[Sensory stimulations for the treatment of idiopathic apneas of prematurity]. / Traitement des apnées idiopathiques du prématuré par stimulations sensorielles.

Idiopathic apneas of prematurity remain an alarming problem in neonatology. Long-term effects on neurological development cannot be excluded. Two principal treatments are currently applied to prevent apneic attacks: analeptics (methylxanthines and doxapram) and continuous positive airway pressure. Several studies suggest the use of sensory stimulations for curative or preventive treatments. Protocols of tactile, kinesthetic and auditory stimulations are described and their respective efficacy is discussed. We also resume a recent preliminary study which suggested the use of an olfactory stimulation as preventive treatment. A beneficial effect, specifically on apnea associated with severe bradycardia, was observed. Complementary studies are necessary to confirm the benefit of such sensory stimulations for apneas of prematurity, and, if so, to allow clinical applications.

[The olfactory sensitivity of the premature newborn]. / La sensibilité olfactive du nouveau-né prématuré.

This document reviews the main data relating to the structural and functional organisation of olfactory perception in the premature newborn. The chemoreceptive systems (main olfactory, trigeminal, vomeronasal and terminal systems) develop in different chronological orders but quite at very early stage during ontogeny. The premature newborn, despite being immature, has been shown to react to a wide variety of olfactory stimuli. Moreover, the infant seems capable of distinguishing odours of different qualities and intensities, memorising stimuli to which he is regularly exposed to, and categorising different odours based on their hedonic valence. An inventory of the olfactory stimuli to which the infant is regularly exposed to in the incubator is carried out. Several attempts to use pleasant and familiar odours to reduce stress due to separation of the infant from its mother, to promote oral feeding, to make medical procedures more acceptable, and more so, to reduce the respiratory instability of the premature infant, are described. If sustained attention is directed to the olfactory characteristics dwelling inside the incubator, the well-being, health and development of the premature newborn could be improved.

NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro.

AIMS/HYPOTHESIS: Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody. METHODS: The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR)) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140(Trk-A) required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis. RESULTS: We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140(Trk-A). Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced. CONCLUSION/INTERPRETATION: We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.

Nerve growth factor inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation, and cytochrome c release.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.

Neurotrophins and neurotrophin receptors mRNAs expression in pancreatic islets and insulinoma cell lines.

CONTEXT: It is worth noting that islets and betaTC6-F7 cells share a common pattern of expression of neurotrophins and neurotrophin receptors. Recently, several studies have hypothesized a role for nerve growth factor in pancreatic development and maturation, suggesting that nerve growth factor may be a survival factor for pancreatic beta-cells. OBJECTIVE: The aim of the present study was to investigate the pattern of expression of neurotrophins and their relative receptors both in rat pancreatic islets and in a wide panel of insulinoma cell lines. MAIN OUTCOME MEASURES: A semi-quantitative reverse-transcription polymerase chain reaction analysis was performed on ribonucleic acids extracted from these cells. RESULTS: Reverse transcription-polymerase chain reaction analysis demonstrates that brain-derived neurotrophic factor, as well as neurotrophins 3 and 4, are expressed both in islets and in all insulinoma cells, while nerve growth factor is expressed only in islets, betaTC6-F7 cells and, at a low level, in RIN 1046-38 cells. Receptors protein tyrosine kinase A and C are ubiquitously expressed both in islets and insulinoma cells. Tyrosine kinase B is absent in HIT-T15 cells. CONCLUSIONS: These data indicate that betaTC6-F7 cells are a suitable model for studying the role of neurotrophins in the survival of beta-cells.

Human foetuses learn odours from their pregnant mother's diet.

Olfactory responsiveness was assessed in 24 neonates born to mothers who had or had not consumed anise flavour during pregnancy. Both groups of infants were followed-up for behavioural markers of attraction and aversion when exposed to anise odour and a control odour immediately after birth and on day 4. Infants born to anise-consuming mothers evinced a stable preference for anise odour over this period, whereas those born to anise non-consuming mothers displayed aversion or neutral responses. This study provides the first clear evidence that through their diet human mothers influence the hedonic polarity of their neonates' initial olfactory responses. The findings have potential implications for the early mother-to-infant transmission of chemosensory information relative to food and addictive products.

Molecular and functional characterization of pituitary adenylate cyclase-activating polypeptide (PACAP-38)/vasoactive intestinal polypeptide receptors in pancreatic beta-cells and effects of PACAP-38 on components of the insulin secretory system.

It has been previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) regulates insulin secretion. PACAP exerts its biological action by binding to at least three different receptor subtypes coupled to different signal transduction mechanisms. The signaling pathways underlying the insulinotropic effect of PACAP involve mainly the activation of adenylate cyclase to form cAMP, which directly and indirectly, through increased intracellular Ca2+, stimulates insulin exocytosis. In the present study we have characterized the functional and molecular expression of PACAP/vasoactive intestinal polypeptide receptors isoforms and subtypes and its isoforms in a beta-cell line and in isolated rat pancreatic islets. Although insulinoma cells express the messenger RNA encoding PAC1 (-R and -hop variants), VPAC1 and VPAC2, binding experiments indicate the preponderance of PAC1 over VPAC 1-2 receptors. We have also shown that the main signaling pathway of PACAP in beta-cells is mediated by adenylate cyclase, whereas the inositol 1,4,5-trisphosphate pathway is almost inactive. Furthermore, we have demonstrated that PACAP exerts long-term effects on beta-cells, such as transcriptional regulation of the insulin gene and genes of the glucose-sensing system (GLUT1 and hexokinase 1).

GLUT2 and glucokinase expression is coordinately regulated by sulfonylurea.

In the present study we examined the effect of sulfonylurea on the expression of the glucose transporter GLUT2 and the glucose phosphorylating enzyme Glucokinase (GK) in betaTC6-F7 cells; furthermore, we studied the modifications induced by sulfonylurea on glucose-responsiveness and -sensitivity. Results demonstrate that sulfonylurea increases GLUT2 and GK mRNA expression after 24 h in a dose-dependent manner. On the contrary, after 48 and 72 h a time-dependent reduction of both GLUT2 and GK mRNA occurs. GLUT2 and GK protein expression follow the same modifications. Therefore, GLUT2 and GK are coordinately regulated by sulfonylurea, probably by a common mechanism. Glucose-induced insulin release is increased by sulfonylurea as well as glucose sensitivity. Our study suggests that short-term effect of sulfonylurea increases while long-term effect reduces the expression of glucose sensing elements. The long-term inhibitory effect on glucose sensing elements would explain the reduced insulin secretion occurring after chronic sulfonylurea treatment.

Olfactory alliesthesia in human neonates: prandial state and stimulus familiarity modulate facial and autonomic responses to milk odors.

This study examines the effects of a shift in the motivational state (from hunger to satiety) of human neonates on their behavioral and autonomic responsiveness to artificial and food-related odors as a function of stimulus familiarity. In Experiment 1, videotaped facial movements and autonomic (respiration rate: RR, heart rate: HR) responses to five olfactory stimuli (familiar regular formula, unfamiliar regular formula, protein hydrolysate formula, vanillin, control) are recorded in 3-day-old neonates (n = 14) during episodes of irregular sleep. The infants are tested on average 50 min. before and after bottle feeding. RR discriminates the odor stimuli from the control stimulus, indicating clear olfactory detection. Furthermore, neonates react with higher HR change only when exposed to their familiar formula milk during the postprandial condition. The measurement of facial movements with the Baby-Facial Action Coding System indicates that disgust and aversive actions are more often evoked by the odor of regular formulas (familiar or unfamiliar) than by the other olfactory stimuli during the postprandial condition. In Experiment 2, untrained adult observers, presented with the videotapes of the infants' facial responses to the odors, are able to decode differential hedonic signals from the sender faces as a function of the infants' motivational states. The present findings are in line with the concept of olfactory alliesthesia as defined in adults.

Corticosterone treatment differentially affects adrenocorticoid receptors expression and binding in the hippocampus and spinal cord of the rat.

We have studied the immediate and long-term effects of high doses of corticosterone (CORT) on mRNA expression and binding properties of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and spinal cord of rats. Animals were treated with corticosterone (10 mg/d subcutaneously) for 21 consecutive days, and mineralocorticoid and glucocorticoid receptors were studied either 24 h or 2 wk after the last injection. Major results show that corticosterone treatment reduces mineralocorticoid and glucocorticoid receptor maximum binding capacity (Bmax) in both the hippocampus and spinal cord and that this reduction is partially reversed after cessation of treatment. With respect to mRNA expression, in the hippocampus recovery after cessation of treatment is complete. By contrast, in the spinal cord, mineralocorticoid receptor mRNA expression is irreversibly increased after treatment, but the glucocorticoid receptor mRNA level remains unaffected during and after treatment. Thus, these data suggest the presence of distinct regulatory mechanisms for adrenocorticoid receptors in rat brain and spinal cord, in response to long-term exposure to high levels of circulating corticosterone and after recovery from treatment.

Bottle-fed neonates prefer an odor experienced in utero to an odor experienced postnatally in the feeding context.

The head-orientation response of 2- and 4-day-old bottle-feeding neonates was studied in paired-choice odor tests. Three tests were conducted at Days 2 and 4 after birth to assess the development of the relative response between two salient odors from the prenatal and postnatal environments: (a) amniotic fluid (AF) versus formula milk (FM), (b) FM versus control stimulus (distilled water), and (c) AF versus control stimulus. At both ages, AF and FM elicited positive orientation when presented simultaneously with the control stimulus, indicating that both odors were detectable and attractive to the infants. However, when AF and FM were presented concurrently, the infants expressed significantly longer orientation response toward AF odor than toward FM odor at the age of 2 and 4 days. Within the first 4 days of life, bottle-feeders thus display olfactory preference for a prenatal substrate over a postnatal substrate to which they were recurrently exposed in the feeding situation.

Maternal and paternal perception of individual odor signatures in human amniotic fluid--potential role in early bonding?

Both human mothers and fathers are able to discriminate the odors of 2 samples of amniotic fluid (AF), one from their own newborn infant and one from an unrelated infant. Moreover, both parents are able to accurately identify the odor of the AF from their own infant. They report qualitative similarity descriptions of their infant's AF odor to the odor of the actual newborn infant and to the odor of the mother, especially at the end of gestation. These data indicate that human AF carries individualized odor properties, the roles of which in the initiation of parent-infant interactions are hypothesized.

Neonatal responsiveness to the odor of amniotic and lacteal fluids: a test of perinatal chemosensory continuity.

The head-orientation response of 2- and 4-day-old breast-feeding neonates was studied in paired-choice odor tests. Three tests were conducted on day 2 (amniotic fluid [AF] versus Colostrum; AF versus Control; Colostrum versus Control) and on day 4 (AF versus Milk; AF versus Control; Milk versus Control). At 2 days, both AF and Colostrum elicited positive orientation when presented simultaneously with the control stimulus, indicating that both odors were detectable to the infants. However, no differential responses were noted when AF and colostrum were presented concurrently, suggesting that both of these substrates were treated as similar sensorily and/or hedonically. On day 4, the odors of AF and transitional milk elicited attraction responses when presented in competition with a control stimulus. When the odor of milk was presented simultaneously with the odor of AF, the former elicited longer head orientation. Thus, within the first 4 days of life olfactory selectivity changes from a null preference between cues carried in AF and in colostrum to a positive preference for cues carried in postamniotic odors, that is, breast milk. An additional experiment indicated that 3-day-old neonates orient longer toward the odor of their own AF than toward the odor of alien AF, showing that prenatal odors elicit selective responding for some time after birth. Altogether these results were interpreted as supporting the hypotheses that prenatal experience might influence the earliest odor preferences in the breast-feeding human neonate and that these preferences rapidly evolve according to postnatal experience.

Olfactory function in the human fetus: evidence from selective neonatal responsiveness to the odor of amniotic fluid.

This study was aimed at characterizing the level of specificity of the human newborn's response to an odor experienced in utero. Three groups of breast-fed infants and 3 groups of bottle-fed infants were examined on Postnatal Day 3 for their differential head-turning response when exposed to paired-choice tests contrasting the odors of either familiar (f) amniotic fluid (AF) or nonfamiliar (nf) AF or either of these AF odors and a control (C) stimulus. In fAF versus nfAF tests, the infants oriented preferentially to the odor of fAF, regardless of their feeding regimen (i.e., of their postnatal reexposure to AF-like cues in maternal milk). The fAF or nfAF versus C tests showed that this response pattern was caused by a true positive orientation toward fAF and not by avoidance from nfAF odor. This highly selective neonatal response to fAF odor is consistent with the hypothesis that the human fetus can detect and store the unique chemosensory information available in the prenatal environment and that this information becomes coupled with positive control of behavior.

Facial and autonomic responses to biological and artificial olfactory stimuli in human neonates: re-examining early hedonic discrimination of odors.

Responses of awake and asleep 3-day-old human neonates were recorded to the presentation of artificial (vanillin, butyric acid, formula milks) and biological (breast milk, amniotic fluid) odorants matched on subjective intensity and trigeminal dimensions. The responses included behavioral (facial and oral movements) and autonomic (respiration, differential skin temperature) measures. The neonates reacted with significant facial and respiratory changes to low concentrations of olfactory stimuli during the various behavioral states. The analysis of olfacto-facial configurations revealed that behavioral markers of disgust (nose wrinkling, upper lip raising) discriminated between some odors judged as being pleasant and unpleasant by adult raters (vanillin vs. butyric acid). However, although some early predisposition to process the affective significance of stimuli may be suggested, no convincing evidence was obtained that neonates discriminated the hedonic valence of odors within the same perceptual space as adults. Finally, neonates evinced a differential pattern of respiratory responding to the presentation of milk odors according to the mode of feeding (breast vs. bottle feeding), suggesting that early olfactory discrimination may be mediated by stimuli with high ecological salience.

Distribution of adrenocorticoid receptors in the rat CNS measured by competitive PCR and cytosolic binding.

Combined quantitative polymerase chain reaction (PCR) and cytosolic binding assay techniques are used to measure mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, Kd, and Bmax in various rat central nervous system (CNS) regions, namely amygdala, hypothalamus, hippocampus, cortex, pituitary, and cervical, thoracic, and lumbar spinal cord. Two internal standards (i.s.) cDNA were cloned for quantitative PCR purposes. The i.s. templates differed from the respective wild-type (wt) templates for a single base-pair mutation introduced by PCR that generated a unique restriction site, thus allowing amplification products arising from coamplification of wt and i.s. to be distinguished. Results show that cerebellum, which displayed average Bmax values for both receptors, contained the highest level of MR and GR mRNA. Hippocampus also had a high level of MR mRNA. Low mRNA content was found in the hypothalamus for MR and GR as well as in the cortex for GR. High Bmax values for both MR and GR were found in the lumbar spinal cord, despite a modest mRNA content. The lowest Bmax values were found in the cortex for both receptors. It is, therefore, concluded that mRNA content and Bmax are not closely correlated in the rat CNS. These data suggest a differential regulation of various adrenocorticoid receptor isoforms. Moreover, this quantitative PCR method is very sensitive and can be used to assay small amounts of material in order to obtain absolute measurements of mRNA expression.

The effects of pregnancy steroids on adaptation of beta cells to pregnancy involve the pancreatic glucose sensor glucokinase.

Pregnancy is associated with adaptive changes including increased number and size of beta cells and enhanced gap-junctional coupling among beta cells, increased glucose-induced insulin response and decreased glucose stimulation threshold. The role exerted by pregnancy steroids and lactogenic hormones in the development of islets upregulation during pregnancy has been widely investigated. In the present study we studied the possibility that pregnancy steroids induce functional modifications of beta cells involving the expression and function of glucokinase. Our results indicate that estradiol and progesterone do not influence significantly glucokinase mRNA expression, while they induce a dose-dependent and time-dependent increase of glucokinase activity in RIN 1046-38 cells. The increased enzymatic activity results in an increased glucose-induced insulin release. Therefore it is possible to hypothesize that pregnancy steroids influence glucokinase expression in beta cells at a post-transcriptional level and that this effect contributes to the development of hyperinsulinemia during pregnancy.

Orientation responses to biological odours in the human newborn. Initial pattern and postnatal plasticity.

The initial pattern and development of odour preference was studied in infants simultaneously exposed to amniotic fluid (AF) and maternal lacteal secretion (L). Five groups of varying age (range: 1-5 days) and breast-feeding experience (range: 0-32 feeds) were studied. Before postnatal day 3, no evidence of differentiation of AF and L was apparent. After 3 days and 7-12 breast-feeding episodes, a significant preference for L arised. The initial stage (days 1-3) may reflect fetal acquisition of AF odour and sensory/motivational equivalence of AF and L odours. The second stage (days 4-5) may reflect the infants' perception of change in milk quality and increasing experience with milk. This sequential development attests to a high plasticity in the initial stage of human olfactory development.