A. Mechanisms of injury of surfactant proteins by reactive oxygen-nitrogen intermediates: structure-function relationships. Electrospray mass spectrometry and a variety of biochemical (including site directed mutagenesis) and physiological techniques have been used to show that exposure of surfactant proteins A (SP-A), and D (SP-D), to reactive oxygen nitrogen species in vitro and in vivo, causes post translational modification (such as nitration of specific tyrosines in the carbohydrate recognition domains). We assess how changes in structure correlate with the decreased ability of SP-A and SP-D to facilitate aggregation and killing of pathogens. These studies impact our ability to understand the fundamental mechanisms of innate immunity. Publications: Myeloperoxidase-dependent inactivation of surfactant protein D in vitro and in vivo. J Biol Chem. 2010 May 28;285(22):16757-70. Epub 2010 Mar 12.PMID: 20228064. Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo. FASEB J. 2009 May;23(5):1415-30. Epub 2009 Jan 6.PMID: 19126597

B. Modification of lung epithelial ion transporters and lung fluid balance by reactive oxygen-nitrogen intermediates. Nitric oxide and reactive oxygen nitrogen intermediates (formed by the reactions of nitric oxide with partially reduced oxygen species), generated in close proximity of epithelial cell membranes by activated inflammatory cells, modulate the activity of amiloride sensitive epithelial sodium (ENaC) and cAMP-activated chloride (CFTR) channels via signal transduction mechanisms (such as activating PKG and PKC) or by post-translational oxidative modifications of sodium channel proteins and their chaperones. Channel activity (and vectorial sodium transport) may be either increased or decreased depending on levels of reactive intermediates and length of exposure. The following techniques are utilized in these experiments: patching primary or cultured cells in the cell attached and whole cell mode; expressing various subunits of ENaC and CFTR in oocytes; immunoprecipation of ion transporting proteins from whole lungs or epithelial cells; measuring the ion transport properties of epithelial cells forming confluent monolayers in Ussing chambers; measuring ion transport across the alveolar and airway epithelial of anesthetized mice; patching epithelial cells in situ. Publications: Inhibition of lung fluid clearance and epithelial Na+ channels by chlorine, hypochlorous acid, and chloramines. J Biol Chem. 2010 Mar 26;285(13):9716-28. PMID: 20106988. Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione. J Biol Chem. 2006 Apr 7;281(14):9190-9. Epub 2006 Jan 17.PMID: 16421103

C. Viral induced injury to the mammalian alveolar epithelium. 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 tenfold increase in epidemic and pandemic scenarios. Respiratory Syncytial Virus (RSV) is a member of the pneumovirus genus of the paramyxoviridae, and has a negative-sense, non-segmented, single-stranded RNA genome. It is the most common cause of lower respiratory tract disease in infants and children worldwide, is a frequent initiator of acute asthma exacerbations in young children, and has a disease impact comparable to that of non-pandemic influenza A in the elderly. We are currently using a variety of biophysical, molecular biology and physiological techniques to assess the basic mechanisms by which viral proteins and active replicating viruses interact with and modulate key functions of the alveolar epithelium both in vitro and in vivo. Results of these studies help us formulate new strategies for decreasing rhinnorhea and pulmonary edema, common consequences of viral infections. Publications: Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species. FASEB J. 2009 Nov;23(11):3829-42. Epub 2009 Jul 13.PMID: 19596899. Respiratory syncytial virus inhibits lung epithelial Na+ channels by up-regulating inducible nitric-oxide synthase. J Biol Chem. 2009 Mar 13;284(11):7294-306. Epub 2009 Jan 8.

D. Developing countermeasures against oxidant gases. 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 during acts of terrorism, has resulted in significant morbidity and mortality to both humans and animals. Physiological and biophysical and biochemical studies utilizing in vitro systems and animals exposed to chlorine gas have helped us elucidate the biochemical mechanisms responsible for chlorine injury to pulmonary and extrapulmonary targets Publications: Elucidating mechanisms of chlorine toxicity: reaction kinetics, thermodynamics, and physiological implications. Am J Physiol Lung Cell Mol Physiol. 2010; 299(3):L289-300. Epub 2010 Jun 4. Review.PMID: 20525917; Inhibition of lung fluid clearance and epithelial Na+ channels by chlorine, hypochlorous acid and chloramines. J Biol Chem. 2010 Mar 26;285(13):9716-28. PMID: 20106988; Mechanisms and modification of chlorine-induced lung injury in animals. Proc Am Thorac Soc. 2010 Jul;7(4):278-83. Review.PMID: 20601632; Chlorine Gas Exposure Causes Systemic Endothelial Dysfunction by Inhibiting eNOS-dependent Signaling. Am J Respir Cell Mol Biol. 2010 Dec 3. [Epub ahead of print]PMID: 21131444; Post Exposure Administration of a {beta}2-Agonist Decreases Chlorine Induced Airway Hyper-Reactivity in Mice. Am J Respir Cell Mol Biol. 2010 Sep 20. [Epub ahead of print]; PMID:20855648. Based on these studies we have formulated effective approaches to counteract this injury. Post exposure administration of antioxidants, nitrite or β2 agonists decreased pulmonary injury and improved survival in rodents exposed to Cl2 gas by a variety of mechanisms. Publications Ascorbate and Deferoxamine Administration Post Chlorine Exposure Decrease Mortality and Lung Injury in Mice. Am J Respir Cell Mol Biol. 2010 Dec 3. [Epub ahead of print]PMID: 21131440; PMID:21148791