Single nucleotide polymorphisms in surfactant protein A1 are not associated with a lack of responsiveness to antenatal steroid therapy in a pregnant sheep model

Treatment with antenatal steroids (ANS) is standard practice for reducing the risk of respiratory distress in the preterm infant. Despite clear overall benefits when appropriately administered, many fetuses fail to derive benefit from ANS therapies. In standardized experiments using a pregnant sheep model, we have demonstrated that around 40% of ANS-exposed lambs did not have functional lung maturation significantly different from that of saline-treated controls. Surfactant protein A is known to play an important role in lung function. In this genotyping study, we investigated the potential correlation between polymorphisms in SFTPA1, messenger RNA and protein levels, and ventilation outcomes in animals treated with ANS. 45 preterm lambs were delivered 48 h after initial ANS therapy and 44 lambs were delivered 8 days after initial ANS therapy. The lambs were ventilated for 30 min after delivery. SFTPA1 mRNA expression in lung tissue was not correlated with arterial blood PaCO2 values at 30 min of ventilation in lambs delivered 48 h after treatment. SFTPA1 protein in lung tissue was significantly correlated with PaCO2 at 30 min of ventilation in lambs ventilated both 48 h and 8 days after ANS treatment. Six different single nucleotide polymorphisms (SNPs) in the Ovis aries SFTPA1 sequence were detected by Sanger Sequencing. No individual SNPs or SNP haplotypes correlated with alterations in PaCO2 at 30 min of ventilation or SFTPA1 protein levels in the lung. For the subset of animals analyzed in the present study, variable lung maturation responses to ANS therapy were not associated with mutations in SFTPA1

Betamethasone phosphate reduces the efficacy of antenatal steroid therapy and is associated with lower birth weights when administered to pregnant sheep in combination with betamethasone acetate

Background Antenatal corticosteroid (ACS) therapy is standard of care for women at imminent risk of preterm labour. Despite this, much remains to be understood regarding an optimal (maximum benefit, minimal risk of side effects) ACS dosing strategy. Although conveying overall benefit when given to the right patient at the right time, ACS treatment efficacy is highly variable, and is not risk-free. Building on earlier findings, we hypothesized that when administered in combination with slow-release betamethasone acetate, betamethasone phosphate and the high materno-fetal betamethasone concentrations it generates are redundant for fetal lung maturation. Objective Using an established sheep model of prematurity and post-natal ventilation of the preterm lamb, we aimed to compare the pharmacodynamic effects of a low-dose treatment with betamethasone acetate only against a standard dose of betamethasone phosphate and betamethasone acetate as recommended by the American College of Obstetricians and Gynaecologists for women at risk of imminent preterm delivery between 24 and 35+6 weeks’ gestation. Methods Ewes carrying a single fetus at 122±1 d gestational age (term=150d) were randomized to receive either: i) maternal intramuscular injections of sterile saline (the Saline Negative Control Group, n=12), ii) two maternal intramuscular injections of 0.25 mg/kg betamethasone phosphate + acetate spaced by 24h (the Beta-P+Ac Group, n=12); or iii) two maternal intramuscular injections of 0.125 mg/kg betamethasone acetate spaced by 24h (the Beta-Ac Group, n=11). Fetuses were surgically delivered 48h after treatment initiation and ventilated for 30 minutes to determine functional lung maturation. Fetuses were euthanized after ventilation and lung were collected for analysis using quantitative polymerase chain reaction and western blot assays. Fetal plasma ACTH levels were measured in the cord blood samples taken at delivery. Results Preterm lambs were defined as either ACS treatment responders or non-responders using an arbitrary cut-off, being a PaCO2 level at 30 minutes of ventilation being more extreme than two standard deviations from the mean value of the normally-distributed Saline Control Group values. Relative to Saline Control Group animals, both ACS treatment group animals showed significantly improved lung physiological responses (blood gas and ventilation data) and had a biochemical signature (mRNA and surfactant protein assays) consistent with functional maturation. However, the Beta-Ac Group had a significantly higher treatment response rate than the Beta-P+Ac Group. These physiological results were strongly correlated to the amount of surfactant protein A. Birth weight was lower in Beta-P+Ac Group and the fetal HPA axis was supressed to a greater extent in the Beta-P+Ac Group. Conclusion Low dose ACS therapy solely employing Beta-Ac was sufficient for fetal lung maturation. The elevated materno-fetal betamethasone concentrations associated with the co-administration of betamethasone phosphate did not additionally improve lung maturation, but were associated with greater HPA axis suppression, a lower ACS treatment response rate, and lower birth weight – outcomes not desirable in a clinical setting. These data warrant a clinical investigation of sustained, low-dose ACS treatments that avoid high materno-fetal betamethasone exposures

Numbat nirvana: the conservation ecology of the endangered numbat Myrmecobius fasciatus (Marsupialia: Myrmecobiidae) reintroduced to Scotia and Yookamurra Sanctuaries, Australia

Despite a vigorous reintroduction program between 1985 and 2010, numbat populations in Western Australia are either static or declining. This study aimed to document the population ecology of numbats at two sites that are going against this trend: Scotia Sanctuary in far western New South Wales and Yookamurra Sanctuary in the riverland of South Australia. Scotia (64 659 ha) and Yookamurra (5026 ha) are conservation reserves owned and managed by the Australian Wildlife Conservancy and where numbats were reintroduced in 1999 and 1993 respectively. Both sites have large conservation-fence-protected introduced-species-free areas where there are no cats (Felis catus) or red foxes (Vulpes vulpes). Numbats were sourced from both wild and captive populations. From small founder populations, the Scotia numbats are now estimated to number 169 (113–225) with 44 at Yookamurra. Radio-collared individuals at Scotia were active between 13 and 31°C. Females had home ranges of 28.3 ± 6.8 ha and males 96.6 ± 18.2 ha, which leads to an estimated sustainable population or carrying capacity of 413–502 at Scotia. Captive-bred animals from Perth Zoo had a high mortality rate upon reintroduction at Scotia due to predation by raptors and starvation. The habitat preferences for mallee with a shrub understorey appear to be driven by availability of termites, and other reintroduced ecosystem engineers appear to have been facilitators by creating new refuge burrows for numbats. This study shows that numbats can be successfully reintroduced into areas of their former range if protected from introduced predators, and illustrates the difficulties in monitoring such cryptic species.</jats:p

Chorioamnionitis induces hepatic inflammation and time-dependent changes of the enterohepatic circulation in the ovine fetus

Chorioamnionitis, inflammation of fetal membranes, is an important cause of preterm birth and a risk factor for the development of adverse neonatal outcomes including sepsis and intestinal pathologies. Intestinal bile acids (BAs) accumulation and hepatic cytokine production are involved in adverse intestinal outcomes. These findings triggered us to study the liver and enterohepatic circulation (EHC) following intra-amniotic (IA) lipopolysaccharide (LPS) exposure. An ovine chorioamnionitis model was used in which circulatory cytokines and outcomes of the liver and EHC of preterm lambs were longitudinally assessed following IA administration of 10 mg LPS at 5, 12 or 24h or 2, 4, 8 or 15d before preterm birth. Hepatic inflammation was observed, characterized by increased hepatic cytokine mRNA levels (5h – 2d post IA LPS exposure) and increased erythropoietic clusters (at 8 and 15 days post IA LPS exposure). Besides, 12h after IA LPS exposure, plasma BA levels were increased, whereas gene expression levels of several hepatic BA transporters were decreased. Initial EHC alterations normalized over time. Concluding, IA LPS exposure induces significant time-dependent changes in the fetal liver and EHC. These chorioamnionitis induced changes have potential postnatal consequences and the duration of IA LPS exposure might be essential herein

Chorioamnionitis induces enteric nervous system injury: Effects of timing and inflammation in the ovine fetus

Background Chorioamnionitis, inflammation of the chorion and amnion, which often results from intrauterine infection, is associated with premature birth and contributes to significant neonatal morbidity and mortality, including necrotizing enterocolitis (NEC). Recently, we have shown that chronic chorioamnionitis is associated with significant structural enteric nervous system (ENS) abnormalities that may predispose to later NEC development. Understanding time point specific effects of an intra-amniotic (IA) infection on the ENS is important for further understanding the pathophysiological processes and for finding a window for optimal therapeutic strategies for an individual patient. The aim of this study was therefore to gain insight in the longitudinal effects of intrauterine LPS exposure (ranging from 5 h to 15 days before premature delivery) on the intestinal mucosa, submucosa, and ENS in fetal lambs by use of a well-established translational ovine chorioamnionitis model. Methods We used an ovine chorioamnionitis model to assess outcomes of the fetal ileal mucosa, submucosa and ENS following IA exposure to one dose of 10 mg LPS for 5, 12 or 24 h or 2, 4, 8 or 15 days. Results Four days of IA LPS exposure causes a decreased PGP9.5- and S100β-positive surface area in the myenteric plexus along with submucosal and mucosal intestinal inflammation that coincided with systemic inflammation. These changes were preceded by a glial cell reaction with early systemic and local gut inflammation. ENS changes and inflammation recovered 15 days after the IA LPS exposure. Conclusions The pattern of mucosal and submucosal inflammation, and ENS alterations in the fetus changed over time following IA LPS exposure. Although ENS damage seemed to recover after prolonged IA LPS exposure, additional postnatal inflammatory exposure, which a premature is likely to encounter, may further harm the ENS and influence functional outcome. In this context, 4 to 8 days of IA LPS exposure may form a period of increased ENS vulnerability and a potential window for optimal therapeutic strategies

Immunophenotypic analysis reveals differences in circulating immune cells in the peripheral blood of patients with segmental and nonsegmental vitiligo

Accumulating studies have indicated immune-based destruction of melanocytes in both segmental vitiligo (SV) and non-SV (NSV). Whereas SV often occurs unilaterally during childhood and stabilizes after an initial period of activity, the disease course of NSV is usually slowly progressive, with new lesions occurring bilaterally during life. This suggests an involvement of distinct pathophysiology pathways, specifically increased systemic immune activation in patients with NSV but not in patients with SV. This research aimed to identify the differences in immune cells in the blood of patients with SV and NSV through immunophenotyping of circulating cells. Regulatory T cells were unaffected in patients with SV compared with that in healthy controls but decreased in patients with NSV. In patients with NSV, the reduction in regulatory T cells was associated with the presence of other systemic autoimmune comorbidities, which were less present in SV. Similarly, the absence of a melanocyte-specific antibody response in patients with SV suggests less involvement of B-cell immunity in SV. These data show that in contrast to patients with NSV, no increased systemic immunity is found in patients with SV, indicating that SV pathogenesis is associated with a localized cytotoxic reaction targeting epidermal melanocytes

The Paradoxical Effects of Chronic Intra-Amniotic Ureaplasma parvum Exposure on Ovine Fetal Brain Development

Chorioamnionitis is associated with adverse neurodevelopmental outcomes in preterm infants. Ureaplasma spp. are the microorganisms most frequently isolated from the amniotic fluid of women diagnosed with chorioamnionitis. However, controversy remains concerning the role of Ureaplasma spp. in the pathogenesis of neonatal brain injury. We hypothesize that re-exposure to an inflammatory trigger during the perinatal period might be responsible for the variation in brain outcome of preterms following Ureaplasma driven chorioamnionitis. To investigate these clinical scenarios, we performed a detailed multi-modal study in which ovine neurodevelopmental outcomes were assessed following chronic intra-amniotic Ureaplasma parvum (UP) infection, either alone or combined with subsequent lipopolysaccharide (LPS) exposure. We show that chronic intra-amniotic UP exposure during the second trimester provoked a decrease of astrocytes, increased oligodendrocyte numbers and elevated 5-methylcytosine levels. In contrast, short-term LPS exposure before preterm birth induced increased microglial activation, myelin loss, elevation of 5-hydroxymethylcytosine levels and lipid profile changes. These LPS-induced changes were prevented by chronic pre-exposure to UP (preconditioning). These data indicate that chronic UP exposure provokes dual effects on preterm brain development in utero. On one hand, prolonged UP exposure causes detrimental cerebral changes which may predispose to adverse postnatal clinical outcomes. On the other, chronic intra-amniotic UP exposure preconditions the brain against a second inflammatory hit. This study demonstrates that microbial interactions, timing and duration of inflammatory insults will determine the effects on the fetal brain. Therefore, this study helps to understand the complex and diverse postnatal neurological outcomes following UP driven chorioamnionitis

Variability in the efficacy of a standardized antenatal steroid treatment is not due to maternal or fetal plasma drug levels. Evidence from a sheep model of pregnancy.

Background Antenatal steroids (ANS) are standard of care for women judged to be at imminent risk of preterm delivery. Worldwide, there is significant variation in ANS dosing strategy, selection for treatment criteria, and agent choice. This, combined with very limited optimization of ANS use per se means that treatment efficacy is highly variable and the rate of respiratory distress syndrome is decreased perhaps as little as 40%. In some cases, ANS use is associated with limited benefit and potential harm. Objective We hypothesized that individual differences in maternal and fetal steroid exposure would contribute to observed variability in ANS treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between materno-fetal steroid exposure and ANS treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. Methods Ewes carrying a single fetus had surgery to catheterize a fetal and maternal jugular vein at 119 days’ gestation. Animals recovered for 24h before being randomized to either: i) a single maternal intramuscular injection (IM) of 2ml saline (Negative Control Group, n=10); or ii) a single maternal IM of 0.25mg/kg betamethasone phosphate + acetate (ANS Group, n=20). Serial maternal and fetal plasma samples were collected from each animal over 48h before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. Results One animal of the Control Group and one animal from the ANS Group had did not complete their treatment protocol and were removed from analyses. Animals in the ANS Group were divided into a Responder (n=12/19) Sub-Group and a Non-Responder Sub-Group (n=7/19) using a cut-off of a PaCO2 at 30 minutes ventilation within 2SD of the mean value from saline-treated Negative Control Group animals. While ANS improved fetal lung maturation in the undivided ANS group, and in the Responder Sub-Group both physiologically (blood gas and ventilation related data) and biochemically (mRNA expression related to fetal lung maturation), these values were not improved relative to saline-treated Control Group animals in the ANS Non-Responder Sub-Group. Interestingly, no differences in betamethasone distribution, clearance, or protein binding were identified between the ANS Responder and Non-Responder Sub-Groups. Conclusion This study correlated individual materno-fetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation not significantly different to saline-treated Control Group animals. These non-responsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of ANS therapy is not solely determined by materno-fetal drug levels, and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid-driven signaling

Direct administration of the non-competitive interleukin-1 receptor antagonist rytvela transiently reduced intrauterine inflammation in an extremely preterm sheep model of chorioamnionitis

Background Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks’ gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. Methods Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. Results LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1β in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. Conclusion A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response

Ex-Vivo Uterine Environment (EVE) Therapy Induced Limited Fetal Inflammation in a Premature Lamb Model

Introduction Ex-vivo uterine environment (EVE) therapy uses an artificial placenta to provide gas exchange and nutrient delivery to a fetus submerged in an amniotic fluid bath. Development of EVE may allow us to treat very premature neonates without mechanical ventilation. Meanwhile, elevations in fetal inflammation are associated with adverse neonatal outcomes. In the present study, we analysed fetal survival, inflammation and pulmonary maturation in preterm lambs maintained on EVE therapy using a parallelised umbilical circuit system with a low priming volume. Methods Ewes underwent surgical delivery at 115 days of gestation (term is 150 days), and fetuses were transferred to EVE therapy (EVE group; n = 5). Physiological parameters were continuously monitored; fetal blood samples were intermittently obtained to assess wellbeing and targeted to reference range values for 2 days. Age-matched animals (Control group; n = 6) were surgically delivered at 117 days of gestation. Fetal blood and tissue samples were analysed and compared between the two groups. Results Fetal survival time in the EVE group was 27.0 ± 15.5 (group mean ± SD) hours. Only one fetus completed the pre-determined study period with optimal physiological parameters, while the other 4 animals demonstrated physiological deterioration or death prior to the pre-determined study end point. Significant elevations (p0.05) in surfactant protein mRNA expression level between the two groups. Conclusion In this study, we achieved limited fetal survival using EVE therapy. Despite this, EVE therapy only induced a modest fetal inflammatory response and did not promote lung maturation. These data provide additional insight into markers of treatment efficacy for the assessment of futur