[CONFAMI study: Effects of home confinement during the COVID-19 epidemic on the lives of children and their families]. / Étude CONFAMI : effets du confinement durant l'épidémie de la COVID-19 sur la vie des enfants et leur famille.

OBJECTIVES: The aim of this study is to understand the changes within families during confinement motivated by the COVID-19 pandemic and to explore the psycho-emotional experiences of children and their parents in this new situation. Confinement necessarily induced significant changes in daily family routines, particularly for work, education, leisure and social activities. In the more vulnerable pediatric population, several authors have warned of the need to consider the impact of lockdown measures during COVID-19 on the psychological impact and well-being. METHOD: This is an anonymous online survey with methodology combining quantitative and qualitative analyses. The questions targeted several themes such as life context, emotional experience and the impact on daily habits in children and adolescents, as perceived by parents. Participants are adults and parents of at least one child. They were recruited through social media and email. RESULTS: A total of 439 parents responded to the questionnaire. The families generally stayed in their usual place of residence and managed to adapt well. On average, the children's level of worry (as estimated by parents) was lower than the level of worry parents attributed to themselves. For the majority, the parents did not observe any change, the psychological state of the children and adolescents was generally stable, but for those who experienced more negative emotions than usual, it was an increase in boredom, irritability and anger. A decrease in the quality of sleep was also observed by a third of the respondents. On the other hand, an increase in autonomy was noted. Regarding the quality of family cohabitation, an important result showed that confinement had improved family relationships for 41% parents but at the expense of usual social ties inducing a feeling of deprivation. Indeed, the participants evoke a lack of "social link" and "social contact with friends". Lack became synonymous with absence, a feeling of loneliness and separation. CONCLUSION: Our results confirm European and international data collected in children in countries where strict lockdown measures have been applied. Despite the negative emotions felt in some children, confinement has helped develop new resources in most families. Families seem to have been successful in maintaining a stable and secure routine which has certainly been a protective factor against anxiety. Some reported factors, such as bonding, could be protective factors and constitute good leads in interventions to be offered to children and their families.

Thiazide diuretics affect osteocalcin production in human osteoblasts at the transcription level without affecting vitamin D3 receptors.

Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1-100 microM) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced OC release by these cells (maximal effect, -40-50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-47.2+/-4.0%; p < 0.0001 by paired ANOVA with 100 microM 13.6+/-0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5+/-6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1-100 microM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate.

Dual role for nitric oxide in the regulation of plasma volume and albumin escape during endotoxin shock in conscious rats.

To assess the role of nitric oxide (NO) produced by the constitutive (cNOS) and inducible NO synthase (iNOS) in the regulation of vascular functions, we compared the effects of aminoguanidine, a relatively selective inhibitor of iNOS, and NG-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor on blood pressure, plasma volume, and albumin escape during the early and delayed phases of endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium-51-tagged erythrocytes and iodine-125-labeled albumin, respectively. Injection of lipopolysaccharide (LPS) 10 mg/kg i.v. resulted in a fall in blood pressure, hemoconcentration, and increased total-body albumin escape, which is reflected by a 25% reduction in plasma volume. When LPS was injected into animals pretreated with L-NAME (7.4 mumol/kg i.v. 15 minutes before LPS), losses in plasma volume and albumin escape were significantly greater than in rats that received LPS alone, despite that L-NAME attenuated the hypotensive action of LPS. Aminoguanidine pretreatment (162 mumol/kg) had no effect on the early responses to LPS, whereas it was as potent as L-NAME in reversing hypotension when injected 70 minutes after LPS. Aminoguanidine treatment also prevented further losses in plasma volume and markedly attenuated total-body and organ albumin escape rates elicited by LPS. L-NAME produced only a slight attenuation of LPS-induced losses in plasma volume and albumin escape in most organs studied, whereas it potentiated albumin extravasation in the lung. These results demonstrate that inhibition of cNOS potentiates, whereas inhibition of iNOS markedly attenuates, losses in plasma volume and albumin escape elicited by LPS, and suggest that selective inhibitors of iNOS may be more effective than nonselective inhibitors of all forms of NOS in the therapy of septic shock.

Glucocorticoid receptor regulates expression of L-selectin and CD11/CD18 on human neutrophils.

BACKGROUND: Recent studies have raised the hypothesis that glucocorticoids could diminish the ability of endothelial cells to direct leukocyte traffic into inflamed tissues by inhibiting expression of the adhesion molecules endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. The aim of the present study was to investigate whether glucocorticoids also regulate the expression of L-selectin and CD11/CD18 integrins on human neutrophil granulocytes. METHODS AND RESULTS: Incubation of human whole blood with platelet-activating factor (PAF, 1 mumol/L) evoked downregulation of L-selectin and upregulation of CD11/CD18 adhesion receptors on neutrophils as measured by flow cytometry. While dexamethasone (0.1 nmol/L to 100 mumol/L) did not affect expression of adhesion molecules on resting neutrophils, it attenuated the PAF-induced changes in L-selectin and CD18 expression in a time- and concentration-dependent fashion with IC50 values of 31 and 13 nmol/L, respectively. These effects of dexamethasone were completely aborted by RU-486 (10 mumol/L), which blocks transcriptional activation of the glucocorticoid receptor, and by the protein synthesis inhibitor cycloheximide (35.5 mumol/L). Dexamethasone, up to a concentration of 1 mumol/L, neither affected significantly the release of granule enzymes nor interfered with PAF binding to its membrane receptors. CONCLUSIONS: Our results show that glucocorticoids at clinically relevant concentrations exert specific actions on expression of adhesion molecules on activated neutrophils, which are mediated through ligation of glucocorticoid receptors and induction of protein synthesis, and suggest a novel mechanism by which anti-inflammatory corticosteroids may inhibit leukocyte accumulation.

Dopaminergic receptors and angiotensinogen gene expression in opossum kidney cells.

To investigate whether expression of the renal angiotensinogen gene is regulated by dopaminergic receptors, we used opossum kidney (OK 27) cells with a fusion gene containing the 5'- flanking regulatory sequence of the rat angiotensinogen gene fused with a human growth hormone (hGH) gene as a reporter [pOGH, angiotensinogen nucleotide (N) -1498/+18], permanently integrated into their genomes. The level of expression of pOGH (angiotensinogen N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive hGH (ir-hGH) secreted into the culture medium. In the absence of 3-isobutyl-1-methylxanthine (IBMX), addition of dopamine (10(-13) to 10(-5)M) had minimal effect on the expression of the pOGH (angiotensinogen N-1498/+18) in OK 27 cells. In the presence of IBMX, addition of low concentrations (10(-13) and 10(-7) M) of dopamine stimulated the expression of pOGH angiotensinogen N-1498/+18) in OK 27 cells in a dose-dependent manner, whereas high concentrations (i.e., > 10(-7) M) had minimal effect. The stimulatory effect of dopamine on the expression of pOGH (angiotensinogen N-1498/+18) was inhibited by the presence of SCH-23390 (D1-dopaminergic receptor antagonist) and spiperone (D2-dopaminergic receptor antagonist), but not by ketanserin (5 HT2/5HT1c-serotonergic receptor antagonist). Moreover, the stimulatory effect of dopamine was inhibited by the presence of U-73122 (an inhibitor of phospholipase C and phospholipase A2) or staurosporine (an inhibitor of protein kinase C) or (R)-p-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP[S]; an inhibitor of cAMP-dependent protein kinase AI and II). Addition of low concentrations (10(-13) to 10(-9)M) of SKF-82958 (D1-dopaminergic receptor agonist) or PPHT (D2-dopaminergic receptor agonist) also stimulated the expression of pOGH (angiotensinogen N-1498/+18). The stimulatory effect of SKF-82958 was inhibited by the presence of SCH-23390 or Rp-cAMP[S], whereas the effect of PPHT was inhibited by the presence of spiperone or staurosporine. These studies demonstrate that the expression of pOGH (angiotensinogen N-1498/+18) in OK 27 cells is modulated by dopaminergic receptor agonists.

Alpha-adrenoceptors and angiotensinogen gene expression in opossum kidney cells.

To investigate whether alpha (alpha)-adrenoceptor agonists have a stimulatory effect on the expression of the angiotensinogen (Ang) gene in opossum kidney (OK) cells, we used OK 27 cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat angiotensinogen gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (Ang N-1498/+18), permanently integrated into their genomes. The level of expression of the pOGH (Ang N-1498/+18) was quantitated by the amount of immunoreactive-human growth hormone (IR-hGH) secreted into the medium. The addition of iodoclonidine (alpha 2-adrenoceptor agonist, 10(-13) to 10(-9) M) and phorbol 12-myristate 13-acetate (PMA, 10(-13) to 10(-5) M) stimulated the expression of pOGH (Ang N-1498/+18) in a dose-dependent manner, whereas the addition of phenylephrine (alpha 1-adrenoceptor agonist, 10(-13) to 10(-5) M) had no effect. The stimulatory effect of iodoclonidine was blocked by the presence of yohimbine (alpha 2-adrenoceptor antagonist) and staurosporine (an inhibitor of protein kinase C) but not blocked by the presence of prazosin (alpha 1-adrenoceptor antagonist) or Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A). The addition of iodoclonidine, phenylephrine or PMA had no effect on the expression of pTKGH in OK 13 cells, an OK cell line, into which had been stably integrated a fusion gene, pTKGH containing the promoter/enhancer DNA sequence of the viral thymidine-kinase (TK) gene fused with a human growth hormone gene as a reporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenergic receptors and angiotensinogen gene expression in mouse hepatoma cells in vitro.

We have previously reported that addition of 8-bromocyclic AMP enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells in vitro. Isoproterenol is known to stimulate the synthesis of hepatic intracellular cyclic AMP via beta-adrenergic receptors. To study the possible effect of beta-adrenergic receptors on the expression of the angiotensinogen gene in mouse hepatoma cells, we transiently transfected them with a fusion gene with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase coding sequence as a reporter, pOCAT (ANG N-1498/+18). The addition of isoproterenol (10(-9) to 10(-5) mol/L) alone had no stimulatory effect on the expression of pOCAT (ANG N-1498/+18). In the presence of dexamethasone (10(-6) mol/L), however, isoproterenol enhanced the stimulatory effect on the dexamethasone on the expression of pOCAT (ANG N-1498/+18). The enhancing effect of isoproterenol was inhibited by the presence of propranolol (beta 1- and beta 2-adrenergic receptor antagonist) and ICI 118,551 (beta 2-adrenergic receptor antagonist) but not by the presence of atenolol (beta 1-adrenergic receptor antagonist). Furthermore, the addition of Rp-cAMP (an inhibitor of protein kinase A I and II) blocked the enhancing effect of isoproterenol. These studies demonstrated that isoproterenol enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells via beta 2-adrenergic receptor and cyclic AMP-dependent protein kinase pathways. Our data may be important in understanding the molecular mechanism(s) of the stimulatory effect of catecholamines/glucocorticoid-induced expression of the angiotensinogen gene in the liver.

Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells.

We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10(-3)-10(-7) M) or forskolin (10(-9)-10(-5) M) stimulated the expression of the plasmid pOCAT [angiotensinogen nucleotide (N) -1498/+18] fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10(-6) M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate [(Rp)-cAMP[S], an inhibitor of cAMP-dependent protein kinase A, I and II] blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10(-3) M) or Dex (10(-6) M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N -35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells.

To investigate whether the expression of the renal angiotensinogen (ANG) gene is regulated by beta-adrenoceptors and the cAMP-dependent protein kinase A pathway, we introduced stably the fusion gene containing the 5'-flanking regulatory sequence of the ANG gene with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), into opossum kidney (OK) cells. We successfully obtained several stable transformants with a high expression of the pOGH (ANG N-1498/+18) fusion gene. One stable transformant (OK 27) that is able to maintain the expression of pOGH (ANG N-1498/+18) in culture for more than a year was used in the present study. The level of expression of the pOGH (ANG N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive-hGH (IR-hGH) secreted into the culture medium. The addition of isoproterenol (10(-11) M to 10(-9) M) stimulated the expression of pOGH (ANG N-1498/+18) and increased the accumulation of intracellular cAMP. Higher concentrations of isoproterenol (that is, greater than 10(-9) M) had low or minimal effect. In contrast, the addition of 8-bromo-cAMP (8-Br-cAMP) and forskolin stimulated the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of isoproterenol was blocked by the presence of propranolol, atenolol and ICI 118,551. The addition of ICI 118,551, however, was less effective than atenolol. Furthermore, the stimulatory effect of isoproterenol and 8-Br-cAMP on the expression of the pOGH (ANG N-1498/+18) was inhibited by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II).(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of expression of the angiotensinogen gene in cultured mouse hepatoma cells.

To investigate the hormonal regulation of expression of the angiotensinogen (ANG) gene in the liver, we constructed fusion genes with various lengths of the 5'-flanking region of the rat ANG gene linked to a bacterial chloramphenicol acetyl transferase (CAT) gene as reporter and introduced them into mouse hepatoma cells (Hepa 1-6). As a negative control, we introduced them into a nonhepatic cell line, a mouse testicular Sertoli (TM4) cell line. The level of expression of ANG-CAT fusion genes, pOCAT (ANG N-1498/+18), pOCAT (ANG N-688/+18), pOCAT (ANG N-110/+18), pOCAT (ANG N-53/+18) and (ANG-35/+18) were 3.7, 4, 1.1, 4, and 3-fold higher than promoterless pOCAT in Hepa 1-6 cells. No significant expression of any of these ANG-CAT fusion genes over the promoterless pOCAT was observed in Sertoli TM4 cells. The addition of dexamethasone (10(-10) to 10(-4) mol/L) stimulated the expression of the pOCAT (ANG N-1498/+18) fusion gene in Hepa 1-6 cells in a dose-dependent manner with a maximum stimulation at 10(-4) mol/L and a half-maximal stimulation at 10(-8) mol/L. A combination of dexamethasone (10(-6) mol/L) and 8-bromo-cyclic AMP (cAMP) (10(-3) mol/L) further enhanced the effect of the dexamethasone alone although cAMP alone had no effect. Testosterone (10(-6) mol/L), estradiol (10(-6) mol/L), progesterone (10(-6) mol/L), and thyroid hormone (L-T3, 10(-6) mol/L) did not have this effect in either the presence or absence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)