Home

Research Interests

Cellular and physiological sequelae of oxidant injury to the mammalian blood-gas barrier; cellular and molecular mechanisms of active and passive solute transport across the alveolar epithelium; modulation of gene transfer by reactive oxygen and nitrogen species.

ACTIVE GRANTS

NITRIC OXIDE MEDIATED INJURY TO ALVEOLAR EPITHELIUM
5R01HL051173-10
PI: MATALON, SADIS
Abstract: 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 des Respiratory syncytial virus (RSV) is commonest cause of lower respiratory tract disease in children worldwide. Pathogenesis of RSV-induced bronchiolitis is poorly understood, and effects of RSV infection on ion transport (a seminal function of respiratory epithelial cells) have not been investigated. I hypothesize that RSV infection of respiratory epithelial cells reduces their Na? transport capacity. Preliminary studies have demonstrated that this hypothesis is correct, both in vitro and in RSV-infected BALB/c mice. My aims for years 01- 03 are to: (1) quantify alterations in Na + transport across airway and alveolar epithelia in vivo and ex vivo, after infection of BALB/c mice with RSV; (2) define changes in Na + currents and amiloride-sensitive channel activity after RSV infection of murine epithelial cells in vitro; and (3) correlate alterations in Na + transport induced by RSV in vitro and in vivo with alterations in ENaC expression by marine respiratory epithelia. My plan for years 04-05 is to determine the role of the ubiquitin/proteasome pathway in mediating reduced Na+ transport after RSV infection of respiratory epithelia, and to identify the role of the RSV small hydrophobic (SH) gene product in modulation of Na + transport. I will use a combined electrophysiologic and biochemical approach to investigate effects of RSV on Na + transport at all levels, from the single cell to the whole animal, and to correlate these effects to Na + channel expression and degradation. My project will emphasize cross-training in diverse techniques, including short-circuit measurements across monolayers, radioisotopic ion flux studies, whole cell and single channel patch-clamp, and measurement of alveolar fluid clearance and nasal potential difference in mice. ENVIRONMENT: This SERCA, with Dr. Matalon (lung physiology) as mentor and Dr. Sullender (respiratory syncytial virus) as co-mentor, will provide the training and setting I require for my maturation into an independent scientist focused on comparative pathophysiologic effects of respiratory viruses on normal epithelial cell function. 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.


MODULATION OF INNATE IMMUNITY IN LUNG TRANSPLANTATION
5R01HL072871-03
PI: MATALON, SADIS
CO INVESTIGATOR: YOUNG, RANDALL; WILLE, KEITH
Abstract: 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.

NITRIC OXIDE MODULATION OF CFTR EXPRESSION AND FUNCTION
1R01HL075540-01A1
PI: MATALON, SADIS
CO INVESTIGATOR: BEBOK, Z; COLLAWN, J.; PATEL, R.; HICKMAN-DAVIS, J.
Abstract: 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.


PATHOPHYSIOLOGY OF SUBLETHAL OXYGEN INJURED LUNGS
5R37HL031197-21
PI: MATALON, SADIS
CO INVESTIGATOR: SULLENDER, WAYNE; DAVIS, IAN
3R37HL031197-21S1
PI: KELLY, OLLIE

Abstract: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in children worldwide. Pathogenesis of RSV-induced bronchiolitis is poorly understood, and effects of RSV infection on ion transport (a seminal function of respiratory epithelial cells) have not been investigated. I hypothesize that RSV infection of respiratory epithelial cells reduces their Na? transport capacity. Preliminary studies have demonstrated that this hypothesis is correct, both in vitro and in RSV-infected BALB/c mice. My aims for years 01- 03 are to: (1) quantify alterations in Na + transport across airway and alveolar epithelia in vivo and ex vivo, after infection of BALB/c mice with RSV; (2) define changes in Na + currents and amiloride-sensitive channel activity after RSV infection of murine epithelial cells in vitro; and (3) correlate alterations in Na + transport induced by RSV in vitro and in vivo with alterations in ENaC expression by marine respiratory epithelia. My plan for years 04-05 is to determine the role of the ubiquitin/proteasome pathway in mediating reduced Na+ transport after RSV infection of respiratory epithelia, and to identify the role of the RSV small hydrophobic (SH) gene product in modulation of Na + transport. I will use a combined electrophysiologic and biochemical approach to investigate effects of RSV on Na + transport at all levels, from the single cell to the whole animal, and to correlate these effects to Na + channel expression and degradation. My project will emphasize cross-training in diverse techniques, including short-circuit measurements across monolayers, radioisotopic ion flux studies, whole cell and single channel patch-clamp, and measurement of alveolar fluid clearance and nasal potential difference in mice. ENVIRONMENT: This SERCA, with Dr. Matalon (lung physiology) as mentor and Dr. Sullender (respiratory syncytial virus) as co-mentor, will provide the training and setting I require for my maturation into an independent scientist focused on comparative pathophysiologic effects of respiratory viruses on normal epithelial cell function.
 

Lab Research

INTERACTIONS OF CFTR AND PROTON-GATED Na CHANNELS
JI04G0
PI: HONG-LONG JI

Abstract: DESCRIPTION (provided by the applicant): A growing body of evidence suggests that the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) down regulates the epithelial Na+ channel (ENaC) and the ENaC up-regulates CFTR activity. A new branch of the ENaC/DEG gene family, the proton-gated Na+ channels (ASICs), has emerged recently. Six isoforms of ASIC channels (ASIC 1a, 1b, 2a, 2b, 3 and 4) have been cloned and kASIC3 is expressed in epithelial tissues. The Principal Investigator’s long-term objective is to stud the physiological function and regulation of CFTR and ASIC3 mediating the interactions between CFTR and ASIC3 channels. Electrophysiological techniques (patch clamp and two-electrode voltage clamp) combined with molecular biological and biochemical methods (mutagenesis, immunoblot, immunolocalization, pull-down assays) will be the main techniques for this project. Expression of ASIC3 in epithelial CFTR cell lines (Calu-3, 16HBE14o, T84, CFPAC), and murine epithelial tissues will be detected by RT-PCR, western blotting, and immunostaining. Biophysical properties of ASIC3 in Calu-3 cells will be characterized. Functional interactions between ASIC3 and CFTR will be examined using patch clamp analysis. Key findings from epithelial cells will be corroborated in the oocyte expression system by co-expression of CFTR and ASIC3. Mutations that modify ASIC3 gating (degenerin site and pre-MI domain) and the first nucleotide binding domain (NBF1) of CFTR will be constructed. Finally, the physical interactions between CFTR and ASIC3 proteins (wild type and mutations) will be tested using in vitro co-immunoprecipitation and pull-down assays. The effects of long-term exposure to acidity on CFTR and ASIC3 expression in epithelial cells will also be tested using western blot. The combined use of these biophysical, biochemical, and molecular techniques will permit a detailed analysis of the interactions between CFTR and ASIC3 channels. Cystic fibrosis is a genetic disease in Caucasians characterized by dysfunctional airway mucosa, pancreas, sweat glands, and reproductive system. The abnormal interactions between defective CFTR and ENaC which result in less base and fluid secretion via CFTR and more absorption via ENaC is a hallmark of cystic fibrosis epithelia. With acidic luminal liquid, ASIC is further activated. However, the role of epithelial ASIC channels and their interactions with CFTR are not clear. In contrast to the function of neural ASIC channels, native epithelial ASIC3 has not been studied. Up-regulation of ASIC channels by acidic extracellular pH, hypoxia, inflammation, and mechanic stimuli implies that ASIC may work in parallel with ENaC in CF epithelial tissues. The proposal intends to investigate the interactions between CFTR and ASIC channels according to their co-distribution in epithelial tissues. Thin information provided by this proposal will contribute to understanding the role of the interactions between epithelial CFTR and ASIC channels in the basic etiology and pathogenesis of cystic fibrosis. The results will also shed light on the molecular basis for the interactions between CFTR and ENaC.


Na+ TRANSPORT INHIBITION BY RESPIRATORY SYNCYTIAL VIRUS
5K01RR017626-02
PI: DAVIS, IAN

Abstract: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in children worldwide. Pathogenesis of RSV-induced bronchiolitis is poorly understood, and effects of RSV infection on ion transport (a seminal function of respiratory epithelial cells) have not been investigated. I hypothesize that RSV infection of respiratory epithelial cells reduces their Na? transport capacity. Preliminary studies have demonstrated that this hypothesis is correct, both in vitro and in RSV-infected BALB/c mice. My aims for years 01- 03 are to: (1) quantify alterations in Na + transport across airway and alveolar epithelia in vivo and ex vivo, after infection of BALB/c mice with RSV; (2) define changes in Na + currents and amiloride-sensitive channel activity after RSV infection of murine epithelial cells in vitro; and (3) correlate alterations in Na + transport induced by RSV in vitro and in vivo with alterations in ENaC expression by marine respiratory epithelia. My plan for years 04-05 is to determine the role of the ubiquitin/proteasome pathway in mediating reduced Na+ transport after RSV infection of respiratory epithelia, and to identify the role of the RSV small hydrophobic (SH) gene product in modulation of Na + transport. I will use a combined electrophysiologic and biochemical approach to investigate effects of RSV on Na + transport at all levels, from the single cell to the whole animal, and to correlate these effects to Na + channel expression and degradation. My project will emphasize cross-training in diverse techniques, including short-circuit measurements across monolayers, radioisotopic ion flux studies, whole cell and single channel patch-clamp, and measurement of alveolar fluid clearance and nasal potential difference in mice. ENVIRONMENT: This SERCA, with Dr. Matalon (lung physiology) as mentor and Dr. Sullender (respiratory syncytial virus) as co-mentor, will provide the training and setting I require for my maturation into an independent scientist focused on comparative pathophysiologic effects of respiratory viruses on normal epithelial cell function.


MODIFICATION OF ENaC EXPRESSION AND FUNCTION DURING RESPIRATORY MYCOPLASMA INFECTION
RG-9928-N
PI: JUDY HICKMAN DAVIS

Abstract: Mycoplasma pneumoniae accounts for 20 to 35% of community-acquired pneumonias in the general population and up to 56% of pneumonias among susceptible populations. Likewise, mycoplasmas are known to exacerbate the pathophysiology of asthma, adult respiratory distress syndrome and chronic obstructive pulmonary disease. Man is the only host of M. pneumoniae, but murine respiratory infection with M. pulmonis reproduces the essential features of human respiratory mycoplasmosis. Previously we utilized this model to demonstrate that mycoplasma infection stimulates significant production of reactive oxygen-nitrogen species (RONS) by inflammatory cells and that RONS are essential for mycoplasma killing. However, despite significant RONS production in response to mycoplasma infection, susceptible individuals may still develop serious respiratory disease. Although adherence of M. pulmonis to the respiratory epithelium is necessary for colonization and development of disease, interactions of mycoplasmas with the pulmonary epithelium remain poorly understood. A primary function of alveolar epithelial cells is the reabsorption of fluid to keep the alveoli dry and ensure normal gas exchange. Active transport of Na+ through the amiloride-sensitive Na+ channel (ENaC) on epithelial cells is vital for normal clearance of fluid from the airspaces. We hypothesize that mycoplasma infection impairs alveolar epithelial cell ion transport by upregulating the production of RONS and altering ENaC function by post-translational modifications. We have designed a series of experiments to test this hypothesis in vivo utilizing the mouse respiratory pathogen M. pulmonis and mycoplasma-resistant C58BL/6 (B6) mice, B6 mice lacking surfactant protein A [B6.SP-A(-/-)], B6 mice lacking the inducible form of nitric oxide synthase [B6.iNOS (-/-)], and B6 mice lacking myeloperoxidase [B6.MPO (-/-)]. Specifically, we plan to (1) Define the mechanisms by which mycoplasmas damage ion transport in vivo by evaluating the contribution of RONS to ATII cell dysfunction during infection. We will instill M. pulmonis intranasally into B6, B6iNOS(-/-) and B6.MPO(-/-) mice and measure (i) production of RONS in whole lung homogenates, bronchoalveolar lavage (BAL) fluid and by cells in the BAL fluid, (ii) measurement of RONS production by ATII cells, and (iii) alveolar fluid clearance; and (2) Quantify the extent to which infection by mycoplasmas modifies ENaC function. Whole lung homogenates and isolated ATII cells from M. pulmonis infected B6, B6.SP-A(-/-) and B6.MPO(-/-) mice will be utilized for determination of (i) cell surface expression of ENaC by biotinylation and immunofluorescence, and (ii) oxidative modification of ENaC by Western blot and ELISA. A better understanding of basic mechanisms of host pathogen interactions may lead to the development of novel therapies, which may extend to other pathogens.