Later Stages Of Gestation Research Articles

Polycythemia of Mice with Human Gain-of-Function EPOR (mtHEPOR) Is Transiently Corrected in Perinatal Life in Association with Low Epo and Increased Erythrocyte Phosphatidylserine Exposure

Gain-of-function mutations of erythropoietin receptor (EPOR) resulting from truncation of the cytoplasmic domain of EPOR are associated with primary familial and congenital polycythemia (PFCP). We generated and previously reported a PFCP mouse model where the murine EpoR gene (mEpoR) was replaced with wild-type human EPOR (wtHEPOR) or mutant human EPOR (mtHEPOR) genes. We showed that mtHEPOR mice become polycythemic at 3-6 weeks of age, but not at birth, similar to the polycythemia phenotype of affected humans, while the wtHEPOR mice were anemic (Divoky et al, Proc Natl Acad Sci U S A 2001;98:986). In addition, we previously reported (Yoon et al, ASH Annual Meeting Abstracts 2005 106: Abstract 567) that mtHEPOR fetuses are polycythemic at the mid- and late-gestation stages (embryonic days, E12.4-18.5), while wtHEPOR fetuses are anemic, similar to what we observed in adult mice. The mtHEPOR fetal liver cells (FLCs) revealed augmented and sustained activation of Stat5, while wtHEPOR FLCs had significantly reduced Stat5 activation. We also previously presented (Yoon and Prchal, ASH Annual Meeting Abstracts 2007 110: Abstract 3664) that the fetal polycythemia of mtHEPOR mice is associated with a delayed switch from primitive to definitive erythropoiesis, and that polycythemia in mtHEPOR mice is transiently corrected in perinatal life and reappears at 3 weeks after birth. Here, we hypothesized that the change of oxygen tension at delivery (from the uterus to ambient atmosphere) and differences in Epo levels may account for the inexplicable perinatal absence of polycythemia in mtHEPOR mice.Epo levels in adult mice were substantially elevated in wtHEPOR (1271±240 pg/mL) and significantly reduced in mtHEPOR (98±51 pg/mL) when compared to mEpoR mice (265±62 pg/mL). We then proposed that destruction of young red blood cells (RBCs), termed neocytolysis (a mechanism that overcorrects increased RBC mass generated during chronic hypoxemia after restoration of normoxic conditions) contributes to the anemia of newborns (Song et al, J Mol Med 2015;93:857). When adult mice were challenged by rapid conversion from 10 days of hypoxia (12% of O2) to normoxia, the mtHEPOR mice had greater changes of hematocrit (drop of 16.9%) than other genotypes (drop of 14.8% for mEpoR and13.8% for wtHEPOR). Similarly, the most pronounced decrease of hematocrit after birth, at perinatal day 7, (PN7) was detected in mtHEPOR mice (drop of 56%) followed by mEpoR (drop of 48%) and only a relatively moderate decrease of hematocrit in wtHEPOR neonates (drop of 24%). In parallel, PN7 mtHEPOR newborns had the lowest levels of Epo (310±102 pg/mL), while PN7 wtHEPOR neonates had Epo levels markedly increased (2757±912 pg/mL); the Epo levels of control mEpoR at PN7 littermates were 494±102 pg/mL. This suggests that Epo protects from neocytolysis. These changes corresponded to exposure of phosphatidylserine; a stimulus for RBC removal from circulation. There was a gradual increase of phosphatidylserine-positive RBCs in mtHEPOR neonates and, to a lower extent, also in mEpoR between PN0 and PN7 with a maximum positivity at PN7 (mean fluorescence: 6.5±2.8 and 6.2±1.7 for mtHEPOR and mEpoR, respectively). This correlated with a maximal decrease of hematocrit and lowest Epo levels at PN7 in mtHEPOR. The proportion of phosphatidylserine-positive RBCs then declined with age (mean fluorescence of 0.6 in adult mtHEPOR mice). In contrast, wtHEPOR mice had the lowest numbers of phosphatidylserine-positive RBCs, which were comparable at all analyzed time-points (mean fluorescence at PN0=0.7±0.1; PN7=1.3±0.3; in adults=0.6±0.04), consistent with the lowest reduction in perinatal hematocrit and very high Epo levels in these mice.In conclusion, our study describes that transient correction of polycythemia of the mtHEPOR PFCP mouse model in the perinatal period is associated with low Epo levels and increased erythrocyte phosphatidylserine exposure. Increased phosphatidylserine exposure on the membrane of mtHEPOR RBCs is consistent with accelerated destruction of these cells by macrophages, thus contributing to the decrease of hematocrit in mtHEPOR neonatal mice.Authorship: JS and BK: equal credit as first authorsAcknowledgment: VD and MH were supported by the Czech Science Foundation (project P301/12/1503) and by the Ministry of Health Czech Republic (project NT13587). DisclosuresNo relevant conflicts of interest to declare.

Late prenatal immune activation causes hippocampal deficits in the absence of persistent inflammation across aging.

BackgroundPrenatal exposure to infection and/or inflammation is increasingly recognized to play an important role in neurodevelopmental brain disorders. It has recently been postulated that prenatal immune activation, especially when occurring during late gestational stages, may also induce pathological brain aging via sustained effects on systemic and central inflammation. Here, we tested this hypothesis using an established mouse model of exposure to viral-like immune activation in late pregnancy.MethodsPregnant C57BL6/J mice on gestation day 17 were treated with the viral mimetic polyriboinosinic-polyribocytidilic acid (poly(I:C)) or control vehicle solution. The resulting offspring were first tested using cognitive and behavioral paradigms known to be sensitive to hippocampal damage, after which they were assigned to quantitative analyses of inflammatory cytokines, microglia density and morphology, astrocyte density, presynaptic markers, and neurotrophin expression in the hippocampus throughout aging (1, 5, and 22 months of age).ResultsMaternal poly(I:C) treatment led to a robust increase in inflammatory cytokine levels in late gestation but did not cause persistent systemic or hippocampal inflammation in the offspring. The late prenatal manipulation also failed to cause long-term changes in microglia density, morphology, or activation, and did not induce signs of astrogliosis in pubescent, adult, or aged offspring. Despite the lack of persistent inflammatory or glial anomalies, offspring of poly(I:C)-exposed mothers showed marked and partly age-dependent deficits in hippocampus-regulated cognitive functions as well as impaired hippocampal synaptophysin and brain-derived neurotrophic factor (BDNF) expression.ConclusionsLate prenatal exposure to viral-like immune activation in mice causes hippocampus-related cognitive and synaptic deficits in the absence of chronic inflammation across aging. These findings do not support the hypothesis that this form of prenatal immune activation may induce pathological brain aging via sustained effects on systemic and central inflammation. We further conclude that poly(I:C)-based prenatal immune activation models are reliable in their effectiveness to induce (hippocampal) neuropathology across aging, but they appear unsuited for studying the role of chronic systemic or central inflammation in brain aging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0437-y) contains supplementary material, which is available to authorized users.

Comparative morphological characteristic of line WAG fetus and newborn rats’ adrenal cortex

Objective – to compare the morphological status of adrenals of newborns and fetuses of WAG-line rats under normal conditions of life of healthy mothers.Materials and methods. As laboratory animals WAG-line rats were used. Pregnant rats were kept in physiological conditions, wherein part of females was removed from an experiment in the later stages of gestation in order to extract the fetuses for further study and from the rest of females the offspring were obtained, which at the end of the first days after birth was removed from an experiment.Results and discussion. In the adrenal cortex of offspring of WAG-line rats under normal conditions of life of healthy mother during the first days of postnatal life significant morphological changes are generated compared with the adrenal glands of the fetus. After birth the adrenal weight decreases rapidly due to the involution of fetal cortex, despite high levels of corticotropin, which is the regulator of the secretion of the fetal zone steroids.Conclusions. In the early postnatal period in the adrenal cortex of newborn rats occurs frank distensibility of zona fasciculata affected by incarceration of glomerular and reticular zone. In the glomerular zone positive increase in the absolute index of its width, increase of density of cells in 1 mm2, increase of area of endocrine cells and their nuclei were detected (P<0.001). For the zona fasciculata of the adrenal cortex of rats offspring, that lived one day, compared with fetuses, severe hyperplasia is defini­tive. Herewith the area of spongiocytes and their nuclei was significantly reduced (P<0.01).

Perinatal induction of Cre recombination with tamoxifen.

Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreER(T2) transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development.

Alveolar development and disease.

Gas exchange after birth is entirely dependent on the remarkable architecture of the alveolus, its formation and function being mediated by the interactions of numerous cell types whose precise positions and activities are controlled by a diversity of signaling and transcriptional networks. In the later stages of gestation, alveolar epithelial cells lining the peripheral lung saccules produce increasing amounts of surfactant lipids and proteins that are secreted into the airspaces at birth. The lack of lung maturation and the associated lack of pulmonary surfactant in preterm infants causes respiratory distress syndrome, a common cause of morbidity and mortality associated with premature birth. At the time of birth, surfactant homeostasis begins to be established by balanced processes involved in surfactant production, storage, secretion, recycling, and catabolism. Insights from physiology and engineering made in the 20th century enabled survival of newborn infants requiring mechanical ventilation for the first time. Thereafter, advances in biochemistry, biophysics, and molecular biology led to an understanding of the pulmonary surfactant system that made possible exogenous surfactant replacement for the treatment of preterm infants. Identification of surfactant proteins, cloning of the genes encoding them, and elucidation of their roles in the regulation of surfactant synthesis, structure, and function have provided increasing understanding of alveolar homeostasis in health and disease. This Perspective seeks to consider developmental aspects of the pulmonary surfactant system and its importance in the pathogenesis of acute and chronic lung diseases related to alveolar homeostasis.

Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy.

Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral.

Patterns of Embryonic Nutrition and Characteristics of Chorioallontoic Placenta in a Viviparous Racerunner Lizard, Eremias multiocellata : a Preliminary Study

In this study we observed the embryonic development processes and compared the weight of eggs with neonates to identify the form of nutritional provisioning in Eremias multiocellata . We also used light microscopy to determine the features of chorioallontoic placenta during late stages of gestation. Neonatal dry weight was approximately 10.3% smaller than egg dry weight. Chorioallantoic membrane and uterine epithelium exhibited an indirect apposition without any fusion. No specialized region, placentome, has been found for nutrition transfer between these membranes. The chorioallontoic placenta of E. multiocellata is consistent with the simplest and most common type, Weekes type I. We infer that E. multiocellata is predominately lecithotrophic, meaning that nourishment for embryonic development is primarily supplied by the yolk. This kind of embryonic nutrition confirms the pattern found in most squamates with a simple placenta. This simple placenta adds anatomical evidence to understand the weak immune interaction between matrix and fetus in E. multiocellata . Finally, we discussed the implications for interpreting the evolution of viviparity and placentation.

Long-term toxicity of reduced graphene oxide nanosheets: Effects on female mouse reproductive ability and offspring development

Reduced graphene oxide (rGO) nanosheets have emerged as novel materials for cancer therapeutics. Their toxicity has attracted much attention since these nanomaterials may have great potential for clinical cancer treatment. Here we report the influence of rGO exposure on female mouse reproductive ability and offspring development. Mouse dams were injected with small or large rGO nanosheets at different doses and time points, pre- or post-fertilization. The sex hormone levels of adult female mice did not significantly change compared with the control group after intravenous injection with either small or large rGO, even at a high dose (25 mg/kg). Mouse dams could produce healthy offspring after treatment with rGO nanosheets before pregnancy and at an early gestational stage (∼6 days). Despite the successful delivery of offspring, malformed fetuses were found among rGO-injected dam litters. All mice had abortions when injected with low (6.25 mg/kg) or intermediate (12.5 mg/kg) doses at a late gestational stage (∼20 days); the majority of pregnant mice died when injected with the high dose of rGO at this stage of pregnancy. Interestingly, all surviving rGO-injected mouse mothers gave birth to another litter of healthy pups. The results presented in this work are important for a deeper understanding of the toxicity of rGO nanosheets on female reproductivity and their offspring development.

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Contrast imaging in mouse embryos using high-frequency ultrasound.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvβ3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Biochemical composition of allantoic fluid during different stages of gestation in gaddi sheep

A study was carried out to estimate the biochemical profile of allantoic fluid during different stages of gestation in Gaddi sheep. Allantoic fluid (10 ml) was collected from 50 gravid Gaddi sheep uteri, at various stages of gestation. The four stages of gestation (stage-I: 30–60 days; stage-II: 61–90 days; stage-III: 91–120 days and stage-IV: 120 days to full term) were identified based on crown rump length of the fetus. The concentrations of creatinine, albumin, urea, uric acid, total bilirubin and direct bilirubin increased with gestation period while the concentration of glucose and total proteins decreased. The level of cholesterol increased from stage I to stage II and III and then decreased in later stages of gestation. The activities of transaminases (alanine aminotransferase and aspartate aminotransferase) and alkaline phosphatase were recorded during different stages of gestation in Gaddi sheep.

Дифференциальная диагностика субклинического изолированного повышения трансаминаз печени во второй половине беременности

The review examines the problem of increase of liver transaminases in the second half of pregnancy. ALT and AST reflect processes of hepatocyte injury, but are not specific for the diagnosis of a particular disease of the liver. However, determining the cause of changes of liver samples in a short time in pregnant women is extremely important because of the significant differences in prognosis for the mother and the fetus, and the tactics of treatment and management of patients. Authorspresent algorithm of differential diagnosis of obstetric and non-obstetric causes of enlarged alanine aminotransferase and aspartate aminotransferase in the later stages of gestation.

Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/μL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

Salmonella enterica Serovar Enteritidis Enterocolitis during Late Stages of Gestation Induces an Adverse Pregnancy Outcome in the Murine Model

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome.

Heat and Moisture Production Rates of a Modern U.S. Swine Breeding, Gestation, and Farrowing Facility

Abstract. Current recommendations for swine building ventilation system design to maintain an environment conducive to animal productivity and well-being are based on heat and moisture production rates measured in the 1950s and 1970s. Advancements in animal genetics, nutrition, and management practices to increase productivity and pork quality since then have led to considerable changes in heat and moisture production rates of modern swine and their housing systems. This study quantifies the total heat production rate (THP) of the animals, which is partitioned into house-level latent heat or moisture production rate (LHP, MP) and house-level sensible heat production rate (SHP), of a 4300-sow breeding, gestation, and farrowing facility in Iowa for 16 consecutive months. The THP was determined using indirect animal calorimetry, LHP or MP was determined from mass balance, and SHP was calculated as the difference between THP and LHP. A mobile air emission monitoring unit equipped with state-of-the-art gas analyzers and a data acquisition system was used to monitor the deep-pit breeding and early gestation barn [1800 head, 204 ±3.2 kg head -1 (mean ±SE)], the deep-pit late gestation barn (1800 head, 219 ±3.0 kg head -1 ), and two shallow-pit (pull-plug) farrowing rooms (40 sows with litters per room, 223 ±0.4 kg head -1 ). Results from the study show that THP at 20°C averages 1.89 W kg -1 for sows in the breeding and early gestation stage, 1.57 W kg -1 for sows in the late gestation stage, and 3.35 W kg -1 for sows and litters in week 0 of the lactation stage. The corresponding house-level LHP for the three stages averages 0.74 W kg -1 (early gestation), 0.57 W kg -1 (late gestation), and 1.98 W kg -1 (lactation, week 0). Finally, the corresponding house-level SHP for the three stages averages 1.15 W kg -1 (early gestation), 1.00 W kg -1 (late gestation), and 1.37 W kg -1 (lactation, week 0). Compared with the ASABE Standards, the values from the current study for gestation sows in their early and late pregnancy stages showed increases of 35% and 12% in THP, 72% and 34% in LHP, and 19% and 3% in SHP, respectively. Values for lactating sows and litters during the first week after parturition showed changes of 29% in THP, 52% in LHP, and 6% in SHP relative to the ASABE Standards. The reductions of THP from day to night for the three stages were 30% (early gestation), 27% (late gestation), and 6% (lactation). These data will help with updating the standards for ventilation design and operation of modern swine housing.

Abstract 2060: Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging

Abstract Introduction: Molecular imaging has the potential to dramatically impact all facets of patient care, from early disease detection to treatment monitoring and follow-up, as a tool for the characterization and measurement of key biomolecules in vivo. In ultrasound, functional and molecular imaging is possible through the use of microbubbles (MB), a contrast agent that can be transformed into targeting agents that bind to vascular biomarkers of interest. In this study, we evaluate whether targeted ultrasound contrast enhanced imaging can provide a quantitative measure of surface receptor expression in endothelial cell populations. Methods: The biomarker endoglin (Eng) was selected for targeting due to its involvement in the processes of development, vascular regulation and disease, including tumor angiogenesis. Endoglin wildtype (Eng+/+), heterozygous null (Eng+/-) and null (Eng-/-) mouse embryonic endothelial cells were cultured on glass slides and mounted in parallel plate flow chambers. MicroMarker microbubbles (endoglin targeted: MBE, isotype control: MBC or untargeted: MBU at 1x107 MB/mL in PBS) were perfused across the cells at 4 mL/min, corresponding to a shear stress of 2 dynes/cm2. Cell and bubble numbers were determined from bright field and phase images (Nikon, 40x), with adhesion quantified as the number of MB/cell. Binding of microbubbles was also assessed in late-gestational stage, isolated, living embryos (Eng+/+, Eng+/-). The highly regulated and controlled activities of normal angiogenesis and vasculogenesis in the mouse embryo make it an excellent surrogate for complex and heterogeneous tumor microenvironments, while genetic manipulation enables the generation of a variety of useful transgenic models. Nonlinear contrast-specific ultrasound imaging, performed at 21MHz with a Vevo-2100 scanner (VisualSonics Inc.), was used to collect contrast mean power ratios (CMPR, representative measure of MB binding) within the brains of each embryo 4 minutes after a bolus injection of MBE, MBC or MBU. Results: Expression levels in cells and embryos were significantly different across genotypes, with endoglin reduced by half in Eng+/- and totally absent in Eng-/- samples. In vitro, microbubble adhesion was found to vary significantly (p<0.05) across genotype populations, with minimal attachment of MBC and MBU compared to MBE. Endoglin-targeted binding was approximately two-fold higher (median = 0.96 MBE/cell) in Eng+/+ compared to Eng+/- (median = 0.42 MBE/cell) cells. In embryo studies, we observed minimal signal from MBC and MBU, while MBE binding was found to be significantly higher in Eng+/+ embryos (CMPR+/+ = 9.71 + 0.66, 95% CI) compared to Eng+/- embryos (CMPR+/- = 5.51 + 0.64, 95% CI). In conclusion, these results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels. Citation Format: Janet M. Denbeigh, Brian A. Nixon, John J.Y. Lee, Mirjana Jerkic, Philip A. Marsden, Michelle Letarte, Mira C. Puri, F. Stuart Foster. Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2014-2060

Cytokine expression in the placenta of pregnant cattle after inoculation with Neospora caninum

Neospora caninum is recognized as a major cause of reproductive losses worldwide but its pathogenesis is not completely understood. Immune mediated placental pathology has been reported as being responsible for compromising pregnancy probably due to the adverse effects of exacerbated Th1 type response at the maternal–foetal interface. Different clinical outcomes are known to occur following experimental infections of cattle at different stages of gestation, with foetal death being the most common finding during early gestation, and the birth of live congenitally infected calves following infection later in gestation. The aim of the current study was to characterize the cytokine expression in the placenta of cattle experimentally challenged with tachyzoites of the Nc-1 strain during early, mid and late gestation. Moderate to severe infiltration of IL-12, IFN-γ and TNF-α expressing cells was observed in the placentas collected at early gestation and this infiltration was more pronounced in the samples collected from challenged dams carrying non-viable foetuses, compared with the mothers carrying viable foetuses. In contrast, the infiltration of Th1 cytokine expressing-cells was mild following N. caninum infection in mid gestation and scarce during infection in late gestation. Scarce expression of IL-4 was observed in the placentas from N. caninum-challenged and negative control animals throughout gestation. The milder Th1 immune response observed during later stages of gestation following Nc-1 infection could partially explain the less severe clinical outcome when compared to early pregnancy.

Comparison of Anti-pneumococcal Antibodies in Cord Blood From Australian Indigenous and Gambian Neonates and the Implications for Otitis Media

Australian indigenous infants experience the highest incidence of chronic suppurative and acute otitis media in the world with many babies developing disease in the early postnatal period. Streptococcus pneumoniae is the major cause of otitis media in this population. Infants are protected against bacterial disease in the first months of life by passive transfer of maternal antibody across the placenta during the late stages of gestation. We hypothesized that reduced passive immunity may contribute to increased disease risk in this population. We compared the concentrations and function of serotype-specific IgG in cord serum from Australian indigenous neonates and Gambian neonates, the latter experiences a similar socioeconomic status to Australian indigenous neonates. Serotype-specific IgG, IgG1 and IgG2 were measured using a modified 3rd generation enzyme linked immunosorbent assay based on World Health Organization recommendations. Antibody avidity was measured using a modified sodium thiocyanate elution method. Australian indigenous neonates had significantly increased levels of serotype-specific IgG compared with Gambian populations for 6 of 12 serotypes (P < 0.02). There was no significant difference in antibody function, as measured by antibody avidity, between the 2 groups. An increased risk for otitis media in Australian indigenous neonates is not primarily determined by specific antibody titers against pneumococcal bacteria. Further investigation into the possible roles of the innate immune response and Eustachian tube dysfunction in the development of chronic otitis media amongst Australian indigenous infants is warranted.

Metabolic fingerprint of Gestational Diabetes Mellitus

Gestational Diabetes (GDM) is causing severe short- and long-term complications for mother, fetus or neonate. As yet, the metabolic alterations that are specific for the development of GDM have not been fully determined, which also precludes the early diagnosis and prognosis of this pathology. In this pilot study, we determine the metabolic fingerprint, using a multiplatform LC-QTOF/MS, GC-Q/MS and CE-TOF/MS system, of plasma and urine samples of 20 women with GDM and 20 with normal glucose tolerance in the second trimester of pregnancy. Plasma fingerprints allowed for the discrimination of GDM pregnant women from controls. In particular, lysoglycerophospholipids showed a close association with the glycemic state of the women. In addition, we identified some metabolites with a strong discriminative power, such as LPE(20:1), (20:2), (22:4); LPC(18:2), (20:4), (20:5); LPI(18:2), (20:4); LPS(20:0) and LPA(18:2), as well as taurine-bile acids and long-chain polyunsaturated fatty acid derivatives. Finally, we provide evidence for the implication of these compounds in metabolic routes, indicative of low-grade inflammation and altered redox-balance, that may be related with the specific pathophysiological context of the genesis of GDM. This highlights their potential use as prognostic markers for the identification of women at risk to develop severe glucose intolerance during pregnancy. Gestational Diabetes Mellitus (GDM) is increasing worldwide and, although diabetes usually remits after pregnancy, women with GDM have a high risk of developing postpartum type 2-diabetes, particularly when accompanied by obesity. Therefore, understanding the pathophysiology of GDM, as well as the identification of potentially modifiable risk factors and early diagnostic markers for GDM are relevant issues. In the present study, we devised a multiplatform metabolic fingerprinting approach to obtain a comprehensive picture of the early metabolic alternations that occur in GDM, and may reflect on the specific pathophysiological context of the disease. Future studies at later stages of gestation will allow us to validate the discriminant power of the identified metabolites.

Late prenatal dexamethasone and phenotype variations in 46,XX CAH: Concerns about current protocols and benefits for surgical procedures

ObjectiveTo describe the action of prenatal dexamethasone (PreDex) on the anatomy of female congenital adrenal hyperplasia (CAH) genitalia when started at later stages of gestation. Materials and methodsOur group follows a large cohort of French CAH patients who underwent PreDex therapy, of whom 258 were recently reported. Four 46,XX patients with a delayed PreDex treatment presented with a virilized genitalia and required surgical reconstruction. This is a retrospective report on genital phenotyping at the time of surgery of these four patients who began PreDex therapy at 8, 12, 20, and 28 weeks of gestation. ResultsAlthough this series is limited in number, the anatomical description of the length of the genital tubercle, the height of the urethra–vaginal confluence, and the degree of fusion of the genital folds seems to be dependent upon the starting date of PreDex. Most PreDex treatments prescribed up to now have covered the full duration of gestation. ConclusionsOur findings suggest that PreDex therapy could be limited to the period of the partitioning window. It is hoped that further prospective multicentric clinical studies will obtain ethical approval in order to elucidate the place and protocols of PreDex therapy in the management of CAH.