Neuroinflammation is dependent on sex and ovarian hormone presence following acute woodsmoke exposure.

Woodsmoke (WS) exposure is associated with significant health-related sequelae. Different populations can potentially exhibit varying susceptibility, based on endocrine phenotypes, to WS and investigating neurological impacts following inhaled WS is a growing area of research. In this study, a whole-body inhalation chamber was used to expose both male and female C57BL/6 mice (n = 8 per group) to either control filtered air (FA) or acute WS (0.861 ± 0.210 mg/m3) for 4 h/d for 2 days. Neuroinflammatory and lipid-based biological markers were then assessed. In a second set of studies, female mice were divided into two groups: one group was ovariectomized (OVX) to simulate an ovarian hormone-deficient state (surgical menopause), and the other underwent Sham surgery as controls, to mechanistically assess the impact of ovarian hormone presence on neuroinflammation following FA and acute WS exposure to simulate an acute wildfire episode. There was a statistically significant impact of sex (P ≤ 0.05) and statistically significant interactions between sex and treatment in IL-1ß, CXCL-1, TGF-ß, and IL-6 brain relative gene expression. Hippocampal and cortex genes also exhibited significant changes in acute WS-exposed Sham and OVX mice, particularly in TGF-ß (hippocampus) and CCL-2 and CXCL-1 (cortex). Cortex GFAP optical density (OD) showed a notable elevation in male mice exposed to acute WS, compared to the control FA. Sham and OVX females demonstrated differential GFAP expression, depending on brain region. Overall, targeted lipidomics in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serum and brain lipids demonstrated more significant changes between control FA and acute WS exposure in female mice, compared to males. In summary, male and female mice show distinct neuroinflammatory markers in response to acute WS exposure. Furthermore, ovarian hormone deficiency may impact the neuroinflammatory response following an acute WS event.

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure.

BACKGROUND: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. METHODS: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. RESULTS: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1ß, TNF-α, CXCL-1, CCL-5, TGF-ß, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1ß, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1ß, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. CONCLUSIONS: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Serum autoantibodies and exploratory molecular pathways in rural miners: A pilot study.

Introduction: The Southwestern United States (SWUS) has an extensive history of coal and metal mining, including uranium (U) mining. Lung diseases, including but not limited to, lung cancer and pulmonary fibrosis, have been studied extensively in miners due to occupational, dust-related exposures. However, high-throughput autoimmune biomarkers are largely understudied in miners, despite the fact that ore miners, such as U-miners, are at an increased risk for the development of autoimmune diseases such as systemic sclerosis and systemic lupus erythematosus (SLE). Additionally, there are current gaps in knowledge regarding which signaling pathways may play a role in occupational exposure-associated autoimmunity. Methods: Most current and former miners in the SWUS live close to their previous workplaces, in remote areas, with limited access to healthcare. In this pilot study, by leveraging a mobile clinical platform for patient care and clinical outreach, we recruited 44 miners who self-identified as either U (n = 10) or non-U miners (n = 34) and received health screenings. Serum IgG and IgM autoantibodies against 128 antigens were assessed using a high-throughput molecular technique, as a preliminary health screening opportunity. Results: Even when adjusting for age as a covariate, there was a significant (p < 0.05) association between self-reported U-mining exposure and biomarkers including IgM alpha-actinin, histones H2B, and H4, myeloperoxidase (MPO) and myelin basic protein. However, adjusting for age did not result in significant associations for IgG autoantibody production in U-miners. Bioinformatic pathway analysis revealed several altered signaling pathways between IgM and IgG autoantibodies among both U and non-U miners. Conclusions: Further research is warranted regarding the mechanistic connection between U-exposure and autoantibody development, especially regarding histone-related alterations and IgM autoantibody production.

VCAM-1 Is Upregulated in Uranium Miners Compared to Other Miners.

The United States has a rich history of mining including uranium (U)-mining, coal mining, and other metal mining. Cardiovascular diseases (CVD) are largely understudied in miners and recent literature suggests that when compared to non-U miners, U-miners are more likely to report CVD. However, the molecular basis for this phenomenon is currently unknown. In this pilot study, a New Mexico (NM)-based occupational cohort of current and former miners (n = 44) were recruited via a mobile screening clinic for miners. Serum- and endothelial-based endpoints were used to assess circulating inflammatory potential relevant to CVD. Non-U miners reported significantly fewer pack years of smoking than U-miners. Circulating biomarkers of interest revealed that U-miners had significantly greater serum amyloid A (SAA), soluble intercellular adhesion molecule 1 (ICAM-1, ng/mL), soluble vascular cell adhesion molecule 1 (VCAM-1, ng/mL), and VCAM-1 mRNA expression, as determined by the serum cumulative inflammatory potential (SCIP) assay, an endothelial-based assay. Even after adjusting for various covariates, including age, multivariable analysis determined that U-miners had significantly upregulated VCAM-1 mRNA. In conclusion, VCAM-1 may be an important biomarker and possible contributor of CVD in U-miners. Further research to explore this mechanism may be warranted.

Uptake and Toxicity of Respirable Carbon-Rich Uranium-Bearing Particles: Insights into the Role of Particulates in Uranium Toxicity.

Particulate matter (PM) presents an environmental health risk for communities residing close to uranium (U) mine sites. However, the role of the particulate form of U on its cellular toxicity is still poorly understood. Here, we investigated the cellular uptake and toxicity of C-rich U-bearing particles as a model organic particulate containing uranyl citrate over a range of environmentally relevant concentrations of U (0-445 µM). The cytotoxicity of C-rich U-bearing particles in human epithelial cells (A549) was U-dose-dependent. No cytotoxic effects were detected with soluble U doses. Carbon-rich U-bearing particles with a wide size distribution (<10 µm) presented 2.7 times higher U uptake into cells than the particles with a narrow size distribution (<1 µm) at 100 µM U concentration. TEM-EDS analysis identified the intracellular translocation of clusters of C-rich U-bearing particles. The accumulation of C-rich U-bearing particles induced DNA damage and cytotoxicity as indicated by the increased phosphorylation of the histone H2AX and cell death, respectively. These findings reveal the toxicity of the particulate form of U under environmentally relevant heterogeneous size distributions. Our study opens new avenues for future investigations on the health impacts resulting from environmental exposures to the particulate form of U near mine sites.

Mine-site derived particulate matter exposure exacerbates neurological and pulmonary inflammatory outcomes in an autoimmune mouse model.

The Southwestern United States has a legacy of industrial mining due to the presence of rich mineral ore deposits. The relationship between environmental inhaled particulate matter (PM) exposures and neurological outcomes within an autoimmune context is understudied. The aim of this study was to compare two regionally-relevant dusts from high-priority abandoned mine-sites, Claim 28 PM, from Blue Gap Tachee, AZ and St. Anthony mine PM, from the Pueblo of Laguna, NM and to expose autoimmune-prone mice (NZBWF1/J). Mice were randomly assigned to one of three groups (n = 8/group): DM (dispersion media, control), Claim 28 PM, or St. Anthony PM, subjected to oropharyngeal aspiration of (100 µg/50 µl), once per week for a total of 4 consecutive doses. A battery of immunological and neurological endpoints was assessed at 24 weeks of age including: bronchoalveolar lavage cell counts, lung gene expression, brain immunohistochemistry, behavioral tasks and serum autoimmune biomarkers. Bronchoalveolar lavage results demonstrated a significant increase in number of polymorphonuclear neutrophils following Claim 28 and St. Anthony mine PM aspiration. Lung mRNA expression showed significant upregulation in CCL-2 and IL-1ß following St. Anthony mine PM aspiration. In addition, neuroinflammation was present in both Claim 28 and St. Anthony mine-site derived PM exposure groups. Behavioral tasks resulted in significant deficits as determined by Y-maze new arm frequency following Claim 28 aspiration. Neutrophil elastase was significantly upregulated in the St. Anthony mine exposure group. Interestingly, there were no significant changes in serum autoantigens suggesting systemic inflammatory effects may be mediated through other molecular mechanisms following low-dose PM exposures.

Assessment of particulate matter toxicity and physicochemistry at the Claim 28 uranium mine site in Blue Gap, AZ.

Thousands of abandoned uranium mines (AUMs) exist in the western United States. Due to improper remediation, windblown dusts generated from AUMs are of significant community concern. A mobile inhalation lab was sited near an AUM of high community concern ("Claim 28") with three primary objectives: to (1) determine the composition of the regional ambient particulate matter (PM), (2) assess meteorological characteristics (wind speed and direction), and (3) assess immunological and physiological responses of mice after exposures to concentrated ambient PM (or CAPs). C57BL/6 and apolipoprotein E-null (ApoE-/-) mice were exposed to CAPs in AirCARE1 located approximately 1 km to the SW of Claim 28, for 1 or 28 days for 4 hr/day at approximately 80 µg/m3 CAPs. Bronchoalveolar lavage fluid (BALF) analysis revealed a significant influx of neutrophils after a single-day exposure in C57BL/6 mice (average PM2.5 concentration = 68 µg/m3). Lungs from mice exposed for 1 day exhibited modest increases in Tnfa and Tgfb mRNA levels in the CAPs exposure group compared to filtered air (FA). Lungs from mice exposed for 28 days exhibited reduced Tgfb (C57BL/6) and Tnfa (ApoE-/-) mRNA levels. Wind direction was typically moving from SW to NE (away from the community) and, while detectable in all samples, uranium concentrations in the PM2.5 fraction were not markedly different from published-reported values. Overall, exposure to CAPs in the region of the Blue GAP Tachee's Claim-28 uranium mine demonstrated little evidence of overt pulmonary injury or inflammation or ambient air contamination attributed to uranium or vanadium.

Serum-borne factors alter cerebrovascular endothelial microRNA expression following particulate matter exposure near an abandoned uranium mine on the Navajo Nation.

BACKGROUND: Commercial uranium mining on the Navajo Nation has subjected communities on tribal lands in the Southwestern United States to exposures from residual environmental contamination. Vascular health effects from these ongoing exposures are an active area of study. There is an association between residential mine-site proximity and circulating biomarkers in residents, however, the contribution of mine-site derived wind-blown dusts on vascular and other health outcomes is unknown. To assess neurovascular effects of mine-site derived dusts, we exposed mice using a novel exposure paradigm, the AirCARE1 mobile inhalation laboratory, located 2 km from an abandoned uranium mine, Claim 28 in Blue Gap Tachee, AZ. Mice were exposed to filtered air (FA) (n = 6) or concentrated ambient particulate matter (CAPs) (n = 5) for 2 wks for 4 h per day. RESULTS: To assess miRNA differential expression in cultured mouse cerebrovascular cells following particulate matter (PM) exposure (average: 96.6 ± 60.4 µg/m3 for all 4 h exposures), the serum cumulative inflammatory potential (SCIP) assay was employed. MiRNA sequencing was then performed in cultured mouse cerebrovascular endothelial cells (mCECs) to evaluate transcriptional changes. Results indicated 27 highly differentially expressed (p < 0.01) murine miRNAs, as measured in the SCIP assay. Gene ontology (GO) pathway analysis revealed notable alterations in GO enrichment related to the cytoplasm, protein binding and the cytosol, while significant KEGG pathways involved pathways in cancer, axon guidance and Wnt signaling. Expression of these 27 identified, differentially expressed murine miRNAs were then evaluated in the serum. Nine of these miRNAs (~ 30%) were significantly altered in the serum and 8 of those miRNAs demonstrated the same directional change (either upregulation or downregulation) as cellular miRNAs, as measured in the SCIP assay. Significantly upregulated miRNAs in the CAPs exposure group included miRNAs in the let-7a family. Overexpression of mmu-let-7a via transfection experiments, suggested that this miRNA may mediate mCEC barrier integrity following dust exposure. CONCLUSIONS: Our data suggest that mCEC miRNAs as measured in the SCIP assay show similarity to serum-borne miRNAs, as approximately 30% of highly differentially expressed cellular miRNAs in the SCIP assay were also found in the serum. While translocation of miRNAs via exosomes or an alternative mechanism is certainly possible, other yet-to-be-identified factors in the serum may be responsible for significant miRNA differential expression in endothelium following inhaled exposures. Additionally, the most highly upregulated murine miRNAs in the CAPs exposure group were in the let-7a family. These miRNAs play a prominent role in cell growth and differentiation and based on our transfection experiments, mmu-let-7a may contribute to cerebrovascular mCEC alterations following inhaled dust exposure.

Vehicular Particulate Matter (PM) Characteristics Impact Vascular Outcomes Following Inhalation.

Roadside proximity and exposure to mixed vehicular emissions (MVE) have been linked to adverse pulmonary and vascular outcomes. However, because of the complex nature of the contribution of particulate matter (PM) versus gases, it is difficult to decipher the precise causative factors regarding PM and the copollutant gaseous fraction. To this end, C57BL/6 and apolipoprotein E knockout mice (ApoE-/-) were exposed to either filtered air (FA), fine particulate (FP), FP+gases (FP+G), ultrafine particulate (UFP), or UFP+gases (UFP+G). Two different timeframes were employed: 1-day (acute) or 30-day (subchronic) exposures. Examined biological endpoints included aortic vasoreactivity, aortic lesion quantification, and aortic mRNA expression. Impairments in vasorelaxation were observed following acute exposure to FP+G in C57BL/6 animals and FP, UFP, and UFP+G in ApoE-/- animals. These effects were completely abrogated or markedly reduced following subchronic exposure. Aortic lesion quantification in ApoE-/- animals indicated a significant increase in atheroma size in the UFP-, FP-, and FP+G-exposed groups. Additionally, ApoE-/- mice demonstrated a significant fold increase in TNFα expression following FP+G exposure and ET-1 following UFP exposure. Interestingly, C57BL/6 aortic gene expression varied widely across exposure groups. TNFα decreased significantly following FP exposure and CCL-5 decreased in the UFP-, FP-, and FP+G-exposed groups. Conversely, ET-1, CCL-2, and CXCL-1 were all significantly upregulated in the FP+G group. These findings suggest that gas-particle interactions may play a role in vascular toxicity, but the contribution of surface area is not clear.

Toxic Effects of Particulate Matter Derived from Dust Samples Near the Dzhidinski Ore Processing Mill, Eastern Siberia, Russia.

Ambient particulate matter (PM) is associated with increased mortality and morbidity, an effect influenced by the metal components of the PM. We characterized five sediment samples obtained near a tungsten-molybdenum ore-processing complex in Zakamensk, Russia for elemental composition and PM toxicity with regard to pulmonary, vascular, and neurological outcomes. Elemental and trace metals analysis of complete sediment and PM10 (the respirable fraction, < 10 µm mass mean aerodynamic diameter) were performed using inductively coupled plasma optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS). Sediment samples and PM10 consisted largely of silicon and iron and silicon and sodium, respectively. Trace metals including manganese and uranium in the complete sediment, as well as copper and lead in the PM10 were observed. Notably, metal concentrations were approximately 10 × higher in the PM10 than in the sediment. Exposure to 100 µg of PM10 via oropharyngeal aspiration in C56BL/6 mice resulted in pulmonary inflammation across all groups. In addition, mice exposed to three of the five PM10 samples exhibited impaired endothelial-dependent relaxation, and correlative analysis revealed associations between pulmonary inflammation and levels of lead and cadmium. A tendency for elevated cortical ccl2 and Tnf-α mRNA expression was induced by all samples and significant upregulation was noted following exposure to PM10 samples Z3 and Z4, respectively. Cortical Nqo1 mRNA levels were significantly upregulated in mice exposed to PM10 Z2. In conclusion, pulmonary exposure to PM samples from the Zakamensk region sediments induced varied pulmonary and systemic effects that may be influenced by elemental PM composition. Further investigation is needed to pinpoint putative drivers of neurological outcomes.

Metal-Induced Pulmonary Fibrosis.

PURPOSE OF REVIEW: The incidence of pulmonary fibrosis is increasing worldwide and may, in part, be due to occupational and environmental exposures. Secondary fibrotic interstitial lung diseases may be mistaken for idiopathic pulmonary fibrosis with important implications for both disease management and prognosis. The purposes of this review are to shed light on possible underlying causes of interstitial pulmonary fibrosis and to encourage dialogue on the importance of acquiring a thorough patient history of occupational and environmental exposures. RECENT FINDINGS: A recent appreciation for various occupational and environmental metals inducing both antigen-specific immune reactions in the lung and nonspecific "innate" immune system responses has emerged and with it a growing awareness of the potential hazards to the lung caused by low-level metal exposures. Advancements in the contrast and quality of high-resolution CT scans and identification of histopathological patterns of interstitial pulmonary fibrosis have improved clinical diagnostics. Moreover, recent findings indicate specific hotspots of pulmonary fibrosis within the USA. Increased prevalence of lung disease in these areas appears to be linked to occupational/environmental metal exposure and ethnic susceptibility/vulnerability. A systematic overview of possible occupational and environmental metals causing interstitial pulmonary fibrosis and a detailed evaluation of vulnerable/susceptible populations may facilitate a broader understanding of potential underlying causes and highlight risks of disease predisposition.

Respirable Uranyl-Vanadate-Containing Particulate Matter Derived From a Legacy Uranium Mine Site Exhibits Potentiated Cardiopulmonary Toxicity.

Exposure to windblown particulate matter (PM) arising from legacy uranium (U) mine sites in the Navajo Nation may pose a human health hazard due to their potentially high metal content, including U and vanadium (V). To assess the toxic impact of PM derived from Claim 28 (a priority U mine) compared with background PM, and consider the putative role of metal species U and V. Two representative sediment samples from Navajo Nation sites (Background PM and Claim 28 PM) were obtained, characterized in terms of chemistry and morphology, and fractioned to the respirable (≤ 10 µm) fraction. Mice were dosed with either PM sample, uranyl acetate, or vanadyl sulfate via aspiration (100 µg), with assessments of pulmonary and vascular toxicity 24 h later. Particulate matter samples were also examined for in vitro effects on cytotoxicity, oxidative stress, phagocytosis, and inflammasome induction. Claim 28 PM10 was highly enriched with U and V and exhibited a unique nanoparticle ultrastructure compared with background PM10. Claim 28 PM10 exhibited enhanced pulmonary and vascular toxicity relative to background PM10. Both U and V exhibited complementary pulmonary inflammatory potential, with U driving a classical inflammatory cytokine profile (elevated interleukin [IL]-1ß, tumor necrosis factor-α, and keratinocyte chemoattractant/human growth-regulated oncogene) while V preferentially induced a different cytokine pattern (elevated IL-5, IL-6, and IL-10). Claim 28 PM10 was more potent than background PM10 in terms of in vitro cytotoxicity, impairment of phagocytosis, and oxidative stress responses. Resuspended PM10 derived from U mine waste exhibit greater cardiopulmonary toxicity than background dusts. Rigorous exposure assessment is needed to gauge the regional health risks imparted by these unremediated sites.

Surface area-dependence of gas-particle interactions influences pulmonary and neuroinflammatory outcomes.

BACKGROUND: Deleterious consequences of exposure to traffic emissions may derive from interactions between carbonaceous particulate matter (PM) and gaseous components in a manner that is dependent on the surface area or complexity of the particles. To determine the validity of this hypothesis, we examined pulmonary and neurological inflammatory outcomes in C57BL/6 and apolipoprotein E knockout (ApoE-/-) male mice after acute and chronic exposure to vehicle engine-derived particulate matter, generated as ultrafine (UFP) and fine (FP) sizes, with additional exposures using UFP or FP combined with gaseous copollutants derived from fresh gasoline and diesel emissions, labeled as UFP + G and FP + G. RESULTS: The UFP and UFP + G exposure groups resulted in the most profound pulmonary and neuroinflammatory effects. Phagocytosis of UFP + G particles via resident alveolar macrophages was substantial in both mouse strains, particularly after chronic exposure, with concurrent increased proinflammatory cytokine expression of CXCL1 and TNFα in the bronchial lavage fluid. In the acute exposure paradigm, only UFP and UFP + G induced significant changes in pulmonary inflammation and only in the ApoE-/- animals. Similarly, acute exposure to UFP and UFP + G increased the expression of several cytokines in the hippocampus of ApoE-/- mice including Il-1ß, IL-6, Tgf-ß and Tnf-α and in the hippocampus of C57BL/6 mice including Ccl5, Cxcl1, Il-1ß, and Tnf-α. Interestingly, Il-6 and Tgf-ß expression were decreased in the C57BL/6 hippocampus after acute exposure. Chronic exposure to UFP + G increased expression of Ccl5, Cxcl1, Il-6, and Tgf-ß in the ApoE-/- hippocampus, but this effect was minimal in the C57BL/6 mice, suggesting compensatory mechanisms to manage neuroinflammation in this strain. CONCLUSIONS: Inflammatory responses the lung and brain were most substantial in ApoE-/- animals exposed to UFP + G, suggesting that the surface area-dependent interaction of gases and particles is an important determinant of toxic responses. As such, freshly generated UFP, in the presence of combustion-derived gas phase pollutants, may be a greater health hazard than would be predicted from PM concentration, alone, lending support for epidemiological findings of adverse neurological outcomes associated with roadway proximity.

Serum from obstructive sleep apnea patients induces inflammatory responses in coronary artery endothelial cells.

BACKGROUND AND AIMS: Obstructive sleep apnea (OSA) is characterized by intermittent airway obstruction and systemic hypoxia during sleep, which can contribute to an increase in reactive oxygen species, vascular remodeling, vasoconstriction and ultimately cardiovascular disease. Continuous positive airway pressure (CPAP) is a clinical therapy that maintains airway patency and mitigates several symptoms of OSA. However, it is currently unknown whether CPAP therapy also reduces the overall inflammatory potential in the circulation; to address this in an unbiased manner, we applied a novel endothelial biosensor approach, the serum cumulative inflammatory potential (SCIP) assay. METHODS: We studied healthy controls (n = 7), OSA subjects receiving no treatment, (OSA controls) (n = 7) and OSA subjects receiving CPAP for 3 months (n = 8). Serum was obtained from OSA subjects before and after CPAP or no treatment. A battery of quantitative and functional assays was performed to assess the serum inflammatory potential, in terms of endothelial responses. For the SCIP assay, human coronary artery endothelial cells (hCAECs) were incubated with 5% serum in media from individual subjects for 4 h. qPCR was performed to assess endothelial inflammatory transcript (ICAM-1, VCAM-1, IL-8, P-selectin, CCL5, and CXCL12) responses to serum. Additionally, transendothelial resistance was measured in serum-incubated hCAECs following leukocyte challenge. RESULTS: hCAECs exhibited significant increases in VCAM-1, ICAM-1, IL-8 and P-selectin mRNA when incubated with serum from OSA patients compared to serum from healthy control subjects. Furthermore, compared to no treatment, serum from CPAP-treated individuals was less potent at inducing inflammatory gene expression in the SCIP assay. Similarly, in a leukocyte adhesion assay, naïve cells treated with serum from patients who received CPAP exhibited improved endothelial barrier function than cells treated with OSA control serum. CONCLUSIONS: OSA results in greater serum inflammatory potential, thereby driving endothelial activation and dysfunction.

Hypoxia-induced pulmonary arterial hypertension augments lung injury and airway reactivity caused by ozone exposure.

Ozone (O3)-related cardiorespiratory effects are a growing public health concern. Ground level O3 can exacerbate pre-existing respiratory conditions; however, research regarding therapeutic interventions to reduce O3-induced lung injury is limited. In patients with chronic obstructive pulmonary disease, hypoxia-associated pulmonary hypertension (HPH) is a frequent comorbidity that is difficult to treat clinically, yet associated with increased mortality and frequency of exacerbations. In this study, we hypothesized that established HPH would confer vulnerability to acute O3 pulmonary toxicity. Additionally, we tested whether improvement of pulmonary endothelial barrier integrity via rho-kinase inhibition could mitigate pulmonary inflammation and injury. To determine if O3 exacerbated HPH, male C57BL/6 mice were subject to either 3 weeks continuous normoxia (20.9% O2) or hypoxia (10.0% O2), followed by a 4-h exposure to either 1ppm O3 or filtered air (FA). As an additional experimental intervention fasudil (20mg/kg) was administered intraperitoneally prior to and after O3 exposures. As expected, hypoxia significantly increased right ventricular pressure and hypertrophy. O3 exposure in normoxic mice caused lung inflammation but not injury, as indicated by increased cellularity and edema in the lung. However, in hypoxic mice, O3 exposure led to increased inflammation and edema, along with a profound increase in airway hyperresponsiveness to methacholine. Fasudil administration resulted in reduced O3-induced lung injury via the enhancement of pulmonary endothelial barrier integrity. These results indicate that increased pulmonary vascular pressure may enhance lung injury, inflammation and edema when exposed to pollutants, and that enhancement of pulmonary endothelial barrier integrity may alleviate such vulnerability.

Ozone Inhalation Impairs Coronary Artery Dilation via Intracellular Oxidative Stress: Evidence for Serum-Borne Factors as Drivers of Systemic Toxicity.

Ambient ozone (O3) levels are associated with cardiovascular morbidity and mortality, but the underlying pathophysiological mechanisms driving extrapulmonary toxicity remain unclear. This study examined the coronary vascular bed of rats in terms of constrictive and dilatory responses to known agonists following a single O3 inhalation exposure. In addition, serum from exposed rats was used in ex vivo preparations to examine whether bioactivity and toxic effects of inhaled O3 could be conveyed to extrapulmonary systems via the circulation. We found that 24 h following inhalation of 1 ppm O3, isolated coronary vessels exhibited greater basal tone and constricted to a greater degree to serotonin stimulation. Vasodilation to acetylcholine (ACh) was markedly diminished in coronary arteries from O3-exposed rats, compared with filtered air-exposed controls. Dilation to ACh was restored by combined superoxide dismutase and catalase treatment, and also by NADPH oxidase inhibition. When dilute (10%) serum from exposed rats was perfused into the lumen of coronary arteries from unexposed, naïve rats, the O3-induced reduction in vasodilatory response to ACh was partially recapitulated. Furthermore, following O3 inhalation, serum exhibited a nitric oxide scavenging capacity, which may partially explain blunted ACh-mediated vasodilatory responses. Thus, bioactivity from inhalation exposures may be due to compositional changes of the circulation. These studies shed light on possible mechanisms of action that may explain O3-associated cardiac morbidity and mortality in humans.

Mitigation of colitis with NovaSil clay therapy.

BACKGROUND/AIMS: Five million people currently live with Crohn's disease (CD) or ulcerative colitis, the two major forms of inflammatory bowel disease. Available treatments frequently result in side effects that compromise the immune health of the patient. Consequently, alternative therapies that cause fewer systemic effects are needed. Dioctahedral smectite clays have been utilized to treat medical conditions, including diarrheal and enteric disease. Herein, we report the ability of a refined dioctahedral smectite (NovaSil, NS) to sorb inflammatory proteins and reduce inflammation in a TNBS (2,4,6-trinitrobenzenesulfonic acid) mouse model of CD. We also investigated whether NS could rescue gut microbial diversity in TNBS-induced mice. METHODS: ELISA, X-ray diffraction, and transmission electron microscopy were employed to characterize the NS-cytokine interaction in vitro. A TNBS mouse colitis model was utilized to study the efficacy of NS supplementation for 4 weeks. The three treatment groups included control, TNBS, and TNBS + NS. DNA was extracted from feces and sorted for bacterial phylogenetic analysis. RESULTS: Results suggest that NS binds TNFα in vitro. In TNBS-treated mice, supplementation with NS significantly reduced weight loss, and serum proinflammatory cytokine levels (IL-2, IL-6, and IL-12, TNFα, IFNγ) compared with the TNBS group. TNBS-treated mice demonstrated a significant reduction in gut microbiota species richness when compared with the TNBS + NS group and control group. CONCLUSIONS: NovaSil mitigated the effects of TNBS-induced colitis based on reduction in systemic markers of inflammation, significant improvement in weight gain, and intestinal microbial profile.

Short-term safety and efficacy of calcium montmorillonite clay (UPSN) in children.

Recently, an association between childhood growth stunting and aflatoxin (AF) exposure has been identified. In Ghana, homemade nutritional supplements often consist of AF-prone commodities. In this study, children were enrolled in a clinical intervention trial to determine the safety and efficacy of Uniform Particle Size NovaSil (UPSN), a refined calcium montmorillonite known to be safe in adults. Participants ingested 0.75 or 1.5 g UPSN or 1.5 g calcium carbonate placebo per day for 14 days. Hematological and serum biochemistry parameters in the UPSN groups were not significantly different from the placebo-controlled group. Importantly, there were no adverse events attributable to UPSN treatment. A significant reduction in urinary metabolite (AFM1) was observed in the high-dose group compared with placebo. Results indicate that UPSN is safe for children at doses up to 1.5 g/day for a period of 2 weeks and can reduce exposure to AFs, resulting in increased quality and efficacy of contaminated foods.

The effect of aflatoxin-B1 on red drum (Sciaenops ocellatus) and assessment of dietary supplementation of NovaSil for the prevention of aflatoxicosis.

Aflatoxin B1 (AFB1) is a potent carcinogen that causes growth stunting, immunosuppression and liver cancer in multiple species. The recent trend of replacing fishmeal with plant-based proteins in fish feed has amplified the AFB1 exposure risk in farm-raised fish. NovaSil (NS), a calcium montmorillonite clay, has previously been shown to reduce AFB1 bioavailability safely and efficaciously in several mammalian species. This study was designed to: (1) evaluate AFB1 impact on cultured red drum, Sciaenops ocellatus, over the course of seven weeks; and (2) assess NS supplementation as a strategy to prevent aflatoxicosis. Fish were fed diets containing 0, 0.1, 0.25, 0.5, 1, 2, 3, or 5 ppm AFB1. Two additional treatment groups were fed either 5 ppm AFB1 + 1% NS or 5 ppm AFB1 + 2% NS. Aflatoxin B1 negatively impacted red drum weight gain, survival, feed efficiency, serum lysozyme concentration, hepatosomatic index (HSI), whole-body lipid levels, liver histopathological scoring, as well as trypsin inhibition. NovaSil inclusion in AFB1-contaminated diets improved weight gain, feed efficiency, serum lysozyme concentration, muscle somatic index, and intraperitoneal fat ratios compared to AFB1-treated fish. Although not significant, NS reduced AFB1-induced histopathological changes in the liver and decreased Proliferating Cell Nuclear Antigen (PCNA) staining. Importantly, NS supplementation improved overall health of AFB1-exposed red drum.