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Dexamethasone Synchronizes asthmatic Respiratory Epithelium repair in vitro

Dexamethasone Synchronizes asthmatic Respiratory Epithelium repair in vitro. Sabah F Iqbal , MD 1,2, Angela S Benton, BAS 1 , Alan M Watson, PhD, 1,2 Mary C Rose, PhD 1,2 , Robert J Freishtat , MD, MPH 1,2 1 Children’s National Medical Center, Washington, DC

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Dexamethasone Synchronizes asthmatic Respiratory Epithelium repair in vitro

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  1. Dexamethasone Synchronizes asthmatic Respiratory Epithelium repair in vitro Sabah F Iqbal, MD 1,2, Angela S Benton, BAS 1, Alan M Watson, PhD, 1,2 Mary C Rose, PhD 1,2, Robert J Freishtat, MD, MPH 1,2 1Children’s National Medical Center, Washington, DC 2George Washington University, Washington, DC

  2. Disclosure Statement • The authors have documented that they have nothing to disclose.

  3. Background: Asthma • Increasing prevalence and severity • 14.7 million annual lost school days* • 196,000 hospital admissions** • 730,000 ED visits** *National Health Interview Survey, 2002 **National Hospital Medical Care Survey, 2002

  4. The role of glucocorticoids (GC) Inflammation Remodeling ?

  5. Circadian Rhythm

  6. Hypothesis • Injured asthmatic respiratory epithelium is characterized by asynchronous regeneration and will resynchronize in the presence of glucocorticoids

  7. Normal Cell Cycle

  8. Experimental Design: ALI • Primary differentiated HBE cells at an air-liquid interface (ALI) were used(Mattek, Ashland, MA) • ‘Gold standard’ model in airway epithelial research as it mimics the human airway • Heterogeneous mixture of ciliated, goblet and basal cells Stimuli Mucus layer Goblet cell Ciliated cell Collagen

  9. Experimental Design:Timeline Mechanical Wound +/- DEX +/- DEX +/- DEX +/- DEX Photo

  10. Bright –field microscopy (16X) 0 48 hours Normal - DEX Normal + DEX Asthma - DEX Asthma + DEX 1mm

  11. Experimental analysis • Flow cytometry • BrdU (bromodeoxyuridine) • Cellular proliferation/mitosis • 7-AAD (7-amino-actinomycin-D) • DNA label/cell cycle phase

  12. G2 Normal Cell Cycle G1

  13. Cell cycle analysis by flow cytometry

  14. Summary: • Proliferating normal cells were more likely to be found in G1 of the cell cycle • Normal cell cycling • Proliferating asthmatic cells were more evenly distributed among G1 and S • Consistent with asynchronous cell cycling • DEX, given at doses analogous to endogenous steroids, re-synchronized the cell cycle

  15. Discussion • Chronotherapeutictrials (Pincus et al, 1997) • Single late-afternoon dose of GC improves lung function, symptoms, and inflammation

  16. Rethinking glucocorticoids Inflammation Remodeling ?

  17. Future directions • DEX at pharmacologic doses may correct for the asynchronous processes and allow normal rapid healing • Use nasal epithelium cells from pediatric patients with asthma and in controls

  18. Acknowledgements: • K12 Mentoring Committee: • Mary Rose Ph.D. • Robert Freishtat M.D. • Eric Hoffman Ph.D. • AnamarisColberg-Poley Ph.D. • Stephen Teach M.D. • Angela Benton • Alan Watson Ph.D. • Teresa Hawley Ph.D. • Funded by the National Institutes of Health - National Heart, Lung, and Blood Institute grant number K12HL090020

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