Myocardial SERCA2 protects against cardiac damage and dysfunction caused by inhaled bromine.

Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen induced myocardial damage we utilized bromine exposed cardiac specific SERCA2 knockout (KO) mice (tamoxifen administered SERCA(flox/flox) Tg(aMHC-MerCreMer) mice) and compared them to the oil administered controls (FF). We performed echocardiography and hemodynamic analysis to investigate cardiac function 24h after bromine (600 ppm, for 30 min) exposure and measured cardiac injury markers in plasma, proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine exposed KO mice revealed significantly enhanced mean arterial pressure (MAP) and sphericity index, decreased LV end diastolic diameter LVEDD and LV end systolic pressure (LVESP), when compared to the bromine exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures. Significance Statement Due to their increased industrial production and transportation, halogens like chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac specific SERCA2 knockout mice these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.

A Murine Model of Vesicant-Induced Acute Lung Injury.

Burn injuries including those caused by chemicals can result in systemic effects and acute lung injury (ALI). Cutaneous exposure to Lewisite, a warfare and chemical burn agent, also causes ALI. To overcome the limitations in conducting direct research on Lewisite-induced ALI in a laboratory setting, an animal model was developed using phenylarsine oxide (PAO) as a surrogate for Lewisite. Due to lack of a reliable animal model mimicking the effects of such exposures, development of effective therapies to treat such injuries is challenging. We demonstrated that a single cutaneous exposure to PAO resulted in disruption of the alveolar-capillary barrier as evidenced by elevated protein levels in the bronchoalveolar lavage fluid (BALF). BALF supernatant of PAO-exposed animals had increased levels of high mobility group box 1, a damage associated molecular pattern molecule. Arterial blood-gas measurements showed decreased pH, increased PaCO2, and decreased partial pressure of arterial O2, indicative of respiratory acidosis, hypercapnia, and hypoxemia. Increased protein levels of interleukin (IL)-6, CXCL-1, CXCL-2, CXCL-5, granulocyte-macrophage colony-stimulating factor, CXCL-10, leukemia inhibitory factor, leptin, IL-18, CCL-2, CCL-3, and CCL-7 were observed in the lung of PAO-exposed mice. Further, vascular endothelial growth factor levels were reduced in the lung. Pulmonary function evaluated using a flexiVent showed a downward shift in the pressure-volume loop, decreases in static compliance and inspiratory capacity, increases in respiratory elastance and tissue elastance. These changes are consistent with an ALI phenotype. These results demonstrate that cutaneous PAO exposure leads to ALI and that the model can be used as an effective surrogate to investigate vesicant-induced ALI. SIGNIFICANCE STATEMENT: This study presents a robust model for studying ALI resulting from cutaneous exposure to PAO, a surrogate for the toxic vesicating agent Lewisite. The findings in this study mimic the effects of cutaneous Lewisite exposure, providing a reliable model for investigating mechanisms underlying toxicity. The model can also be used to develop medical countermeasures to mitigate ALI associated with cutaneous Lewisite exposure.

Beyond Chemical Preservatives: Enhancing the Shelf-Life and Sensory Quality of Ready-to-Eat (RTE) Hummus with Vinegar and Other Natural Antimicrobials.

Hummus is a traditional and very popular Mediterranean ready-to-eat (RTE) food, with growing popularity worldwide. However, it has a high water activity and is susceptible to microbial growth and post-process contamination that limit its quality and shelf-life. For this purpose, the present study compared the use of several antimicrobials, alone or in combination, for hummus preservation during storage (4 °C), for up to 45 days. The chemical preservative potassium sorbate 0.09% (S) was evaluated, along with three natural antimicrobials: garlic 1.25% (G); vinegar 5% (V); natamycin 0.002% (N); or their combination: garlic 1.25%-vinegar 5% (GV); vinegar 5%-natamycin 0.002% (VN); garlic 1.25%-natamycin 0.002% (GN); and garlic 1.25%-vinegar 5%-natamycin 0.002% (GVN) to increase the shelf-life of hummus. A thymol and carvacrol mixture 0.2% (O) was also assessed to preserve and develop a new oregano-flavored hummus. All treatments that included vinegar used alone or in combination had significantly higher antimicrobial effectiveness than the other treatments. They achieved 2.2-3.2, 1.8-3.1, and 1.4-2.1 log reductions in total aerobic counts (TAC), Pseudomonas spp., and lactic acid bacteria (LAB), respectively, as compared to the control samples © at day 21. Therefore, the shelf-life of C, S, N, G, GN, and O was around (ca.) 19 days, compared to an extended one of ca. 25 days for V and VN, and ca. 30 days for GV and GVN. Sensory analysis showed the highest acceptability for C, N, S, V, and VN, followed by GV and GVN, and the lowest was for G, GN, and finally O. The findings provide potential alternatives to chemical preservatives, which could be used for natural hummus preservation and shelf-life extension.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

Epigenetic underpinnings of inflammation: Connecting the dots between pulmonary diseases, lung cancer and COVID-19.

Inflammation is an essential component of several respiratory diseases, such as chronic obstructive pulmonary disease (COPD), asthma and acute respiratory distress syndrome (ARDS). It is central to lung cancer, the leading cancer in terms of associated mortality that has affected millions of individuals worldwide. Inflammation and pulmonary manifestations are also the major causes of COVID-19 related deaths. Acute hyperinflammation plays an important role in the COVID-19 disease progression and severity, and development of protective immunity against the virus is greatly sought. Further, the severity of COVID-19 is greatly enhanced in lung cancer patients, probably due to the genes such as ACE2, TMPRSS2, PAI-1 and furin that are commonly involved in cancer progression as well as SAR-CoV-2 infection. The importance of inflammation in pulmonary manifestations, cancer and COVID-19 calls for a closer look at the underlying processes, particularly the associated increase in IL-6 and other cytokines, the dysregulation of immune cells and the coagulation pathway. Towards this end, several reports have identified epigenetic regulation of inflammation at different levels. Expression of several key inflammation-related cytokines, chemokines and other genes is affected by methylation and acetylation while non-coding RNAs, including microRNAs as well as long non-coding RNAs, also affect the overall inflammatory responses. Select miRNAs can regulate inflammation in COVID-19 infection, lung cancer as well as other inflammatory lung diseases, and can serve as epigenetic links that can be therapeutically targeted. Furthermore, epigenetic changes also mediate the environmental factors-induced inflammation. Therefore, a better understanding of epigenetic regulation of inflammation can potentially help develop novel strategies to prevent, diagnose and treat chronic pulmonary diseases, lung cancer and COVID-19.

Sex differences in cardiopulmonary effects of acute bromine exposure.

Accidental occupational bromine (Br>2>) exposures are common, leading to significant morbidity and mortality; however, the specific effects of Br>2> inhalation in female victims are unclear. Our studies demonstrated that acute high-concentration Br>2> inhalation is fatal, and cardiac injury and dysfunction play an important role in Br>2> toxicity in males. In this study, we exposed female Sprague Dawley rats, age-matched to those males from previously studied, to 600 ppm Br>2> for 45 min and assessed their survival, cardiopulmonary injury and cardiac function after exposure. Br>2> exposure caused serious mortality in female rats (59%) 48 h after exposure. Rats had severe clinical distress, reduced heart rates and oxygen saturation after Br>2> inhalation as was previously reported with male animals. There was significant lung injury and edema when measured 24 h after exposure. Cardiac injury biomarkers were also significantly elevated 24 h after Br>2> inhalation. Echocardiography and hemodynamic studies were also performed and revealed that the mean arterial pressure was not significantly elevated in females. Other functional cardiac parameters were also altered. Aside from the lack of elevation of blood pressure, all other changes observed in female animals were also present in male animals as reported in our previous study. These studies are important to understand the toxicity mechanisms to generate therapies and better-equip first responders to deal with these specific scenarios after bromine spill disasters.>.

Echocardiographic, Biochemical, and Electrocardiographic Correlates Associated With Progressive Pulmonary Arterial Hypertension.

Background: Pulmonary arterial hypertension (PAH) is a progressive proliferative vasculopathy associated with mechanical and electrical changes, culminating in increased vascular resistance, right ventricular (RV) failure, and death. With a main focus on invasive tools, there has been an underutilization of echocardiography, electrocardiography, and biomarkers to non-invasively assess the changes in myocardial and pulmonary vascular structure and function during the course of PAH. Methods: A SU5416-hypoxia rat model was used for inducing PAH. Biventricular functions were measured using transthoracic two-dimensional (2D) echocardiography/Doppler (echo/Doppler) at disease onset (0 week), during progression (3 weeks), and establishment (5 weeks). Similarly, electrocardiography was performed at 0, 3, and 5 weeks. Invasive hemodynamic measurements and markers of cardiac injury in plasma were assessed at 0, 3, and 5 weeks. Results: Increased RV systolic pressure (RVSP) and rate of isovolumic pressure rise and decline were observed at 0, 3, and 5 weeks in PAH animals. EKG showed a steady increase in QT-interval with progression of PAH, whereas P-wave height and RS width were increased only during the initial stages of PAH progression. Echocardiographic markers of PAH progression and severity were also identified. Three echocardiographic patterns were observed: a steady pattern (0-5 weeks) in which echo parameter changed progressively with severity [inferior vena cava (IVC) expiratory diameter and pulmonary artery acceleration time (PAAT)], an early pattern (0-3 weeks) where there is an early change in parameters [RV fractional area change (RV-FAC), transmitral flow, left ventricle (LV) output, estimated mean PA pressure, RV performance index, and LV systolic eccentricity index], and a late pattern (3-5 weeks) in which there is only a late rise at advanced stages of PAH (LV diastolic eccentricity index). RVSP correlated with PAAT, PAAT/PA ejection times, IVC diameters, RV-FAC, tricuspid systolic excursion, LV systolic eccentricity and output, and transmitral flow. Plasma myosin light chain (Myl-3) and cardiac troponin I (cTnI) increased progressively across the three time points. Cardiac troponin T (cTnT) and fatty acid-binding protein-3 (FABP-3) were significantly elevated only at the 5-week time point. Conclusion: Distinct electrocardiographic and echocardiographic patterns along with plasma biomarkers were identified as useful non-invasive tools for monitoring PAH progression.

Behavioral and Neuronal Effects of Inhaled Bromine Gas: Oxidative Brain Stem Damage.

The risk of accidental bromine (Br2) exposure to the public has increased due to its enhanced industrial use. Inhaled Br2 damages the lungs and the heart; however, adverse effects on the brain are unknown. In this study, we examined the neurological effects of inhaled Br2 in Sprague Dawley rats. Rats were exposed to Br2 (600 ppm for 45 min) and transferred to room air and cage behavior, and levels of glial fibrillary acidic protein (GFAP) in plasma were examined at various time intervals. Bromine exposure resulted in abnormal cage behavior such as head hitting, biting and aggression, hypervigilance, and hyperactivity. An increase in plasma GFAP and brain 4-hydroxynonenal (4-HNE) content also was observed in the exposed animals. Acute and delayed sympathetic nervous system activation was also evaluated by assessing the expression of catecholamine biosynthesizing enzymes, tryptophan hydroxylase (TrpH1 and TrpH2), and tyrosine hydroxylase (TyrH), along with an assessment of catecholamines and their metabolites. TyrH was found to be increased in a time-dependent manner. TrpH1 and TrpH2 were significantly decreased upon Br2 exposure in the brainstem. The neurotransmitter content evaluation indicated an increase in 5-HT and dopamine at early timepoints after exposure; however, other metabolites were not significantly altered. Taken together, our results predict brain damage and autonomic dysfunction upon Br2 exposure.

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase.

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs 1-4 , the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits 5 . Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and ß-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp 6 . In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection 7-9 .

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats.

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

The bio-sonographic index. A novel modality for early detection of acute kidney injury after complex vascular surgery. A protocol for an exploratory prospective study.

OBJECTIVE: Acute kidney injury (AKI) is a common complication of complex aortic surgery with high mortality, morbidity and health care expense. The current definition of AKI does not allow for structural characterization of the kidneys and utilizes functional indices with substantial limitations leading to delayed diagnosis and ineffective interventions. The aim of this study is to develop a method of early detection of structural renal abnormalities that can precede and predict the occurrence of AKI in this population. We propose a novel combined index of ultrasonography (shear wave elastography), biomarkers of renal stress (urinary insulin growth factor binding protein-7, IGFBP-7 and inhibitor of tissue metalloproteinase-2, TIMP-2) and renal injury markers (urinary neutrophil gelatinase-associated lipocalin -NGAL)- the bio-sonographic index (BSI). METHODS: A prospective observational study at a tertiary referral center will be performed enrolling 80 patients undergoing elective open and endovascular repair of the visceral aorta. The BSI will be evaluated at baseline, and at 6 and 24 hours after the procedure. The primary outcome is the occurrence of AKI according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria. Each patient will be his/her own control. A reference group of 15 healthy volunteers who are not undergoing interventions will be enrolled to test the feasibility of and to refine the novel SWE protocol. The BSI will be tested for its predictability of the occurrence of AKI. Comparisons will be made between individual and combined components of the BSI and traditional markers used in the KDIGO definition; serum creatinine and urine output in terms of baseline status of the kidney. Correlations will be made between the BSI and conventional indices of AKI and exploratory analyses will be conducted to identify individual disease patterns using the BSI. DISCUSSION: We hypothesize that the BSI will be a sensitive index of early structural abnormalities that precede and predict the occurrence of AKI as defined by KDIGO in complex vascular surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT04144894. Registered 1/6/2020.

MicroRNA-mediated inflammation and coagulation effects in rats exposed to an inhaled analog of sulfur mustard.

Exposure of rats to 2-chloroethyl ethyl sulfide (CEES), an analog of sulfur mustard, can cause acute lung injury (ALI), resulting in increased inflammation and coagulation and altered levels of plasma microRNAs (miRNAs). Rats were exposed to aerosolized CEES and euthanized 12 h later for collection of tissue and plasma. Profiling of miRNAs in plasma, using a TaqMan-based RT-PCR array, revealed 14 differentially expressed miRNAs. Target gene prediction and pathway analysis revealed miRNA-mediated regulation of organismal injury, inflammation, and respiratory diseases. miR-140-5p, a marker of ALI, was downregulated in the plasma, lung, liver, and kidney of CEES-exposed rats, with a concomitant increase in the expression of the inflammation markers IL-6 and IL-1α and the coagulation marker tissue factor (F3). Exposure of rat airway epithelial cells (RL-65) to CEES (0.5 mM) caused cell death and a decrease in miR-140-5p both in cells and media supernatant. This was accompanied by an increase in cellular mRNA levels of IL-6, IL-1α, and F3, as well as FGF9 and EGR2, putative targets of miR-140. Knockdown of miR-140 by specific oligos in RL-65 cells mimicked the in vivo CEES-mediated effects, leading to significantly increased mRNA levels of IL-6, IL-1α, F3, FGF9, and EGR2. Our study identifies miR-140-5p as a mediator of CEES-induced ALI, which could potentially be targeted for therapy.

Circulating and tissue biomarkers as predictors of bromine gas inhalation.

The threat from deliberate or accidental exposure to halogen gases is increasing, as is their industrial applications and use as chemical warfare agents. Biomarkers that can identify halogen exposure, diagnose victims of exposure or predict injury severity, and enable appropriate treatment are lacking. We conducted these studies to determine and validate biomarkers of bromine (Br2 ) toxicity and correlate the symptoms and the extent of cardiopulmonary injuries. Unanesthetized rats were exposed to Br2 and monitored noninvasively for clinical scores and pulse oximetry. Animals were euthanized and grouped at various time intervals to assess brominated fatty acid (BFA) content in the plasma, lung, and heart using mass spectrometry. Bronchoalveolar lavage fluid (BALF) protein content was used to assess pulmonary injury. Cardiac troponin I (cTnI) was assessed in the plasma to evaluate cardiac injury. The blood, lung, and cardiac tissue BFA content significantly correlated with the clinical scores, tissue oxygenation, heart rate, and cardiopulmonary injury parameters. Total (free + esterified) bromostearic acid levels correlated with lung injury, as indicated by BALF protein content, and free bromostearic acid levels correlated with plasma cTnI levels. Thus, BFAs and cardiac injury biomarkers can identify Br2 exposure and predict the severity of organ damage.

Cutaneous lewisite exposure causes acute lung injury.

Lewisite is a strong vesicating and chemical warfare agent. Because of the rapid transdermal absorption, cutaneous exposure to lewisite can also elicit severe systemic injury. Lewisite (2.5, 5.0, and 7.5 mg/kg) was applied to the skin of Ptch1+/- /SKH-1 mice and acute lung injury (ALI) was assessed after 24 hours. Arterial blood gas measurements showed hypercapnia and hypoxemia in the lewisite-exposed group. Histological evaluation of lung tissue revealed increased levels of proinflammatory neutrophils and a dose-dependent increase in structural changes indicative of injury. Increased inflammation was also confirmed by altered expression of cytokines, including increased IL-33, and a dose-dependent elevation of CXCL1, CXCL5, and GCSF was observed in the lung tissue. In the bronchoalveolar lavage fluid of lewisite-exposed animals, there was a significant increase in HMGB1, a damage-associated molecular pattern molecule, as well as elevated CXCL1 and CXCL5, which coincided with an influx of neutrophils to the lungs. Complete blood cell analysis revealed eosinophilia and altered neutrophil-lymphocyte ratios as a consequence of lewisite exposure. Mean platelet volume and RBC distribution width, which are predictors of lung injury, were also increased in the lewisite group. These data demonstrate that cutaneous lewisite exposure causes ALI and may contribute to mortality in exposed populations.

Extracellular nucleic acid scavenging rescues rats from sulfur mustard analog-induced lung injury and mortality.

Sulfur mustard (SM) is a highly toxic war chemical that causes significant morbidity and mortality and lacks any effective therapy. Rats exposed to aerosolized CEES (2-chloroethyl ethyl sulfide; 10% in ethanol), an analog of SM, developed acute respiratory distress syndrome (ARDS), which is characterized by increased inflammation, hypoxemia and impaired gas exchange. We observed elevated levels of extracellular nucleic acids (eNA) in the bronchoalveolar lavage fluid (BALF) of CEES-exposed animals. eNA can induce inflammation, coagulation and barrier dysfunction. Treatment with hexadimethrine bromide (HDMBr; 10 mg/kg), an eNA neutralizing agent, 2 h post-exposure, reduced lung injury, inhibited disruption of alveolar-capillary barrier, improved blood oxygenation (PaO2/FiO2 ratio), thus reversing ARDS symptoms. HDMBr treatment also reduced lung inflammation in the CEES-exposed animals by decreasing IL-6, IL-1A, CXCL-1 and CCL-2 mRNA levels in lung tissues and HMGB1 protein in BALF. Furthermore, HDMBr treatment also reduced levels of lung tissue factor and plasminogen activator inhibitor-1 indicating reduction in clot formation and increased fibrinolysis. Fibrin was reduced in BALF of the HDMBr-treated animals. This was further confirmed by histology that revealed diminished airway fibrin, epithelial sloughing and hyaline membrane in the lungs of HDMBr-treated animals. HDMBr completely rescued the CEES-associated mortality 12 h post-exposure when the survival rate in CEES-only group was just 50%. Experimental eNA treatment of cells caused increased inflammation that was reversed by HDMBr. These results demonstrate a role of eNA in the pathogenesis of CEES/SM-induced injury and that its neutralization can serve as a potential therapeutic approach in treating SM toxicity.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Acute pulmonary effects of aerosolized nicotine.

Nicotine is a highly addictive principal component of both tobacco and electronic cigarette that is readily absorbed in blood. Nicotine-containing electronic cigarettes are promoted as a safe alternative to cigarette smoking. However, the isolated effects of inhaled nicotine are largely unknown. Here we report a novel rat model of aerosolized nicotine with a particle size (~1 µm) in the respirable diameter range. Acute nicotine inhalation caused increased pulmonary edema and lung injury as measured by enhanced bronchoalveolar lavage fluid protein, IgM, lung wet-to-dry weight ratio, and high-mobility group box 1 (HMGB1) protein and decreased lung E-cadherin protein. Immunohistochemical analysis revealed congested blood vessels and increased neutrophil infiltration. Lung myeloperoxidase mRNA and protein increased in the nicotine-exposed rats. Complete blood counts also showed an increase in neutrophils, white blood cells, eosinophils, and basophils. Arterial blood gas measurements showed an increase in lactate. Lungs of nicotine-inhaling animals revealed increased mRNA levels of IL-1A and CXCL1. There was also an increase in IL-1α protein. In in vitro air-liquid interface cultures of airway epithelial cells, there was a dose dependent increase in HMGB1 release with nicotine treatment. Air-liquid cultures exposed to nicotine also resulted in a dose-dependent loss of barrier as measured by transepithelial electrical resistance and a decrease in E-cadherin expression. Nicotine also caused a dose-dependent increase in epithelial cell death and an increase in caspase-3/7 activities. These results show that the nicotine content of electronic cigarettes may have adverse pulmonary and systemic effects.

Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.