[Comparison of palliative care representations between pediatrician residents and oncologist residents: A qualitative study]. / Comparaison des représentations des soins palliatifs des internes de pédiatrie à celles des internes d'onco-hématologie - étude qualitative.

BACKGROUND: Pediatrics residents treat patients who are particularly vulnerable and they care for many patients in palliative situations. The purpose of this study was to build a typology detailing the representations of pediatrics and oncology residents on palliative care and how these transfer to their practice, and to determine their knowledge of euthanasia and end-of-life legislation. METHODS: To draw up this typology, we used a semidirective interview method. The topics treated were their definition of palliative care, end of life, the emotions involved in these situations, and their daily practice. Then we asked them to speak about their opinions and knowledge of euthanasia and end-of-life legislation. RESULTS: Thirteen residents were interviewed: eight pediatrics residents, two oncologists, and three hemato-oncologists. Interviews lasted around 45min. Pediatrics and oncology residents had common representations based on "care giving." Nevertheless, pediatrics residents remained within the technical aspects to protect themselves from their negative emotions and stayed away from their patients. Oncology residents set their emotions aside to be able to carry on taking care of their patients. CONCLUSION: It seems necessary to disseminate a palliative culture, particularly in pediatrics, to improve management of children in palliative situations and to improve resident's feelings.

Effects of 17beta-estradiol on preadipocyte proliferation in human adipose tissue: Involvement of IGF1-R signaling.

Estrogens are known to stimulate the proliferation of human preadipocytes. However, the molecular mechanisms underlying the increased cell growth by these steroids are poorly understood. In the present study, we have demonstrated that the proliferative effect of 17beta-estradiol involves the induction of both cell cycle gene expressions, c-myc and cyclin D1. Moreover, the mitogenic effects of 17beta-estradiol are suppressed by the pure antagonist ICI 182780 suggesting that estradiol action is mediated by estrogen receptor (ER). We have also shown that 17beta-estradiol is able to inhibit human preadipocyte apoptosis capacity as reflected by DNA fragmentation experiments and the mRNA expression of the pro- and antiapoptotic genes. Finally, 17beta-estradiol significantly induces both mRNA and protein expression of RIGF1 in human preadipose cells via ER and thus reinforces the signaling pathway of the proliferative factor, IGF1. Taken together, these data reinforce the concept of cross-talk between IGF1- and ER-signaling pathways in preadipocytes and indicate that IGFI may be a critical regulator of estrogen-mediated preadipose growth.

Evidence for functional estrogen receptors alpha and beta in human adipose cells: regional specificities and regulation by estrogens.

Adipocytes are estrogen-responsive cells, but the quantitative expression and transcriptional regulation of the estrogen receptors (ER-alpha and ER-beta) in human adipocytes and their precursor cells are unclear. Using real-time quantitative PCR, we have demonstrated that both ER-alpha and ER-beta mRNA are expressed in human mature adipocytes with a large predominance of ER-alpha mRNA. Moreover, ER-alpha mRNA is identically expressed whatever the anatomic origin (intraabdominal and subcutaneous) of the adipocytes and the gender. ER-beta mRNA levels are higher in women compared with men, without regional differences. 17beta-Estradiol in vitro upregulates expression of both ER-alpha and ER-beta mRNA in subcutaneous adipocytes from women but only the ER-alpha mRNA in subcutaneous and intra-abdominal adipocytes from men. In preadipocytes, only the ER-alpha subtype was present. In the latter cells, estrogens in vitro had no influence on ER-alpha expression (mRNA and protein). The present study also shows that estrogens in vitro increase the AP-1, SP-1, and estrogen response element DNA binding activities in differentiated but not in confluent preadipocytes, suggesting that ER become functional during the course of adipogenesis. On the whole, these data are consistent with a predominant role of the ER-alpha subtype in mediating the effects of estrogens on human adipose tissue development and metabolism.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways.

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.

Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptor gamma2.

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17beta-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (x2) of 17beta-estradiol (0.01 microM) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 microM) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17beta-estradiol (0.01 microM), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17beta-estradiol significantly increased (x1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor gamma2 was up-regulated by 17beta-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 beta-estradiol (0.01 microM) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 microM) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor gamma2 expression (estrogens).

In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones.

As a sexual dimorphism appears in plasma leptin levels, the aim of the present study was to investigate, in vivo and in vitro, the influence of sex steroid hormones on ob messenger RNA (mRNA) and leptin expressions in rat fat cells from various anatomical localizations. In male rats, castration resulted in a modulation of ob gene mRNA expression which was increased by 2-fold in perirenal and half-reduced in sc adipocytes. Moreover, in isolated fat cells from both perirenal and s.c. fat depots, ob gene mRNA expression was reduced by 20% after a 24-h in vitro exposure to dihydrotestosterone (10(-8) M). This effect of dihydrotestosterone on ob mRNA was prevented by exposure to the antiandrogen cyproterone acetate and also by actinomycin D. In contrast, leptin secretion from both perirenal and sc adipocytes was unchanged after 24 h exposure to dihydrotestosterone. In female rats, ovariectomy induced a 25% decrease in ob gene mRNA expression in perirenal fat cells. In vitro studies revealed that a 24-h exposure to 17-beta estradiol (10(-8) M) induced a 1.4-, 1.2-, and 1.75-fold increase in ob mRNA expression and a 3.8-, 1.65- and 2-fold increase in leptin secretion in sc, perirenal and parametrial adipocytes, respectively. Moreover, these effects were prevented by the antiestrogen ICI182780 and also by actinomycin D. Altogether, these results demonstrate that in rat adipocytes, estrogens, and androgens modulate ob gene expression at the mRNA level through sex steroid receptor-dependent transcriptional mechanisms.

Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids.

Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.

Enhancement of the expression of the alpha 2-adrenoreceptor protein and mRNA by a direct effect of androgens in white adipocytes.

In vivo, testosterone-treatment of female hamsters for 4 days promotes a doubling of alpha 2-adrenoreceptor protein in parametrial adipocytes, with a concomitant accumulation of the alpha 2A-adrenoreceptor subtype mRNA. During in vitro incubation of minced parametrial fat pads for 6 to 48h with testosterone or dihydrotestosterone (100 nM), alpha 2A-adrenoreceptor protein and mRNA levels were also increased and remained to control levels when an antiandrogen or actinomycin D were added in the medium. It is concluded that in hamster adipocytes, androgens upregulate alpha 2A-adrenoreceptor subtype expression at the mRNA level by an androgen receptor-dependent transcriptional activation.

Androgen receptors in cultured rat adipose precursor cells during proliferation and differentiation: regional specificities and regulation by testosterone.

Different studies suggest that sex hormones affect adipose tissue metabolism and deposition. To investigate the possibility that androgens may play a role in adipose tissue development, we have studied androgen receptors (AR) in rat adipose precursor cells from two different anatomical fat deposits, one deep intraabdominal (epididymal) and one subcutaneous (inguinal) during the proliferation and differentiation processes. AR were quantified by [(3)H]R1881 specific binding in whole cells and the nuclear fraction and were localized by immunocytofluorimetry in both the cytosol and the nucleus. During the proliferative phase, total AR level decreased from D3 to D6. At confluence (D5), AR were higher in epididymal (64±4 fmol/mg protein) than in subcutaneous (33±3 fmoles/mg proteins) preadipocytes and were up-regulated by testosterone but not by 5α-dihydrotestosterone or by 17ß-estradiol. At differentiation (D10-11), nuclear AR decreased by 50% in both precursor fat cell populations when compared to the confluent state (D5) and AR were no more up-regulated but rather down-regulated by testosterone. Because AR are present in preadipocytes and are differently regulated by testosterone depending on the stage of proliferation and differentiation, this study suggests that testosterone may play a role in the control of the adipogenic process.

Water-in-oil emollients as steroid-sparing adjunctive therapy in the treatment of psoriasis.

An open label study of ninety-six patients with chronic plaque-type psoriasis demonstrated the efficacy of the addition of water-in-oil emollients to a topical corticosteroid regimen. Twice daily application of betamethasone dipropionate cream and once daily application of both betamethasone dipropionate cream and either a water-in-oil based moisturizing cream or lotion were equivalent in efficacy (p = 0.05). Once daily application of both betamethasone dipropionate cream and either a water-in-oil based cream or lotion was significantly better than once daily application of betamethasone dipropionate cream alone (p = 0.05). Water-in-oil emollients are useful in the therapy of chronic, plaque-type psoriasis and provide a steroid-sparing effect.

Evidence that testosterone modulates in vivo the adenylate cyclase activity in fat cells.

In male hamster fat cell membranes, the alpha 2-adrenoreceptor-mediated inhibitory response of adenylate cyclase was almost completely suppressed by castration and was restored to control values after testosterone treatment, whereas the cyclase inhibitory response to nicotinic acid was insensitive to androgenic status. Basal and forskolin-, guanylylimidodiphosphate- and isoproterenol-stimulated cyclase activities were decreased by 30-40% after castration and restored to control values after testosterone treatment. In addition, Mn2+ + forskolin-stimulated activity in the presence or absence of GDP beta S was lower (-30%) after castration and normalized after testosterone treatment. Finally, the effects of testosterone described above were completely abolished when the potent androgen receptor antagonist RU 23908 was administered together with testosterone. These results indicate that both the inhibitory and stimulatory responses of adenylate cyclase are promoted by testosterone through an androgen receptor-dependent mechanism; promotion of the inhibitory response concerns specifically the alpha 2-receptor-mediated pathway, whereas promotion of the stimulatory response appears unspecific and mainly due to increased activity of the cyclase catalytic subunit.

Influence of androgenic status on the alpha 2/beta-adrenergic control of lipolysis in white fat cells: predominant alpha 2-antilipolytic response in testosterone-treated-castrated hamsters.

The aim of this study was to evaluate the influence of castration with or without testosterone propionate (TP) administration (one daily injection of 1 mg for 10 days) on the fat cell lipolytic activity in male hamsters. Basal and maximal lipolytic responses to the pure beta-adrenergic agonist isoproterenol, the mixed alpha 2-and beta-adrenergic agonist epinephrine, and the nonadrenergic compounds ACTH and 3-isobutyl-1-methylxanthine were all reduced by half in castrated animals. TP treatment restored these defective responses to control values, except the response to epinephrine which remained paradoxically unchanged. Sensitivity of lipolysis to epinephrine was unimpaired by castration but markedly reduced (10-fold) in TP-treated castrated hamsters. The antilipolytic potencies of the alpha 2-component of epinephrine and of the two alpha 2-agonists, UK 14304 and clonidine, were reduced by half in castrated animals, and returned to a value slightly higher than control after TP treatment. These changes in lipolysis were accompanied by parallel alterations in the stimulated cAMP responses to isoproterenol and forskolin but not to epinephrine. The latter was either unimpaired by castration or was clearly inhibited after TP treatment. Castration also induced a 2-fold decrease in the inhibitory potency of clonidine toward forskolin-stimulated cAMP production. Finally, these changes in the potency of clonidine were accompanied by parallel variations of the number of fat cell alpha 2-adrenoreceptors. These results indicate that testosterone in vivo, while increasing the beta-adrenergic lipolytic action of catecholamines (possibly through enhancement of the adenylate cyclase activity), promotes, to a greater extent, their alpha 2-adrenoreceptor-mediated antilipolytic potency. By providing the first demonstration that the androgenic status controls the functional alpha 2/beta-adrenergic balance in fat cells, this study also emphasizes the potential importance of such a control in the mechanisms underlying the sex-related differences in adipose tissue regional distribution and fat cell size.

Evidence that the lipolytic defect induced by estradiol-treatment in hamster adipocytes is related to an estrogen receptor-mediated defect in the adenylate cyclase catalytic subunit but not in Ns.

This study demonstrates that estradiol-treatment (10 micrograms per day x 5 days) does not impair the level of Ns, the adenylate cyclase stimulatory regulatory protein, in hamster fat cell membranes. In addition, this report shows that the defective cyclic AMP response induced in intact adipocytes by the estradiol-treatment is either unaltered by the administration of alpha-bromocriptine or abolished by tamoxifen- or 4-hydroxytamoxifen-treatment. It can thus be concluded that the reduced lipolytic response found in hamster fat cells after estradiol-treatment is related only to an estradiol-receptor-mediated defect in adenylate cyclase catalytic subunit activity which is independent from increased prolactin secretion.

Estradiol treatment decreases the lipolytic responses of hamster white adipocytes through a reduction in the activity of the adenylate cyclase catalytic subunit.

After 5 days of daily administration of 10 micrograms estradiol to 6-week-old male hamsters, the in vitro maximal lipolytic and cAMP responses of white adipocytes to isoproterenol, epinephrine, ACTH, and theophylline were reduced by one half, with no change in the sensitivity of these responses. In contrast, the antilipolytic response to the alpha 2-adrenergic agonist clonidine was unimpaired. beta-Adrenergic receptor number and affinity, assessed in intact cells with [3H]CGP-12177 binding, showed no difference between control and estradiol-treated hamsters. In adipocyte membranes from estradiol-treated hamsters, maximal adenylate cyclase responses to Mn2+, GTP alone or in combination with isoproterenol, ACTH, or fluoride were all decreased by 30-40% below the values found in controls, but the sensitivity of these responses was unaltered. The maximum velocity (Vmax) of adenylate cyclase was reduced by one half in estrogen-treated animals, but the Michaelis-Menten constant (Km) of the enzyme for ATP was unchanged. Finally, complementation of adipocyte membranes with solubilized human erythrocyte Ns failed to restore to control values the maximal adenylate cyclase response to isoproterenol plus guanosine 5'-[beta, gamma-imido]-triphosphate in the estradiol-treated hamsters. These results indicate that a defect in the catalytic subunit of adenylate cyclase is one of the mechanisms through which estradiol treatment reduces the lipolytic response of hamster white adipocytes.

In vivo desensitization of the beta, but not the alpha 2-adrenoreceptor-coupled-adenylate cyclase system in hamster white adipocytes after administration of epinephrine.

To assess the physiological relevance of the changes in adrenergic receptor observed in adipocyte membranes after in vitro exposure to catecholamines, hamster white adipocytes, which possess both beta- and alpha-adrenergic receptors, were studied after a 6-day in vivo epinephrine administration (1 mg/kg, daily). This treatment resulted in a 3-fold increase in total plasma catecholamine levels and in the following changes in the adipocytes. The lipolytic and cAMP responses to beta-agonists, ACTH, and theophylline were decreased by 55-60%, but the sensitivity of these responses to (-)isoproterenol was unchanged. Although basal adenylate cyclase activity was unaffected, (-)isoproterenol-, ACTH- or fluoride-stimulated activities were reduced by half, a defect which persisted in the presence of guanosine 5'-[beta, gamma-imido]triphosphate. Furthermore, the number of beta-adrenergic receptors was also decreased by 54%. In contrast, epinephrine treatment failed to impair the adipocyte antilipolytic response to the alpha 2-agonist clonidine, the alpha 2-component of epinephrine, and the adenosine analog N6-phenylisopropyladenosine, the adenylate cyclase inhibitory response to these compounds, and the number of alpha 2-adrenergic receptors. These results indicate that in vivo epinephrine administration does not alter the alpha 2-adrenergic system of hamster white adipocyte, but desensitizes the lipolytic response to beta-agonists and also to nonadrenergic lipolytic agents. It is therefore suggested that the likely mechanism(s) responsible for this lipolysis desensitization mainly consists in impaired adenylate cyclase coupling and possibly in altered intracellular processes, rather than in the observed beta-adrenergic receptor down-regulation.

Characterization of the beta-adrenergic receptors of hamster white fat cells. Evidence against an important role for the alpha 2-receptor subtype in the adrenergic control of lipolysis.

The binding characteristics of the beta-adrenergic antagonist, [3H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity (Kd = 1.6 +/- 1.3 nM) but low capacity (32 +/- 17 fmol/mg membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both beta-antagonists and beta-agonists, competition at the low-affinity sites occurred only with beta-antagonists and was non-stereoselective. Thus, the beta-agonist (-)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity (Kd = 1.6 +/- 0.5 nM) binding sites with a binding capacity of 53 +/- 13 fmol/mg membrane protein (corresponding to 4000 +/- 980 sites per cell), and independent kinetic analysis provided a Kd value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a beta 1-receptor subtype, yielding Kd values in good agreement with the Kact and the Ki values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of beta-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [3H]dihydroalprenolol binding sites defined as the binding displaceable by (-)-isoproterenol represent the physiologically relevant beta-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (-)-norepinephrine showed that the inhibitory effect of the alpha 2-component of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the alpha 2-receptors in the adrenergic control of lipolysis in hamster white adipocytes.