OBJECTIVE Uterine overdistention is thought to induce preterm labor in women with twin and multiple pregnancies, but the pathophysiology remains unclear. We investigated for the first time the pathogenesis of preterm birth associated with rapid uterine distention in a pregnant nonhuman primate model. STUDY DESIGN A nonhuman primate model of uterine overdistention was created using preterm chronically catheterized pregnant pigtail macaques (Macaca nemestrina) by inflation of intraamniotic balloons (N = 6), which were compared to saline controls (N = 5). Cesarean delivery was performed due to preterm labor or at experimental end. Microarray, quantitative reverse transcriptase polymerase chain reaction, Luminex (Austin, TX), and enzyme-linked immunosorbent assay were used to measure messenger RNA (mRNA) and/or protein levels from monkey (amniotic fluid, myometrium, maternal plasma) and human (amniocytes, amnion, myometrium) tissues. Statistical analysis employed analysis of covariance and Wilcoxon rank sum. Biomechanical forces were calculated using the law of Laplace. RESULTS Preterm labor occurred in 3 of 6 animals after balloon inflation and correlated with greater balloon volume and uterine wall stress. Significant elevations of inflammatory cytokines and prostaglandins occurred following uterine overdistention in an “inflammatory pulse” that correlated with preterm labor (interleukin [IL]-1β, tumor necrosis factor [TNF]-α, IL-6, IL-8, CCL2, prostaglandin E2, prostaglandin F2α, all P < .05). A similar inflammatory response was observed in amniocytes in vitro following mechanical stretch (IL1β, IL6, and IL8 mRNA multiple time points, P < .05), in amnion of women with polyhydramnios (IL6 and TNF mRNA, P < .05) and in amnion (TNF-α) and myometrium of women with twins in early labor (IL6, IL8, CCL2, all P < .05). Genes differentially expressed in the nonhuman primate after balloon inflation and in women with polyhydramnios and twins are involved in tissue remodeling and muscle growth. CONCLUSION Uterine overdistention by inflation of an intraamniotic balloon is associated with an inflammatory pulse that precedes and correlates with preterm labor. Our results indicate that inflammation is an early event after a mechanical stress on the uterus and leads to preterm labor when the stress is sufficiently great. Further, we find evidence of uterine tissue remodeling and muscle growth as a common, perhaps compensatory, response to uterine distension.

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.

RationaleContinuous performance tests (CPTs) are widely used to assess attentional processes in a variety of disorders including Alzheimer’s disease and schizophrenia. Common human CPTs require discrimination of sequentially presented, visually patterned ‘target’ and ‘non-target’ stimuli at a single location.ObjectivesThe aims of this study were to evaluate the performance of three popular mouse strains on a novel rodent touchscreen test (rCPT) designed to be analogous to common human CPT variants and to investigate the effects of donepezil, a cholinesterase inhibitor and putative cognitive enhancer.MethodsC57BL/6J, DBA/2J and CD1 mice (n = 15–16/strain) were trained to baseline performance using four rCPT training stages. Then, probe tests assessed the effects of parameter changes on task performance: stimulus size, duration, contrast, probability, inter-trial interval or inclusion of flanker distractors. rCPT performance was also evaluated following acute administration of donepezil (0–3 mg/kg, i.p.).ResultsC57BL/6J and DBA/2J mice showed similar acquisition rates and final baseline performance following rCPT training. On probe tests, rCPT performance of both strains was sensitive to alteration of visual and/or attentional demands (stimulus size, duration, contrast, rate, flanker distraction). Relative to C57BL/6J, DBA/2J mice exhibited (1) decreasing sensitivity (d′) across the 45-min session, (2) reduced performance on probes where the appearance of stimuli or adjacent areas were changed (size, contrast, flanking distractors) and (3) larger dose- and stimulus duration-dependent changes in performance following donepezil administration. In contrast, CD1 mice failed to acquire rCPT (stage 3) and pairwise visual discrimination tasks.ConclusionsrCPT is a potentially useful translational tool for assessing attention in mice and for detecting the effects of nootropic drugs.

Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP‐1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that (NFκB) activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP‐1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP‐1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO‐treated controls). Using an LPS (Escherichia coli: serotype O111)‐induced PTL model that selectively activates AP‐1 but not NFkB, we show that myometrial AP‐1 activation drives production of cytokines (Il‐6, Il‐8, and Il‐1β), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox‐2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL‐1β, and IL‐1β cleavage, were increased following LPS‐induced activation of AP‐1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that (NFκB) activation is not a functional requirement for infection/inflammation‐induced preterm labor and that AP‐1 activation is sufficient to drive inflammatory pathways that cause PTL.—MacIntyre, D. A., Lee, Y. S., Migale, R., Herbert, B. R., Waddington, S. N., Peebles, D., Hagberg, H., Johnson, M. R., Bennett, P. R. Activator protein 1 is a key terminal mediator of inflammation‐induced preterm labor in mice. FASEB J. 28, 2358–2368 (2014). http://www.fasebj.org

BackgroundPreterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data.MethodsMyometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data.ResultsWe identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse.ConclusionsLabor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human “functional progesterone withdrawal.” This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-016-0632-4) contains supplementary material, which is available to authorized users.

Objective-To produce transgenic mice expressing the D374Y variant of the human proprotein convertase subtilisin/kexin type 9 (PCSK9) gene at physiological levels to investigate the mechanisms causing hypercholesterolemia and accelerated atherosclerosis. Methods and Results-A bacterial artificial chromosome containing PCSK9 and its flanking regions was modified to introduce the D374Y mutation and a C-terminal myc 2 tag. Transgenic mice that expressed 1 copy of the mutant or wild-type (WT) PCSK9 bacterial artificial chromosome were produced. Human PCSK9 mRNA was expressed at levels comparable to endogenous pcsk9 and with the same tissue specificity. The expression of D374Y or WT human PCSK9 increased the serum cholesterol level and reduced hepatic low-density lipoprotein receptor protein levels in the transgenic mice compared with bacterial artificial chromosome-negative controls; however, the effects were more marked in D374Y mice. The effect of a high-cholesterol diet on increasing serum cholesterol level was greater in D374Y mice, and atherosclerotic plaques after 15 weeks were more extensive in mice expressing D374Y than in WT PCSK9. D374Y mice secreted more triglyceride-rich lipoproteins into the circulation than WT mice. Conclusion-The expression of human D374Y PCSK9 at physiological levels produced a phenotype that closely matched that found in heterozygous D374Y patients and suggested that reduced low-density lipoprotein receptor activity is not the sole cause of their hypercholesterolemia. 1 The gene, which encodes a putative proprotein convertase, was first identified as a gene that was upregulated in neural cells undergoing apoptosis (neural apoptosis-regulated convertase-1) 2 ; thus, no underlying causative mechanism for hypercholesterolemia was apparent. Measurement of lipoprotein turnover in 2 patients heterozygous for a PCSK9 mutation suggested that apolipoprotein B (apoB) synthesis was increased. 3 Also, microarray analyses of global gene expression in mice fed a highcholesterol diet 4 or overexpressing the transcription factor SREBP 5 revealed that expression of this gene was regulated by sterols and that it was, therefore, likely to play some role in cholesterol homeostasis. Adenoviral-mediated expression of PCSK9 in the liver of mice resulted in reduction of hepatic low-density lipoprotein (LDL) receptor protein, with no change in the LDL receptor (LDLR) mRNA 6 ; however, there was no apparent difference between mice expressing wildtype (WT) and mutant PCSK9. 7 On the other hand, genetic ablation of pcsk9 in mice resulted in increased LDLR levels and a reduced plasma cholesterol level 8 ; in the general population, heterozygosity for a null mutation in PCSK9 is associated with reductions in serum cholesterol level and the risk of coronary heart disease. 9 See accompanying article on page 1279Thus, it rapidly became clear that PCSK9 normally regulates LDLR levels and thereby influences serum cholesterol level, and that the rare mutations found in patients with FH are gain of function. These obse...

ObjectivesWe tested the hypothesis that there is a positive association between maternal body mass index (BMI) and the concentration of appetite-regulating hormones leptin, insulin, ghrelin and resistin in breast milk. We also aimed to describe the change in breast milk hormone concentration within each feed, and over time.SettingMothers were recruited from the postpartum ward at a university hospital in London. Breast milk samples were collected at the participants’ homes.ParticipantsWe recruited 120 healthy, primiparous, breastfeeding mothers, aged over 18 years. Mothers who smoked, had multiple births or had diabetes were excluded. Foremilk and hindmilk samples were collected from 105 women at 1 week postpartum and 92 women at 3 months postpartum.Primary and secondary outcome measuresWe recorded maternal and infant anthropometric measurements at each sample collection and measured hormone concentrations using a multiplex assay.ResultsThe concentration of leptin in foremilk correlated with maternal BMI at the time of sample collection, at 7 days (r=0.31, p=0.02) and 3 months postpartum (r=0.30, p=<0.00). Foremilk insulin correlated with maternal BMI at 3 months postpartum (r=0.22, p=0.04). Breast milk ghrelin and resistin were not correlated with maternal BMI. Ghrelin concentrations at 3 months postpartum were increased in foremilk compared with hindmilk (p=0.01). Concentrations of ghrelin were increased in hindmilk collected at 1  week postpartum compared with samples collected at 3 months postpartum (p=0.03). A trend towards decreased insulin concentrations in hindmilk was noted. Concentrations of leptin and resistin were not seen to alter over a feed.ConclusionsA positive correlation between maternal BMI and foremilk leptin concentration at both time points studied, and foremilk insulin at 3 months postpartum was observed. This may have implications for infant appetite regulation and obesity risk.

Intrauterine inflammation is recognized as a key mediator of both normal and preterm birth but is also associated with neonatal neurological injury. Lipopolysaccharide (LPS) is often used to stimulate inflammatory pathways in animal models of infection/inflammation-induced preterm labor; however, inconsistencies in maternal and neonatal responses to LPS are frequently reported. We hypothesized that LPS serotype-specific responses may account for a portion of these inconsistencies. Four different Escherichia coli LPS serotypes (O111:B4, O55:B5, O127:B8, and O128:B12) were administered to CD1 mice via intrauterine injection at gestational day 16. Although control animals delivered at term 60 ± 15 hours postinjection (p.i.), those administered with O111:B4 delivered 7 ± 2 hours p.i., O55:B5 delivered 10 ± 3 hours p.i., O127:B8 delivered 16 ± 10 hours p.i., and O128:B12 delivered 17 ± 2 hours p.i. (means ± SD). A correlation between the onset of preterm labor and myometrial activation of the inflammatory transcription factor, activator protein 1, but not NF-κB was observed. Specific LPS serotypes induced differential activation of downstream contractile and inflammatory pathways in myometrium and neonatal pup brain. Our findings demonstrate functional disparity in inflammatory pathway activation in response to differing LPS serotypes. Selective use of LPS serotypes may represent a useful tool for targeting specific inflammatory response mechanisms in these models.