Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an ARDS Model.

Rationale: Acute Respiratory Distress Syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but to date, no Orai1-specific inhibitors exist. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly-isolated ARDS patient immune cells. A murine acute lung injury model caused by bacterial pneumonia was then used. Mice were infected with P. aeruginosa, S. aureus, methicillin-resistant S. aureus (MRSA) or multidrug-resistant P. aeruginosa (MDR-Pa) and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with an IC50 of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including MRSA. ELD607 worked as an immunomodulator that reduced cytokine levels, lowered neutrophilia and promoted both macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multi-organ inflammation and treat antibiotic-resistant bacteria.

Occupational arsenic exposure and genitourinary cancer: systematic review and meta-analysis.

BACKGROUND: Existing evidence suggests that ingestion of high doses of arsenic through drinking water is associated with an increased risk of genitourinary cancers, while systematic evidence on workers exposed to arsenic is lacking. AIMS: The aim of this study is to systematically review the evidence on the association between occupational exposure to arsenic and genitourinary cancer risk and mortality. METHODS: A systematic literature search was carried out on Pubmed, Web of Science and Embase by including cohort and case-control studies. Study-specific relative risks (RRs) and the corresponding 95% confidence intervals (CIs) were pooled using Mandel-Paule random-effects model. Contour-enhanced funnel plot and Egger's test were used to assess the occurrence of publication bias. RESULTS: A total of 17 studies were included in the meta-analysis, 7 on cancer incidence (n = 161,244 individuals) and 10 on cancer mortality (n = 91,868). Most of them were cohort (71%) and industry-based studies (59%). The meta-analysis failed to detect an increased risk of genitourinary cancers among workers exposed to arsenic, except for a suggestive but not significant positive association for bladder cancer incidence (RR: 1.26, 95% CI: 0.89, 1.80), although this estimate was based on only three studies. No compelling evidence of publication bias was found (P = 0.885). CONCLUSIONS: Our findings did not show an association between occupational exposure to arsenic and genitourinary cancers, although further high-quality studies with detailed exposure assessment at the individual level are needed to clarify this relationship.

SPLUNC1 is a negative regulator of the Orai1 Ca2+ channel.

Orai1 is a ubiquitously-expressed plasma membrane Ca2+ channel that is involved in store-operated Ca2+ entry (SOCE): a fundamental biological process that regulates gene expression, the onset of inflammation, secretion, and the contraction of airway smooth muscle (ASM). During SOCE, Ca2+ leaves the endoplasmic reticulum, which then stimulates a second, amplifying wave of Ca2+ influx through Orai1 into the cytoplasm. Short Palate LUng and Nasal epithelial Clone 1 (SPLUNC1; gene name BPIFA1) is a multi-functional, innate defense protein that is highly abundant in the lung. We have previously reported that SPLUNC1 was secreted from epithelia, where it bound to and inhibited Orai1, leading to reduced SOCE and ASM relaxation. However, the underlying mechanism of action is unknown. Here, we probed the SPLUNC1-Orai1 interactions in ASM and HEK293T cells using biochemical and imaging techniques. We observed that SPLUNC1 caused a conformational change in Orai1, as measured using Forster resonance energy transfer (FRET). SPLUNC1 binding also led to Nedd4-2 dependent ubiquitination of Orai1. Moreover, SPLUNC1 internalized Orai1 to lysosomes, leading to Orai1 degradation. Thus, we conclude that SPLUNC1 is an allosteric regulator of Orai1. Our data indicate that SPLUNC1-mediated Orai1 inhibition could be utilized as a therapeutic strategy to reduce SOCE.

E-Cigarettes and Cardiopulmonary Health: Review for Clinicians.

Electronic cigarettes (e-cigarettes) are battery powered electronic nicotine delivery systems that use a propylene glycol/vegetable glycerin base to deliver vaporized nicotine and flavorings to the body. E-cigarettes became commercially available without evidence regarding their risks, long-term safety, or utility in smoking cessation. Recent clinical trials suggest that e-cigarette use with counseling may be effective in reducing cigarette use but not nicotine dependence. However, meta-analyses of observational studies demonstrate that e-cigarette use is not associated with smoking cessation. Cardiovascular studies reported sympathetic activation, vascular stiffening, and endothelial dysfunction, which are associated with adverse cardiovascular events. The majority of pulmonary clinical trials in e-cigarette users included standard spirometry as the primary outcome measure, reporting no change in lung function. However, studies reported increased biomarkers of pulmonary disease in e-cigarette users. These studies were conducted in adults, but >30% of high school-age adolescents reported e-cigarette use. The effects of e-cigarette use on cardiopulmonary endpoints in adolescents and young adults remain unstudied. Because of adverse clinical findings and associations between e-cigarette use and increased incidence of respiratory diseases in people who have never smoked, large longitudinal studies are needed to understand the risk profile of e-cigarettes. Consistent with the Centers for Disease Control and Prevention recommendations, clinicians should monitor the health risks of e-cigarette use, discourage nonsmokers and adolescents from using e-cigarettes, and discourage smokers from engaging in dual use without cigarette reduction or cessation.

A SPLUNC1 Peptidomimetic Inhibits Orai1 and Reduces Inflammation in a Murine Allergic Asthma Model.

Orai1 is a plasma membrane Ca2+ channel that mediates store-operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signaling, Förster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1-/- mice were exposed to house dust mite (HDM) extract with or without α6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1-/- mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1-/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, α6 inhibited SOCE in multiple immune cell types, and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.

Acute cigarette smoke or extract exposure rapidly activates TRPA1-mediated calcium influx in primary human airway smooth muscle cells.

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.

Reactive Oxygen Species, Mitochondrial Membrane Potential, and Cellular Membrane Potential Are Predictors of E-Liquid Induced Cellular Toxicity.

INTRODUCTION: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear. METHODS: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondrial membrane potential (ΔΨ  m), reactive oxygen species production (ROS), and cellular membrane potential (Vm) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of ≥90 commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry. RESULTS: We found that e-liquids caused a decrease in ΔΨ  m and Vm and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied. CONCLUSION: Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition. IMPLICATIONS: E-liquid cellular toxicity can be predicted using parameters amenable to HTS. Our data suggest that ΔΨ  m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure, and this toxicity is linked to the chemical composition, that is, flavoring components. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.

Cigarette Smoke Exposure Induces Retrograde Trafficking of CFTR to the Endoplasmic Reticulum.

Chronic obstructive pulmonary disease (COPD), which is most commonly caused by cigarette smoke (CS) exposure, is the third leading cause of death worldwide. The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane anion channel that is widely expressed in epithelia throughout the body. In the airways, CFTR plays an important role in fluid homeostasis and helps flush mucus and inhaled pathogens/toxicants out of the lung. Inhibition of CFTR leads to mucus stasis and severe airway disease. CS exposure also inhibits CFTR, leading to the decreased anion secretion/hydration seen in COPD patients. However, the underlying mechanism is poorly understood. Here, we report that CS causes CFTR to be internalized in a clathrin/dynamin-dependent fashion. This internalization is followed by retrograde trafficking of CFTR to the endoplasmic reticulum. Although this internalization pathway has been described for bacterial toxins and cargo machinery, it has never been reported for mammalian ion channels. Furthermore, the rapid internalization of CFTR is dependent on CFTR dephosphorylation by calcineurin, a protein phosphatase that is upregulated by CS. These results provide new insights into the mechanism of CFTR internalization, and may help in the development of new therapies for CFTR correction and lung rehydration in patients with debilitating airway diseases such as COPD.

Increases in cytosolic Ca2+ induce dynamin- and calcineurin-dependent internalisation of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic Ca2+. However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.

Evaluation of e-liquid toxicity using an open-source high-throughput screening assay.

The e-liquids used in electronic cigarettes (E-cigs) consist of propylene glycol (PG), vegetable glycerin (VG), nicotine, and chemical additives for flavoring. There are currently over 7,700 e-liquid flavors available, and while some have been tested for toxicity in the laboratory, most have not. Here, we developed a 3-phase, 384-well, plate-based, high-throughput screening (HTS) assay to rapidly triage and validate the toxicity of multiple e-liquids. Our data demonstrated that the PG/VG vehicle adversely affected cell viability and that a large number of e-liquids were more toxic than PG/VG. We also performed gas chromatography-mass spectrometry (GC-MS) analysis on all tested e-liquids. Subsequent nonmetric multidimensional scaling (NMDS) analysis revealed that e-liquids are an extremely heterogeneous group. Furthermore, these data indicated that (i) the more chemicals contained in an e-liquid, the more toxic it was likely to be and (ii) the presence of vanillin was associated with higher toxicity values. Further analysis of common constituents by electron ionization revealed that the concentration of cinnamaldehyde and vanillin, but not triacetin, correlated with toxicity. We have also developed a publicly available searchable website (www.eliquidinfo.org). Given the large numbers of available e-liquids, this website will serve as a resource to facilitate dissemination of this information. Our data suggest that an HTS approach to evaluate the toxicity of multiple e-liquids is feasible. Such an approach may serve as a roadmap to enable bodies such as the Food and Drug Administration (FDA) to better regulate e-liquid composition.

Chronic E-Cigarette Exposure Alters the Human Bronchial Epithelial Proteome.

RATIONALE: E-cigarettes vaporize propylene glycol/vegetable glycerin (PG/VG), nicotine, and flavorings. However, the long-term health effects of exposing lungs to vaped e-liquids are unknown. OBJECTIVES: To determine the effects of chronic vaping on pulmonary epithelia. METHODS: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers) and obtained bronchial brush biopsies and lavage samples from these subjects for proteomic investigation. We further employed in vitro and murine exposure models to support our human findings. MEASUREMENTS AND MAIN RESULTS: Visual inspection by bronchoscopy revealed that vaper airways appeared friable and erythematous. Epithelial cells from biopsy samples revealed approximately 300 proteins that were differentially expressed in smoker and vaper airways, with only 78 proteins being commonly altered in both groups and 113 uniquely altered in vapers. For example, CYP1B1 (cytochrome P450 family 1 subfamily B member 1), MUC5AC (mucin 5 AC), and MUC4 levels were increased in vapers. Aerosolized PG/VG alone significantly increased MUC5AC protein in human airway epithelial cultures and in murine nasal epithelia in vivo. We also found that e-liquids rapidly entered cells and that PG/VG reduced membrane fluidity and impaired protein diffusion. CONCLUSIONS: We conclude that chronic vaping exerts marked biological effects on the lung and that these effects may in part be mediated by the PG/VG base. These changes are likely not harmless and may have clinical implications for the development of chronic lung disease. Further studies will be required to determine the full extent of vaping on the lung.

E-Liquid Autofluorescence can be used as a Marker of Vaping Deposition and Third-Hand Vape Exposure.

In the past 5 years, e-cigarette use has been increasing rapidly, particularly in youth and young adults. Due to the novelty of e-cigarettes (e-cigs) and e-cigarette liquids (e-liquids), research on their chemo-physical properties is still in its infancy. Here, we describe a previously unknown and potentially useful property of e-liquids, namely their autofluorescence. We performed an emission scan at 9 excitation wavelengths common to fluorescent microscopy and found (i) that autofluorescence differs widely between e-liquids, (ii) that e-liquids are most fluorescent in the UV range (between 350 and 405 nm) and (iii) fluorescence intensity wanes as the emission wavelength increases. Furthermore, we used the autofluorescence of e-liquids as a marker for tracking e-cig aerosol deposition in the laboratory. Using linear regression analysis, we were able to quantify the deposition of a "vaped" e-liquid onto hard surfaces. Using this technique, we found that every 70 mL puff of an e-cigarette deposited 0.019% e-liquid (v/v) in a controlled environment. Finally, we vaped a surface in the laboratory and used our method to detect e-cig aerosol third-hand exposure. In conclusion, our data suggest that e-cigarette autofluorescence can be used as a marker of e-cigarette deposition.

Tobacco Smoke Constituents Trigger Cytoplasmic Calcium Release.

Cytosolic Ca2+ is a universal second messenger that is involved in many processes throughout the body, including the regulation of cell growth/cell division, apoptosis, and the secretion of both ions, and macromolecules. Tobacco smoke exerts multiple effects on airway epithelia and we have previously shown that Kentucky reference cigarette smoke exposure elevated the second messenger Ca2+, leading to dysfunctional ion secretion. In this study, we tested whether little cigar and commercial cigarette smoke exposure exerts similar effects on intracellular Ca2+ levels. Indeed, Swisher Sweets, Captain Black, and Cheyenne little cigars, as well as Camel, Marlboro, and Newport cigarettes, triggered a comparable increase in intracellular Ca2+ as seen with Kentucky reference cigarettes in human bronchial epithelia. We also found that Kentucky reference cigarette smoke exposure caused increases in Ca2+ in HEK293T cells and that similar increases in Ca2+ were seen with the tobacco smoke metabolites 1-NH2-naphthalene, formaldehyde, nicotine, and nicotine-derived nitrosamine ketone. Given the large number of physiological processes governed by changes in cytosolic Ca2+, our data suggest that Ca2+ signaling is a useful and reproducible assay that can be used to probe the propensity of tobacco products and their constituents to cause toxicity.

N-Benzyl-5-methoxytryptamines as Potent Serotonin 5-HT2 Receptor Family Agonists and Comparison with a Series of Phenethylamine Analogues.

A series of N-benzylated-5-methoxytryptamine analogues was prepared and investigated, with special emphasis on substituents in the meta position of the benzyl group. A parallel series of several N-benzylated analogues of 2,5-dimethoxy-4-iodophenethylamine (2C-I) also was included for comparison of the two major templates (i.e., tryptamine and phenethylamine). A broad affinity screen at serotonin receptors showed that most of the compounds had the highest affinity at the 5-HT2 family receptors. Substitution at the para position of the benzyl group resulted in reduced affinity, whereas substitution in either the ortho or the meta position enhanced affinity. In general, introduction of a large lipophilic group improved affinity, whereas functional activity often followed the opposite trend. Tests of the compounds for functional activity utilized intracellular Ca(2+) mobilization. Function was measured at the human 5-HT2A, 5-HT2B, and 5-HT2C receptors, as well as at the rat 5-HT2A and 5-HT2C receptors. There was no general correlation between affinity and function. Several of the tryptamine congeners were very potent functionally (EC50 values from 7.6 to 63 nM), but most were partial agonists. Tests in the mouse head twitch assay revealed that many of the compounds induced the head twitch and that there was a significant correlation between this behavior and functional potency at the rat 5-HT2A receptor.

Insulin normalization as an approach to the pharmacological treatment of obesity.

Hyperinsulinemia and exaggerated insulin response to glucose are among the hallmarks of obesity. However, the role of hyperinsulinemia in the etiology and maintenance of obesity has been controversial. If hyperinsulinemia plays a critical role as proposed, then its reversal may have therapeutic potential. To test this hypothesis, the activity of Ro 23-7637, (4-(2,2-diphenylethenyl)-1-[1-oxo-9-(3-pyridinyl) nonyl]piperidine), which partially normalizes plasma insulin by an action on pancreatic islets from obese rats, was assessed. When islets were cultured for 2 days with 10 microM Ro 23-7637, a significant reduction in the exaggerated glucose-induced insulin secretion was observed. When islets from lean rats were exposed to Ro 23-7637, no reduction in insulin secretion was observed. The effects of oral administration of Ro 23-7637 were assessed in Zucker and diet-induced obese rats in doses ranging from 5 to 90 mg/kg/day. Dose-related reductions were observed in: 1) glucose-induced insulin secretion; 2) basal insulin concentration; 3) daily food intake; and 4) bodyweight gain. In diet-induced obese rats, selective mobilization of fat, maintenance of body protein, and decreased energetic efficiency were also observed. An association between the partial normalization of glucose-induced insulin responses and reductions of basal insulin, reduced rates of body weight gain or body weight loss and decreased food intake was observed in obese rats. Therefore, these studies indicate that Ro 23-7637 is an orally active, efficacious antiobesity agent.

Molecular cloning of a recombinant retrovirus carrying a mutated envAKR-mycMH2 fusion gene immortalizing cells of the monocytic-macrophage lineage.

The VN-11 recombinant retroviruses, originally generated by co-transfection of the avian MH2 and AKRv viral genomes, were molecularly cloned from an infected mouse cell line named N11. The analysis of the proviral genome sequence from one of these recombinants showed a possible envAKR-mycMH2 fusion. Point mutations were also found in this envAKR-mycMH2 gene. The cloned viral genome was co-transfected with the neo gene into the psi 2 packaging cell line. Selected clones were shown to transcribe the viral genome and supernatants from these cultures, containing C-type particles, were used to infect primary cultures from mouse lymphoid tissues and brain. Proliferating macrophages and microglial cell clones were obtained, indicating that various types of cells of the mouse monocytic-macrophage lineage can be immortalized in spite of the absence of selection or special growth conditions.