1. Dr Hasib Ahmadzai
Dr Jeanie Chui
Dr Barbara Cameron
Prof Denis Wakefield
Prof Andrew Lloyd
Prof Paul S. Thomas
Measurement of exhaled
markers of granulomatous
inflammation in sarcoidosis

2.  Multi-system
granulomatous disorder,
uncertain aetiology
 Often acute, self-limiting
but progressive pulmonary
fibrosis can result
 Death from pulmonary,
cardiac or neurological
involvement

3.  Clinical presentation
 Imaging
 BAL lymphocyte profile
 Serum ACE
 Histopathology

4.  Exaggerated T Helper type-1 (TH1) lymphocyte processes
causing localised granulomatous inflammation
 Elevated ratio of CD4+ to CD8+ T cells in tissues
Sarcoid granuloma with epithelioid cells
and multinucleate giant cells Kumar et al. (2007).

5. Sarcoid Immunopathogenesis
Unknown sarcoid antigen
HLA-DRB1, HLA-DQB1, HLA-B8
BTNL2
APCT cell
TNF-α, Interleukin-1, IL-12, MIP, MCP-1
CD4+
T cell
TH1 cells TH2 cells
IFN-γ, IL-2 IL-4, IL-5, IL-10, IL-13
Resolution Fibrosis
Granuloma Granuloma derived factors:
ACE, TNF- α, Neopterin, TGF-β1,
IL-6, osteopontin
↑TH2 responses↑TH1 responses
CD28,
CD152
CD80,
CD86
TH17 cells
IL-17

6.  Approach for identifying
inflammatory markers in
respiratory disease
 Simple, non-invasive method
of sampling lower respiratory
tract fluids
 Less invasive than BAL

7.  Few studies investigating EBC in sarcoidosis
 IL-6, TNF-α, TGF-β detected in sarcoid EBC
 Previous studies indicate TGF-β & neopterin
measurable in EBC in other diseases
 Other cytokines & ACE have never been measured
in sarcoid EBC

8.  Hypothesis:
◦ ACE and neopterin will be elevated in EBC and TGF-β
will be associated with sarcoid pulmonary fibrosis
 Aim:
◦ To identify markers of granulomatous inflammation in
EBC of sarcoidosis patients compared to controls

9.  Observational, cross-sectional study
 Recruit patients & gender matched non-
smoking control subjects
 Obtain lung function tests: FEV1, FVC
 Sarcoid radiological stage
 Serum ACE levels
 Medications
 Smoking history
 Collect EBC using validated method

10. Collect EBC from sarcoid patients & controls
Mouthpiece with unidirectional valve, subject
exhales here
Polyethylene
Tube
Ice chamber
Exhaled breath condensate
Saliva trap tubing

11. EBC sample
Measure markers of macrophage activity: Neopterin
using ELISA , total protein & ACE activity with
colorimetric assay
Measure active TGF-β1 with ELISA following acid activation
Correlate with lung function tests and degree of pulmonary fibrosis

12. Sarcoidosis
(n = 16)
Healthy controls
(n = 22)
p - value
Sex, Female/male 5/11 9/13 ns
Age (year) median (min,
max)
48.5 (32, 83) 37 (21, 82) ns
Smoking status
(current/ex/never)
0/9/7 0/5/17
X-ray stage (0/I/II/III/IV) 1/6/6/1/2 N/A
Serum ACE (U/L) median
(min, max)
(Normal<42U/L)
59.4 (32.4, 107) N/A
Extra-pulmonary disease
(yes/no)
15/1 N/A
FEV1 (% predicted) 86.1 ± 17.8 98.8 ± 9.7 <0.05
FVC (% predicted) 93.7 ± 15.3 98.4 ± 9.5 ns
FEV1/FVC ratio (%
predicted)
93.3 ± 15.6 102.8 ± 4.4 <0.05
Immunosuppressive
treatment (yes/no)
4/12 None

13.  Total protein in EBC samples was 8.1g/ml ±
2.3g/ml (mean ± SD) in healthy controls
 In patients with sarcoidosis total protein was
17.8g/ml ± 38.5g/ml (p = 0.27)

17.  ACE activity was low in EBC
 Only detectable in 3/20 healthy controls and
2/16 patients with sarcoidosis (p = 0.91)

18.  EBC levels of neopterin & TGF-β1 increased
in patients with sarcoidosis compared with
healthy controls
 Levels of active TGF-β1 did not correlate
with radiological disease stage
 ACE activity in EBC appears to have limited
value

19.  EBC may be a useful simple, non-invasive approach
for disease monitoring & identifying those at risk of
pulmonary fibrosis
 Assess other biomarkers of granulomatous
inflammation
 Larger-scale studies & development of sensitive
assays are warranted

20.  The authors would like to thank:
◦ The study participants
◦ Sandra Jeffrey and Amir Ghanbari, Prince of Wales
Hospital Lung Function Laboratory

21.  Diagnosis of sarcoidosis according to WASOG;
Symptoms: fatigue, dry cough, chest pain, sweats,
dyspnoea on exertion
Radiology findings
Pulmonary function testing
Non-caseating granulomas on biopsy with absence of
known cause (mycobacterial/fungal infection or foreign
body reaction)
Löfgren’s syndrome
BAL mononuclear cell alveolitis with elevated CD4/CD8
ratio
Mantoux skin test negative
Excluded patients on ACE inhibitors or angiotensin II
receptor I antagonists

22.  Can assess both activated & non-activated
TGF-β1
 Activation requires acidification and
neutralisation of the EBC samples- this is
critical.
 Assay sensitivity for TGF-β1 is 0.01pg/ml
 EBC TGF-β1 levels are 20.1±22.7pg/ml in
pulmonary fibrosis

24.  Neopterin is measurable in EBC
 Our research group were the first to show
this, with levels found to be ~1.2nmol/l in
EBC of healthy patients

26.  Iannuzzi, M.C., Rybicki, B.A., Teirstein, A.S. (2007). Sarcoidosis. The New
England Journal of Medicine. 357(21): 2153-2165.
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