Caci, E.

Galietta, L.J.V.

Gianotti, A.

Melani, R.

Zegarra-Moran, O.

Elsevier - Publisher Connector 

S70 4. New Therapies Posters
55 Secretory leukoprotease inhibitor (SLPI) as a novel therapeutic
targeting neutrophil chemotaxis in cystic ﬁbrosis
N. Banville1, D. Ryan1, D.A. Bergin1, O. McElvaney1, S.J. O’Neill1, E.P. Reeves1,
N.G. McElvaney1. 1RCSI, Respiratory Medicine, Dublin, Ireland
Cystic ﬁbrosis (CF) is generally diagnosed early in childhood, it is characterised by
sustained neutrophil dominated inﬂammation from a very young age. Conversely,
recruited neutrophils are ineffective in bacteria clearance and play a signiﬁcant
role in bronchiectasis development. Identiﬁcation of agents modulating neutrophil
chemotaxis constitutes a relevant goal.
Studies have shown that SLPI is anti-inﬂammatory in such disorders as athero-
sclerosis and lung emphysema. This study aimed to enhance knowledge of SLPI’s
anti-inﬂammatory effects and in the context of tissue homeostasis, we investigated
SLPI’s ability to moderate neutrophil chemotaxis.
Neutrophils were puriﬁed from whole blood and subcellular fractionation performed
using ultracentrifugation techniques. Cell migration was assessed using a boyden
chamber. The inhibitory effect of SLPI on F-actin polymerisation and talin cleavage
post fMLP (10−6M) and IL-8 (10 ng) stimulation was assessed by western blot anal-
ysis. Activity of calpain, a cysteine protease involved in cytoskeletal rearrangements
required for neutrophil chemotaxis, was determined using a ﬂuorometric assay.
SLPI was localised to the cytosol and secondary granules of resting neutrophils.
Experimental results indicate that SLPI is not a physiological substrate for calpain.
Instead, SLPI inhibited neutrophil calpain activity in a dose dependent manner (Ki =
70 nM) and prevented fMLP and IL-8 induced chemotaxis, F-actin polymerisation
and talin cleavage.
Inhibition of cytoskeletal rearrangements by SLPI may represent a novel anti-
chemotactic mechanism, strengthening SLPI’s attraction as a potential therapeutic
in inﬂammatory lung disease.
55A Combined approaches to restore airways hydration in cystic
ﬁbrosis
O. Zegarra-Moran1, A. Gianotti1, R. Melani1, E. Caci1, L.J.V. Galietta1. 1Istituto
Giannina Gaslini, Laboratory of Molecular Genetics, Genoa, Italy
The CF airway disease, characterized by the increased viscosity of mucus, infection
and colonization of the lungs, is a consequence of the incapacity of mutated CFTR
to secrete Cl−. This in turn, disrupts the balance between absorption and secretion
and leads to a prevalence of Na+ absorption. We have hypothesized that to restore
airway hydration it is necessary either to recover Cl− secretion from mutated CFTR,
to reduce Na+ absorption or both.
A short-interfering RNA (siRNA) approach targeting the epithelial sodium channel
(ENaC) subunits was used to reduce Na+ absorption, while to increase Cl− secretion
we utilized CFTR potentiators on CF and non-CF bronchial primary epithelial cells.
To evaluate the effects of these actions, we measured transepithelial Na+ and Cl−
short-circuit currents and the height of the Texas Red-coloured surface ﬂuid layer
with confocal microscopy.
Our results indicate that (i) siRNA sequences complementary to any of the ENaC
subunit are able to reduce ENaC transcripts and Na+ channel activity but only
silencing alpha and beta ENaC subunits at the same time increases the airways
surface ﬂuid layer; (ii) the hydration obtained in this way was similar to that mea-
sured after maximal CFTR stimulation on non-CF epithelia; (iii) CFTR potentiators
increased airway hydration of corrected F508del epithelia.
In conclusion, both CFTR potentiators and ENaC silencing produce a signiﬁcant
and long-lasting increase of the airway hydration in vitro.
Supported by the Italian CF Foundation grant FFC#7/2009 with contributions
from Work in progress Communication and Brooks Brothers Retail Brand Alliance
Europe. R.M. and A.G. are Italian CFF contract holders.


55A Combined approaches to restore airways hydration in cystic fibrosis 

file:///data/core-remote/dit/data/elsevier/pdf/f54/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMTU2OTE5OTMxMjYwMjI1Mg==.pdf

55A Combined approaches to restore airways hydration in cystic fibrosis

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector