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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.
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