Uterine NK cells are critical in shaping DC immunogenic functions compatible with pregnancy progression.

Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression

Chronic Naltrexone Therapy Is Associated with Improved Cardiac Function in Volume Overloaded Rats

Purpose: Myocardial opioid receptors were demonstrated in animals and humans and seem to colocalize with membranous and sarcolemmal calcium channels of the excitation-contraction coupling in the left ventricle (LV). Therefore, this study investigated whether blockade of the cardiac opioid system by naltrexone would affect cardiac function and neurohumoral parameters in Wistar rats with volume overload-induced heart failure. Methods: Volume overload in Wistar rats was induced by an aortocaval fistula (ACF). Left ventricular cardiac opioid receptors were identified by immunohistochemistry and their messenger ribonucleic acid (mRNA) as well as their endogenous ligand mRNA quantified by real-time polymerase chain reaction (RT-PCR). Following continuous delivery of either the opioid receptor antagonist naltrexone or vehicle via minipumps (n = 5 rats each), hemodynamic and humoral parameters were assessed 28 days after ACF induction. Sham-operated animals served as controls. Results: In ACF rats mu-, delta-, and kappa-opioid receptors colocalized with voltage-gated L-type Ca2+ channels in left ventricular cardiomyocytes. Chronic naltrexone treatment of ACF rats reduced central venous pressure (CVP) and left ventricular end-diastolic pressure (LVEDP), and improved systolic and diastolic left ventricular functions. Concomitantly, rat brain natriuretic peptide (rBNP-45) and angiotensin-2 plasma concentrations which were elevated during ACF were significantly diminished following naltrexone treatment. In parallel, chronic naltrexone significantly reduced mu-, delta-, and kappa-opioid receptor mRNA, while it increased the endogenous opioid peptide mRNA compared to controls. Conclusion: Opioid receptor blockade by naltrexone leads to improved LV function and decreases in rBNP-45 and angiotensin-2 plasma levels. In parallel, naltrexone resulted in opioid receptor mRNA downregulation and an elevated intrinsic tone of endogenous opioid peptides possibly reflecting a potentially cardiodepressant effect of the cardiac opioid system during volume overload

Perinatal and 2-year neurodevelopmental outcome in late preterm fetal compromise: the TRUFFLE 2 randomised trial protocol

Introduction: Following the detection of fetal growth restriction, there is no consensus about the criteria that should trigger delivery in the late preterm period. The consequences of inappropriate early or late delivery are potentially important yet practice varies widely around the world, with abnormal findings from fetal heart rate monitoring invariably leading to delivery. Indices derived from fetal cerebral Doppler examination may guide such decisions although there are few studies in this area. We propose a randomised, controlled trial to establish the optimum method of timing delivery between 32 weeks and 36 weeks 6 days of gestation. We hypothesise that delivery on evidence of cerebral blood flow redistribution reduces a composite of perinatal poor outcome, death and short-term hypoxia-related morbidity, with no worsening of neurodevelopmental outcome at 2 years. Methods and analysis: Women with non-anomalous singleton pregnancies 32+0 to 36+6 weeks of gestation in whom the estimated fetal weight or abdominal circumference is <10th percentile or has decreased by 50 percentiles since 18-32 weeks will be included for observational data collection. Participants will be randomised if cerebral blood flow redistribution is identified, based on umbilical to middle cerebral artery pulsatility index ratio values. Computerised cardiotocography (cCTG) must show normal fetal heart rate short term variation (≥4.5 msec) and absence of decelerations at randomisation. Randomisation will be 1:1 to immediate delivery or delayed delivery (based on cCTG abnormalities or other worsening fetal condition). The primary outcome is poor condition at birth and/or fetal or neonatal death and/or major neonatal morbidity, the secondary non-inferiority outcome is 2-year infant general health and neurodevelopmental outcome based on the Parent Report of Children's Abilities-Revised questionnaire. Ethics and dissemination: The Study Coordination Centre has obtained approval from London-Riverside Research Ethics Committee (REC) and Health Regulatory Authority (HRA). Publication will be in line with NIHR Open Access policy. Trial registration number: Main sponsor: Imperial College London, Reference: 19QC5491. Funders: NIHR HTA, Reference: 127 976. Study coordination centre: Imperial College Healthcare NHS Trust, Du Cane Road, London, W12 0HS with Centre for Trials Research, College of Biomedical & Life Sciences, Cardiff University. IRAS Project ID: 266 400. REC reference: 20/LO/0031. ISRCTN registry: 76 016 200

A targeted amplicon sequencing panel to simultaneously identify mosquito species and Plasmodium presence across the entire Anopheles genus

International audienceAbstract Anopheles is a diverse genus of mosquitoes comprising over 500 described species, including all known human malaria vectors. While a limited number of key vector species have been studied in detail, the goal of malaria elimination calls for surveillance of all potential vector species. Here, we develop a multilocus amplicon sequencing approach that targets 62 highly variable loci in the Anopheles genome and two conserved loci in the Plasmodium mitochondrion, simultaneously revealing both the mosquito species and whether that mosquito carries malaria parasites. We also develop a cheap, nondestructive, and high‐throughput DNA extraction workflow that provides template DNA from single mosquitoes for the multiplex PCR, which means specimens producing unexpected results can be returned to for morphological examination. Over 1000 individual mosquitoes can be sequenced in a single MiSeq run, and we demonstrate the panel’s power to assign species identity using sequencing data for 40 species from Africa, Southeast Asia, and South America. We also show that the approach can be used to resolve geographic population structure within An. gambiae and An. coluzzii populations, as the population structure determined based on these 62 loci from over 1000 mosquitoes closely mirrors that revealed through whole genome sequencing. The end‐to‐end approach is quick, inexpensive, robust, and accurate, which makes it a promising technique for very large‐scale mosquito genetic surveillance and vector control

Combined depletion of DC and NK cells arrests decidual development leading to early pregnancy loss.

<p>(A) Experimental design: upon cohabitation with Balb/c males, plug-positive CD11c-DTR females were injected i.p. with either DT, anti-asialo GM1 or a combination of both for DC and/or NK cell depletion on gd 4.5, as described in Methods. Depletion of both subsets was confirmed using FACS analysis of uterine cell suspensions obtained during gd 5.5. (B) DT and anti-asialo GM1 treatment on gd 4.5 efficiently deplete DC and NK cells from the uterus. Left panels: Percentage of CD11c⁺ cells, which in CD11c-DTR mice co-express a GFP transgene, was analysed by FACS in control (Ctrl) and DT injected (ØDC) mice. Right panels: representative flow cytometric analysis of uterine cell suspensions obtained during gd 5.5 for the presence of NK cells. (C) Microscopical assessment of H&E stained serial sections revealed abnormalities in the decidual architecture of DC depleted (ØDC) and double DC-NK depleted (ØDCØNK) implantation sites (IS), with abnormal development of the antimesometrial and mesometrial compartments and signs of embryo arrest on gd 6.5. (D) Morphometric analysis of the IS diameter at gd 5.5 and 6.5. Sizes in the different groups analysed are presented as percentage of Ctrl IS. (E) Uterine IL-11 mRNA levels on gd 5.5 and 6.5, as measured by RT-PCR. The progressive increase on IL-11 expression in Ctrl females was abrogated by all treatments, with significantly reduced levels observed in ØDCØNK on gd 6.5. (F) Immunohistochemical staining of phosphorylated histone H3 (PHH3) in the mouse uterus during decidualization on gd 5.5 and 6.5. Right panel: quantification of PHH3⁺ stromal cells at the AM and M regions of the implantation sites. PHH3⁺ cells were counted per mm² using magnification ×400. (G) Immunofluorescence analysis of connexin 43 (Cx-43) on gd 5.5 and 6.5. The photomicrographs of representative uterine sections are shown at 50×. Abbreviations: M = mesometrial pole, em = embryo and AM = anti-mesometrial pole of the implantation sites. Arrows indicate the regions where Cx-43 is differentially expressed in ØNK, ØDC, ØDCØNK and control implantations on gd 5.5 and 6.5. In all figures, the bars denote the means for each group in which 7 mice/group were analysed. *, ** and *** denote <i>p<0.05</i>, <i>p<0.01</i> and <i>p<0.001</i> respectively, as analysed by the Tukey's test. Scale bars: 200 µm.</p

NK cells regulate DC function during early gestation.

<p>(A) Confirmation of differential gene expression on ↑DCØNK implantation sites. The pictures display qPCR results confirming up-regulated expression of chemokine (C-X-C motif) receptor 2 (Cxcr2), protein tyrosine phosphatase, non-receptor type 5 (Ptpn5), Interferon regulatory factor 7 (Irf7), signal-regulatory protein beta 1A and B (Sirpb1), and secreted phosphoprotein 1 (Spp1), as was noted on microarray analysis comparing ↑DC <i>vs</i> ↑DCØNK transcription profiles on gd 5.5. In all figures, *, ** and *** denote <i>p<0.05</i>, <i>p<0.01</i> and <i>p<0.001</i> respectively, as analysed by Mann–Whitney rank-sum test. (B) Up-regulation of progesterone receptor (Pgr) was analysed on IHC staining of uterine sections obtained during gd 5.5. (C–D) Selected enrichment plot of Gene Set Enrichment Analysis (GSEA). All detectable genes in the microarray analysis were ranked according to fold change and adjusted p-value (see Material & Methods) and compared to sets of differentially expressed genes (DEGs) from other study as well as gene sets from the GSEA database. Selected enrichment plot for gene sets enriched towards the genes up-regulated in ↑DCØNK are shown. X-axis represents the ranked data set with the most significantly up-regulated genes on the left. Black vertical bars represent the positions of the genes of the compared gene sets. The references for the gene sets can be found in the text.</p

Summary of the results obtained in microarray experiments comparing the transcription profile of ↑DC and ↑DCØNK implantation sites obtained during gd 5.5.

<p>Differential expression with respect to control (↑DC) mice is expressed as fold change in mRNA levels. Putative functions associated to each gene are indicated based on the Gene Ontology project database (<a href="http://www.geneontology.org/" target="_blank">http://www.geneontology.org/</a>). The complete list of differentially expressed genes (FDR 10%) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046755#pone.0046755.s002" target="_blank">Table S2</a>.</p

NK cells become critical for normal pregnancy progression upon DC expansion.

<p>(A) Model for DC expansion during early pregnancy. Upon plug detection; female mice were injected daily with 10 µg FL i.p. This strategy was combined with a single anti-asialo GM1 injection on gd 4.5 to analyse effects of NK depletion upon expansion of DC (↑DCØNK mice). (B) Representative flow cytometric analysis of uterine cell suspensions obtained on gd 5.5 to confirm DC expansion in the uterus upon FL treatment. FL treated (↑DC) mice exhibited percentages of CD11c⁺ cells approximately 3-fold higher than controls. (C) Phenotypic characterization of uterine DC expanded upon FL treatment. Uterine cells from ↑DC and control females were isolated on gd 4.5, and subject to FACS analysis for the expression of CD11c, PDCA-1, CD4, CD8, MHC-II, CD80, and DNGR-1. FL treatment was associated with increased percentages of CD11c⁺DNGR-1⁺ cells, whereas other markers exhibited similar expression levels with respect to control mice. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046755#s3" target="_blank">Results</a> correspond to at least three independent experiments using three to five animals/group. (D) Histological analysis of DC expanded implantation sites. While implantation was normal upon DC expansion, a regression of embryo development is observed on gd 6.5 when both strategies were combined ↑DCØNK mice. Scale bar: 200 µm. (E) Size of the IS registered in ↑DC and ↑DCØNK mice at gd 5.5 and 6.5 expressed as percentage of control. (F) Macroscopical appearance of the uterus and IS registered during gd 7.5. The pictures show the normal phenotype of control and ↑DC IS in contrast to that observed in ↑DCØNK mice, which exhibit completely resorbed embryos.</p