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SP-B Detection and Gene Expression in Chronic Rhinosinusitis

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

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SP-B Detection and Gene Expression in Chronic Rhinosinusitis

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  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

  2. Grant support • Cystic Fibrosis Foundation

  3. Surfactant • Secreted in lungs by type II pneumocytes and Clara cells • Phospholipids (lamellar bodies): 80-90% • Used for premature infants • Decrease surface tension • Decrease viscosity of mucus • 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 • Decreases the viscosity of mucus • Increases mucociliary clearance • 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 • SP-A and D found at mucosal and epithelial surfaces throughout the body • SP-B originally thought to be limited to the lungs • Recent studies show expression in the Eustachian tube mucosa

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

  7. Prior Studies • Phospholipid lamellar bodies in sinonasal epithelium

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

  9. Hypothesis 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 • 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. Methods – Cycle threshold (Ct) • 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

  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 42 kDa Where is the protein produced and secreted? Detection of the proprotein and intermediate forms confirms translated product

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

  16. Discussion • SP-B was significantly upregulated in CF CRS mucosa • Pseudomonas produces proteases known to degrade SPs (Malloy et al). • SP-B upregulated in response to degradation of SP-B and surfactant byPseudomonas. • 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. • 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 • In vivo and in vitro models • Further delineation of protein expression and specific localization with immunoelectron microscopy

  19. Thank You

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