Langerhans cell (LC) proliferation mediates neonatal development, homeostasis, and inflammation-associated expansion of the epidermal LC network.

Most tissues develop from stem cells and precursors that undergo differentiation as their proliferative potential decreases. Mature differentiated cells rarely proliferate and are replaced at the end of their life by new cells derived from precursors. Langerhans cells (LCs) of the epidermis, although of myeloid origin, were shown to renew in tissues independently from the bone marrow, suggesting the existence of a dermal or epidermal progenitor. We investigated the mechanisms involved in LC development and homeostasis. We observed that a single wave of LC precursors was recruited in the epidermis of mice around embryonic day 18 and acquired a dendritic morphology, major histocompatibility complex II, CD11c, and langerin expression immediately after birth. Langerin+ cells then undergo a massive burst of proliferation between postnatal day 2 (P2) and P7, expanding their numbers by 10–20-fold. After the first week of life, we observed low-level proliferation of langerin+ cells within the epidermis. However, in a mouse model of atopic dermatitis (AD), a keratinocyte signal triggered increased epidermal LC proliferation. Similar findings were observed in epidermis from human patients with AD. Therefore, proliferation of differentiated resident cells represents an alternative pathway for development in the newborn, homeostasis, and expansion in adults of selected myeloid cell populations such as LCs. This mechanism may be relevant in locations where leukocyte trafficking is limited.

Bisphenol-A promotes antiproliferative effects during neonatal prostate development in male and female gerbils

Conselho Nacional de Desenvolvimento Científico e Tecnol[ogico (CNPq)

Childbirth care: contributing to the debate on human development

Com a finalidade de subsidiar gestores da área de saúde da mulher, na formulação de políticas públicas, voltadas ao desenvolvimento humano, realizou-se esta investigação, cujo objetivo foi avaliar a estrutura e o processo da atenção ao parto e ao neonato desenvolvido em região do interior paulista. Estudo epidemiológico, voltado para avaliação dos serviços de saúde, baseou-se na observação da assistência prestada pelo Sistema Único de Saúde em 12 maternidades e 134 partos, adotando-se padrões estabelecidos pelo Ministério da Saúde ou Organização Mundial de Saúde para comparação. Os resultados apontam problemas relacionados à estrutura em algumas maternidades, mostrando que práticas úteis ao parto normal ainda são pouco utilizadas, enquanto que outras prejudiciais ou ineficazes ainda são utilizadas rotineiramente. Reverter esse quadro será essencial para oferecer atendimento de qualidade às mulheres, com conseqüente redução nas taxas de mortalidade materna e neonatal, para que a região atinja as metas estabelecidas para ampliação do desenvolvimento humano no milênio.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Copper homeostasis in human mammary epithelial cells

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. This thesis analysed copper transporting proteins in mammary epithelial cells and the impact of copper and lactational hormones upon the regulation these proteins was measured.

Developmental origins of obesity and the metabolic syndrome : the role of maternal obesity

Obesity and its sequelae may prove to be the greatest threat to human lifestyle and health in the developed world this century. The so called obesity epidemic has seen the incidence of obesity and overweight almost double in Western societies and the trend is mirrored in nations that are transitioning to first world economies. There is no doubt that much of the rise in obesity can be attributed to lifestyle factors such as the excess consumption of energy-dense foods and the decline in physical activity. However, the ‘fetal origins’hypothesis, first proposed by Barker and colleagues and elaborated by several groups over the past 15 years to be termed the ‘Developmental Origins of Adult Health and Disease’ (DOHaD), provides an alternative explanation for the rising rates of obesity. The DOHaD hypothesis states that exposure to an unfavourable environment during development (either in utero or in the early postnatal period) programmes changes in fetal or neonatal development such that the individual is then at greater risk of developing adulthood disease. This chapter discusses the effects of maternal obesity on fetal development and birth outcomes as well as the manner in which DOHaD may contribute to the obesity epidemic.

Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting

Impact of gestational diabetes and lactational insulin replacement on structure and secretory function of offspring rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Il contributo della psicopatologia ansiosa materna prenatale sul temperamento infantile e la relazione precoce madre-bambino

La sintomatologia ansiosa materna nel periodo prenatale risulta influire negativamente non sullo stato materno ma anche sul successivo sviluppo infantile, Tuttavia, sono limitati gli studi che hanno considerato lo specifico contributo dei disturbi d’ansia nel periodo prenatale. L’obiettivo generale dello studio è quello di indagare nel primo periodo post partum la relazione tra psicopatologia ansiosa materna e: temperamento e sviluppo neonatale, qualità del caregiving materno e dei pattern interattivi madre-bambino. 138 donne sono state intervistate utilizzando SCID-I (First et al., 1997) durante il terzo trimestre di gravidanza. 31 donne (22,5%) presentano disturbo d’ansia nel periodo prenatale. A 1 mese post partum il comportamento del neonato è stato valutato mediante NBAS (Brazelton, Nugent, 1995), mentre le madri hanno compilato MBAS (Brazelton, Nugent, 1995). A 3 mesi postpartum, una sequenza interattiva madre-bambino è stata videoregistrata e codificata utilizzando GRS (Murray et al., 1996). La procedura dello Stranger Episode (Murray et al., 2007) è stata utilizzata per osservare i pattern interattivi materni e infantili nell’interazione con una persona estranea. I neonati di madri con disturbo d’ansia manifestano alle NBAS minori capacità a livello di organizzazione di stati comportamentali, minori capacità attentive e di autoregolazione. Le madri ansiose si percepiscono significativamente meno sicure nell’occuparsi di loro, valutando i propri figli maggiormente instabili e irregolari. Nell’interazione face to face, esse mostrano comportamenti significativamente meno sensibilI, risultando meno coinvolte attivamente con il proprio bambino. Durante lo Stranger Episode, le madri con fobia sociale presentano maggiori livelli di ansia e incoraggiando in modo significativamente inferiore l’interazione del bambino con l’estraneo. I risultati sottolineano l’importanza di valutare in epoca prenatale la psicopatologia ansiosa materna. Le evidenze confermano la rilevanza che può assumere un modello multifattoriale di rischio in cui i disturbi d’ansia prenatali e la qualità del caregiving materno possono agire in modo sinergico nell’influire sugli esiti infantili.

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

Acetylcholine receptor ɛ-subunit deletion causes muscle weakness and atrophy in juvenile and adult mice

In mammalian muscle a postnatal switch in functional properties of neuromuscular transmission occurs when miniature end plate currents become shorter and the conductance and Ca2+ permeability of end plate channels increases. These changes are due to replacement during early neonatal development of the γ-subunit of the fetal acetylcholine receptor (AChR) by the ɛ-subunit. The long-term functional consequences of this switch for neuromuscular transmission and motor behavior of the animal remained elusive. We report that deletion of the ɛ-subunit gene caused in homozygous mutant mice the persistence of γ-subunit gene expression in juvenile and adult animals. Neuromuscular transmission in these animals is based on fetal type AChRs present in the end plate at reduced density. Impaired neuromuscular transmission, progressive muscle weakness, and atrophy caused premature death 2 to 3 months after birth. The results demonstrate that postnatal incorporation into the end plate of ɛ-subunit containing AChRs is essential for normal development of skeletal muscle.

Visualization of α9 acetylcholine receptor expression in hair cells of transgenic mice containing a modified bacterial artificial chromosome

The α9 acetylcholine receptor (α9 AChR) is specifically expressed in hair cells of the inner ear and is believed to be involved in synaptic transmission between efferent nerves and hair cells. Using a recently developed method, we modified a bacterial artificial chromosome containing the mouse α9 AChR gene with a reporter gene encoding green fluorescent protein (GFP) to generate transgenic mice. GFP expression in transgenic mice recapitulated the known temporal and spatial expression of α9 AChR. However, we observed previously unidentified dynamic changes in α9 AChR expression in cochlear and vestibular sensory epithelia during neonatal development. In the cochlea, inner hair cells persistently expressed high levels of α9 AChR in both the apical and middle turns, whereas both outer and inner hair cells displayed dynamic changes of α9 AChR expression in the basal turn. In the utricle, we observed high levels of α9 AChR expression in the striolar region during early neonatal development and high levels of α9 AChR in the extrastriolar region in adult mice. Further, simultaneous visualization of efferent innervation and α9 AChR expression showed that dynamic expression of α9 AChR in developing hair cells was independent of efferent contacts. We propose that α9 AChR expression in developing auditory and vestibular sensory epithelia correlates with maturation of hair cells and is hair-cell autonomous.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Neonatal palliative care attitude scale : development of an instrument to measure the barriers to and facilitators of palliative care in neonatal nursing

OBJECTIVE The aim of this research project was to obtain an understanding of the barriers to and facilitators of providing palliative care in neonatal nursing. This article reports the first phase of this research: to develop and administer an instrument to measure the attitudes of neonatal nurses to palliative care. METHODS The instrument developed for this research (the Neonatal Palliative Care Attitude Scale) underwent face and content validity testing with an expert panel and was pilot tested to establish temporal stability. It was then administered to a population sample of 1285 neonatal nurses in Australian NICUs, with a response rate of 50% (N 645). Exploratory factor-analysis techniques were conducted to identify scales and subscales of the instrument. RESULTS Data-reduction techniques using principal components analysis were used. Using the criteria of eigenvalues being 1, the items in the Neonatal Palliative Care Attitude Scale extracted 6 factors, which accounted for 48.1% of the variance among the items. By further examining the questions within each factor and the Cronbach’s of items loading on each factor, factors were accepted or rejected. This resulted in acceptance of 3 factors indicating the barriers to and facilitators of palliative care practice. The constructs represented by these factors indicated barriers to and facilitators of palliative care practice relating to (1) the organization in which the nurse practices, (2) the available resources to support a palliative model of care, and (3) the technological imperatives and parental demands. CONCLUSIONS The subscales identified by this analysis identified items that measured both barriers to and facilitators of palliative care practice in neonatal nursing. While establishing preliminary reliability of the instrument by using exploratory factor-analysis techniques, further testing of this instrument with different samples of neonatal nurses is necessary using a confirmatory factor-analysis approach.