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Burn pits are a method of open-air waste management that was common during military operations in Iraq, Afghanistan, and other regions in Southwest Asia. Veterans returning from deployment have reported respiratory symptoms, potentially from exposure to burn pit smoke, yet comprehensive assessment of such exposure on pulmonary health is lacking. We have previously shown that exposure to condensates from burn pit smoke emissions causes inflammation and cytotoxicity in mice. In this study, we explored the effects of burn pit smoke condensates on human airway epithelial cells (HAECs) to understand their impact on cellular targets in the human lung. HAECs were cultured at the air-liquid interface (ALI) and exposed to burn pit waste smoke condensates (plywood, cardboard, plastic, mixed, and mixed with diesel) generated under smoldering and flaming conditions. Cytotoxicity was evaluated by measuring transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) release; toxicity scores (TSs) were quantified for each exposure. Pro-inflammatory cytokine release and modulation of gene expression were examined for cardboard and plastic condensate exposures. Burn pit smoke condensates generated under flaming conditions affected cell viability, with flaming mixed waste and plywood exhibiting the highest toxicity scores. Cardboard and plastic smoke condensates modulated cytokine secretion, with GM-CSF and IL-1ß altered in more than one exposure group. Gene expression of detoxifying enzymes (ALDH1A3, ALDH3A1, CYP1A1, CYP1B1, NQO1, etc.), mucins (MUC5AC, MUC5B), and cytokines was affected by several smoke condensates. Particularly, expression of IL6 was elevated following exposure to all burn pit smoke condensates, and polycyclic aromatic hydrocarbon acenaphthene was positively associated with the IL-6 level in the basolateral media of HAECs. These observations demonstrate that exposure to smoke condensates of materials present in burn pits adversely affects HAECs and that aberrant cytokine secretion and altered gene expression profiles following burn pit material smoke exposure could contribute to the development of airway disease.
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Células Epiteliais , Fumaça , Humanos , Fumaça/efeitos adversos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Cultivadas , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Linhagem Celular , Queima de Resíduos a Céu AbertoRESUMO
Rationale: Numerous studies have demonstrated that e-cigarettes can impact respiratory immune homeostasis; however, the extent of these effects remains an active area of investigation, and most previous studies were conducted with model systems or subjects exposed to third-generation e-cigarettes, such as vape pens and box mods. Objectives: Given the rise in popularity of nicotine-salt-containing pods and disposable e-cigarettes (fourth generation), we set out to better understand the respiratory effects of these newer e-cigarettes and compare their effects to early-generation devices. Methods: We collected induced sputum samples from a cohort of nonsmokers, smokers, third-generation e-cigarette users, and fourth-generation e-cigarette users (n = 20-30 per group) and evaluated the cellular and fluid-phase composition for markers of inflammation, host defense, and lung injury. Measurements and Main Results: Fourth-generation e-cigarette users had significantly more bronchial epithelial cells in the sputum, suggestive of airway injury. Concentrations of soluble intercellular adhesion molecule 1 (sICAM1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were significantly lower in fourth-generation e-cigarette users in comparison with all other groups, and CRP (C-reactive protein), IFN-γ, MCP-1 (monocyte chemoattractant protein-1), MMP-2 (matrix metalloproteinase 2), uteroglobin, and VEGF (vascular endothelial growth factor) were significantly lower in fourth- versus third-generation e-cigarette users, suggestive of overall immune suppression in fourth-generation e-cigarette users. Predictive modeling also demonstrated clear separation between exposure groups, indicating that the overall mediator milieu is different between groups, particularly fourth-generation e-cigarette users. Conclusions: Our results indicate disrupted immune homeostasis in fourth-generation e-cigarette users and demonstrate that the biological effects of fourth-generation e-cigarette use are unique compared with those associated with previous-generation e-cigarettes.
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Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Vaping , Humanos , Vaping/efeitos adversos , Metaloproteinase 2 da Matriz , Fator A de Crescimento do Endotélio Vascular , Biomarcadores , HomeostaseRESUMO
Inhalation exposure to cigarette smoke and e-cigarette aerosol is known to alter the respiratory immune system, particularly cytokine signaling. In assessments of health impacts of tobacco product use, cytokines are often measured using a variety of sample types, from serum to airway mucosa. However, it is currently unclear whether and how well cytokine levels from different sample types and the airway locations they represent are correlated, making comparing studies that utilize differing sample types challenging. To address this challenge, we compared baseline cytokine signatures in upper and lower airways and systemic samples and evaluated how groups of coexpressed cytokines change with tobacco product use. Matched nasal lavage fluid (NLF), nasal epithelial lining fluid (NELF), sputum, and circulating serum samples were collected from 14 nonsmokers, 13 cigarette smokers, and 17 e-cigarette users and analyzed for levels of 22 cytokines. Individual cytokine signatures were first compared across each sample type, followed by identification of cytokine clusters within each sample type. Identified clusters were then evaluated for potential alterations following tobacco product use using eigenvector analyses. Individual cytokine signatures in the respiratory tract were significantly correlated (NLF, NELF, and sputum) compared with randomly permutated signatures, whereas serum was not significantly different from random permutations. Cytokine clusters that were similar across airway sample types were modified by tobacco product use, particularly e-cigarettes, indicating a degree of uniformity in terms of how cytokine host defense and immune cell recruitment responses cooperate in the upper and lower airways. Overall, cluster-based analyses were found to be especially useful in small cohort assessments, providing higher sensitivity than individual signatures to detect biologically meaningful differences between tobacco use groups. This novel cluster analysis approach revealed that eigencytokine patterns in noninvasive upper airway samples simulate cytokine patterns in lower airways.
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Sistemas Eletrônicos de Liberação de Nicotina , Produtos do Tabaco , Tabagismo , Citocinas , Humanos , Sistema Respiratório , Produtos do Tabaco/efeitos adversos , Uso de TabacoRESUMO
BACKGROUND: Chronic lung disease (CLD) is the most common pulmonary morbidity in extremely preterm infants. It is unclear to what extent prenatal exposures influence the risk of CLD. Epigenetic variation in placenta DNA methylation may be associated with differential risk of CLD, and these associations may be dependent upon sex. METHODS: Data were obtained from a multi-center cohort of infants born extremely preterm (<28 weeks' gestation) and an epigenome-wide approach was used to identify associations between placental DNA methylation and CLD (n = 423). Associations were evaluated using robust linear regression adjusting for covariates, with a false discovery rate of 0.05. Analyses stratified by sex were used to assess differences in methylation-CLD associations. RESULTS: CLD was associated with differential methylation at 49 CpG sites representing 46 genes in the placenta. CLD was associated with differential methylation of probes within genes related to pathways involved in fetal lung development, such as p53 signaling and myo-inositol biosynthesis. Associations between CpG methylation and CLD differed by sex. CONCLUSIONS: Differential placental methylation within genes with key roles in fetal lung development may reflect complex cell signaling between the placenta and fetus which mediate CLD risk. These pathways appear to be distinct based on fetal sex. IMPACT: In extremely preterm infants, differential methylation of CpG sites within placental genes involved in pathways related to cell signaling, oxidative stress, and trophoblast invasion is associated with chronic lung disease of prematurity. DNA methylation patterns associated with chronic lung disease were distinctly based on fetal sex, suggesting a potential mechanism underlying dimorphic phenotypes. Mechanisms related to fetal hypoxia and placental myo-inositol signaling may play a role in fetal lung programming and the developmental origins of chronic lung disease. Continued research of the relationship between the placental epigenome and chronic lung disease could inform efforts to ameliorate or prevent this condition.
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Doenças do Prematuro , Pneumopatias , Ilhas de CpG , Metilação de DNA , Feminino , Humanos , Lactente Extremamente Prematuro , Recém-Nascido , Inositol , Pneumopatias/genética , Placenta/metabolismo , GravidezRESUMO
Macrophages are pivotal innate immune cells which exhibit high phenotypic plasticity and can exist in different polarization states dependent on exposure to external stimuli. Numerous methods have been employed to simulate macrophage polarization states to test their function in vitro. However, limited research has explored whether these polarization methods yield comparable populations beyond key gene, cytokine, and cell surface marker expression. Here, we employ an unbiased comprehensive analysis using data organized through the all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database, which compiles all RNAseq data deposited into the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). In silico analyses were carried out demonstrating that commonly employed macrophage polarization methods generate distinct macrophage subsets that remained undescribed until now. Our analyses confirm existing knowledge on macrophage polarization, while revealing nuanced differences between M2a and M2c subpopulations, suggesting non-interchangeable stimuli for M2a polarization. Furthermore, we identify divergent gene expression patterns in M1 macrophages following standard polarization protocols, indicating significant subset distinctions. Consequently, equivalence cannot be assumed among polarization regimens for in vitro macrophage studies, particularly in simulating diverse pathogen responses.
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Wildfire events are becoming increasingly common across many areas of the United States, including North Carolina (NC). Wildfires can cause immediate damage to properties, and wildfire smoke conditions can harm the overall health of exposed communities. It is critical to identify communities at increased risk of wildfire events, particularly in areas with that have sociodemographic disparities and low socioeconomic status (SES) that may exacerbate incurred impacts of wildfire events. This study set out to: (1) characterize the distribution of wildfire risk across NC; (2) implement integrative cluster analyses to identify regions that contain communities with increased vulnerability to the impacts of wildfire events due to sociodemographic characteristics; (3) provide summary-level statistics of populations with highest wildfire risk, highlighting SES and housing cost factors; and (4) disseminate wildfire risk information via our online web application, ENVIROSCAN. Wildfire hazard potential (WHP) indices were organized at the census tract-level, and distributions were analyzed for spatial autocorrelation via global and local Moran's tests. Sociodemographic characteristics were analyzed via k-means analysis to identify clusters with distinct SES patterns to characterize regions of similar sociodemographic/socioeconomic disparities. These SES groupings were overlayed with housing and wildfire risk profiles to establish patterns of risk across NC. Resulting geospatial analyses identified areas largely in Southeastern NC with high risk of wildfires that were significantly correlated with neighboring regions with high WHP, highlighting adjacent regions of high risk for future wildfire events. Cluster-based analysis of SES factors resulted in three groups of regions categorized through distinct SES profiling; two of these clusters (Clusters 2 and 3) contained indicators of high SES vulnerability. Cluster 2 contained a higher percentage of younger (<5 years), non-white, Hispanic and/or Latino residents; while Cluster 3 had the highest mean WHP and was characterized by a higher percentage of non-white residents, poverty, and less than a high school education. Counties of particular SES and WHP-combined vulnerability include those with majority non-white residents, tribal communities, and below poverty level households largely located in Southeastern NC. WHP values per census tract were dispersed to the public via the ENVIROSCAN application, alongside other environmentally-relevant data.
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Populações Vulneráveis , Incêndios Florestais , North Carolina/epidemiologia , Humanos , Incêndios Florestais/estatística & dados numéricos , Populações Vulneráveis/estatística & dados numéricos , Fatores Socioeconômicos , Análise por Conglomerados , Justiça SocialRESUMO
Toxicology research has rapidly evolved, leveraging increasingly advanced technologies in high-throughput approaches to yield important information on toxicological mechanisms and health outcomes. Data produced through toxicology studies are consequently becoming larger, often producing high-dimensional data. These types of data hold promise for imparting new knowledge, yet inherently have complexities causing them to be a rate-limiting element for researchers, particularly those that are housed in "wet lab" settings (i.e., researchers that use liquids to analyze various chemicals and biomarkers as opposed to more computationally focused, "dry lab" researchers). These types of challenges represent topics of ongoing conversation amongst our team and researchers in the field. The aim of this perspective is to i) summarize hurdles in analyzing high-dimensional data in toxicology that require improved training and translation for wet lab researchers, ii) highlight example methods that have aided in translating data analysis techniques to wet lab researchers; and iii) describe challenges that remain to be effectively addressed, to date, in toxicology research. Specific aspects include methodologies that could be introduced to wet lab researchers, including data pre-processing, machine learning, and data reduction. Current challenges discussed include model interpretability, study biases, and data analysis training. Example efforts implemented to translate these data analysis techniques are also mentioned, including online data analysis resources and hands-on workshops. Questions are also posed to continue conversation in the toxicology community. Contents of this perspective represent timely issues broadly occurring in the fields of bioinformatics and toxicology that require ongoing dialogue between wet and dry lab researchers.
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Extracellular vesicle (EV)-mediated intercellular communication significantly influences pulmonary cell health and disease, yet in vitro methods to investigate these mechanisms are limited. We hypothesize that organotypic models of the airway can be leveraged to investigate EV-mediated intercellular signaling, focusing on EV proteomic content as a case study. Two in vitro airway culture models were evaluated by mass spectrometry-based proteomics analysis: a tri-culture model consisting of alveolar epithelial, fibroblast, and lung microvascular endothelial cells and a co-culture model of alveolar epithelial and fibroblasts. EVs isolated from the tri-culture model were enriched with EV proteins regulating RNA-to-protein translation. EVs isolated from the co-culture model were enriched with EV biogenesis and extracellular matrix signaling proteins. These model-specific differences suggest that different pulmonary cell types uniquely affect EV composition and the biological pathways influenced by the EV proteome in recipient cells. These findings can inform future studies surrounding EV-related pulmonary disease pathogenesis and therapeutics.
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BACKGROUND: Ozone (O3) exposure causes respiratory effects including lung function decrements, increased lung permeability, and airway inflammation. Additionally, baseline metabolic state can predispose individuals to adverse health effects from O3. For this reason, we conducted an exploratory study to examine the effect of O3 exposure on derivatives of cholesterol biosynthesis: sterols, oxysterols, and secosteroid (25-hydroxyvitamin D) not only in the lung, but also in circulation. METHODS: We obtained plasma and induced sputum samples from non-asthmatic (n = 12) and asthmatic (n = 12) adult volunteers 6 hours following exposure to 0.4ppm O3 for 2 hours. We quantified the concentrations of 24 cholesterol precursors and derivatives by UPLC-MS and 30 cytokines by ELISA. We use computational analyses including machine learning to determine whether baseline plasma sterols are predictive of O3 responsiveness. RESULTS: We observed an overall decrease in the concentration of cholesterol precursors and derivatives (e.g. 27-hydroxycholesterol) and an increase in concentration of autooxidation products (e.g. secosterol-B) in sputum samples. In plasma, we saw a significant increase in the concentration of secosterol-B after O3 exposure. Machine learning algorithms showed that plasma cholesterol was a top predictor of O3 responder status based on decrease in FEV1 (>5%). Further, 25-hydroxyvitamin D was positively associated with lung function in non-asthmatic subjects and with sputum uteroglobin, whereas it was inversely associated with sputum myeloperoxidase and neutrophil counts. CONCLUSION: This study highlights alterations in sterol metabolites in the airway and circulation as potential contributors to systemic health outcomes and predictors of pulmonary and inflammatory responsiveness following O3 exposure.
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Ozônio , Adulto , Humanos , Ozônio/efeitos adversos , Projetos Piloto , Esteróis/farmacologia , Cromatografia Líquida , Espectrometria de Massas em Tandem , Pulmão , Inflamação/induzido quimicamente , Vitaminas/farmacologia , Vitamina D/farmacologiaRESUMO
Extracellular vesicles (EVs) represent small, membrane-enclosed particles that are derived from parent cells and are secreted into the extracellular space. Once secreted, EVs can then travel and communicate with nearby or distant cells. Due to their inherent stability and biocompatibility, these particles can effectively transfer RNAs, proteins, and chemicals/metabolites from parent cells to target cells, impacting cellular and pathological processes. EVs have been shown to respond to disease-causing agents and impact target cells. Given that disease-causing agents span environmental contaminants, pathogens, social stressors, drugs, and other agents, the translation of EV methods into public health is now a critical research gap. This paper reviews approaches to translate EVs into exposure science, toxicology, and public health applications, highlighting blood as an example due to its common use within clinical, epidemiological, and toxicological studies. Approaches are reviewed surrounding the isolation and characterization of EVs and molecular markers that can be used to inform EV cell-of-origin. Molecular cargo contained within EVs are then discussed, including an original analysis of blood EV data from Vesiclepedia. Methods to evaluate functional consequences and target tissues of EVs are also reviewed. Lastly, the expanded integration of these approaches into future public health applications is discussed, including the use of EVs as promising biomarkers of exposure, effect, and disease. IMPACT STATEMENT: Extracellular vesicles (EVs) represent small, cell-derived structures consisting of molecules that can serve as biomarkers of exposure, effect, and disease. This review lays a novel foundation for integrating EVs, a rapidly advancing molecular biological tool, into the field of public health research including epidemiological, toxicological, and clinical investigations. This article represents an important advancement in public health and exposure science as it is among the first to translate EVs into this field.
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Vesículas Extracelulares , Saúde Pública , Biomarcadores/análise , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , HumanosRESUMO
INTRODUCTION: Wildfires are a threat to public health world-wide that are growing in intensity and prevalence. The biological mechanisms that elicit wildfire-associated toxicity remain largely unknown. The potential involvement of cross-tissue communication via extracellular vesicles (EVs) is a new mechanism that has yet to be evaluated. METHODS: Female CD-1 mice were exposed to smoke condensate samples collected from the following biomass burn scenarios: flaming peat; smoldering peat; flaming red oak; and smoldering red oak, representing lab-based simulations of wildfire scenarios. Lung tissue, bronchoalveolar lavage fluid (BALF) samples, peripheral blood, and heart tissues were collected 4 and 24 h post-exposure. Exosome-enriched EVs were isolated from plasma, physically characterized, and profiled for microRNA (miRNA) expression. Pathway-level responses in the lung and heart were evaluated through RNA sequencing and pathway analyses. RESULTS: Markers of cardiopulmonary tissue injury and inflammation from BALF samples were significantly altered in response to exposures, with the greatest changes occurring from flaming biomass conditions. Plasma EV miRNAs relevant to cardiovascular disease showed exposure-induced expression alterations, including miR-150, miR-183, miR-223-3p, miR-30b, and miR-378a. Lung and heart mRNAs were identified with differential expression enriched for hypoxia and cell stress-related pathways. Flaming red oak exposure induced the greatest transcriptional response in the heart, a large portion of which were predicted as regulated by plasma EV miRNAs, including miRNAs known to regulate hypoxia-induced cardiovascular injury. Many of these miRNAs had published evidence supporting their transfer across tissues. A follow-up analysis of miR-30b showed that it was increased in expression in the heart of exposed mice in the absence of changes to its precursor molecular, pri-miR-30b, suggesting potential transfer from external sources (e.g., plasma). DISCUSSION: This study posits a potential mechanism through which wildfire exposures induce cardiopulmonary responses, highlighting the role of circulating plasma EVs in intercellular and systems-level communication between tissues.
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Vesículas Extracelulares , MicroRNAs , Incêndios Florestais , Animais , Biomassa , Vesículas Extracelulares/metabolismo , Feminino , Hipóxia , Camundongos , SoloRESUMO
Birth weight (BW) represents an important clinical and toxicological measure, indicative of the overall health of the newborn as well as potential risk for later-in-life outcomes. BW can be influenced by endogenous and exogenous factors and is known to be heavily impacted in utero by the health and function of the placenta. An aspect that remains understudied is the influence of genomic and epigenomic programming within the placenta on infant BW. To address this gap, we set out to test the hypothesis that genes involved in critical placental cell signaling are associated with infant BW, and are likely regulated, in part, through epigenetic mechanisms based on microRNA (miRNA) mediation. This study leveraged a robust dataset based on 390 infants born at low gestational age (ranged 23-27 weeks) to evaluate genome-wide expression profiles of both mRNAs and miRNAs in placenta tissues and relate these to infant BW. A total of 254 mRNAs and 268 miRNAs were identified as associated with BW, the majority of which showed consistent associations across placentas derived from both males and females. BW-associated mRNAs were found to be enriched for important biological pathways, including glycoprotein VI (the major receptor for collagen), human growth, and hepatocyte growth factor signaling, a portion of which were predicted to be regulated by BW-associated miRNAs. These miRNA-regulated pathways highlight key mechanisms potentially linking endogenous/exogenous factors to changes in birth outcomes that may be deleterious to infant and later-in-life health.