamic vasopressin mRNA (Muglia et al., 2000), have normal basal pituitary POMC mRNA expression, ACTH peptide content within the pituitary, and plasma ACTH concentrations, compared to their Crh / littermates, suggesting that CRH is not necessary either for the development of corticotrophs or for the maintenance of basal levels of POMC mRNA expression and peptide secretion (Muglia et al., 1995; Muglia et al., 2000). Following adrenalectomy, both Crh / and Crh / show a robust increase in pituitary POMC mRNA content, with the latter having slightly lower POMC mRNA levels (Muglia et al., 1995; Muglia et al., 2000). Replacement of adrenalectomized mice with corticosterone at physiological non-stress levels suppresses the above increases in POMC mRNA in both Crh / and Crh / mice (Muglia et al., 2000). Despite the almost normal rise in POMC mRNA content in Crh / mice following adrenalectomy, their plasma ACTH levels remain low. However, when adrenalectomy is combined with CRH infusion, plasma levels of ACTH in Crh / mice reach the same levels (over 10-fold above basal) found in wild type adrenalectomized mice (Muglia et al., 2000). The above data suggest that the normal POMC mRNA content in adrenalectomized Crh / mice is dependent on glucocorticoid secretion, and POMC mRNA can increase without the corticotroph being stimulated by CRH. They also suggest that CRH is necessary to stimulate ACTH secretion, which normally occurs during glucocorticoid withdrawal. It had been previously postulated that CRH stimulates Pomc transcription and POMC synthesis, and that vasopressin stimulates ACTH
In rodents hypothalamic CRH content and secretion change as a function of the time of the day, and the development of a rhythm in CRH coincides with the development of the daily rhythm in plasma corticosterone (Kwak et al., 1993; Watts and Swanson, 1989). Indeed, CRH mRNA expression increases from morning to afternoon in rats but decreases rapidly near the onset of dark as ACTH and glucocorticoid reach peak concentrations in plasma, suggesting that the adrenal glucocorticoid circadian rhythm may modulate CRH secretion (Watts and Swanson, 1989), rather than CRH driving this rhythm. However, other studies in rats with invariant levels of corticosterone continue to express a diurnal rhythm in CRH mRNA expression, suggesting that diurnal variation in plasma corticosterone is not required for diurnal ¯uctuation in CRH mRNA (Kwak et al., 1993). The Crh-de®cient mouse has been informative in understanding the role of CRH in the generation and establishment of diurnal adrenal rhythmicity and corticosterone production. Crh / mice, despite their normal light-entrained and free-running circadian locomotor activity have an absent (males), or markedly impaired (females) circadian rise in blood corticosterone (Muglia et al., 1997). Furthermore, the normal evening increase in ACTH levels does not occur in adrenalectomized Crh / mice (Muglia et al., 1997). When Crh / mice were given a constant infusion of CRH, the diurnal rhythm in corticosterone production was restored by the second day of treatment (Muglia et al., 1997) indicating that diurnal variation of CRH secretion is not necessary to drive rhythmic adrenal glucocorticoid secretion. The adrenal insuf®ciency of Crh / mice together with the absence of diurnal rhythmicity in plasma ACTH concentration in intact or adrenalectomized mice suggest that the normal evening elevation in ACTH levels might be required to maintain adrenal cortex integrity and function, possibly by preventing adrenal cortical apoptosis (Wyllie et al., 1973).
Neuropeptides (2002) 36(2^3), 96^102
STRESS RESSPONSES IN Crh ^/^ MICE Role of CRH in the HPA axis response following various stressors The HPA axis represents one of the efferent limbs of the stress system. Following stress, activation of the HPA axis leads to increased secretion of glucocorticoid from the adrenal gland. Since several neuropeptides have been implicated in the activation of the HPA axis during stress, the role of CRH has been uncertain. To further elucidate ß 2002 Elsevier Science Ltd. All rights reserved.
Lessons from the CRH knockout mice 99
the importance of CRH in the regulation of the stress response several physiological stress paradigms were applied in Crh / mice. Following restraint, Crh / mice have absent (males) or markedly impaired (females) production of both ACTH and corticosterone compared to Crh / mice (Muglia et al., 1995). Similar diminished HPA axis responses are observed in Crh / mice following fasting for 36 hr, a stimulus, which results in a robust rise in both ACTH and corticosterone levels in wild type mice ( Jeong et al., 1999). Similarly, CRH-de®cient mice have markedly attenuated, but not absent, responses to hemorrhage and hypoglycemia induced by insulin ( Jacobson et al., 2000). These ®ndings suggest that CRH is required for the normal pituitary and adrenal response to psychological and physiological stressful stimuli, and that other hypothalamic neuropeptides such as vasopressin and oxytocin cannot compensate for the loss of CRH in maintaining the normal response to stress. In addition, the above data indicate that gender-speci®c differences in adrenal responsiveness are CRH-independent. Role of CRH during immune stress Hypothalamic CRH acts as a potent anti-in¯ammatory factor via the stimulation of adrenal glucocorticoid secretion (Munck and Guyre, 1986). Recent studies have proposed that CRH, expressed outside of the central nervous system in immune and in¯amed tissues (Stephanou et al., 1990; Karalis et al., 1991; Aird et al., 1993; Karalis et al., 1997), serves as a potent proin¯ammatory factor (Karalis et al., 1991; Crofford et al., 1995). The relative pro- and antiin¯ammatory actions of CRH were evaluated using two different in¯ammatory models: the subcutaneous (s.c.) granuloma formation by administration of carrageenin (Karalis et al., 1999) and the intramuscular (i.m.) abscess formation by injection of turpentine (Venihaki et al., 2001). Following administration of carrageenin, Crh / mice have a signi®cantly greater cellular in®ltration than that of Crh / mice, likely due to the lower circulating levels of glucocorticoid in Crh / mice (Karalis et al., 1999). However, when glucocorticoid levels in Crh / mice are clamped to mimic those of Crh / mice, the in¯ammatory response of Crh / mice is much less than that in Crh / mice (Karalis et al., 1999), supporting the previously described proin¯ammatory role for CRH (Karalis et al., 1991; Crofford et al., 1993; Chrousos, 1995). Intramuscular injection of turpentine oil, which creates a localized abscess, causes a similar rise in circulating corticosterone levels in both normal and Crh / genotypes (Venihaki et al., 2001). This suggests that the adrenal corticosterone response following turpentine injection is independent of the presence of CRH, in contrast to the need for CRH for adrenal responsiveness to other types of stressors. The signi®cant rise in corticosterone levels in ß 2002 Elsevier Science Ltd. All rights reserved.
Crh / mice following turpentine- or LPS-induced in¯ammation (Bethin et al., 2000; Venihaki et al., 2001) may be explained by the 2±3 fold higher plasma concentrations of IL-6 (a potent stimulator of adrenal glucocorticoid secretion) measured in Crh / mice following in¯ammation (Bethin et al., 2000; Venihaki et al., 2001). Finally, despite this similar level of glucocorticoid response in the two genotypes, a signi®cantly lower local in¯ammatory response was observed in Crh / mice compared to that in Crh / mice, consistent with a pro-in¯ammatory role for CRH (Venihaki et al., 2001). BEHAVIORAL RESPONSES OF CRH ^/^ MICE The wide distribution of CRH in brain regions has led to the hypothesis that it may play a signi®cant role in behavioral responses to stress. Several studies have been conducted to investigate its potential role in these behaviors. Indeed CRH, when administered icv, produces a signi®cant increase in locomotor activity, rearing, and grooming in rat (Britton et al., 1982; Sutton et al., 1982) while administration of peptide and nonpeptide antagonists such as alpha helical CRH and antalarmin cause reduced anxiety in rodents, as measured using several behavioral paradigms (Berridge and Dunn, 1987; Kalin et al., 1988; Deak et al., 1999). In support to the above pharmacological studies, transgenic animals with overexpression of CRH show increased anxiety as a result of increased expression of CRH in several brain regions (Stenzel-Poore et al., 1994). Surprisingly, Crh / mice exhibit normal behavior under basal conditions and following 30 min of restraint, as assessed using the multicompartment chamber and the elevated plus maze stress paradigms (Weninger et al., 1999). Furthermore, both peptide (alpha helical CRH) and non peptide (CP-154, 526) CRH receptor 1 (CRHR1) antagonists effectively block the normal stress-induced behavior of both Crh / and Crh / mice in the foot shock test (Weninger et al., 1999), providing evidence that this receptor is involved in anxiety-related behaviors. Indeed, mice with speci®c deletion of the CRHR1 receptor show decreased anxiogenic behavior in the elevated plus maze test and the light-dark emergence task (Smith et al., 1998). The pharmacological studies in the Crh / mice together with the studies using the Crhr1 / mice as well as mice carrying speci®c deletion of the CRH receptor 2 (Bale et al., 2000; Kishimoto et al., 2000), which show increased anxiety, suggest the possibility that another CRH-like molecule mediates stress-induced behaviors. Many studies have proposed that CRH is a potent anorexiogenic factor (Dunn and Berridge, 1990; Koob and Heinrichs, 1999). Indeed, intracerebroventricular injection of CRH results in reduced food intake in rodents, Neuropeptides (2002) 36(2^3), 96^102
100 Venihaki and Majzoub
while administration of CRH antagonists attenuates hypophagic responses to many stressors. However, Crh / mice do not differ from Crh / in food intake following three chronic stressors (daily restraint, turpentine abscess, and surgical stress) (Weninger et al., 1999; Venihaki et al., 2001) or administration of mouse interleukin-1beta, lipopolysaccharide, or the serotonergic agonist d-fen¯uramine (Swiergiel and Dunn, 1999), suggesting that CRH is not necessary for stress-induced decreases in food intake. CRH RELATED PEPTIDES Three CRH related peptides have been recently identi®ed: Urocortin, Urocortin II (UCNII) and Urocortin III (UCNIII) (Vaughan et al., 1995; Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001). Urocortin was identi®ed based on its similarity with the ®sh analog urotensin (Vaughan et al., 1995). Urocortin binds both CRHR1 and CRHR2 with higher af®nity than does CRH. The normal stress-induced behavior of Crh / mice led to the hypothesis that urocortin may mediate anxiety-related behaviors. Although mRNA expression of urocortin is 2±3 fold upregulated in the Edinger-Westphal (EW) nucleus of the Crh / mice (Weninger et al., 2000), its restricted expression in this nucleus, a brain region not known to project to any other brain regions which may play a role in anxiety like behavior, suggests that urocortin does not mediate these behaviors (Weninger et al., 2000). The Edinger-Wesphal nucleus may play a role in the autonomic nervous system and has projections to various nuclei that express CRH receptors (Bittencourt et al., 1999). Since urocortin RNA expression in the EW nucleus is increased following restraint it is likely that urocortin may play a role in the regulation of the autonomic nervous system during stress (Weninger et al., 2000). Urocortin II (UCNII) was recently identi®ed based on its similarity with members of the CRH peptide family (Hsu and Hsueh, 2001; Reyes et al., 2001). UCNII binds to and activates speci®cally the CRHR2 (Reyes et al., 2001). It is expressed in several brain regions and a plethora of peripheral tissues. Although initial studies in rats showed that icv administration of UCNII attenuates food intake in rats, however, no other effect was found on locomotor activity or anxiety-related paradigms (Reyes et al., 2001). Therefore, it is unlikely that UCNII is involved in stress-induced behaviors. Urocortin III (UCNIII) was identi®ed recently based on its similarity with a CRH-like peptide identi®ed in the Takifugu rubripes (Hsu and Hsueh, 2001; Lewis et al., 2001). We had also identi®ed UCNIII (Venihaki, unpublished data) by searching a commercial mouse database (Celera). UCNIII, like UCNII, binds speci®cally to and activates CRHR2 (Hsu and Hsueh, 2001; Lewis et al., 2001). It is expressed in several brain regions including Neuropeptides (2002) 36(2^3), 96^102
the hypothalamus, amygdala, and the bed nucleus stria terminalis, regions known to be involved in stress-induced behaviors (Lewis et al., 2001). The mRNA expression of UCNIII in Crh / mice is similar to that found in Crh / mice (Venihaki, unpublished data). The role of UCN III in stress-induced behaviors is under investigation. CONCLUSIONS-FUTURE DIRECTIONS The studies performed in the Crh / mice have been useful in understanding the role of CRH and glucocorticoid in normal physiology, as well as in endocrine and behavioral stress responses. CRH is not required for basal ACTH secretion, although it is necessary for adrenal development and function. It is a major mediator of HPA axis activation following exposure to stressors, but it is not required for the activation of the HPA axis by immune stimuli. There are several issues however, that remain to be resolved, including the characterization of the CRHrelated molecules that mediate stress-induced behaviors, and the interactions of CRH, if any, with these molecules. The inactivation of the other CRH-related peptides and the development of better models for behavioral analysis will be useful tools to further illuminate the ways in which these peptides affect normal and pathologic function in mammals. ACKNOWLEDGEMENTS The authors thank members of Dr. Majzoub's and Dr. Karalis' laboratories and Dr. Muglia of Washigton University, St. Louis for helpful discussions during the course of these studies. REFERENCES Affolter HU, Reisine T (1985) Corticotropin releasing factor increases proopiomelanocortin messenger RNA in mouse anterior pituitary tumor cells. J Biol Chem 260: 15477±15481. Aguilera G, Harwood JP, Wilson JX, Morell J, Brown JH, Catt KJ (1983) Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells. J Biol Chem 258: 8039±8045. Aird F, Clevenger CV, Prystowsky MB, Redei E (1993) Corticotropinreleasing factor mRNA in rat thymus and spleen. Proc Natl Acad Sci USA 90: 7104±7108. Aoki Y, Iwasaki Y, Katahira M, Oiso Y, Saito H (1997) Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I: Effects of the common secretagogues. Endocrinology 138: 1923±1929. Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, Vale WW, Lee KF (2000) Mice deficient for corticotropinreleasing hormone receptor-2 display anxiety-like behaviour and are hypersensitive to stress. Nat Genet 24(4): 410±414. Berridge CW, Dunn AJ (1987) A corticotropin-releasing factor antagonist reverses the stress-induced changes of exploratory behavior in mice. Horm Behav 21: 393±401.
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