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

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.

Angiotensin II receptor imbalance associated with neonatal cardiac growth restriction is a prelude to adult cardiac hypertrophy

The Hypertrophic Heart Rat (HHR) displays spontaneous cardiomyocyte hypertrophy in association with an apparent reduction in myocyte number in adulthood. This suggests the possibility of reduced hyperplasia or increased apoptosis during early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, but the precise mechanisms are unclear. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with the Normal Heart Rat (NHR) control strain. Cardiac tissues were freshly harvested from male HHR and NHR at several developmental stages (p2 and 4, 6, 8, 12wks). HHR cardiac weight indices were considerably smaller than NHR at day 2 (4.330.19 vs 5.010.08 mg/g), but ‘caught-up’ to NHR by 4 weeks (5.100.15 vs 5.160.11 mg/g). By 12 weeks, HHR hearts were 27% larger than NHR. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited a 4.6-fold higher AT2/AT1 mRNA expression ratio. Cultured neonatal cardiomyocytes were infected with AT1A and/or AT2 receptor-expressing adenoviruses to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to emulate receptor expression in neonatal HHR hearts, cells were co-infected with AT1A and AT2 receptors at a 4:1 ratio. Apoptosis incidence was studied by morphological analysis after 72 hours exposure to 0.1 M AngII. When infected with the AT1A receptor alone, a higher proportion of HHR myocytes appeared apoptotic than NHR (22.7 4.1% vs 1.1 0.6%, P 0.001). This implies that intrinsic differences predispose HHR cells to accentuated AT1-mediated apoptosis. Interestingly, the bax-1/bcl-2 mRNA expression ratio was significantly higher (50%) in HHR neonatal hearts. When cells were co-infected with AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4 0.1%). These findings suggest a novel capacity of AT2 receptors to counteract accentuated AT1A receptor-induced apoptosis in the HHR in early cardiac growth.

Neonatal growth outcomes at birth and one month postpartum following in utero exposure to antidepressant medication

Objective: There is evidence of increasing prescription of antidepressant medication in pregnant women. This has arisen from the recognition of the importance of treating maternal depression. This must be balanced, however, with information on outcomes for infants and children exposed to antidepressants in pregnancy. The aim of the present study was to examine whether neonatal outcomes including gestational age at birth, neonatal growth outcomes at birth and then at 1 month postpartum were altered by in utero exposure to antidepressant medication using a prospective and controlled design.

Method: A prospective case–control study recruited 27 pregnant women taking antidepressant medication and 27 matched controls who were not taking antidepressant medication in pregnancy at an obstetric hospital in Melbourne, Australia. Of the 27 women taking medication, 25 remained on medication in the third trimester. A purpose-designed self-report questionnaire and the Beck Depression Inventory-II were completed in pregnancy, after birth and at one month postpartum. In addition information was collected on exposed and non-exposed infants including Apgar scores, birthweight/length/head circumference and gestational age at birth. Weight/length/head circumference was again collected at 1 month of age.

Results: Infants exposed to antidepressants in utero were eightfold more likely to be born at a premature gestational age, had significantly lower birthweight and were smaller in length and head circumference than non-exposed infants. There was no association between birth outcomes and maternal depression. At 1 month, the difference in weight in the exposed group became significantly greater than the control group.

Conclusion: Antidepressant exposure in utero may affect gestational age at birth and neonatal outcomes independently of antenatal maternal depression. Further studies are needed to examine whether these findings vary according to the type of antidepressant prescribed and follow up growth and development in exposed infants beyond 1 month.

Researching maternal, neonatal and child health service use in rural and pastoralist Ethiopia: A key informant research approach

Like many nations in sub-Saharan Africa, Ethiopia has both a high neonatal mortality rate and maternal mortality ratio and is unlikely to meet Millennium Development Goals 4 and 5 by 2015. This working paper examines how Key Informant Research (KIR) in rural and pastoralist Ethiopia will identify facilitators and barriers to the use of maternal, neonatal and child health services. The methodology is informed by Participative Ethnographic Evaluation Research (PEER) and Key Informant Monitoring (KIM). Key Informant Research (KIR) training will provide research skills to Health Extension Workers (HEWs) and Non-government organisation (NGO) staff to enable them to develop research questions, collect data and participate in preliminary data analysis. This will enable the identification of strategies that improve the identification of risk, enhance early referral, increase access, affordability and acceptability of skilled birthing services in rural and pastoralist Ethiopia.

Global Surgery 2030: evidence and solutions for achieving health, welfare, and economic development.

Remarkable gains have been made in global health in the past 25 years, but progress has not been uniform. Mortality and morbidity from common conditions needing surgery have grown in the world’s poorest regions, both in real terms and relative to other health gains. At the same time, development of safe, essential, life-saving surgical and anaesthesia care in low-income and middle-income countries (LMICs) has stagnated or regressed. In the absence of surgical care, case-fatality rates are high for common, easily treatable conditions including appendicitis, hernia, fractures, obstructed labour, congenital anomalies, and breast and cervical cancer. In 2015, many LMICs are facing a multifaceted burden of infectious disease, maternal disease, neonatal disease, non-communicable diseases, and injuries. Surgical and anaesthesia care are essential for the treatment of many of these conditions and represent an integral component of a functional, responsive, and resilient health system. In view of the large projected increase in the incidence of cancer, road traffic injuries, and cardiovascular and metabolic diseases in LMICs, the need for surgical services in these regions will continue to rise substantially from now until 2030. Reduction of death and disability hinges on access to surgical and anaesthesia care, which should be available, affordable, timely, and safe to ensure good coverage, uptake, and outcomes. Despite growing need, the development and delivery of surgical and anaesthesia care in LMICs has been nearly absent from the global health discourse. Little has been written about the human and economic effect of surgical conditions, the state of surgical care, or the potential strategies for scale-up of surgical services in LMICs. To begin to address these crucial gaps in knowledge, policy, and action, the Lancet Commission on Global Surgery was launched in January, 2014. The Commission brought together an international, multi- disciplinary team of 25 commissioners, supported by advisors and collaborators in more than 110 countries and six continents. We formed four working groups that focused on thedomains of health-care delivery and management; work-force, training, and education; economics and finance; and information management. Our Commission has five key messages, a set of indicators and recommendations to improve access to safe, affordable surgical and anaesthesia care in LMICs, and a template for a national surgical plan.