2.2.1 Light-scattering particle size analysis
Laser diffractometry was carried out on a 1mg/ml aqueous peptide solution using a Spraytech Laser Light Scattering System (Malvern,
UK). Briefly, 250µl of the 1mg/ml solution was nebulised into the
apparatus using an Aeroneb® Solo vibrating mesh nebuliser (Aerogen, Ireland). An extraction flow of 15l/min was applied to
prevent aerosol re-entry into the measurement zone. Particle sizing of the aerosol spray was carried out measuring the intensity of the light scattered from the laser beam as it passes through the spray. This allowed the calculation of the volume median diameter (VMD), where half the volume of the spray contains particles of larger and smaller diameter, and was carried out four separate times.
2.2.2 Impaction particle size analysis
A next generation impactor was also used to assess the droplet diameter of the nebulised spray. The nebuliser (Aeroneb® Solo) was attached to the apparatus via a plastic connector and metal inlet (throat). The intake was 15l/min and 1ml of the 1mg/ml solution in
dH2O was nebulised. After nebulisation was complete, the apparatus
was dismantled and the eight plates, connector, throat, nebuliser, and end filter (Respigard 303, Baxter, Ireland) were all thoroughly rinsed
with dH2O to collect the impacted peptide. The washings of each stage
were analysed by HPLC and compared to a standard line of peptide concentration versus peak area. The % deposition in each stage was used to calculate the mass median aerodynamic diameter (MMAD) of the spray and the mass balance of each stage. This was carried out three times for each peptide. Samples of low concentration were
lyophilised and reconstituted in 1/10th the original volume to increase
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The Aeroneb® Solo was used to nebulise 1ml of a 1mg/ml solution in
dH2O into a breathing apparatus. The conditions of a healthy human
breathing pattern was simulated using a Salter valved facemask 81070-0 (Salter, USA) and an ASL 5000 active servo lung (IngMar Medical, USA). The parameters were 15 breathes/min,
inhalation:expiration ratio of 1:1, tidal volume of 500ml, and 2l of supplementary gas flow. The nebulised peptides were collected below the model on a Respigard 303 filter (Baxter, Ireland) which was
washed afterwards with 10ml dH2O. The nebuliser was also washed.
The washings were analysed by HPLC and the % of the original dose delivered to the filter was calculated, giving the % deliverable to the lung. This was carried out three times for each peptide.
2.3 In vivo studies
2.3.1 Transgenic mice
All in vivo studies were granted ethical approval by the
Regierungspräsidium (Regional Council) of Karlsruhe, Baden-
Württemberg, Germany (reference G-284/14) and were carried out in collaboration with the laboratory of Prof Dr. Marcus Mall, University of Heidelberg, Germany. The animal handling licence was obtained from Laboratory Animal Science and Training, Ireland. The mouse strains
used were Scnn1b-Tg (β-ENaC), and Scnn1b-Tg NE-/- (β-ENaC NE-
knockout) on a C57BL/6 background. All mice used in infection studies
were 8-10 weeks old. For toxicity studies wildtype and NE-/- C57BL/6
mice aged 11-15 weeks were used.
2.3.2 Bacterial challenge
Mice were sedated using 3% v/v isoflurane in O2 at a flow rate of
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attached to an upright examination board. 50µl of a P. aeruginosa
PAO1 inoculum (1 - 2.5 x 107 CFU/mouse) was pipetted into their
throat and their nose was covered until the mice fully inhaled the bacterial suspension. After 6h the mice were then treated in a similar
manner with either 50µl of PBS or a 1mg/ml peptide solution. Mice
were sacrificed 24h after initial infection.
2.3.3 Lung lavage
Mice were euthanized by intraperitoneal injection of 120mg/kg ketamine and 16mg/kg xylazine and exsanguination. The midline anterior was incised and the trachea isolated, cannulated with a 22G needle, and tied. 0.035ml cold PBS/g (bodyweight) was used to lavage the lungs. Bacterial counts were carried out on each BAL fluid using the plate count method, after which the BAL fluids were centrifuged at 600xg for 5min and the supernatant removed. Murine NE was
quantified in each BAL supernatant using MeOSuc-AAPV-AMC, measuring the change in fluorescence at 460nm after excitation at 380nm. Recombinant murine NE was used as a standard (R & D systems, Germany), this was pre-activated by incubating for 4h at
37oC with murine cathepsin C (R & D systems, Germany). This step is
required to cleave the activation dipeptide from the amino terminal of the proenzyme (168). Please note that this is a different protocol to that used above for the determination of human NE levels in CF BAL fluid.
2.3.4 Toxicity studies
Wildtype mice were sedated and suspended by their incisors as with
the infection studies. The mice were treated intratracheally with 50µg
of peptide in the morning and evening and sacrificed 24h after the first dose. Any mice that were found to have died were not processed further. In living mice, lung lavage was carried out on half lungs while the other half was stored in 4% formaldehyde solution overnight,
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washed three times in dH2O, and stored in 70% ethanol at -20oC for
histological processing and analysis.
2.3.5 BAL cell count, total and differential
BAL fluids were centrifuged at 600xg at 4oC for 5min. The cell pellet
was resuspended in 50µl of cold PBS. The BAL fluid cell count was
carried out microscopically using Trypan blue staining and a
haemocytometer. The cell suspension was then then spread onto a
slide by centrifugation using a Cytospin (3 x 104 cells per slide). After
air-drying overnight the slides were stained using May-Grünwald- Giemsa staining to count the relative proportion of each cell type. Briefly, slides were placed in May-Grünwald solution for 3min, rinsed
in dH2O, placed in Giemsa solution for 7min and rinsed in dH2O. After
drying the slides were placed in xylene for 5s and then mounting media and a coverslip were added. The proportion of each cell type was quantified using oil-immersion microscopy at 100x magnification.
2.3.6 BAL cytokine release assay
BAL supernatants were analysed for cytokine levels using a V-PLEX Plus Proinflammatory Panel 1 (mouse) Kit (MSD, Ireland), analysing
levels of interferon γ (IFN-γ), IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-
6, keratinocyte chemoattractant (KC), and TNF-α. Briefly, BAL
samples were diluted and incubated on the plate for 2h at RT. Wells were washed three times with PBS/0.05% TWEEN-20 and then incubated for 2h at RT with the secondary antibodies. Plates were washed again and then read by chemiluminescence on a MESO QuickPlex SQ 120 (MSD, Ireland). Analysis was carried out on BAL fluids from mice in the toxicity study and from mice infected with 2.5 x
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