Effects of sevoflurane on lung alveolar epithelial wound healing and survival in a sterile in vitro model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a serious lung condition that often leads to hospitalization in intensive care units and a high mortality rate. Sevoflurane is a volatile anesthetic with growing interest for sedation in ventilated patients with ARDS. It has been shown to have potential lung-protective effects, such as reduced inflammation and lung edema, or improved arterial oxygenation. In this study, we investigated the effects of sevoflurane on lung injury in cultured human carcinoma-derived lung alveolar epithelial (A549) cells. We found that sevoflurane was associated with improved wound healing after exposure to inflammatory cytokines, with preserved cell proliferation but no effect on cell migration properties. Sevoflurane exposure was also associated with enhanced cell viability and active autophagy in A549 cells exposed to cytokines. These findings suggest that sevoflurane may have beneficial effects on lung epithelial injury by promoting alveolar epithelial wound healing and by influencing the survival and proliferation of A549 epithelial cells in vitro. Further research is needed to confirm these findings and to investigate the key cellular mechanisms explaining sevoflurane's potential effects on lung epithelial injury.

CX3CL1/Fractalkine as a biomarker for early pregnancy prediction of preterm premature rupture of membranes.

OBJECTIVES: The objective of our study was to evaluate serum CX3CL1/Fractalkine, a monocyte/macrophage chemoattractant expressed in cytotrophoblasts and decidual cells, as a predictive biomarker for the occurrence of preterm premature rupture of membranes (PPROM). METHODS: A case-control study of 438 pregnancies including 82 PPROM cases and 64 preterm labor with intact membranes cases with blood samples collected at first trimester, second trimester and delivery was conducted. The predictive ability of CX3CL1 and maternal risk factors for the occurrence of PPROM was assessed by receiver operating characteristic curve analysis. A second, independent cohort was prospectively constituted to confirm the case-control study results. RESULTS: First trimester CX3CL1 was significantly increased in PPROM cases when compared to matched controls. Multivariate regression analysis highlighted a significant difference for CX3CL1 measured during the first trimester (p<0.001). Alone, CX3CL1 predicts PPROM with a 90 % sensitivity and a specificity around 40 %. The area under the receiver operating characteristic curve for PPROM prediction were 0.64 (95% confidence interval: 0.57-0.71) for first trimester CX3CL1, and 0.61 (95% confidence interval: 0.54-0.68) for maternal risk factors (body mass index<18.5 kg/m2, nulliparity, tobacco use and the absence of high school diploma). The combination of CX3CL1 and maternal risk factors significantly improved the area under the curve: 0.72 (95% confidence interval: 0.66-0.79) (p<0.001). The results were confirmed on a second independent cohort. CONCLUSIONS: CX3CL1 is a promising blood biomarker in the early (first trimester) prediction of PPROM.

Effects of sevoflurane on lung epithelial permeability in experimental models of acute respiratory distress syndrome.

BACKGROUND: Preclinical studies in acute respiratory distress syndrome (ARDS) have suggested that inhaled sevoflurane may have lung-protective effects and clinical trials are ongoing to assess its impact on major clinical outcomes in patients with ARDS. However, the underlying mechanisms of these potential benefits are largely unknown. This investigation focused on the effects of sevoflurane on lung permeability changes after sterile injury and the possible associated mechanisms. METHODS: To investigate whether sevoflurane could decrease lung alveolar epithelial permeability through the Ras homolog family member A (RhoA)/phospho-Myosin Light Chain 2 (Ser19) (pMLC)/filamentous (F)-actin pathway and whether the receptor for advanced glycation end-products (RAGE) may mediate these effects. Lung permeability was assessed in RAGE-/- and littermate wild-type C57BL/6JRj mice on days 0, 1, 2, and 4 after acid injury, alone or followed by exposure at 1% sevoflurane. Cell permeability of mouse lung epithelial cells was assessed after treatment with cytomix (a mixture of TNFɑ, IL-1ß, and IFNγ) and/or RAGE antagonist peptide (RAP), alone or followed by exposure at 1% sevoflurane. Levels of zonula occludens-1, E-cadherin, and pMLC were quantified, along with F-actin immunostaining, in both models. RhoA activity was assessed in vitro. RESULTS: In mice after acid injury, sevoflurane was associated with better arterial oxygenation, decreased alveolar inflammation and histological damage, and non-significantly attenuated the increase in lung permeability. Preserved protein expression of zonula occludens-1 and less increase of pMLC and actin cytoskeletal rearrangement were observed in injured mice treated with sevoflurane. In vitro, sevoflurane markedly decreased electrical resistance and cytokine release of MLE-12 cells, which was associated with higher protein expression of zonula occludens-1. Improved oxygenation levels and attenuated increase in lung permeability and inflammatory response were observed in RAGE-/- mice compared to wild-type mice, but RAGE deletion did not influence the effects of sevoflurane on permeability indices after injury. However, the beneficial effect of sevoflurane previously observed in wild-type mice on day 1 after injury in terms of higher PaO2/FiO2 and decreased alveolar levels of cytokines was not found in RAGE-/- mice. In vitro, RAP alleviated some of the beneficial effects of sevoflurane on electrical resistance and cytoskeletal rearrangement, which was associated with decreased cytomix-induced RhoA activity. CONCLUSIONS: Sevoflurane decreased injury and restored epithelial barrier function in two in vivo and in vitro models of sterile lung injury, which was associated with increased expression of junction proteins and decreased actin cytoskeletal rearrangement. In vitro findings suggest that sevoflurane may decrease lung epithelial permeability through the RhoA/pMLC/F-actin pathway.

Inflammation of Dry Eye Syndrome: A Cellular Study of the Epithelial and Macrophagic Involvement of NFAT5 and RAGE.

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation.

Characterization of RAGE and CK2 Expressions in Human Fetal Membranes.

At the feto-maternal interface, fetal membranes (FM) play a crucial role throughout pregnancy. FM rupture at term implicates different sterile inflammation mechanisms including pathways activated by the transmembrane glycoprotein receptor for advanced glycation end-products (RAGE) belonging to the immunoglobulin superfamily. As the protein kinase CK2 is also implicated in the inflammation process, we aimed to characterize the expressions of RAGE and the protein kinase CK2 as a candidate regulator of RAGE expression. The amnion and choriodecidua were collected from FM explants and/or primary amniotic epithelial cells throughout pregnancy and at term in spontaneous labor (TIL) or term without labor (TNL). The mRNA and protein expressions of RAGE and the CK2α, CK2α', and CK2ß subunits were investigated using reverse transcription quantitative polymerase chain reaction and Western blot assays. Their cellular localizations were determined with microscopic analyses, and the CK2 activity level was measured. RAGE and the CK2α, CK2α', and CK2ß subunits were expressed in both FM layers throughout pregnancy. At term, RAGE was overexpressed in the amnion from the TNL samples, whereas the CK2 subunits were expressed at the same level in the different groups (amnion/choriodecidua/amniocytes, TIL/TNL), without modification of the CK2 activity level and immunolocalization. This work paves the way for future experiments regarding the regulation of RAGE expression by CK2 phosphorylation.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1ß and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1ß by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- ("pro-inflammatory") alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE-TXNIP-NLRP3 interactions during macrophage activation in acute lung injury.

The receptor for advanced glycation end-products enhances lung epithelial wound repair: An in vitro study.

Re-epithelialization of the alveolar surface is a key process of lung alveolar epithelial barrier repair after acute lung injury. The receptor for advanced glycation end-products (RAGE) pathway plays key roles in lung homeostasis, and its involvement in wound repair has been already reported in human bronchial epithelial cells. However, its effects on lung alveolar epithelial repair after injury remain unknown. We investigated whether RAGE stimulation with its ligands high-mobility group box 1 protein (HMGB1) or advanced glycation end-products (AGEs), alone or associated with RAGE inhibition using RAGE antagonist peptide, affects in vitro wound healing in human alveolar epithelial A549 cells. We further asked whether these effects could be associated with changes in cell proliferation and migration. We found that treatment of A549 cells with HMGB1 or AGEs promotes RAGE-dependent wound healing after a scratch assay. In addition, both RAGE ligands increased cell proliferation in a RAGE-dependent manner. Treatment with HMGB1 increased migration of alveolar epithelial cells at 12 h, independently of RAGE, whereas AGEs stimulated migration as measured 48 h after injury in a RAGE-dependent manner. Taken together, these results suggest that RAGE pathway is involved in lung alveolar epithelial wound repair, possibly through enhanced cell migration and proliferation.

Cigarette Smoke Condensate Exposure Induces Receptor for Advanced Glycation End-Products (RAGE)-Dependent Sterile Inflammation in Amniotic Epithelial Cells.

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.

Driving pressure and acute respiratory distress syndrome in critically ill patients.

BACKGROUND AND OBJECTIVE: Elevated driving pressure (ΔP) may be associated with increased risk of acute respiratory distress syndrome (ARDS) in patients admitted via the emergency department and with post-operative pulmonary complications in surgical patients. This study investigated the association of higher ΔP with the onset of ARDS in a high-risk, intensive care unit (ICU) population. METHODS: This is a secondary analysis of a prospective multicentre observational study. Data for this ancillary study were obtained from intubated adult patients with at least one ARDS risk factor upon ICU admission enrolled in a previous multicentre observational study. Patients were followed up for the development of ARDS within 7 days (primary outcome). Univariate and multivariate analyses tested the association between ΔP (measured at ICU admission (baseline) or 24 h later (day 1)) and the development of ARDS. RESULTS: A total of 221 patients were included in this study, among whom 34 (15%) developed ARDS within 7 days. These patients had higher baseline ΔP than those who did not (mean ± SD: 12.5 ± 3.1 vs 9.8 ± 3.4 cm H2 O, respectively, P = 0.0001). The association between baseline ΔP and the risk of developing ARDS was robust to adjustment for baseline tidal volume, positive-end expiratory pressure, illness severity, serum lactate and sepsis, pneumonia, severe trauma and shock as primary ARDS risk factors (odds ratio: 1.20; 95% CI: 1.03-1.41; P = 0.02). The same results were found with day 1 ΔP. CONCLUSION: Among at-risk ICU patients, higher ΔP may identify those who are more likely to develop ARDS.

Retinoic acid and tracheal occlusion for diaphragmatic hernia treatment in rabbit fetuses.

INTRODUCTION: Lung hypoplasia and pulmonary arterial hypertension in congenital diaphragmatic hernia lead to a high perinatal mortality. Although sustained fetoscopic tracheal occlusion (TO) improves lung development, a major side effect is abnormal pneumocyte differentiation. This study evaluated the potential ability of intratracheal retinoic acid (RA) administration to reduce adverse effects of sustained TO in a rabbit model of diaphragmatic hernia. METHODS: A left diaphragmatic defect was created on day 23 in time-dated pregnant rabbits. On day 28, the same rabbits underwent sham surgery or TO, with an injection of empty or RA-loaded liposomes. On day 30, the fetuses were harvested, and the lungs were processed for histology, immunohistochemistry, and gene expression quantification. RESULTS: A tracheal RA injection at the time of TO had no effect on the lung-to-body-weight ratio, radial alveolar count or lung connective tissue composition. Retinoic acid plus TO had synergic effects on vascular measurements, proportional medial thickness, and endothelin-1 receptor type-A gene expression. The most noticeable effect was recovery of normal pneumocyte differentiation. CONCLUSION: Retinoic acid plus TO prevented abnormal pneumocyte differentiation and seemed to have a beneficial effect on pulmonary vascularization.

Myocilin expression is regulated by retinoic acid in the trabecular meshwork-derived cellular environment.

Glaucoma is the leading cause of irreversible blindness and is usually classified as angle closure and open angle glaucoma (OAG). Primary open angle glaucoma represents the most frequent clinical presentation leading to ganglion cell death and optic nerve degeneration as a main consequence of an intraocular pressure' (IOP) increase. The mechanisms of this IOP increase in such pathology remain unclear but one protein called Myocilin could be a part of the puzzle in the trabecular meshwork (TM). Previously described to be transcriptionally regulated by glucocorticoids, the comprehension of the trabecular regulation of Myocilin' expression has only weakly progressed since 15 years. Due to the essential molecular and cellular implications of retinoids' pathway in eye development and physiology, we investigate the potential role of the retinoic acid in such regulation and expression. This study demonstrates that the global retinoids signaling machinery is present in immortalized TM cells and that Myocilin (MYOC) expression is upregulated by retinoic acid alone or combined with a glucocorticoid co-treatment. This regulation by retinoic acid acts through the MYOC promoter which contains a critical cluster of four retinoic acid responsive elements (RAREs), with the RARE-DR2 presenting the strongest effect and binding the RARα/RXRα heterodimer. All together, these results open up new perspectives for the molecular understanding glaucoma pathophysiology and provide further actionable clues on Myocilin gene regulation.

Effects of tracheal occlusion with retinoic acid administration on normal lung development.

INTRODUCTION: Tracheal occlusion (TO) is an investigational therapy for severe congenital diaphragmatic hernia that decreases pulmonary hypoplasia, but sustained TO also induces deficient surfactant synthesis. Intramuscular maternal administration of retinoic acid (RA) in a surgical rabbit model of congenital diaphragmatic hernia showed a beneficial effect on lung maturation. We evaluated the potential of RA delivery into the trachea and studied the combined effects of TO and RA on normal lung development. METHODS: Experiments were performed on normal rabbit fetuses. Liposomes and capric triglyceride (Miglyol® ), alone and with RA, were administered in the trachea just before TO (d26). Lung morphology and surfactant production were studied at term (d30). RESULTS: Tracheal occlusion increased lung weight and enhanced alveolar development but increased apoptotic activity and decreased surfactant expression. Tracheal injection of RA improved surfactant production to levels of normal controls. CONCLUSION: We established the potential of liposome and Miglyol as RA vehicle for delivering this bioactive molecule in the fetal airways. Tracheal RA injection seems to oppose the effects of TO in fetuses with normal lungs. © 2017 John Wiley & Sons, Ltd.

Nuclear retinoid receptors and pregnancy: placental transfer, functions, and pharmacological aspects.

Animal models of vitamin A (retinol) deficiency have highlighted its crucial role in reproduction and placentation, whereas an excess of retinoids (structurally or functionally related entities) can cause toxic and teratogenic effects in the embryo and foetus, especially in the first trimester of human pregnancy. Knock-out experimental strategies-targeting retinoid nuclear receptors RARs and RXRs have confirmed that the effects of vitamin A are mediated by retinoic acid (especially all-trans retinoic acid) and that this vitamin is essential for the developmental process. All these data show that the vitamin A pathway and metabolism are as important for the well-being of the foetus, as they are for that of the adult. Accordingly, during this last decade, extensive research on retinoid metabolism has yielded detailed knowledge on all the actors in this pathway, spurring the development of antagonists and agonists for therapeutic and research applications. Natural and synthetic retinoids are currently used in clinical practice, most often on the skin for the treatment of acne, and as anti-oncogenic agents in acute promyelocytic leukaemia. However, because of the toxicity and teratogenicity of retinoids during pregnancy, their pharmacological use needs a sound knowledge of their metabolism, molecular aspects, placental transfer, and action.

Metabolic disturbances of the vitamin A pathway in human diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common life-threatening congenital anomaly resulting in high rates of perinatal death and neonatal respiratory distress. Some of the nonisolated forms are related to single-gene mutations or genomic rearrangements, but the genetics of the isolated forms (60% of cases) still remains a challenging issue. Retinoid signaling (RA) is critical for both diaphragm and lung development, and it has been hypothesized that subtle disruptions of this pathway could contribute to isolated CDH etiology. Here we used time series of normal and CDH lungs in humans, in nitrofen-exposed rats, and in surgically induced hernia in rabbits to perform a systematic transcriptional analysis of the RA pathway key components. The results point to CRPBP2, CY26B1, and ALDH1A2 as deregulated RA signaling genes in human CDH. Furthermore, the expression profile comparisons suggest that ALDH1A2 overexpression is not a primary event, but rather a consequence of the CDH-induced lung injury. Taken together, these data show that RA signaling disruption is part of CDH pathogenesis, and also that dysregulation of this pathway should be considered organ specifically.

DeepMPTB: a vaginal microbiome-based deep neural network as artificial intelligence strategy for efficient preterm birth prediction.

In recent decades, preterm birth (PTB) has become a significant research focus in the healthcare field, as it is a leading cause of neonatal mortality worldwide. Using five independent study cohorts including 1290 vaginal samples from 561 pregnant women who delivered at term (n = 1029) or prematurely (n = 261), we analysed vaginal metagenomics data for precise microbiome structure characterization. Then, a deep neural network (DNN) was trained to predict term birth (TB) and PTB with an accuracy of 84.10% and an area under the receiver operating characteristic curve (AUROC) of 0.875 ± 0.11. During a benchmarking process, we demonstrated that our DL model outperformed seven currently used machine learning algorithms. Finally, our results indicate that overall diversity of the vaginal microbiota should be taken in account to predict PTB and not specific species. This artificial-intelligence based strategy should be highly helpful for clinicians in predicting preterm birth risk, allowing personalized assistance to address various health issues. DeepMPTB is open source and free for academic use. It is licensed under a GNU Affero General Public License 3.0 and is available at https://deepmptb.streamlit.app/ . Source code is available at https://github.com/oschakoory/DeepMPTB and can be easily installed using Docker ( https://www.docker.com/ ).

Ontogeny of aquaporins in human fetal membranes.

It has been proposed that four members of the aquaporin family (AQPs 1, 3, 8, and 9) are involved in the control of amniotic fluid (AF) homeostasis, as illustrated by their differential expression patterns in normal and pathological human term fetal membranes. However, there are no data available to date on their ontogeny throughout pregnancy. Our objective was to determine spatiotemporal expression profiles of the mRNA and proteins of all 13 members of this transmembrane channel family. For this purpose, we used healthy fetal membranes from the first, second, and third trimesters of pregnancy. Total mRNA and proteins were extracted from total membranes and from separated amnion and chorion. Quantitative PCR, Western blot, and immunohistochemistry experiments were carried out to determine the presence of AQPs and to quantify their spatiotemporal expression patterns throughout pregnancy. The WISH cell line was tested to propose a cellular model for the role of AQPs in the amnion compartment. AQP11 expression was established in amniotic membranes at term. Aquaporins 1, 3, 8, 9, and 11 mRNA and proteins were present in amnion and chorion throughout human gestation. Each AQP has a time-specific expression pattern, with AQP1 presenting the highest variation in terms of mRNA and protein levels. The WISH cell line also expressed the same five AQPs. Taken together, these results indicate that AQPs are expressed and potentially involved in the regulation of AF homeostasis throughout pregnancy. This also clearly supports the hypothesis that abnormal expression could occur at any time during pregnancy, ultimately leading to obstetrical pathologies such as polyhydramnios or oligohydramnios.

The Polycomb group protein EZH2 directly controls DNA methylation.

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.

Oxidative and antioxidative stress markers in dry eye disease: A systematic review and meta-analysis.

PURPOSE: To conduct a systematic review and meta-analysis on the levels of oxidative stress markers and antioxidants in dry eye disease (DED) compared with healthy subject. METHOD: The PubMed, Cochrane Library, Embase, Science Direct and Google Scholar databases were searched on 10 January 2021 for studies reporting oxidative and antioxidative stress markers in DED and healthy controls. Main meta-analysis was stratified by type of biomarkers, type of samples (tears, conjunctival cells or biopsies), Sjögren's syndrome (SS) (patients with or without SS) and by geographical zones (Asia or Europe). RESULTS: We included nine articles, for a total of 333 patients (628 eye samples) with DED and 165 healthy controls (451 eye samples). There is an overall increase in oxidative stress markers in DED compared with healthy controls (standard mean deviation = 2.39, 95% confidence interval 1.85-2.94), with a significant increase in lipid peroxide (1.90, 0.69-3.11), myeloperoxidase (2.17, 1.06-3.28), nitric oxide synthase 3 (2.52, 0.95-4.08), xanthine oxidase/oxidoreductase (2.41, 1.40-5.43), 4-hydroxy-2-nonenal (4HNE) (4.75, 1.67-7.84), malondialdehyde (3.00, 2.55-3.45) and reactive oxygen species (1.31, 0.94-1.68). Oxidative stress markers were higher in tears, conjunctival cells and conjunctival biopsies of DED than controls. Even if small number of studies were included for antioxidants, catalase seemed to be decreased in DED compared with healthy controls (-2.17, -3.00 to -1.34), with an increase of antioxidants in tears of DED patients without SS (1.13, 0.76-1.49). CONCLUSION: Oxidative stress markers, and probably antioxidants, were dysregulated in DED, establishing a local oxidative environment in tears, conjunctival cells and tissues.

Dysregulation of the Amniotic PPARγ Pathway by Phthalates: Modulation of the Anti-Inflammatory Activity of PPARγ in Human Fetal Membranes.

Phthalates are reprotoxic pollutants that are omnipresent in the environment. Detectable in amniotic fluid, these compounds (with the most concentrated being mono-2-ethylhexyl phthalate (MEHP)) are in direct contact with fetal membranes (FMs). They can lead to the premature rupture of FMs by deregulating cellular and molecular pathways, such as, for example, the nuclear transcription factor peroxysome proliferator-activated receptor gamma (PPARγ) pathway. The objective was to study the impact of MEHP on the PPARγ pathway in FMs using amnion and choriodecidua across the three trimesters of pregnancy and the amniotic epithelial AV3 cell model by analyzing (i) PPARγ expression (mRNA and proteins) using RT-qPCR and Western blot assays; (ii) cytotoxicity and cell viability following MEHP treatment by lactate dehydrogenase (LDH) measurement and using Cell-counting Kit 8; and (iii) modulation by MEHP of PPARγ transcriptional activity (using a reporter gene assay) and PPARγ anti-inflammatory properties (by measuring IL6 and IL8 levels). PPARγ is expressed in the human amnion and choriodecidua during the three trimesters of pregnancy and in amniotic cells. In the AV3 cell line, MEHP is not cytotoxic and does not reduce cell viability, but it reduces PPARγ activity, here induced by a classical agonist without influencing its expression. MEHP also reduces PPARγ's anti-inflammatory properties. In conclusion, PPARγ signaling is dysregulated by MEHP; this paves the way for future explorations to highlight the hypothesis of phthalates as an amniotic PPARγ disruptor that can explain the premature rupture of FMs.

Physiological TLR4 regulation in human fetal membranes as an explicative mechanism of a pathological preterm case.

The integrity of human fetal membranes is crucial for harmonious fetal development throughout pregnancy. Their premature rupture is often the consequence of a physiological phenomenon that has been exacerbated. Beyond all the implied biological processes, inflammation is of primary importance and is qualified as 'sterile' at the end of pregnancy. In this study, complementary methylomic and transcriptomic strategies on amnion and choriodecidua explants obtained from the altered (cervix zone) and intact fetal membranes at term and before labour were used. By cross-analysing genome-wide studies strengthened by in vitro experiments, we deciphered how the expression of toll-like receptor 4 (TLR4), an actor in pathological fetal membrane rupture, is controlled. Indeed, it is differentially regulated in the altered zone and between both layers by a dual mechanism: (1) the methylation of TLR4 and miRNA promoters and (2) targeting by miRNA (let-7a-2 and miR-125b-1) acting on the 3'-UTR of TLR4. Consequently, this study demonstrates that fine regulation of TLR4 is required for sterile inflammation establishment at the end of pregnancy and that it may be dysregulated in the pathological premature rupture of membranes.