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METHODS

Patients

Subjects with CF had the diagnoses made on either a positive sweat test or detection of

2 CF disease causing mutations [1]; included patients with PCD were diagnosed on

conventional criteria[2] .

Indications for bronchoscopy

All fibreoptic bronchoscopies (FOB) were carried out for clinical indications. The

investigation is routinely performed in our centre shortly after a diagnosis of CF is made

in order to establish microbiological status and to allow treatment for pathogens where

present; this group of patients made up part of the CF cohort. The remainder of the CF

group and the PCD and bronchiectasis patients were undergoing the procedure because

of unexplained decline in respiratory status or clinical symptoms and, in the majority, an

inability to expectorate sputum for culture. Healthy controls were bronchoscoped for

other reasons such as upper airway examination or a history of haemoptysis.

Consent

Written informed consent was obtained from parents/guardians of children undergoing

clinically indicated FOB. Specific consent (and assent where appropriate) was obtained

to retain samples surplus to requirement, and obtain additional samples for research.

Bronchoscopy Protocol

The procedure was carried out under general anaesthetic as is usual practice within the

centre and the specific anaesthetic technique was determined by the consultant

anaesthetist. Most children have anaesthetic induction by inhaled sevofluorane via face

mask, with the remaining children receiving intravenous propofol. Anaesthetic

maintenance is with sevofluorene and intravenous propofol if needed. The airway was

most commonly maintained by facemask and manual chin lift. Laryngeal mask airway

(LMA) and endotracheal tube were used if deemed appropriate by the anaesthetic

consultant.

2.8 internal diameter Olympus BF-XP40, 3.6 mm BF-3C20 or 3C40, 4.0 mm BF-MP60 and

4.6mm BF-P2OD bronchoscopes were used, the choice being dictated by the size of the

child. An initial inspection of upper and lower airways was made prior to samples being

obtained. Where possible, no suctioning was carried out in the upper airway to prevent

contamination of samples with upper airway flora.

BALF collection and processing

BALF samples were obtained by instilling 3 aliquots of 1ml/kg (maximum aliquot volume

of 40ml) 0.9% saline at room temperature to the right middle lobe or most affected

lobe(s). The aliquots were pooled. 1ml aliquots were sent to clinical laboratories for

microbiology ,processed in accordance with CF trust guidelines [3] , and cytology where

cell differential was performed. Cellular differential was performed following

cytospinning for 3 minutes at 200 g, air drying then fixation with methanol and May-

Grünwald-Geimsa staining. The research aliquot was put immediately on to ice and used

for research purposes. A 50 μl aliquot of whole BALF was mixed 1:1 with trypan blue

(Sigma-Aldrich, USA) and a total cell count performed using a dual chamber Neubauer

haemocytometer (Assisten, Sondheim, Germany). The remaining BALF was centrifuged

at 4˚C, 2000 g for 10 minutes and the supernatant stored in aliquots at -80˚C. As per

current research governance stipulations, samples were coded and individual aliquots

item tracked.

Blood samples

Venepuncture was clinically indicated and an additional aliquot of ≤3 ml of blood was

taken for research (total volume never greater than 1 ml/kg). Blood samples were

obtained through venepuncture whilst under general anaesthetic. These samples were

centrifuged at 4˚C, 2200 g for 10 minutes and the serum carefully pipetted and stored in

aliquots of between 125 and 500μl at -80˚C.

Measurements

25(OH)D2 Vitamin D was measured in serum. Assays were performed by the

biochemistry department at Royal Brompton and Harefield NHS Foundation Trust. The

samples were analysed using mass spectrometry with high pressure liquid

chromatography (HPLC).

HBD-2 HBD-2 was measured in BALF samples using ELISA (Phoenix

pharmaceuticals, USA). This was performed as per manufacturers’ instructions and

detects HBD-2 from 7.8 – 500 pg/ml [4]. Samples were diluted 1:1 with assay buffer

making the detectable range 15.6 pg/ml – 1000 pg/ml. 14 samples were measured in

duplicate and paired values very similar (CoV median (IQR) 4% (0.3-10%). The remainder

were run in singlicate.

LL-37 LL-37 was measured in BALF using ELISA (Hyocult biotech). Samples were

undiluted and the ELISA performed as per manufacturer’s instructions hyocult [5].

Samples were initially diluted, but serial dilution experiments revealed that dilution

effect was not linear and therefore all reported results were performed on neat

samples. Samples could not be run induplicate due to resources available. The assay

allowed measurement over the range 0.1 to 100 ng/ml. Two samples fell outside the

range of the kit and so the lower limit of detection, 0.1 ng/ml, was used for these

samples.

Cytokines

Meso scale discovery (MSD) was used for measurements of cytokines in the BALF. IL-1β,

IL-2, IL-6, IL-8, IL-10, IL-12, IFN-γ, TNF-α, GM-CSF and IL-17a were measured using a

combination of single and multiplex cytokine assays (Meso Scale Discovery, Rockville).

Samples were run in singlicate. These cytokines were chosen due to their actions of

regulation of inflammation in the airway [6].

In order to measure the cytokines, an antibody is coated onto a well, or for the

multiplex assays, a range of antibodies are coated in a specific pattern. The samples are

prepared by the addition of a solution containing detection antibodies (anti-IL-2, anti-IL-

8 etc.) each of which is labelled with an electrochemiluminescent compound. During

incubation periods, binding occurs and the final solutions are used for the readings. The

MSD instrument measures the quantity of emitted light which provides a quantative

measurement of the desired compound.

BALF samples were processed as per the assay protocol with a few minor changes, as

the protocol was not written for BALF. 12 μl 10% BSA solution was added to each of the

samples (108μl) to achieve a concentration of 1% BSA within each sample. The

standards were made up in 1% BAS and not the diluent as stated in the protocol to allow

for increased protein content within BALF.

The upper limit of detection for all cytokines was 2500pg/ml. The lower limit of

detection was determined by the individual assay run and varied from 0.013 to

4.86pg/ml. Samples were run in singlicate due to financial resources available

Viral PCR

The numbers of the group with viral PCR performed was smaller because the clinical

laboratory changed its protocol during the study period and previously viral analysis was

performed using immunofluorescence technique; which has been proven insensitive

and so these data have not been analysed.

Clinical data collection

Clinical data were collected including patient demographics, height, weight and clinical

symptoms at the time of the bronchoscopy, and spirometry results and microbiology

surveillance cough swabs/sputum samples over a 3 year period.

Statistical analysis

Mann Whitney for 2 groups, Kruskal-Wallis with Dunn’s correction for multiple groups,

logistic regression for binary outcomes and Chi squared test was used for comparison of

categorical data. SPSS v21 (IBM Corporation, New York) and Graphpad prism v6

(Graphpad software, San Diego) were used.

Definition of vitamin D deficiency:

Original cut-off values [7]

Insufficient

<50 nmol/L

Relatively sufficient≥50 nmol/L

Alternative 1[7]

Insufficient< 75 nmol/L

Sufficient≥75 nmol/L

Alternative 2[8]

Insufficient

<50 nmol/L

Relatively sufficient50–74 nmol/L

Sufficient

≥75 nmol/L

Alternative 3 [9-11]

Deficient<25 nmol/L

Insufficient25–74 nmol/L

Sufficient≥75 nmol/L

Alternative 4 Deficient

<25 nmol/L

Insufficient

25–49 nmol/L

Relatively sufficient50–74 nmol/L

Sufficient

≥75 nmol/L

Table 1: Post hoc analyses were performed using different cut-off values for vitamin D sufficiency and insufficiency. Irrespective of which cut-off value was used, no difference was seen between sufficient and insufficient patients.

RESULTS

Vitamin D level and age

The median ages were not different across the 3 groups although the proportion of

children <1 year of age was significantly higher in the CF group (p<0.01)(Table 2)

reflecting the practice of routine FOB in newly diagnosed infants with CF in our centre;

the 6 healthy controls were also older. For the patients with CF, vitamin D correlated

inversely with age (r=-0.35, p=0.001) and therefore multiple regression was undertaken

where relevant. Given this finding, post-hoc analyses were performed excluding all

children under 2 years of age, and again including only the children 2 years of age and

above, with no alteration to outcomes. Multivariate analyses accounted for this age

effect.

TABLES AND FIGURES

Table 2 CF patients with bacteria isolated in their BALF had higher BALF total cell count, neutrophil count, neutrophil differential, blood neutrophils, and higher BALF LL-37, IL-10, IL-12p70, IL-1B, IL-2, IL-8 and TNF-a. Values shown are median (95% CI).

BALFculture +ve

BALFculture -ve

p-value P value <0.01

BALF absolute count (x 103/L) 1,035 (730 – 1,880) 368 (273– 525) < 0.0001 ****BALF neutrophil differential (%) 58 (42 – 71) 14 (8 – 23) < 0.0001 ****BALF neutrophil count ( x 103/L) 521 (209– 1142) 39 (23.2 – 81.6) < 0.0001 ****

Serum neutrophils ( x 109/L) 5.3 (4.3 – 6.3) 3.5 (2.7 – 4.3) < 0.0001 ****BALF LL-37 ng/ml 0.82 (0.49 – 1.13) 0.35 (0.30 – 0.43) < 0.0001 ****BALF HBD-2 (pg/ml) 166 (123 – 231) 119 (65.5 – 203.5) 0.16 nsTNF-α (pg/ml) 3.4 (1.53 – 9.11) 0.5 (0.15 – 0.80) < 0.0001 ****GM-CSF (pg/ml) 0.7 (0.26 – 1.90) 0.2 (0.13 – 0.81) 0.02 nsINF-γ (pg/ml) 0.1 (0.00 – 1.6) 0.0 (0.0 – 0.11) 0.02 nsIL-10 (pg/ml) 1.2 (0.43 – 2.17) 0.2 (0.11 – 0.42) < 0.0001 ****IL-12p70 (pg/ml) 0.6 (0.14 – 1.46) 0.06 (0.00 – 0.13) 0.0004 ***IL-1β (pg/ml) 43 (15.3 – 77.4) 4.2 (1.06 – 8.68) < 0.0001 ****IL-2 (pg/ml) 0.5 (0.11 – 1.48) 0.1 (0.02 – 0.47) 0.002 **IL-6 (pg/ml) 21 (9.30 – 31.3) 6.4 (4.21- 14.2) 0.04 nsIL-8 (pg/ml) 2,347 (1,307– 4,163) 442 (234– 6995) 0.0004 ***IL-17 (pg/ml) 0.07 (0.02 – 0.18) 0.03 (0.01 – 0.07) 0.09 ns

Table 3 CF patients with viral detection in their BALF by PCR did not differ from those with no viral detection in vitamin D or antimicrobial levels. Values shown are median (95% CI).

BALFViral PCR +ve

BALFViral PCR -ve

p-value P value <0.01

n 8 16Vitamin D (nmol/L) 69 (37 – 105) 47 (34 – 71) 0.13 nsBALF LL-37 ng/ml 0.71 (0.31 – 3.94) 0.42 (0.37 – 1.16) 0.8 nsBALF HBD-2 (pg/ml) 70.2 (15.6 – 456) 68.3 (15.6 – 176) 0.99 ns

OLS Figure 1:

The median (95% CI) serum 25(OH)D2 levels were 57 (52 – 66), 42 (26 – 51) and 57 (24 – 74) nmol/L in the CF, non-CF CSLD and the healthy control groups respectively. The non-CF CSLD group had a significantly lower vitamin D level than the CF group (p<0.01). Vitamin D levels of each of the 3 groups were examined according to their microbiological status; BAL culture positive (red circles) vs. BAL culture negative (black triangles) and no differences were seen within each group (see OLS for further details and viral data)

OLS Figure 2: CF patients who isolated bacterial or fungal organisms from their BALF had higher levels of LL37 than those with sterile BALF. Median (95% CI) 0.82 (0.49 – 1.13) vs. 0.35 (0.30 – 0.43 ng/ml (p<0.0001).

OLS figure 3: A significant (p < 0.001) positive correlation was seen between LL-37 and BALF absolute cell count, BALF neutrophil count and differential and inflammatory cytokines.

OLS Figure 4: In CF patients, BALF LL-37 correlated with cellular and soluble markers of inflammation

OLS Figure 5: In CF patients, there was no correlation between cellular and soluble markers of inflammation and BALF HBD-2

OLS figure 6: There was no relationship seen between FEV1 nor FVC and either of the antimicrobial peptides in CF patients. LL37 did not correlate with FEV1 (r = - 0.14, ns) or FVC (r = - 0.15, ns). Similarly HBD-2 did not correlate with FEV 1 (r = 0.01, ns) and FVC (r = - 0.07, ns).

OLS Figure 7: CF patients with positive bacterial culture of their BALF had a higher BALF absolute cell counts than CF patients in whom no bacteria was isolated (p<0.0001)(figure a). These patients also had higher BALF neutrophil count (b), neutrophil differential (c), serum neutrophils, IL-10 (d), IL-1β (e) ,TNF-α (p < 0.0001), IL-8 (p=0.0004)(figure f)

OLS Figure 8: Vitamin D levels of CF patients who isolated any organisms in their BALF were compared with CF patients with sterile BALF. No difference was seen between the culture negative and culture positive groups (median (CI); 63 (52-68) vs. 56 (47 – 63)

nmol/L, ns). This was also true for the non-CF CSLD group (median (CI); 43 (27 – 59) vs. 33 (21 – 60) nmol/L, ns).

OLS Figure 9: FEV1 values taken from the annual assessments (AA) closet to the time of the FOB (AA FOB), the AA 12 months prior to this (AA pre) and the AA 12 months later. (AA post). No correlation was seen between vitamin D and FEV1 at any of the time points.

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