1. SP-B Detection and Gene Expression in Chronic Rhinosinusitis Bradford A. Woodworth, MD Noam A. Cohen, MD, PhD Rachel Wood, BS Geeta Bhargave, BS John E. Baatz, PhD Rodney J. Schlosser, MD Department of Otorhinolaryngology – HNS University of Pennsylvania Health System & Medical University of South Carolina & Medical University of South Carolina

2. Grant support Cystic Fibrosis Foundation Cystic Fibrosis Foundation

3. Surfactant Secreted in lungs by type II pneumocytes and Clara cells Secreted in lungs by type II pneumocytes and Clara cells Phospholipids (lamellar bodies): 80-90% Phospholipids (lamellar bodies): 80-90% –Used for premature infants –Decrease surface tension –Decrease viscosity of mucus Proteins: 10-15% Proteins: 10-15% –SP B and C: hydrophobic, PL processing & trafficking, anti-microbial properties –SP A and D: hydrophilic, immune functions

4. Surfactant in Airway Mucus Coats surface of gel layer to reduce surface tension Coats surface of gel layer to reduce surface tension Decreases the viscosity of mucus Decreases the viscosity of mucus Increases mucociliary clearance Increases mucociliary clearance SP-B facilitates properties of surfactant and is also shown to have direct anti-microbial properties SP-B facilitates properties of surfactant and is also shown to have direct anti-microbial properties

5. SP-B Extremely hydrophobic protein with multiple post-translational modifications Extremely hydrophobic protein with multiple post-translational modifications SP-A and D found at mucosal and epithelial surfaces throughout the body SP-A and D found at mucosal and epithelial surfaces throughout the body SP-B originally thought to be limited to the lungs SP-B originally thought to be limited to the lungs Recent studies show expression in the Eustachian tube mucosa Recent studies show expression in the Eustachian tube mucosa

6. LB Tubular Myelin SP-B SP-B in Pulmonary Surfactant What role does surfactant play in the sinuses?

7. Prior Studies Phospholipid lamellar bodies in sinonasal epithelium Phospholipid lamellar bodies in sinonasal epithelium

8. Prior Studies Is there a role for SP-B? Hydrophilic surfactant proteins A and D in sinus mucosa Hydrophilic surfactant proteins A and D in sinus mucosa –Localize to epithelium and submucosal glandular elements –Upregulated in cystic fibrosis CRS mucosa

9. Hypothesis SP-B is present in sinonasal mucosa and expression is altered in several types of CRS when compared to healthy controls. SP-B is present in sinonasal mucosa and expression is altered in several types of CRS when compared to healthy controls.

10. Methods Sinus mucosal biopsies Sinus mucosal biopsies –Allergic Fungal Rhinosinusitis (n=7) –Cystic Fibrosis (n = 4) –Non-atopic CRS with nasal polyps (n=5) –Healthy controls (n=5) –Quantitative RT-PCR, immunoblot, immunohistochemistry

11. Methods – Cycle threshold (Ct)

12. Delta Ct (∆Ct) - Ct for mRNA subtracted from Ct of internal control (18s rRNA). Delta Ct (∆Ct) - Ct for mRNA subtracted from Ct of internal control (18s rRNA). –Eliminates effect of differences in sample concentration on Ct. Individual ∆Ct values of each subtype of CRS are compared to the healthy control tissue Individual ∆Ct values of each subtype of CRS are compared to the healthy control tissue Methods – Cycle threshold (Ct)

13. SP-B Quantitative RT-PCR * p = 0.004 167-fold elevation in CF patients compared to healthy controls *

14. Detection of SP-B Proprotein Where is the protein produced and secreted? 42 kDa Detection of the proprotein and intermediate forms confirms translated product

15. Immunofluorescence SP-B localizes to the epithelium and submucosal glands Sinus EpitheliumSubmucosal Glands SP-B expression – green, Nuclear stain in blue

16. Discussion SP-B was significantly upregulated in CF CRS mucosa SP-B was significantly upregulated in CF CRS mucosa Pseudomonas produces proteases known to degrade SPs (Malloy et al). Pseudomonas produces proteases known to degrade SPs (Malloy et al). SP-B upregulated in response to degradation of SP-B and surfactant by Pseudomonas. SP-B upregulated in response to degradation of SP-B and surfactant by Pseudomonas. Alternatively, increased submucosal glands in CF mucosa contribute more mRNA transcripts to sample Alternatively, increased submucosal glands in CF mucosa contribute more mRNA transcripts to sample

17. Conclusion SP-B is upregulated in cystic fibrosis CRS and is produced by the epithelium and submucosal glands of the sinonasal cavities. SP-B is upregulated in cystic fibrosis CRS and is produced by the epithelium and submucosal glands of the sinonasal cavities. Further studies indicated to investigate the role that SP-B and surfactant have in CRS. Further studies indicated to investigate the role that SP-B and surfactant have in CRS.

18. Future directions Anti-microbial properties of SP-B as a potential therapy for infectious CRS Anti-microbial properties of SP-B as a potential therapy for infectious CRS –In vivo and in vitro models Further delineation of protein expression and specific localization with immunoelectron microscopy Further delineation of protein expression and specific localization with immunoelectron microscopy

19. Thank You