Sadis Matalon - US grants

Oxygen administration, in concentrations of 60% or less, is being widely used in clinical medicine for the correction of arterial hypoxemia, as if it were innocuous. However, animals exposed to 60% O2 for seven days, developed microscopic lesions in the pulmonary capillary endothelium identified by light and electron microscopy. The physiological implications of these findings have not been documented. Our general objective is to correlate structural alterations with physiological changes in lungs of animals exposed to sublethal levels of O2. We will further establish whether these changes are permanent or reversible when the animals resume breathing air, and quantify whether the presence of acute and chronic lung injury exacerbates or delays the onset of severity of the oxygen induced damage. Conscious, unanesthetized, chronically instrumented sheep, will be exposed to 45 and 60% O2 at 1 ata for two weeks and then returned to air for an additional two weeks. Acute and chronic lung injury will be produced by the intratracheal instillation of bleomycin. The following variables will be measured periodically throughout the exposure: Minute ventilation and respiratory frequency, arterial and mixed venous oxygen and carbon dioxide tensions and pH, cardiac output, vascular and pleural pressures, functional residual capacity, the distribution of the pulmonary blood flow, the permeability of the alveolar epithelium and capillary endothelium to solute, and the pulmonary extravascular volumes of distribution of albumin and cyanocobalamin. These variables will be correlated with simultaneous histological, morphological and gravimetric measurements of lung tissue. Our results will establish whether exposure of the normal and injured lung to sublethal oxygen concentrations causes physiological damage (i.e. derangement of lung fluid balance, mechanics and hemodynamics) before or in the absence of overt clinical symptoms (i.e. impairment of gas exchange).

Mycoplasma pneumoniae (mycoplasmas) account for 20 to 30 percent of all pneumonias in humans, and exacerbate the pathophysiology of asthma, chronic obstructive disease and other pulmonary diseases. Man is the only host of M. pneumoniae, but Mycoplasma pulmonis infection in mice provides an excellent animal model that reproduces the essential features of human respiratory mycoplasmosis. When C3H/He mice are infected with mycoplasmas they develop a clinical condition similar to human respiratory mycoplasmosis. On the other hand, C57BL/6 mice are resistant to mycoplasmas. Presently, the basic mechanisms by which some hosts, but not others, kill mycoplasmas in vivo have not been elucidated. Based on our preliminary data, we hypothesize that in the early stages of infection (8-72 h), mycoplasmas are killed by reactive oxygen-nitrogen intermediates (ROS) produced by activated alveolar macrophages (AM). Surfactant protein A (SP-A) is essential and necessary or this killing to occur by (i) upregulating production of nitric oxide by activated AM, and (ii) stimulating phagocytosis of mycoplasmas by AM. Furthermore, injury to SP-A by reactive oxygen-nitrogen species abrogates its host-defense functions. We have designed a series of experiments to test this hypothesis in vitro, using AM isolated from the lungs of these mice, and in vivo using congenic germ-free knock-out mice which we are currently developing. Specifically, we plan to: (1) Identify the mechanisms by which normal but not nitrated SP-A mediates killing of mycoplasmas by resistant C57BL/6 AM; (2) Quantify the extent of killing of intanasally instilled mycoplasmas in the lungs of germ-free congenic C57BL/6 SP-A (-/-) and C57BL/6: NOS (-/-) mice in vivo and (3) Identify the mechanisms responsible for decreased mycoplasmal killing by AM from C3H/He mice. A better understanding of basic mechanisms of innate lung defenses may lead to the development of novel therapies, which may extend to other pathogens.

Active sodium (Na+) transport across the adult alveolar epithelium plays an important role in the maintenance of lung fluid balance, especially after sublethal hyperoxic injury to the blood-gas barrier, when the effectiveness of the passive Starling forces is diminished. Presently, the mechanisms by which Na+ ions enter the apical membranes of normal and oxygen-injured alveolar epithelial cells, have not been elucidated. Based on preliminary data, we hypothesize that alveolar type II cells (ATII) contain Na+ channels with low affinity to amiloride and that the properties and spatial distribution of these channels may be altered by exposure to sublethal hyperoxia. Since sodium channels conduct at rates far exceeding that of any other transporter, and their activities may be upregulated by a number of agents, they may form a major pathway for the entry of Na+ ions into alveolar epithelial cells. The overall goal of this research project is to assess the distribution of these ion channels in the alveolar epithelium of normal and hyperoxic-injured rats, characterize their properties at the single cell level and study some of their fundamental regulatory mechanisms. Sublethal hyperoxic injury will be induced by exposing rats to 60 h of 100% 02 and returning them to room air for 24 h and 72 h, an exposure period known to increase the activity of lung Na+-K+ ATPase, and the rate of fluid removal across the alveolar epithelium. These studies will be conducted in both freshly isolated and cultured ATII cells to assess changes in the properties of these channels with time in culture. The specific aims of this application are: (1) to define the selectivity, single ion current, open and close time probabilities, sensitivity to amiloride and other pharmacological agents by patch-clamp techniques; (2) to establish the existence of amiloride-binding protein(s) in ATII cells by Western blotting techniques and determine their spatial localization by immunocytochemical studies at both the light and electron microscopic level; and (3) to investigate whether phosphorylation of these channels via the cAMP-dependent protein kinase (PKA) correlated with increased Na+ transport at both the whole cell and single channel level. Completion of these specific aims will provide new and fundamental knowledge on the possible mechanisms of fluid clearance across the alveolar space of both the normal and injured mammalian lung.

laboratory mouse; transfection /expression vector

DESCRIPTION (provided by applicant): A multi-center clinical trial sponsored by Fujisawa Healthcare, Inc, was planned to compare the efficacy of treating lung transplant patients with tacrolimus and sirolimus versus tacrolimus and azathioprine in reducing the incidence of acute rejection during the first twelve months after lung transplantation. Infection is a secondary endpoint and is assessed throughout the trial (i.e. for 3 years after randomization). Presently the mechanisms by which these agents may modify lung innate immunity have not been identified. Herein, we are proposing to isolate SP-A and AMs from the bronchoalveolar lavage fluid (BALF) of patients participating in this clinical trial to identify differences in the ability of AMs to kill gram positive and gram-negative bacterial pathogens and to identify differences in quantity of SP-A and modifications thereof. These data will be correlated with incidences of infection and rejection in patients participating in the clinical trial. We are also proposing to identify basic mechanisms by which normal but not nitrated SP-A enhances phagocytosis. These goals will be accomplished by completing the set of measurements outlined in the following specific aims: (1) Measure levels of surfactant lipids and SP-A in bronchoalveolar lavage (BAL) samples from patients treated with tacrolimus and sirolimus vs. tacrolimus and azathioprine. Oxidative modification to SP-A (oxidation and nitration) will be assessed by Western blotting, ELISA and mass spectrometry analysis using techniques already established in our laboratory; (2) Quantitate levels of inflammatory cytokines (TNFa, INFgamma, IL-6 and IL-lb), as well as levels of nitrate and nitrite, the stable end products of NO metabolism, and nitrotyrosine in the BAL of these patients; (3) Assess the extent of pathogen killing (Klebsiella pneumoniae, a gram negative bacterium and Staphylococcus aureus, a gram positive bacterium) by AMs isolated from the lungs of these patients in the presence of SP-A and surfactant lipids, and (4) Identify putative mechanisms responsible for decreased ability of oxidized or nitrated SP-A to mediate pathogen killing by AMs. We propose that SP-A binding to AM receptors leads to activation of phospholipase C (PLC) which releases 1,4,5 inositol triphosphate (IP3) resulting in Ca+2 release from the endoplasmic reticulum. SP-A nitration may lead to decreased binding, diminishing or abrogating intracellular Ca+2 changes. Our data may provide mechanistic insight into why some patients may develop clinical infection and acute and chronic rejection and thereby form the rationale basis for choosing between these two immunosuppressive regiments for the management of patients with lung transplantation.

DESCRIPTION (provided by applicant): The cystic fibrosis trans-membrane conductance regulator (CFTR), a 1480 amino acid protein, is a member of the traffic ATPase family (60) and functions as a cAMP-regulated CI channel. Based on our published results and preliminary data, we hypothesize that chronic exposure of mice and airway cells to agents which increase concentrations of reactive species (RONS), formed by the interaction of nitric oxide (NO) with partially reduced oxygen intermediates, results in oxidative modifications (oxidation, nitration and/or nitrosation) of key CFTR amino acids. These changes may: (1) decrease apical levels of CFTR by targeting it for ubiquitination and endoplasmic reticulum associated degradation by proteasomes and (2) impair Cl- secretion across the airway and alveolar epithelial cells following cAMP-stimulation by decreasing CFTR phosphorylation. These hypotheses will be tested both in vitro, by exposing Calu-3, primary human airway epithelial cells and mouse tracheocytes (MTE) to NO and RONS, as well as C57BL/6 mice to NO (1-10 ppm); nitrogen dioxide (NO2:1-10 ppm); intratracheal instillation of Mycoplasma pulmonis and measure the extent of oxidative modification and ubiquitination of CFTR as well as microscopic (single channel Cl currents) and macroscopic (whole cell Cl currents, nasal potential differences and alveolar fluid clearance) indices of its ability to act as a cAMP-activated Cl- channel. To identify specific amino acids modifications leading to loss of CFTR function, we will construct CFTR mutants by substituting each of the 40 CFTR tyrosines with alanine, express each cRNA in oocytes, and measure basal and cAMP-activated whole cell and single channel Cl- currents before and after exposure of oocyte to the RONS. Because of the well demonstrated vital importance of CFTR in both the hydration of airway fluid, as well as in cAMP-activated Na+ transport across the alveolar epithelium, the results of these studies may offer significant new insight into the pathophysiology of a number of pulmonary, non cystic fibrosis inflammatory diseases such as asthma, chronic obstructive lung disease and adult respiratory distress syndrome.

DESCRIPTION (provided by applicant): Chlorine (C12) is a moderately soluble, highly reactive oxidant gas, used extensively for water purification, manufacturing of Pharmaceuticals and chemicals and as a potent disinfectant. Persons exposed to chlorine gas, may experience mild symptoms for the first 6-24 hours (h). However, following this latency period, severe lung injury, characterized by protein-rich edema and the onset of hypoxemia may develop. Presently, the cellular and biochemical events leading to this injury have not been elucidated. We propose that reactive oxygen-chloride and nitrogen intermediates (RONS), formed by the interaction of C12 and its hydrolysis products with nitric oxide (NO), initiate self-propagating chain reactions, the products of which damage alveolar epithelial cells decreasing their ability to produce and secrete surfactant, actively transport sodium (Na+) ions and maintain a tight, semi-permeable barrier. Thus, systemic administration of reactive species scavengers (such as ascorbate, N-acetyl-cysteine (NAC), and deferoxamine, as well as agents that augment surfactant levels, ion transport and paracellular resistance (such as albuterol (a long acting b-agonist) and a recently described peptide based on the lectin region of TNFa (tip peptide), shortly after exposure to C12 will decrease lung injury, morbidity and mortality. This hypothesis will be tested by exposing either confluent monolayers of rat alveolar type II (ATII) epithelial cells (SPECIFIC AIM # 1) or rats (SPECIFIC AIMS #2) to C12 (50-200 ppm for 30 min) and measure the following indices at 0.5, 6, 12 and 24 h post exposure: physiological and biochemical indices of lung function (including surfactant function and composition), ability of the lungs to transport ions in vivo and in vitro and clear pulmonary edema in vivo, levels of inflammatory cytokines in the rat alveolar space and in the plasma, arterial blood gases and pH, as well as levels of low reactive species scavengers (ascorbate, NAC) at 0.5, 6, 12, 24 and 48 h post exposure. These measurements will be repeated following intravenous injections of NAC, ascorbate and deferoxamine as well as albuterol and the tip peptide, every 6 h post exposure for 48 h. In SPECIFIC AIM #3 , we will assess the efficacy of intratracheally instilled ascorbate, NAC, deferoxamine, Infasurf (a surfactant replacement mixture), albuterol and the tip peptide, as well as aerosolized albuterol, in prolonging survival of rats with respiratory failure post C12 exposure. The subject matter of this research is both timely and important: more than 25 million tons of chlorine is manufactured annually in the United States and the majority of this gas is transported by rail and can be used as a chemical weapon.

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Administrative Core Description: The Administrative Core (directed by Sadis Matalon, PhD) will provide the proper framework that will support, encourage and focus the research efforts of the three projects and of the Exposure Core Facility. This will ensure that all investigators work together as a group, the research efforts remain focused and well integrated and the individual projects generate high quality research to complete their milestones in a timely fashion. Dr. Matalon will also ensure that Dr. Sven Jordt, in spite of the geographical separation, remains well integrated with the rest of the members. In addition the Administrative Core will be responsible for the establishment and maintenance of a Center Website, as specified by the CounterAct Network regulations. Finally, the Director will be in constant touch with NIH personnel and will update the investigators and co-investigators on recent developments.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This Research Center of Excellence (RCE) entitled: "Novel Treatments of Chlorine Induced Injury to the Cardio-Respiratory Systems" consists of three projects and two cores. The unifying theme spanning all projects is that exposure of animals to C12 results in the formation of reactive intermediates which deplete ascorbate and reduced glutathione in the lung epithelial fluids, damage key components of the respiratory and alveolar epithelial [such as transient receptor protein (TRP) and epithelial sodium channels (ENaC)] and then, via inhibition of eNOS signaling compromise seminal functions of the pulmonary and systemic vasculatures. Furthermore, we propose that these toxic effects of C12 will be heightened in animals infected with respiratory syncytial virus or challenged with ova albumin. In our first series of experiments we will perform a number of state of the art biochemical, biophysical, physiological and morphometric measurements in RSV infected and ova albumin challenged mice as well as normal rats prior to and following C12 exposure to document the onset and progression of injury to lung epithelia and pulmonary and systemic vasculature. We will then treat them with antioxidants, TRP antagonists, /32 agonists and nitrite administered at various intervals post C12 exposure either intra-tracheally or via aerosolization or intraperitoneally (antioxidants and nitrite) and quantify recovery by specific functional measurements. Strong points of the RCE include the diverse talents of the investigators, the unique facilities, and the novelty of the preliminary data. The three projects (two of which build on novel findings generated by existing UO1 grants) are supported by an administrative core and the exposure core, which play key roles by both providing essential functions (such as exposure of animals to C12) and helping to integrate the team into a cohesive entity. [unreadable]

DESCRIPTION (provided by applicant): Influenza (flu) is a contagious respiratory illness caused by flu viruses, leading to about 36,000 deaths every year in the United States alone, with the potential for at least a ten fold increase in epidemic and pandemic scenarios. During attachment of flu viruses to epithelial cells, hemagglutinin, one of its surface proteins, binds to sialic acid residues, initiating a series of events leading to activation of PKC, which in turn, down-regulates the activity of amiloride sensitive epithelial Na+ channels (ENaC) of tracheal and alveolar cells. It has been thought that these events are responsible for flu-induced rhinorrhea and life-threatening alveolar edema in humans. However, events occurring during the attachment of influenza virus to epithelial cells are likely to be transient and relatively few cells will be initially affected. We propose that M2, a transmembrane protein that plays a critical role in viral replication, enhances intracellular production of reactive oxygen-nitrogen species (RONS) which (i) oxidize and nitrate ENaC; and (ii) activate PKC(. Both processes enhance ENaC ubiquitination and subsequent destruction by the proteasome or lysosome systems. These hypotheses will be tested by completing the following comprehensive in vitro and in vivo studies listed in four specific aims: (1) Identify regions and specific amino acids of the influenza strain A/Udorn/72 M2 proton (H+) channel responsible for ENaC down-regulation in Xenopus oocytes microinjected with 1-,2-, and 3-ENaC. (2) Identify the mechanisms by which M2 decreases ENaC protein levels and function. We propose that M2 enhances intracellular production of reactive oxygen-nitrogen species (RONS) which (i) oxidize and nitrate ENaC; and (ii) activate PKC(. Both processes enhance ENaC ubiquitination and subsequent destruction by the proteasome or lysosome systems (3) Identify the mechanisms by which M2 inhibits amiloride sensitive Na+ currents in human airway (H441) and rat alveolar type II (rATII) cells, expressing native ENaC and (4) Establish the contribution of M2 in the inhibition of lung fluid clearance of mice infected by replicating flu viruses and identify the mechanisms involved. The results of our studies may provide the rational basis for the development of new therapeutic strategies, against a highly conserved region of the viral genome to knockdown M2 expression, and thus broadly and effectively decrease flu induced pulmonary edema and rhinorrhea. Due to the public health impact of influenza, there is a strong need to investigate and develop therapies that address the host response to viral infection, which may contribute to the morbidity and mortality of pathogenic respiratory viruses. PUBLIC HEALTH RELEVANCE: The results of our studies will provide the rational basis for the development of new therapeutic strategies, such as administration of agents to decrease M2 expression, and thus broadly and effectively decrease the flu-induced rhinorrhea, alveolar edema and hypoxemia. Due to the public health impact of influenza, there is a strong need to investigate and develop therapies that address the host response to viral infection, which may contribute to the morbidity and mortality of pathogenic respiratory viruses.

DESCRIPTION (provided by applicant): Chlorine (Cl2) is a highly irritant and reactive gas produced in large quantities throughout the world. Exposure to Cl2 released into the atmosphere during transportation and industrial accidents, as well as acts of terrorism, has resulted in significant lung injury leading to death from respiratory failure or significant morbidty. Our recent data show that there may be benefit to treating subjects exposed to Cl2 with inhaled local anesthetics. The present studies will examine the effects of this treatment as monotherapy or in combination with a beta adrenergic agonist on measures of pain and inflammation in murine models.

DESCRIPTION (provided by applicant): Chlorine (Cl2) is a highly irritant and reactive gas produced in large quantities throughout the world and used extensively for pulp bleaching, waste sanitation and in the manufacturing of various pharmaceuticals. It also poses a significant threat to public health when inhaled. Exposure to Cl2, released into the atmosphere during transportation and industrial accidents, as well as acts of terrorism resulted in significant morbidity and mortality to both humans and animals. There is no safe exposure to Cl2: Even domestic exposure to low levels of Cl2 may result in wheezing and exacerbate the clinical outcome of asthma and chronic obstructive pulmonary disease. When inhaled, Cl2 first reacts with antioxidants in the lung epithelial lining fluid (ELF); when antioxidants are depleted, it fors relatively stable adducts with proteins, components of the extracellular matrix and unsaturated fatty acids which then proceed to prolong the toxicity of the initial Cl2 exposure and contribute t the long term pathology. In this proposal we will test the hypothesis that these secondary reactive species target the mitochondrion and so decrease mitochondrial quality and cause bioenergetic dysfunction which delays tissue recovery and repair. Based upon these data we hypothesize that mitochondria are a critical target for Cl2 toxicity in lung epithelial cells and te combined strategy of preventing mitochondrial oxidative damage by mitochondrial targeted antioxidants (such as MitoQ) with enhancing mitophagy (by rapamycin and trehalose), will be beneficial in ameliorating Cl2 toxicity. This hypothesis will be tested by completing the in vitro and in vivo studies highlighted in these two highly integrated specific aims: SA-1: Determine the mechanisms and physiological sequelae of mitochondria injury and autophagy following exposure of human airway cells to Cl2 in vitro. SA-2: Determine if post Cl2 administration of MitoQ, rapamycin and trehalose in mice decreases Cl2 induced mortality and lung injury and improves mitochondrial bioenergetics function. Completion of these experiments will provide the rational basis for additional studies to establish effective therapies for a major environmental and public health threat to humans.

DESCRIPTION (provided by applicant): Bromine (Br2) is a halogen used as a water disinfectant, for bleaching fibers, in the manufacture of antiepileptic drugs, dyestuffs, flame retardants, insecticides, drilling fluids, and gasoline additives When inhaled at higher concentrations, as may occur during transportation accidents, transfers among containers or acts of terrorisms, Br2 has caused death from respiratory failure. There are very few published studies evaluating acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Our exciting and highly novel preliminary data show that mice overexpressing the human form of the heme oxygenase (HO)-1 gene and protein (hHO-1 BAC) exhibit significantly lower mortality when returned to room air post Br2 exposure as compared to their wild-type littermate controls. In contrast, mice deficient in the HO-1 gene (HO-1-/-), display markedly increased mortality following Br2 exposure. We have also identified five compounds that are potent inducers of the human HO-1 gene using high-throughput screening. The goals of this application are: (1) to establish the role of HO-1 in protecting mice from Br2 induced injury and (2) test the efficacy of these compounds, administered in mice systemically, post Br2 exposure, to upregulate HO-1, and decrease mortality and lung injury. SA #1. We will expose hHO-1 BAC, HO-1-/- mice and their wild-type littermates to Br2 (600 ppm for 30 min) in environmental chambers, and return them to room air and measure mortality and lung injury for two weeks. SA #2: (i) We will expose confluent monolayers of human airway Clara-cell like cells (H441) to Br2 and return them to room air; we will then test the efficacy of the five compounds identified by high throughput screening, to increase HO-1 and decrease Br2 induced cellular necrosis and apoptosis. (ii) We will profile these compounds to determine those with the best pharmacokinetic properties related to intramuscular dosing. (iii) We will inject hHO-1 BAC and wild-type mice intramuscularly with the compound that exhibits the best cytoprotective effects in vitro and measure lung mRNA and hHO-1 activity at two, 24 and 72 post injection. (iv) We will expose hHO-1 BAC, HO- 1-/- mice and their wild-type littermates (all in C57BL/6 background) to Br2 (600 ppm for 30 min), return them to room air and inject them intramuscularly starting at 2 h post-exposure with this compound. We will measure mortality in all groups and indices of lung injury and lung hHO1 activity for 14 d as described in SA #1.

? DESCRIPTION (provided by applicant): Industrial accidents, urban environmental disasters and terrorist attacks involving bromine gas (Br2) are of great concern to public safety. Br2 is used extensively in manufacturing and, therefore, needs to be transported and stored in large quantities. Accidental and intentional discharges of Br2 with consequent large number of casualties have been reported. We are only beginning to understand the mechanisms and potential countermeasures of Br2 toxicity and the identification of particularly vulnerable populations is completely lacking. Our preliminary studies show that pregnant mice exposed to 600 ppm Br2 for 30 minutes at gestation day 15 (E15) and returned to room air, exhibit 75% mortality. This mortality rate is considerably higher than what is seen in non-pregnant female or male mice at the same exposure level (25% mortality). Fetuses of surviving mothers are severely growth restricted at E19 and the mothers exhibit symptoms of preeclampsia. Placentae of pregnant mice exposed to Br2 express high levels of the short form of fms-like tyrosine kinase 1 (sFLT1), a known mediator of systemic endothelial dysfunction and preeclampsia. We propose the following hypotheses: 1) Preeclampsia, as a pregnancy-specific condition is responsible for the increased vulnerability of pregnant mothers and their unborn fetuses to Br2 toxicity. 2) Intervention with inhibitors of type 5 cyclic nucleotide-specific phosphodiesterases (PDE5i) will mitigate systemic endothelial dysfunction, which is a shared mechanism of halogen toxicity and preeclampsia; they will save the lives of mothers and improve fetal growth. We are proposing to conduct the following experiments grouped in two well integrated specific aims: SA #1. Identify the mechanisms by which exposure of pregnant mice to Br2 gas (600 or 400 ppm for 30 min) leads to significantly higher mortality than non-pregnant mice and causes fetal growth restriction/fetal demise (FGR/FD) when returned to room air. We propose that maternal mortality and FGR/FD are due to systemic hypoxemia and damage to endothelial NOS (eNOS), causing the placenta to produce anti-angiogenic factors, leading to the development of preeclampsia/eclampsia.SA #2. Investigate the efficacy of post Br2 exposure administration of (PDE5i) to decrease preeclampsia, FGR/FD and mortality in pregnant mice. Following exposure to Br2, tadalafil (1 mg*kg-1*day-1; a PDE5i with a long half-life) or placebo will be administered by gavage every 24 h starting at 1 h post exposure. Maternal death, fetal growth, and hallmark symptoms of systemic endothelial dysfunction and preeclampsia will be measured in pregnant and non-pregnant mice as discussed in SA#1. In summary, this exploratory translational research project will begin delineating the mechanisms of Br2 toxicity in pregnant mice and will test the efficacy of highly promising countermeasures (PDE5i) which are already in clinical trials for the treatment of preeclampsia. Thus, we believe that this project is consistent with the overarching goal of the CounterACT program to design better countermeasures for vulnerable populations.

? DESCRIPTION (provided by applicant): Bromine (Br2) is a highly toxic dark-reddish liquid, which evaporates readily to a red vapor with a suffocating odor. World production of Br2 exceeds 300,000 tons per year. Exposure to Br2 causes acute lung injury, death from respiratory failure, and fibrosis. Because of the potential for industrial and transportation accidents to release of large amounts of Br2 in populated areas, Br2 presents a clear and present danger to public health. Few published studies have evaluated the acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Similar to human pathology, exposure of mice to Br2 causes reactive airway disease syndrome (RADS), increased permeability of the blood gas barrier to plasma proteins, and inflammation followed by sub-epithelial airway fibrosis and significant mortality. The overall purpose of this application is to identify the biochemical and molecular mechanisms responsible for these events and develop appropriate countermeasures. We propose that Br2 and hypobromous acid (HOBr-) interact with and fragment high molecular weight hyaluronan (H-HA), a ubiquitous matrix glycosaminoglycan, to generate highly inflammatory low molecular weight hyaluronan fragments (L-HA). L- HA binds to CD44 and Toll like receptor (TLR)-4, increases intracellular Ca+2 and activates TGF-?1, and RhoA in lung epithelial and airway smooth muscle cells. These events lead to RADS, increased epithelial permeability to plasma proteins, epithelial-mesenchymal cell transition (EMT) of airway cells, sub-epithelial fibrosis, an death from respiratory failure. In addition, we demonstrate for the first time the formation of brominated lipids in the lungs and plasma of mice exposed to Br2. These compounds, formed by the interaction of Br2 with lung plasmalogens, mediate and amplify Br2 lung injury and act as biomarkers of Br2 exposure. Based on solid data we posit that post-Br2 exposure administration of aerosolized Yabro? (a form of H-HA, currently in clinical trials in Europe for asthma), attenuates lung damage, enhances repair and decreases mortality. Experiments proposed in the first specific aim will assess physiological, biochemical, and morphological changes in mice exposed to Br2 and returned to room air for up to three weeks and test the effectiveness of aerosolized Yabro? administered post exposure to decrease lung injury and mortality. We will then identify the mechanisms by which Br2 damages rodent and human airway smooth muscle (ASM), bronchial and alveolar type II (ATII) cells. We posit that Br2, brominated lipids, and L-HA increase intracellular Ca2+ and activate RhoA, which lead to increased airway contractility and epithelial permeability. Experiments will: (i) determine membrane potentials by patch clamp; (ii) intracellular Ca+2 by fura-2 fluorescence; (iii) RhoA and ROCK activation; (iv) myosin light chain phosphorylation; and (v) (for epithelial cells) permeability to fluorescent dextrans. Finally, we wll isolate mouse tracheal rings at 1, 24, and 72 hr. post Br2 exposure and measure smooth muscle contraction in response to methacholine.

The halogen bromine (Br2) is used as water disinfectant, for bleaching fibers, for manufacturing antiepileptic drugs, dyestuffs, flame-retardants, insecticides, drilling fluids, and gasoline additives. When inhaled, it causes exposure-level-dependent acute and chronic pulmonary and systemic injuries ranging from mild eye and airway irritation, to significant damage to cardiopulmonary system and other organs, which can lead to death. Survivors may develop reactive airway disease syndrome, pulmonary fibrosis as well as restrictive and obstructive pulmonary diseases. Presently, there are no studies evaluating acute and chronic sequelae of Br2 inhalation in pregnant rodent and non-rodent models, even though US census bureau data predicts two of every 100 people in the US being pregnant. Exposure of pregnant mice at gestational day 15 (E15) to Br2 (600 ppm for 30 min.) results in 75% mortality over four days, in contrast to 25% mortality in males or non-pregnant females (p<0001). When delivered at E19, fetuses of surviving Br2-exposed mice exhibit severe fetal growth restriction (FGR) and fetal demise (FD). Placentas are poorly developed and express increased levels of short-FMS-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic mediator and biomarker of both preeclampsia and pulmonary hypertension. When born naturally, none of the fetuses survive. Oral administration of an FDA- approved type 5 cyclic nucleotide-specific phosphodiesterase inhibitor (PDE5i; tadalafil) to the dams post- exposure, dramatically improved maternal survival, fetal growth restriction and neonatal survival. We hypothesize that brominated intermediates, formed by the reaction of Br2 and HOBr with plasmalogens cause injury to the endothelium and the placenta, inducing the release of vasoconstrictor and anti-angiogenic mediators which in turn mediate pulmonary vasoconstriction, increased pulmonary artery pressure and right ventricular dysfunction. Tadalafil restores pulmonary and uterine vasodilation, preserves heart function and improves uterine/placental blood supply resulting in maternal and fetal survival. We will test these proposed mechanisms and we will perform the necessary efficacy studies to identify the optimum therapeutic regimen of tadalafil to decrease maternal morbidity and mortality, improve fetal growth restriction and increase fetal survival when administered orally post exposure. Specific Aim #1. To test the hypothesis that exposure of pregnant mice to Br2 at E15 causes extensive pulmonary injury as well as systemic endothelial injury, placental injury, pulmonary hypertension, right heart failure resulting in maternal mortality, fetal growth restriction and fetal demise/stillbirth. Specific Aim #2: To identify the sequence of events and mechanisms involved in the development of maternal vasoconstriction, pulmonary hypertension and right heart failure. Specific Aim #3. To investigate the efficacy of post halogen exposure administration of tadalafil to decrease maternal and fetal death and morbidity and to develop a rabbit (non-rodent) model of Br2 toxicity in pregnancy.

The halogen bromine (Br2) is used as water disinfectant, for bleaching fibers, for manufacturing antiepileptic drugs, dyestuffs, flame-retardants, insecticides, drilling fluids, and gasoline additives. When inhaled, it causes exposure-level-dependent acute and chronic pulmonary and systemic injuries ranging from mild eye and airway irritation, to significant damage to cardiopulmonary system and other organs, which can lead to death. Survivors may develop reactive airway disease syndrome, pulmonary fibrosis as well as restrictive and obstructive pulmonary diseases. Presently, there are no studies evaluating acute and chronic sequelae of Br2 inhalation in pregnant rodent and non-rodent models, even though US census bureau data predicts two of every 100 people in the US being pregnant. Exposure of pregnant mice at gestational day 15 (E15) to Br2 (600 ppm for 30 min.) results in 75% mortality over four days, in contrast to 25% mortality in males or non-pregnant females (p<0001). When delivered at E19, fetuses of surviving Br2-exposed mice exhibit severe fetal growth restriction (FGR) and fetal demise (FD). Placentas are poorly developed and express increased levels of short-FMS-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic mediator and biomarker of both preeclampsia and pulmonary hypertension. When born naturally, none of the fetuses survive. Oral administration of an FDA- approved type 5 cyclic nucleotide-specific phosphodiesterase inhibitor (PDE5i; tadalafil) to the dams post- exposure, dramatically improved maternal survival, fetal growth restriction and neonatal survival. We hypothesize that brominated intermediates, formed by the reaction of Br2 and HOBr with plasmalogens cause injury to the endothelium and the placenta, inducing the release of vasoconstrictor and anti-angiogenic mediators which in turn mediate pulmonary vasoconstriction, increased pulmonary artery pressure and right ventricular dysfunction. Tadalafil restores pulmonary and uterine vasodilation, preserves heart function and improves uterine/placental blood supply resulting in maternal and fetal survival. We will test these proposed mechanisms and we will perform the necessary efficacy studies to identify the optimum therapeutic regimen of tadalafil to decrease maternal morbidity and mortality, improve fetal growth restriction and increase fetal survival when administered orally post exposure. Specific Aim #1. To test the hypothesis that exposure of pregnant mice to Br2 at E15 causes extensive pulmonary injury as well as systemic endothelial injury, placental injury, pulmonary hypertension, right heart failure resulting in maternal mortality, fetal growth restriction and fetal demise/stillbirth. Specific Aim #2: To identify the sequence of events and mechanisms involved in the development of maternal vasoconstriction, pulmonary hypertension and right heart failure. Specific Aim #3. To investigate the efficacy of post halogen exposure administration of tadalafil to decrease maternal and fetal death and morbidity and to develop a rabbit (non-rodent) model of Br2 toxicity in pregnancy.

Chlorine (Cl2) is an irritant and reactive gas produced in large quantities throughout the world. Humans and animals exposed to Cl2 from industrial accidents or acts of terrorism, develop severe reactive airway disease, pulmonary edema and even death from respiratory failure. Those that survive are at risk of developing chronic lung diseases, such as pulmonary fibrosis and emphysema and be susceptive to infections. However, the mechanism(s) involved and the countermeasures required to ameliorate acute and chronic lung injury remain elusive. Herein we show that exposure of mice to Cl2 damages their mitochondria DNA (mtDNA) that administration of the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1), attached to the mitochondrial targeting signal (mitoOGG1 or OGG1 fusion protein) ameliorated mitochondrial dysfunction and Cl2-induced acute and chronic lung injury. The goals of our R21 application are: (1) to establish that exposure of mice to Cl2 damages their mtDNA; (2) administration of mitoOGG1 post Cl2 exposure decrease acute and chronic lung injury and mortality by repairing mtDNA and (3) that mitoOGG1 repairs the mitochondria bioenergetics in vitro. These hypotheses will be tested by completing the comprehensive set of experiments outlined in the following two Specific Aims: 1. Assess the efficacy of mitoOGG1 administered in mice post Cl2 exposure to decreases acute lung injury, mortality and the development of pulmonary fibrosis in vivo. 2. To demonstrate that mitoOGG1, administered to lung cells post Cl2 exposure in vitro, reverses, or at least mitigates, mitochondria bioenergetics and cell injury by repairing their mtDNA. Injury to mtDNA will compromise mitochondria respiration and membrane potential resulting in apoptosis and necrosis. mitoOGG1, administered post-heme, will reverse, or at least mitigate, these effects by repairing mtDNA. Previous studies have shown that injury to mtDNA plays a key role in the development of acute lung injury. Overexpression of mitoOGG1 attenuated mtDNA damage and preserved cardiac function in heart failure), protected against ventilator-induced lung injury in intact mice and limited human lung injury after circulatory death. Thus, these published studies, in addition to our highly exciting preliminary data, indicated that mitoOGG1 may prove to be a therapeutic for acute and chronic lung injury in patients exposed to Cl2, as well as other halogens (such as Bromine) and chlorine-containing compounds (such as Phosgene). These studies combine the expertise of two senior investigators (Drs. Matalon and Gillespie) with significant expertise in Cl2 induced lung injury and repair and mitochondrial function.