Annual General Meeting of the Irish Thoracic Society - IJMS | Irish ...

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SESSION FOUR ONE
ANNUAL MEETING OF THE IRISH THORACIC SOCIETY • 11 - 12 November 2005 • WESTWOOD HOUSE HOTEL, GALWAY
Session 4: Lung Injury / Inflammation
ANNUAL MEETING OF THE IRISH THORACIC SOCIETY • 11 - 12 November 2005 • WESTWOOD HOUSE HOTEL, GALWAY
SESSION
4SESSION FOUR ONE
4.1
Hypercapnia does not attenuate oxidation in the endotoxin
induced acute lung injury
Introduction
Acute Lung Injury (ALI) is a major problem
causing thousands of deaths annually worldwide.
Hypercapnic acidosis (HCA) has protective effects in
endotoxin-induced ALI and may have therapeutic
potential. The mechanism of this protective action is
unknown; it has been shown in vitro that high CO2
can inhibit peroxynitrite-mediated oxidation, an
important mechanism of damage in ALI. We tested
the hypothesis that HCA attenuates ALI in vivo by
inhibiting peroxynitrite-mediated oxidation.
Methods
Adult rats were anaesthetized, mechanically
ventilated and ALI induced by intra-tracheal
instillation of endotoxin. We measured the oxidative
formation of rhodamine from dihydrorhodamine in
bronchoalveolar fluid (BAL). NOS inhibition
(L-NMMA) was used to prevent peroxynitrite
formation.
Results
The formation of rhodamine was increased above
controls following endotoxin injury, however there
was no difference between the endotoxin groups
(n=10 per group).
Conclusions
Oxidation was not inhibited by HCA in endotoxininduced
ALI. The failure of NOS inhibition to reduce
rhodamine formation, suggests that peroxynitrite
was not an essential mediator of oxidation.
A Nichol, D O’Croinin,
F Naughton,
P McLoughlin
University College
Dublin, School of
Medicine and Medical
Sciences
Supported by
ICSI, IRCSET
4.2
Up-regulation of matrix metalloproteases and cathepsins
by neutrophile elastase: a novel hierarchy in protease
regulation
Introduction
Matrix metalloprotease (MMP), cathepsin and
neutrophil elastase (NE) activities were measured in
bronchoalveolar lavage (BAL) from individuals with
Alpha-1 Antitrypsin (AAT) deficiency, pneumonia
and healthy controls. We observed correlation
between NE activity and cathepsin and MMP activity
postulating that NE might upregulate expression of
these key proteases and that this upregulation could
be inhibited by treatment with aerosolised plasma
purified AAT (ppAAT).
Methods
Macrophage supernatants were assessed for
cathepsin and MMP expression and activity
following stimulation with NE. Cathepsin and MMP
activities were assessed in pneumonia and control
BAL and in BAL from AAT deficient patients pre- and
post- aerosolisation of ppAAT.
Results
Cathepsin and MMP activity are significantly higher
in BAL samples in AAT deficiency and pneumonia
compared to controls and correlate significantly
with NE activity. Treatment with ppAAT significantly
decreases cathepsin and MMP activity and
expression in BAL from AAT deficient patients in vivo
and pneumonia patients in vitro.
Conclusions
We describe a novel protease cascade whereby
NE upregulates cathepsin and MMP expression/
activation. Neutralization of NE by the antiprotease,
AAT, inhibits this up-regulation. This
new insight into protease function may facilitate
targeted anti-protease therapy in diseases such as
COPD, CF, pneumonia and A1AT deficiency where NEinduced
cathepsin / MMP-mediated inflammation
are a feature.
M Rogan, P Geraghty,
C Greene, M Brantly,
C Taggart, S O’Neill,
NG McElvaney
Pulmonary research
Division, RCSI,
Beaumont Hospital,
Dublin and Dept of
Medicine, University of
Florida, USA
4.3
Endoplasmic reticulum stress-induced apoptosis and
its inhibition by Tauroursodeoxycholic acid in Alpha-1
antitryspsin deficiency
BAL NEUTROPHIL [*103/ML] PAO2[MMHG] RHODAMINE [PM]
Control 9.1 (2.5) 136.4 (7.5) 23.3 (5.1)
Normocapnia 5230* (991) 89.1* (6.0) 55.1* (4.8)
Hypercapnia 5142* (1079) 105.4* (6.4) 48.7* (5.1)
Normocapnia LNMMA 6065* (828) 103.3* (5.0) 46.4* (4.5)
Hypercapnia LNMMA 5204* (1602) 92.4* (92.4) 52.9* (4.0)
Values are mean (SEM). * significantly different from control [ANOVA]..
Introduction
Z alpha-1-antitrypsin (AAT) deficiency is a
genetic disease associated with accumulation
of misfolded AAT in the endoplasmic reticulum
(ER). We investigated both the effect of ZAAT on
apoptosis, using an in vitro model system of ZAAT ER
accumulation, and the mechanism of inhibition of
this apoptosis by tauroursodeoxycholic acid (TUDCA).
Method
Apoptosis was induced in HEK293 cells using
thapsigargin, etoposide or by transfection with
a ZAAT cDNA. Cleavage of caspases-3,-4 and -7,
cytochrome c release and phosphorylation of the
Bcl-2 family member Bad were assessed by western
immunoblotting. Caspase activities were quantified
by fluorimetry or luminometry, as appropriate.
Apoptosis was demonstrated using TUNEL staining
and cell viability assays were performed. The
inhibitory effects of TUDCA were also assessed.
Results
ER accumulation of ZAAT, but not normal MAAT,
leads to cleavage and activation of caspases -3,-4
and -7. Similar effects were also induced using the
ER agonist thapsigargin. TUNEL staining of ZAATexpressing
cells confirmed the presence of apoptosis.
Inhibition studies using TUDCA demonstrated its
ability to inhibit caspase-4 and caspase-3/7 activation,
mitochondrial release of cytochrome c and apoptosis
induced by ZAAT. TUDCA increased phosphorylation
of Bad. Cell viability assays confirmed the beneficial
effect of TUDCA on cell survival.
Conclusions
Our data shows a new mechanism of cell death
in ZAAT deficiency: Activation of the caspase
cascade and apoptosis via the ER-specific caspase-
4. Inhibition studies using TUDCA demonstrate
its ability to inhibit caspase activation via the
phosphorylation of Bad and implicate it as a
potential therapeutic agent in AAT deficiency.
S Miller, C Greene,
C Taggart, C McElvaney,
S O’Neill
Dept of Medicine,
Respiratory Research
Division, RCSI
Education and
Research Centre,
Beaumont Hospital,
Dublin
46 IRISH JOURNAL OF MEDICAL SCIENCE • VOLUME 174 • NUMBER 4 • SUPPLEMENT 3
IRISH JOURNAL OF MEDICAL SCIENCE • VOLUME 174 • NUMBER 4 • SUPPLEMENT 3 47