Pyroptosis: A Promising Mechanism Linking SARS-CoV-2 Infection to Adverse Pregnancy Outcomes.

Pregnancy is characterized by a delicate immune balance; therefore, infectious diseases might increase the risk of adverse pregnancy outcomes (APOs). Here, we hypothesize that pyroptosis, a unique cell death pathway mediated by the NLRP3 inflammasome, could link SARS-CoV-2 infection, inflammation, and APOs. Two blood samples were collected from 231 pregnant women at 11-13 weeks of gestation and in the perinatal period. At each time point, SARS-CoV-2 antibodies and neutralizing antibody titers were measured by ELISA and microneutralization (MN) assays, respectively. Plasmatic NLRP3 was determined by ELISA. Fourteen miRNAs selected for their role in inflammation and/or pregnancy were quantified by qPCR and further investigated by miRNA-gene target analysis. NLRP3 levels were positively associated with nine circulating miRNAs, of which miR-195-5p was increased only in MN+ women (p-value = 0.017). Pre-eclampsia was associated with a decrease in miR-106a-5p (p-value = 0.050). miR-106a-5p (p-value = 0.026) and miR-210-3p (p-value = 0.035) were increased in women with gestational diabetes. Women giving birth to small for gestational age babies had lower miR-106a-5p and miR-21-5p (p-values = 0.001 and 0.036, respectively), and higher miR-155-5p levels (p-value = 0.008). We also observed that neutralizing antibodies and NLRP3 concentrations could affect the association between APOs and miRNAs. Our findings suggest for the first time a possible link between COVID-19, NLRP3-mediated pyroptosis, inflammation, and APOs. Circulating miRNAs might be suitable candidates to gain a comprehensive view of this complex interplay.

PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are key participants in both early- and late-stage atherosclerosis and influence neighbouring cells possibly by means of bioactive molecules, some of which are packed into extracellular vesicles (EVs). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed and secreted by VSMCs. This study aimed to unravel the role of PCSK9 on VSMCs-derived EVs in terms of content and functionality. EVs were isolated from human VSMCs overexpressing human PCSK9 (VSMCPCSK9-EVs) and tested on endothelial cells, monocytes, macrophages and in a model of zebrafish embryos. Compared to EVs released from wild-type VSMCs, VSMCPCSK9-EVs caused a rise in the expression of adhesion molecules in endothelial cells and of pro-inflammatory cytokines in monocytes. These acquired an increased migratory capacity, a reduced oxidative phosphorylation and secreted proteins involved in immune response and immune effector processes. Concerning macrophages, VSMCPCSK9-EVs enhanced inflammatory milieu and uptake of oxidized low-density lipoproteins, whereas the migratory capacity was reduced. When injected into zebrafish embryos, VSMCPCSK9-EVs favoured the recruitment of macrophages toward the site of injection. The results of the present study provide evidence that PCSK9 plays an inflammatory role by means of EVs, at least by those derived from smooth muscle cells of vascular origin.

Cord blood androgen levels of females from same sex and opposite sex twins - A pilot study.

OBJECTIVE: Opposite-sex twins have shown behavioural and reproductive differences between females and males. These differences may be determined by higher intrauterine levels of androgens among females that were exposed to a male co-twin. The aim of this study was to compare cord blood androgen levels in females from same-sex and opposite-sex twins. DESIGN: A prospective study. In this pilot study, we compared cord blood androgens (DHEA-S, Δ-4 androstenedione, total testosterone-TT) and sex hormone-binding globulin (SHBG) levels in 20 females from same sex and 20 females from opposite-sex dichorionic diamniotic twins. We used generalized estimating equation (GEE) modelling to assess differences in cord blood androgens between females from same-sex twin pregnancies and females from opposite-sex twin pregnancies. PATIENTS: Twenty opposite-sex twin pairs (female-male twins) and 20 same-sex twin pairs (female-female). MEASUREMENTS: Cord blood total testosterone, Δ-4 androstenedione, DHEA-S and sex hormone-binding globulin (SHBG) levels. RESULTS: No difference in the levels of androgens as Δ-4 androstenedione, total testosterone and SHBG was identified between females that were exposed to a female co-twin compared with females that were exposed to a male co-twin. DHEA-S levels were significantly lower among females from opposite-sex twins compared with females from same-sex twins. CONCLUSIONS: Our preliminary data do not support the hypothesis that females exposed to male co-twins are exposed to higher levels of androgens in utero compared with females exposed to female co-twins. Further studies are needed to explain the reported behavioural and reproductive differences among opposite-sex twins.

Effect of environmental exposures on cancer risk: Emerging role of non-coding RNA shuttled by extracellular vesicles.

Environmental and lifestyle exposures have a huge impact on cancer risk; nevertheless, the biological mechanisms underlying this association remain poorly understood. Extracellular vesicles (EVs) are membrane-enclosed particles actively released by all living cells, which play a key role in intercellular communication. EVs transport a variegate cargo of biomolecules, including non-coding RNA (ncRNA), which are well-known regulators of gene expression. Once delivered to recipient cells, EV-borne ncRNAs modulate a plethora of cancer-related biological processes, including cell proliferation, differentiation, and motility. In addition, the ncRNA content of EVs can be altered in response to outer stimuli. Such changes can occur either as an active attempt to adapt to the changing environment or as an uncontrolled consequence of cell homeostasis loss. In either case, such environmentally-driven alterations in EV ncRNA might affect the complex crosstalk between malignant cells and the tumor microenvironment, thus modulating the risk of cancer initiation and progression. In this review, we summarize the current knowledge about EV ncRNAs at the interface between environmental and lifestyle determinants and cancer. In particular, we focus on the effect of smoking, air and water pollution, diet, exercise, and electromagnetic radiation. In addition, we have conducted a bioinformatic analysis to investigate the biological functions of the genes targeted by environmentally-regulated EV microRNAs. Overall, we draw a comprehensive picture of the role of EV ncRNA at the interface between external factors and cancer, which could be of great interest to the development of novel strategies for cancer prevention, diagnosis, and therapy.

Digital RT-PCR Chip method for detection of SARS-CoV-2 virus.

The "gold standard" method for detection of SARS-CoV-2 is the real time reverse transcription-polymerase chain reaction, but due to pre-analytical and technical limitations, biological samples with low viral load are not sometimes detected. For this purpose a digital RT-PCR method on-chip was developed for detection of the SARS-CoV-2 virus, using two TaqMan™ Assays for quantification of the N Protein (Nucleocapsid) and the S Protein (Spike), and the QuantStudio™ 3D Digital PCR instrument. The method was applied to assess the nasopharyngeal swabs of asymptomatic subjects recruited in the UNICORN Study. The digital RT-PCR method is characterized by a higher sensitivity than the RT-qPCR method, even if performed with the same TaqMan™, and could be a promising tool for SARS-CoV-2 viral load quantification.

Upper Respiratory Microbiome in Pregnant Women: Characterization and Influence of Parity.

During pregnancy, the woman's immune system changes to support fetal development. These immunological modifications can increase the risk of respiratory diseases. Because the respiratory microbiome is involved in airway homeostasis, it is important to investigate how it changes during pregnancy. Additionally, since parity is associated with immune system alterations and cohabitants shared a similar microbiome, we investigated whether having a child may influence the respiratory microbiome of pregnant women. We compared the microbiome of 55 pregnant with 26 non-pregnant women using 16S rRNA gene sequencing and analyzed taxonomy, diversity, and metabolic pathways to evaluate the differences among nulliparous, primiparous, and multiparous women. The microbiome was similar in pregnant and non-pregnant women, but pregnant women had higher alpha diversity (Chao1 p-value = 0.001; Fisher p-value = 0.005) and a lower abundance of several metabolic pathways. Multiparous pregnant women had a higher relative abundance of Moraxella (p-value = 0.003) and a lower abundance of Corynebacterium (p-value = 0.002) compared with primiparous women. Both multiparous (pregnant) and primiparous/multiparous (non-pregnant) women reported a higher abundance of Moraxella compared with primiparous (pregnant) or nulliparous ones (p-value = 0.001). In conclusion, we characterized for the first time the upper airway microbiome of pregnant women and observed the influence of parity on its composition.

Nasopharyngeal Bacterial Microbiota Composition and SARS-CoV-2 IgG Antibody Maintenance in Asymptomatic/Paucisymptomatic Subjects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), ranging from asymptomatic conditions to severe/fatal lung injury and multi-organ failure. Growing evidence shows that the nasopharyngeal microbiota composition may predict the severity of respiratory infections and may play a role in the protection from viral entry and the regulation of the immune response to the infection. In the present study, we have characterized the nasopharyngeal bacterial microbiota (BNM) composition and have performed factor analysis in a group of 54 asymptomatic/paucisymptomatic subjects who tested positive for nasopharyngeal swab SARS-CoV-2 RNA and/or showed anti-RBD-IgG positive serology at the enrolment. We investigated whether BNM was associated with SARS-CoV-2 RNA positivity and serum anti-RBD-IgG antibody development/maintenance 20-28 weeks after the enrolment. Shannon's entropy α-diversity index [odds ratio (OR) = 5.75, p = 0.0107] and the BNM Factor1 (OR = 2.64, p = 0.0370) were positively associated with serum anti-RBD-IgG antibody maintenance. The present results suggest that BNM composition may influence the immunological memory against SARS-CoV-2 infections. To the best of our knowledge, this is the first study investigating the link between BNM and specific IgG antibody maintenance. Further studies are needed to unveil the mechanisms through which the BNM influences the adaptive immune response against viral infections.

Extracellular vesicles and their miRNA contents counterbalance the pro-inflammatory effect of air pollution during physiological pregnancy: A focus on Syncytin-1 positive vesicles.

The impact of exposure to respirable particulate matter (PM) during pregnancy is a growing concern, as several studies have associated increased risks of adverse pregnancy and birth outcomes, and impaired intrauterine growth with air pollution. The molecular mechanisms responsible for such effects are still under debate. Extracellular vesicles (EVs), which travel in body fluids and transfer microRNAs (miRNAs) between tissues (e.g., pulmonary environment and placenta), might play an important role in PM-induced risk. We sought to determine whether the levels of PM with aerodynamic diameters of ≤10 µm (PM10) and ≤2.5 µm (PM2.5) are associated with changes in plasmatic EV release and EV-miRNA content by investigating 518 women enrolled in the INSIDE study during the first trimester of pregnancy. In all models, we included both the 90-day averages of PM (long-term effects) and the differences between the daily estimate of PM and the 90-day average (short-term effects). Short-term PM10 and PM2.5 were associated with increased concentrations of all seven EV types that we assayed (positive for human antigen leukocyte G (HLA-G), Syncytin-1 (Sync-1), CD14, CD105, CD62e, CD61, or CD25 determinants), while long-term PM10 showed a trend towards decreased EV concentrations. Increased Sync-1 + EV levels were associated with the plasmatic decrease of sVCAM-1, but not of sICAM-1, which are circulating biomarkers of endothelial dysfunction. Thirteen EV-miRNAs were downregulated in response to long-term PM10 and PM2.5 variations, while seven were upregulated (p-value < 0.05, false discovery rate p-value (qFDR) < 0.1). Only one EV-miRNA (hsa-miR-221-3p) was downregulated after short-term variations. The identified PM-modulated EV-miRNAs exhibited putative roles in inflammation, gestational hypertension, and pre-eclampsia, as highlighted by miRNA target analysis. Our findings strongly support the hypothesis that EVs have an important role in modulating PM exposure effects during pregnancy, possibly through their miRNA cargo.

Brain-Derived Neurotrophic Factor and Extracellular Vesicle-Derived miRNAs in an Italian Cohort of Individuals With Obesity: A Key to Explain the Link Between Depression and Atherothrombosis.

Background: Obesity and depression are intertwined diseases often associated with an increased risk of cardiovascular (CV) complications. Brain-Derived Neurotrophic Factor (BDNF), altered in the brain both of subjects with depression and obesity, provides a potential link between depression and thrombosis. Since the relationship among peripheral BDNF, depression and obesity is not well-defined, the aim of the present report has been to address this issue taking advantage of the contribution played by extracellular vesicle (EV)-derived miRNAs. Research Process: Associations among circulating BDNF, depression and EV-derived miRNAs related to atherothrombosis have been evaluated in a large Italian cohort of obese individuals (n = 743), characterized by the Beck Depression Inventory (BDI-II) score. Results: BDI-II was negatively associated with BDNF levels without a significant impact of the rs6265 BDNF polymorphism; this association was modified by raised levels of IFN-γ. BDNF levels were linked to an increase of 80 EV-derived miRNAs and a decrease of 59 miRNAs related to atherosclerosis and thrombosis. Network analysis identified at least 18 genes targeted by these miRNAs, 7 of which involved in depression and CV risk. The observation of a possible link among BDNF, depression, and miRNAs related to atherothrombosis and depression in obesity is novel and may lead to a wider use of BDNF as a CV risk biomarker in this specific subject group.

Extracellular Vesicles: Footprints of environmental exposures in the aging process?

PURPOSE OF THE REVIEW: Extracellular vesicles (EVs) are nano-sized lipid particles that participate in intercellular signaling through the trafficking of bioactive molecules from parental cells to recipient ones. This well-orchestrated communication system is crucial for the organism to respond to external cues in a coordinated manner; indeed, environmental and lifestyle exposures can modify both EV number and content, with consequences on cellular metabolism and homeostasis. In particular, a growing body of evidence suggests that exposome-induced changes in EV profile could regulate the aging process, both at the cellular and organismal levels. Here, we provide an overview of the role played by ambient-induced EVs on aging and age-related diseases. Among the several environmental factors that can affect the communication network operated by EVs, we focused on air pollution, ultraviolet light, diet, and physical exercise. Moreover, we performed a miRNA target analysis, to support the role of EV-miRNA emerging from the literature in the context of aging. RECENT FINDINGS: The overall emerging picture strongly supports a key regulatory role for EVs at the interface between external stimuli and cellular/organismal aging, thus providing novel insights into the molecular mechanisms linking a "healthy exposome" to well-being in old age. In addition, this knowledge will pave the way for research aimed at developing innovative antiaging strategies based on EVs.

Particulate Matter Exposure and Allergic Rhinitis: The Role of Plasmatic Extracellular Vesicles and Bacterial Nasal Microbiome.

Particulate matter (PM) exposure is linked to the worsening of respiratory conditions, including allergic rhinitis (AR), as it can trigger nasal and systemic inflammation. To unveil the underlying molecular mechanisms, we investigated the effects of PM exposure on the release of plasmatic extracellular vesicles (EV) and on the complex cross-talk between the host and the nasal microbiome. To this aim, we evaluated the effects of PM10 and PM2.5 exposures on both the bacteria-derived-EV portion (bEV) and the host-derived EVs (hEV), as well as on bacterial nasal microbiome (bNM) features in 26 AR patients and 24 matched healthy subjects (HS). In addition, we assessed the role exerted by the bNM as a modifier of PM effects on the complex EV signaling network in the paradigmatic context of AR. We observed that PM exposure differently affected EV release and bNM composition in HS compared to AR, thus potentially contributing to the molecular mechanisms underlying AR. The obtained results represent the first step towards the understanding of the complex signaling network linking external stimuli, bNM composition, and the immune risponse.

The association between prenatal concentrations of polybrominated diphenyl ether and child cognitive and psychomotor function.

Previous studies suggest a negative association between prenatal polybrominated diphenyl ethers (PBDEs) exposure and child cognitive and psychomotor development. However, the timing of the relationship between PBDE exposure and neurodevelopment is still unclear. We examined the association between PBDE concentration at two different prenatal times (early and late pregnancy) and cognitive function in children 6-8 years of age. METHODS: Eight hundred pregnant women were recruited between 2007 and 2009 from Sherbrooke, Canada. Four PBDE congeners (BDE-47, -99, -100, and -153) were measured in maternal plasma samples collected during early pregnancy (12 weeks of gestation) and at delivery. At 6-8 years of age, 355 children completed a series of subtests spanning multiple neuropsychologic domains: verbal and memory skills were measured using the Wechsler Intelligence Scale for Children, Fourth Edition; visuospatial processing using both Wechsler Intelligence Scale for Children, Fourth Edition and Neuropsychological Assessment second edition; and attention was assessed through the Test of Everyday Attention for Children. Additionally, parents completed subtests from the Developmental Coordination Disorder Questionnaire to measure child motor control. We used linear regression and quantile g-computation models to estimate associations of PBDE congener concentrations and psychologic test scores. RESULTS: In our models, no significant associations were detected between PBDE mixture and any of the child psychologic scores. BDE-99 concentration at delivery was nominally associated with higher scores on short-term and working memory while a decrease in spatial perception and reasoning was nominally associated with higher BDE-100 concentration at delivery. CONCLUSION: Overall, our results did not show a significant association between PBDEs and child cognitive and motor development.

Associations Among PCSK9 Levels, Atherosclerosis-Derived Extracellular Vesicles, and Their miRNA Content in Adults With Obesity.

Background: Extracellular vesicles (EV) concentration is generally increased in patients with cardiovascular diseases, although the protective role of EVs in atherosclerosis has been reported. Among the specific cargo of EVs, miRNAs contribute to different stages of atherosclerosis. Aim of the present report has been to investigate, in individuals with obesity, the interplay among EVs derived from cells relevant for the atherosclerotic process (i.e., platelets, endothelium, monocytes/macrophages, and neutrophils), their miRNA content and proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the main regulators of low-density lipoprotein receptor (LDLR). Methods and Results: EVs have been isolated from 936 individuals with obesity (body mass index = 33.6 ± 5.6 Kg/m2) and a raised cardiovascular risk (e.g., LDL-C = 131.6 ± 36.4 mg/dL, HOMA-IR = 3.1, and roughly 50% on anti-hypertensive medications). PCSK9 levels were negatively associated with EV count in the range 150-400 nm and with those derived from macrophages (CD14+), endothelium (CD105+), and neutrophils (CD66+). The association between PCSK9 and platelet-derived EVs (CD61+) was modified by platelet counts. PCSK9 was significantly associated with five EV-derived miRNAs (hsa-miRNA-362-5p,-150,-1244,-520b-3p,-638). Toll-like receptor 4 and estrogen receptor 1 were targeted by all five miRNAs and LDLR by four. The effect on LDLR expression is mainly driven by hsa-miR-150. Considering the implication of EV in atherosclerosis onset and progression, our findings show a potential role of PCSK9 to regulate EV-derived miRNAs, especially those involved in inflammation and expression of low-density lipoprotein receptor (LDLR) receptor.

DNA Methylation in Nasal Epithelium: Strengths and Limitations of an Emergent Biomarker for Childhood Asthma.

Asthma is one of the most widespread chronic respiratory conditions. This disease primarily develops in childhood and is influenced by different factors, mainly genetics and environmental factors. DNA methylation is an epigenetic mechanism which may represent a bridge between these two factors, providing a tool to comprehend the interaction between genetics and environment. Most epidemiological studies in this field have been conducted using blood samples, although DNA methylation marks in blood may not be reliable for drawing exhaustive conclusions about DNA methylation in the airways. Because of the role of nasal epithelium in asthma and the tissue specificity of DNA methylation, studying the relationship between DNA methylation and childhood asthma might reveal crucial information about this widespread respiratory disease. The purpose of this review is to describe current findings in this field of research. We will present a viewpoint of selected studies, consider strengths and limitations, and propose future research in this area.