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The Final Presentation (1) (1)

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The Final Presentation (1) (1)

  1. 1. K R I S T E N T R I P I C C H I O STUDY OF NITROGEN MUSTARD VESICANT INJURY IN BASAL KERATINOCYTES USING THE SCRATCH WOUND MODEL
  2. 2. INTRODUCTION: MUSTARDS AS VESICANTS • Vesicant chemical warfare agent – causes blisters to the eyes, skin, and mucosal membranes • Structure & analogs • Alkylating agent via the carbenium ion6 Sulfur mustard Nitrogen mustard 2-chloroethyl ethyl sulfide (CEES)
  3. 3. INTRODUCTION: MUSTARDS AS VESICANTS • Blistering occurs in a delayed fashion – apoptotic death6 • Injury occurs because the epidermis basal keratinocytes detach from the basement membrane or dermal layer6 • Delayed reepithelialization2
  4. 4. INTRODUCTION: WOUND HEALING & LAMININ 332 • Basal keratinocytes are anchored to the basement membrane via hemidesmosomes7 • Anchoring molecule - holds cells via integrins to collagen fibers in the BM4,8 • Trimeric protein – previous studies on γ2 chain of the trimer1,2
  5. 5. THE SCRATCH WOUND ASSAY • “Simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity.”3 • Different approaches to analysis: change in length, area, track one particular cell Make scratch in each well Seed cells into plate Take images at regular intervals Data Analysis
  6. 6. SCRATCH WOUND: PARAMETERS TESTED • Initial seeding concentration – % confluency • What seeding number will lead to consistent results and will lead to a nice monolayer after 24 hrs of growth? • Passage number • Does passage number have an effect on the migration rate of the cells? • Human variation in seeding & scratching • If the same cells are seeded at the same density, does it matter who seeds or scratches?
  7. 7. SEEDING CONCENTRATION/CONFLUENCY • Wells were plated with 1.25x10⁵, 2.5x10⁵, 3.75x10⁵, & 5x10⁵ cells/mL • Wells with 3.75x10⁵ & 2.5x10⁵ saw significantly less variation in migration rate than the higher seeded 5x10⁵ cells/mL concentration • Wells plated with 2.5x10 ⁵ cells/mL did not result in a complete monolayer • Therefore, 3.75x10 ⁵ cells/mL was chosen as the ideal seeding conc. 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 WoundClosure(%) Time (hrs) Average Area Migrated vs. Time (5x10⁵ cells/mL) 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 WoundClosure(%) Time (hrs) Average Area Migrated vs. Time (3.75x10⁵ cells/mL) 2.5x10 ⁵ cells/mL 3.75x10 ⁵ cells/mL 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 WoundClosure(%) Time (hrs) Control A1B A1T A2B A2T A3B A3T
  8. 8. PASSAGE NUMBER • Compared PAM212 cells with a passage number of p5 & p23 • Similar rate of migration and amount of variation among samples • Average rates of migration were similar (5.5 & 6.3%/hr) & not statistically significant when including the std. deviations 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 Percent Area Migrated (p5) A1B A1T A2B A2T A3B A3T 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 Percent Area Migrated (p23) B1B B1T B2B B2T B3B B3T y = 0.0551x + 0.0105 R² = 0.9964 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 p5 Avg Migration Rate y = 0.0633x + 0.0004 R² = 0.9979 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 p23 Avg Migration Rate
  9. 9. HUMAN VARIATION: SEEDING & SCRATCHING • Lab tech, Amy, and I each seeded our own 6-well plates • We then scratched half of each plate • No notable difference 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 WoundClosure(%) Time (hrs) Average Migration Rates (Scratch comparison) Amy scratch KT scratch y = 0.0151x + 0.0051 y = 0.0172x + 0.0064 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 Woundclosure(%) Time (hrs) Average migration rates (Seeding comparison) Amy seeding KT seeding
  10. 10. EXPERIMENTAL SETUP Control 0.1 µM NM 10 µM NM 1 µM NM Non-Coated Coated (2 µg/mL laminin 332) Control 0.1 µM NM 1 µM NM 10 µM NM NitrogenMustardDoseStudy HYPOTHESIS: As dose increases, migration will decrease. Coated wells will decrease migration.
  11. 11. RESULTS 0 0.2 0.4 0.6 0.8 1 Control 0.1 1 10 WoundClosure(%) Dose of NM (µM) Average Wound Closure after 9 hrs for coated and non-coated wells Non-coated Coated • Chose 9-hr time point because this was the longest time point prior to the wounds closing • Overall, coated wells have slower closure rate • 10 µM non-coated average changed the trend…
  12. 12. RESULTS • Compared to the 1 µM data, there was a lot of variation present resulting in a large std deviation • Effect of scratch size • 3.4% deviation vs. 38.5%! • Redoing the results with the two wells with similar sizes resulted in… 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 Noncoated 10 µM NM 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 Noncoated 1 µM NM
  13. 13. RESULTS 0 0.2 0.4 0.6 0.8 1 Control 0.1 1 10 WoundClosure(%) Dose of NM (µM) Average Wound Closure after 9 hrs for coated & non-coated wells Noncoated Coated
  14. 14. DISCUSSION/CONCLUSIONS • Preliminary study • Validated assay – confluency & similar scratch sizes are critical • Increasing dose of NM resulted in increased migration rate – implications, what does this mean? • The coated wells resulted in a decreased migration rate – this may better represent how the tissue acts in vivo
  15. 15. FUTURE EXPERIMENTS • Perform experiment with higher doses of NM • Add different parts of the laminin trimer to the media to see if they result in faster migration • Coat plate with other EM proteins: fibronectin, collagen, etc. • Develop assay to be high throughput & efficient – 96-well plates (triplicate resulted in large std devs)
  16. 16. ACKNOWLEDGEMENTS • I would like to thank Dr. Chang, Amy, and Dr. Gerecke for offering endless guidance over the last few weeks and being quick to answer any questions • I would also like to thank Dr. Aleksunes, Erin, and Jorge for putting in all of your time and effort to making the SURF program so successful • Lastly, thanks to the NIH Graduation School of Biomedical Sciences for funding my research experience this summer.
  17. 17. BIBLIOGRAPHY 1. Chang et al. Upregulation of Gamma-2 Laminin 332 in the mouse ear vesicant wound model. J Biochem Molecular Toxicology, 2009, 23, p. 172-184. 2. Chang et al. Sulfur mustard induces an endoplasmic reticulum stress response in the mouse ear vesicant model. Toxicology and Applied Pharmacology, 2013, 268, p. 178-187. 3. Cory, Giles. Scratch-Wound Assay. Chapter 2 in Methods in Molecular Biology, 2011, 769, p. 25-30. 4. Decline, F; Rousselle, P. Keratinocyte migration requires α2β1 integrin-mediated interaction with the laminin 5 γ2 chain. Journal of Cell Science, 2001, 114, p 811-823. 5. Jourdan, MM et al. Laminin 332 deposition is diminished in irradiated skin in an animal model of combined radiation and wound skin injury. Radiation Research, 2011, 176, p. 636-648. 6. Kehe, K; Balszuweit, F; Steinritz, D; Thiermann, H. Molecular toxicology of SM-induced cutaneous inflammation and blistering. Toxicology, 2009, 263, p. 12-19. 7. Kirfel, G; Herzog, V. Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance. Protoplasma, 2004, 223, p 67-78. 8. Nguyen et al. Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion. Current Opinion in Cell Biology, 2000, 12, p. 554-562.
  18. 18. QUESTIONS?

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