Effect of maternal administration of betamethasone on peripheral arterial development in fetal rabbit lungs

Objectives: Glucocorticoids promote lung maturation and reduce the incidence of respiratory distress syndrome in premature newborns. We hypothesized that betamethasone (BM), which is known to induce thinning of the alveolar walls, would also thin the arterial media and adventitia of intra-parenchymatic vessels in developing rabbit lungs. Study Design: 112 fetuses from 21 time-mated, pregnant, giant white rabbits received maternal injections of BM at either 0.05 or 0.1 mg/kg/day on days 25-26 of gestational age. Controls received either saline (10 does, 56 fetuses) or no injection (10 does, 59 fetuses). Fetuses were harvested from day 27 onwards until term (day 31). 44 additional fetuses (8 does) were harvested between days 23 and 26. Endpoints were wet lung-to-body weight ratio, vascular morphometric indices and immunohistochemistry staining for α-smooth muscle actin, Flk-1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). ANOVA (Tukey's test) and independent t test (p < 0.05) were used for comparison between BM and saline groups. Results: Maternal BM injected on days 25-26 to pregnant rabbits induced a significant decrease in fetal body and lung weight and the lung-to-body weight ratio in the preterm pups shortly after injection. BM led to a dose-dependent thinning of the arterial media and adventitia (pulmonary arteries with an external diameter (ED) of <100 μm), to an increase in the percentage of non-muscularized peripheral vessels (ED <60 μm), in eNOS and VEGF immunoreactivity of the endothelial and smooth muscle cells in the pulmonary vessels and to an increase in Flk-1-positive pulmonary epithelial cell density. Conclusions: Maternal administration of BM caused thinning of the arterial wall of pulmonary vessels (ED <100 μm) and a decrease in muscularization in peripheral vessels (ED <60 μm). This coincided with increased expression of Flk-1 in the endothelium and smooth muscle cells of the pulmonary arteries. All the effects studied were dose-dependent. Copyrigh

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP. KEYWORDS: Craniopharyngioma; IL1-β; Inflammasome; MAPK/ERK pathway; Odontogenesis; Paracrine signalling; Trametini

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Signaling molecules in the fetal rabbit model for congenital diaphragmatic hernia.

RATIONALE AND OBJECTIVES: Little is known about molecular changes in lungs of fetal rabbits with surgically induced diaphragmatic hernia (DH). Therefore, we examined in this model gene expressions of pivotal molecules for the developing lung. METHODS: At day 23 of gestation, DH was created in 12 fetuses from 4 does. Both lungs from six live DH fetuses and from six unoperated controls were harvested and weighed at term. Transcription of 15 genes involved in alveolarization, angiogenesis, regulation of vascular tone, or epithelial maturation was investigated by real-time quantitative polymerase chain reaction. MAIN RESULTS: DH decreased lung-to-body weight ratio (P < 0.001). A bilateral downregulation was seen for genes encoding for tropoelastin (P < 0.01), lysyl oxidase (P < 0.05), fibulin 5 (P < 0.05), and cGMP specific phosphodiesterase 5 (P < 0.05). Lower mRNA levels for endothelial nitric oxide synthase occurred in the ipsilateral lung (P < 0.05). CONCLUSIONS: Experimental DH in fetal rabbits disrupted transcription of genes implicated in lung growth and function. Similarities with the human disease make this model appropriate for investigation of new prenatal therapies. Pediatr Pulmonol. © 2012 Wiley Periodicals, Inc.JOURNAL ARTICLEFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Markers of extracellular matrix and epithelial cell maturation after fetal tracheal occlusion in newborn rabbits with lung hypoplasia induced by diaphragmatic hernia.

Background and aims: Tracheal occlusion (TO), a prenatal treatment promoting lung growth, improves defective elastogenesis in congenital diaphragmatic hernia (CDH). Because the transforming growth factor-ß (TGF-ß) regulates extracellular matrix (ECM) during late lung development, we hypothesized that the effects of TO on ECM would be mediated by the TGF-ß/Smad2 pathway. We therefore investigated the effects of TO on lung connective tissue composition and function, and on TGF-ß signaling expression in an experimental model of CDH. Methods: Rabbit fetuses were randomized twice during gestation: at day 23 (pseudoglandular phase), for a DH or a sham thoracotomy, and five days later (canalicular phase) for a tracheal ligation or a sham operation. Non-operated littermates served as controls. At term (day 31), live fetuses underwent an evaluation of global and peripheral lung mechanics, followed by pulmonary tissue sampling for morphometric and biological studies. Results: TO normalized the lung to body weight ratio and morphometry of airways in DH fetuses compared to controls. TO did not improve dynamic resistance and compliance in DH fetuses, and even resulted in worsening of lung function in sham DH fetuses. TO had no effect on peripheral lung mechanics assessed by forced oscillation technique. In DH fetuses, gene expressions for tropoelastin and lysyl-oxidase were downregulated (by 50%) and restored by TO. Besides, TO stimulates gene expressions for fibulin-5, types I and III procollagens, integrins (respectively by 700%, 180% and 200%), and the density of elastic fibers was increased. These effects on ECM composition co-occurred with an increase in gene and protein expressions for TGF-ß, and a rise of the phosphorylation of Smad2 (by 100%). Conclusions: In surgically induced CDH, sustained TO restores elastogenesis, but also enhances the synthesis of many ECM components, coinciding with an activation of the TGF-ß/Smad2 signaling pathway. These biological effects, together with decreased level of surfactant after TO, may contribute to the impairment of neonatal lung mechanics.info:eu-repo/semantics/nonPublishe

Alveolarization genes modulated by fetal tracheal occlusion in the rabbit model for congenital diaphragmatic hernia: a randomized study.

BACKGROUND: The mechanisms by which tracheal occlusion (TO) improves alveolarization in congenital diaphragmatic hernia (CDH) are incompletely understood. Therefore transcriptional and histological effects of TO on alveolarization were studied in the rabbit model for CDH. The question of the best normalization strategy for gene expression analysis was also addressed. METHODS: Fetal rabbits were randomized for CDH or sham operation on gestational day 23/31 and for TO or sham operation on day 28/31 resulting in four study groups. Untouched littermates were added. At term and before lung harvest, fetuses were subjected to mechanical ventilation or not. Quantitative real-time PCR was performed on lungs from 4-5 fetuses of each group with and without previous ventilation. Stability of ten housekeeping genes (HKGs) and optimal number of HKGs for normalization were determined, followed by assessment of HKG expression levels. Expression levels of eleven target genes were studied in ventilated lungs, including genes regulating elastogenesis, cell-environment interactions, and thinning of alveolar walls. Elastic staining, immunohistochemistry and Western blotting completed gene analysis. RESULTS: Regarding HKG expression, TO increased β-actin and β-subunit of ATP synthase. Mechanical ventilation increased β-actin and β2-microglobulin. Flavoprotein subunit of succinate dehydrogenase and DNA topoisomerase were the most stable HKGs. CDH lungs showed disorganized elastin deposition with lower levels for tropoelastin, fibulin-5, tenascin-C, and α6-integrin. After TO, CDH lungs displayed a normal pattern of elastin distribution with increased levels for tropoelastin, fibulin-5, tenascin-C, α6-integrin, ß1-integrin, lysyl oxidase, and drebrin. TO increased transcription and immunoreactivity of tissue inhibitor of metalloproteinase-1. CONCLUSIONS: Experimental TO might improve alveolarization through the mechanoregulation of crucial genes for late lung development. However part of the transcriptional changes involved genes that were not affected in CDH, raising the question of TO-induced disturbances of alveolar remodeling. Attention should also be paid to selection of HKGs for studies on mechanotransduction-mediated gene expressions.info:eu-repo/semantics/publishedJournal articl

Assessment of diffusion-weighted MRI in predicting response to neoadjuvant chemotherapy in breast cancer patients

Abstract To compare region of interest (ROI)-apparent diffusion coefficient (ADC) on diffusion-weighted imaging (DWI) measurements and Ki-67 proliferation index before and after neoadjuvant chemotherapy (NACT) for breast cancer. 55 women were enrolled in this prospective single-center study, with a final population of 47 women (49 cases of invasive breast cancer). ROI-ADC measurements were obtained on MRI before and after NACT and were compared to histological findings, including the Ki-67 index in the whole study population and in subgroups of “pathologic complete response” (pCR) and non-pCR. Nineteen percent of women experienced pCR. There was a significant inverse correlation between Ki-67 index and ROI-ADC before NACT (r = − 0.443, p = 0.001) and after NACT (r = − 0.614, p < 0.001). The mean Ki-67 index decreased from 45.8% before NACT to 18.0% after NACT (p < 0.001), whereas the mean ROI-ADC increased from 0.883 × 10–3 mm2/s before NACT to 1.533 × 10–3 mm2/s after NACT (p < 0.001). The model for the prediction of Ki67 index variations included patient age, hormonal receptor status, human epidermal growth factor receptor 2 status, Scarff-Bloom-Richardson grade 2, and ROI-ADC variations (p = 0.006). After NACT, a significant increase in breast cancer ROI-ADC on diffusion-weighted imaging was observed and a significant decrease in the Ki-67 index was predicted. Clinical trial registration number: clinicaltrial.gov NCT02798484, date: 14/06/2016

Antenatal management and neonatal outcomes of monochorionic twin pregnancies in a tertiary teaching hospital: a 10-year review

Monochorionic (MC) pregnancy is a high risk pregnancy with well-defined specific complications, such as twin-to-twin transfusion syndrome (TTTS) and twin anaemia-polycythaemia sequence (TAPS). Laser photocoagulation (LPC) is an effective treatment for both complications. In the current retrospective study, we determined the incidence of MC pregnancy complications in a tertiary care centre during a 10-year period. Single foetal death (FD) beyond 14 weeks’ gestation was significantly higher when complicated by either TTTS, TAPS or selective foetal growth restriction (21.4%, 16.7% and 9.1% versus 1.6%, p20% is an independent risk factor for single or double FD after LPC. Consequently, prior to LPC, patients should be counselled that early diagnosis of TTTS, advanced Quintero stages and weight discordances >20% are potential risk factors for FD. Further studies are needed to identify additional risk factors for TTTS and TAPS outcome after LPC.Impact Statement What is already known on this subject? Monochorionic (MC) pregnancy is a high risk pregnancy with well-defined specific complications, such as twin-twin transfusion syndrome (TTTS) and twin anaemia-polycythaemia sequence (TAPS). Laser photocoagulation (LPC) is an effective treatment for both complications. What the results of this study add? The results of the current study determined the incidence of MC pregnancy complications in a tertiary care centre in Brussels, and identified that twins’ weight discordance >20% is an independent risk factor for single or double foetal death after LPC. What the implications are of these findings for clinical practice and/or further research? Prior to laser coagulation, patients should be counselled that early diagnosis of TTTS, Quintero stages 3 or 4 and weight discordances >20% are potential risk factors for foetal demise. Further studies are needed to identify additional risk factors for TTTS and TAPS outcome after LPC