2. Background
• Microparticles from different cellular sources have varying effects
-example: monocyte MPs stimulate the release of inflammatory
cytokines such as IL-8 in airway epithelial cells while MPs from
endothelial cells can induce angiogenesis1
• Neutrophil oxidative bursts play an important role in host defense
function against pathogenic microbes2
• Acid Sphingomyelinase catalyzes the production of ceramide from
sphingomyelin, increased ceramide aids in pathogen invasion into
host cells, however, acid ceramidase produces sphingosine from
ceramide, a potent antimicrobial agent3
• Sphingosine can be present in the MP bilayer
3. • Aim 1: identify differentials in microparticle dosing on
inhibiting bacteria growth
• Rationale: MPs have antimicrobial effects as
demonstrated from previous killing assays
• Hypothesis: a larger dose of MPs will be more effective at
inhibiting CFU formation, specifically, a 10E8 dose will
have a greater antimicrobial effect when compared to
10E6 and 10E7 doses.
4. Methods
• Dosing Assays
-Isolate MPs from lungs via BAL
-Obtain aliquots of 10E6, 10E7, and 10E8 MPs for
dosing assays
-Place MP doses against 2,000 CFUs and incubate for 1
hr
-+ Control= bacteria and saline; - control= bacteria and
K5 -Plate 200 CFUs and incubate overnight
-Count CFUs the next day
6. Summary
• 10E6 dose does not show a significant reduction in CFU
(p= 0.485)
• 10E7 and 10E8 doses show a significant reduction in
CFU (p= 0.005, p= 0.003, respectively)
7. • Aim 2: determine whether microparticles from neutrophils
or tracheal epithelial cells are more effective at inhibiting
bacterial growth
• Rationale: MPs are generated in different locals of the
body, for example, the bone marrow and trachea, and
consequently posses varying levels of antimicrobial
activity1,2
• Hypothesis: MPs derived from neutrophils (NDMPs) are
more effective at inhibiting bacteria growth than those
derived from tracheal epithelial cells.
8. Methods
• Bacterial Killing Assays
-Isolate MPs from tracheal epithelial cells and
neutrophils -Obtain aliquots of 10E7 MPs for killing
assays
-Place MPs against 2,000 CFUs of P. aeruginosa and
incubate for 1 hr
-+ Control= bacteria and saline; - control= bacteria and
K5
-Plate 200 CFUs and incubate overnight
-Count CFUs the next day
9. Results
94.2475
73.592875
0
0
20
40
60
80
100
120
C
F
U
s
Trachea MP Killing Assay
Series1
Series2
Series3
1= + Control (Bact. + Saline, n=4)
2= Trachea MPs, p= 0.154 (n=8)
3= - Control (Bact. + K5, n=2)
181
121.25
0
0
20
40
60
80
100
120
140
160
180
200
C
F
U
s
NDMP Killing Assay
Series1
Series2
Series3
1= + Control (Bact. + Saline, n=3)
2= NDMP, p=0.032 (n= 4)
3= - Control (Bact. + K5, n=1)
10. Summary
• NDMPs appear to have a greater antimicrobial effect (p=
0.032) when compared to MPs from tracheal epithelial
cells (p=0.154)
11. • Aim 3: identify microparticle efficacy from 2 genetically
different groups
• Rationale: Acid Sphingomyelinase deficient (ASM) mice
have decreased levels of ceramide and subsequently
lower levels of sphingosine
• Hypothesis: ASM BAL MPs will have a lower antimicrobial
effect relative to WT
Sphingomyelin
ASM
Ceramide
Acid Ceramidase
Spingosine3
12. Methods
• Bacterial Killing Assays
-Isolate MPs from lungs via BAL
-Obtain aliquots of 10E7 MPs for killing assays
-Place MPs against 2,000 CFUs of P. aeruginosa and
incubate for 1 hr
-+ Control= bacteria and saline; - control= bacteria and
K5 -Plate 200 CFUs and incubate overnight
-Count CFUs the next day
14. Summary
• ASM MPs appeared to have a greater antimicrobial effect
in this particular experiment
• However, neither WT nor ASM MPs produced a
significant reduction in CFUs for these samples (p= 0.705,
p= 0.384, respectively).
15. Conclusions
• Preliminary results show that NDMPs are more effective
at inhibiting bacterial growth when compared to tracheal
epithelial cell MPs
• ASM mice surprisingly had a greater antimicrobial effect
when compared to WT
• 10E7 MP dose is effective at significantly reducing CFUs
(p= 0.005)
-If MPs are limited, use a 10E7 dose
-Use 10E7 dose place of 10E8 dose, this will save
more MPs for additional replicates
16. Future Directions
• Compare antimicrobial efficacy of MPs from other locals
• Repeat ASM vs WT BAL MP killing assays to confirm
preliminary results
-investigate explanations if results stay consistent
• Use 10E7 MP dose for effective bacterial killing assays
17. Acknowledgements
• Department of Surgery, University of Cincinnati
-Charles Caldwell, PhD, mentor
-Brynne Whitacre, taught killing assays
-Holly Goetzman, assisted with FACS
-Lisa England, helped perform BALS
-Amanda Pugh, MD, assisted with killing assays
-Brent Xia, MD, assisted with FACS and NDMP prep
-Ellis Green, taught trachea prep and NDMP prep
Editor's Notes
1. Reid, V. L., & Webster, N. R. (2012). Role of microparticles in sepsis. British Journal of
Anaesthesia, 503-513.
2. Braga, P. C., Sasso, M. D., & Zuccotti, T. (2000). Assessment of the Antioxidant Activity of the SH
Metabolite I of Erdosteine on Human Neutrophil Oxidative Bursts. Arzneimittelforschung,
739-746.
3. Seitz, A. P., Grassme, H., Edwards, M. J., Pewzner-Jung, Y., & Gulbins, E. (2015). Ceramide and
sphingosine in pulmonary infections. Biol. Chem., 611-620.
Day 1: Plate bacteria overnight for growth
Obtain TSB plate from 4 C cold room and allow to warm to room temperature
Obtain Pseudomonas aeruginosa from -80 C freezer and use inoculating loop to gently spread one scrape of frozen bacteria onto TSB plate, place bacteria back into freezer immediately to avoid freeze-thawing
Incubate overnight at 37 C
Day 2: BAL killing assay
1.) Perform BAL, collect in 15 mL conical vial
2.) Centrifuge at 450G for 10 minutes at 21 C, collect supernatant
3.) Centrifuge at 10,000G for 10 minutes at 21 C, collect supernatant
4.) Obtain 5 vials of 8 mL BBL Trypticase Soy Broth from 4 C cold room, pre warm, and combine them in a sterile 125 mL flask, poke hole in tin foil cover
5.) Use inoculating loop to scrape plated bacteria from day 1 into flask
6.) Pipette up and down using 10 mL pipette to break up any bacteria clumps 7.) Incubate in C25 Incubator Shaker at 37 C and 125 rpm for 1 hour with tin foil cover on 8.) Centrifuge BAL MPs at 25,000G for 30 minutes at 21 C 9.) Turn on Photospectrometer to warm up (15 minutes minimum needed) 10.) Resuspend MP pellet in 5 mL of MP free saline 11.) Take 100 microliter aliquot to enumerate on NTA 12.) Calculate volume needed for 10E7 MPs 13.) Collect bacterial solution from 125 mL flask and pour into 50 mL conical vial 14.) Centrifuge at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 15.) Resuspend bacteria pellet in 40 mL PBS 16.) Centrifuge again at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 17.) Resuspend bacteria pellet in 40 mL PBS 18.) Open plastic wrapped, sterile, round bottomed test tube, serial number 1451500, for cuvette (avoid fingerprinting cuvette by carrying at the top) and add 2 mL PBS 19.) Calibrate a blank standard on the Photospectrometer, set blank to 600 nm with 100 transmittance and 0 absorbance 20.) Dropwise, add bacteria solution to cuvette using transfer pipette and vortex, continue until an absorbance of 0.04 is reached (1 to 2 drops is typically sufficient). If surpassed 0.04, add PBS dropwise to decrease the absorbance until desirable. This absorbance corresponds to 4.80E6 CFUs per 2 mL. 21.) Obtain 24 well clear flat bottom plate and add 100 microliters of bacteria to top row of wells (each well holds 2 mL). This provides 2.4E5 CFUs per well. 22.) Add 900 microliters of sterile saline to top wells with bacteria. Also add 900 microliters of sterile saline to rows 2 and 3. 23.) Take 100 microliters from the top row of wells and add to row 2 wells directly below, pipette up and down gently.
24.) Take 100 microliters from row 2 wells and add to row 3 wells directly below, pipette up and down gently. 25.) Add 10E7 microparticle dose to row 3 wells, pipette up and down gently, for positive control, add equivalent volume of sterile saline, and for negative control add equivalent volume of K5, then begin time course killing assay 26.) Perform time course killing assay by incubating at 37 C for 5, 15, 30, and 60 minutes 27.) Once the time course begins running, remove TSB plates from 4 C cold room and allow them to cool to room temp 28.) After time course, add 100 microliters from row 3 wells to row 4 wells. 29.) Add 900 microliters of sterile saline to row 4 wells, pipette up and down 30.) Place 1 mL from row 4 wells onto TSB plates and spread evenly using inoculating loop 31.) Incubate all samples at 37 C overnight
Day 3: Count CFUs and quantify bacteria
Count CFUs by marking each CFU with a dot using a sharpie, keep track of your dot count for each TSB plate
Create excel spreadsheet comparing CFU formation from experimental versus control groups. Include standard error, standard deviation, and averages.
1. Reid, V. L., & Webster, N. R. (2012). Role of microparticles in sepsis. British Journal of
Anaesthesia, 503-513.
2. Braga, P. C., Sasso, M. D., & Zuccotti, T. (2000). Assessment of the Antioxidant Activity of the SH
Metabolite I of Erdosteine on Human Neutrophil Oxidative Bursts. Arzneimittelforschung,
739-746.
BAL Protocol… comparable to NDMP and TEC MP killing assay Day 1: Plate bacteria overnight for growth
Obtain TSB plate from 4 C cold room and allow to warm to room temperature
Obtain Pseudomonas aeruginosa from -80 C freezer and use inoculating loop to gently spread one scrape of frozen bacteria onto TSB plate, place bacteria back into freezer immediately to avoid freeze-thawing
Incubate overnight at 37 C
Day 2: BAL killing assay
1.) Perform BAL, collect in 15 mL conical vial
2.) Centrifuge at 450G for 10 minutes at 21 C, collect supernatant
3.) Centrifuge at 10,000G for 10 minutes at 21 C, collect supernatant
4.) Obtain 5 vials of 8 mL BBL Trypticase Soy Broth from 4 C cold room, pre warm, and combine them in a sterile 125 mL flask, poke hole in tin foil cover
5.) Use inoculating loop to scrape plated bacteria from day 1 into flask
6.) Pipette up and down using 10 mL pipette to break up any bacteria clumps 7.) Incubate in C25 Incubator Shaker at 37 C and 125 rpm for 1 hour with tin foil cover on 8.) Centrifuge BAL MPs at 25,000G for 30 minutes at 21 C 9.) Turn on Photospectrometer to warm up (15 minutes minimum needed) 10.) Resuspend MP pellet in 5 mL of MP free saline 11.) Take 100 microliter aliquot to enumerate on NTA 12.) Calculate volume needed for 10E7 MPs 13.) Collect bacterial solution from 125 mL flask and pour into 50 mL conical vial 14.) Centrifuge at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 15.) Resuspend bacteria pellet in 40 mL PBS 16.) Centrifuge again at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 17.) Resuspend bacteria pellet in 40 mL PBS 18.) Open plastic wrapped, sterile, round bottomed test tube, serial number 1451500, for cuvette (avoid fingerprinting cuvette by carrying at the top) and add 2 mL PBS 19.) Calibrate a blank standard on the Photospectrometer, set blank to 600 nm with 100 transmittance and 0 absorbance 20.) Dropwise, add bacteria solution to cuvette using transfer pipette and vortex, continue until an absorbance of 0.04 is reached (1 to 2 drops is typically sufficient). If surpassed 0.04, add PBS dropwise to decrease the absorbance until desirable. This absorbance corresponds to 4.80E6 CFUs per 2 mL. 21.) Obtain 24 well clear flat bottom plate and add 100 microliters of bacteria to top row of wells (each well holds 2 mL). This provides 2.4E5 CFUs per well. 22.) Add 900 microliters of sterile saline to top wells with bacteria. Also add 900 microliters of sterile saline to rows 2 and 3. 23.) Take 100 microliters from the top row of wells and add to row 2 wells directly below, pipette up and down gently.
24.) Take 100 microliters from row 2 wells and add to row 3 wells directly below, pipette up and down gently. 25.) Add 10E7 microparticle dose to row 3 wells, pipette up and down gently, for positive control, add equivalent volume of sterile saline, and for negative control add equivalent volume of K5, then begin time course killing assay 26.) Perform time course killing assay by incubating at 37 C for 5, 15, 30, and 60 minutes 27.) Once the time course begins running, remove TSB plates from 4 C cold room and allow them to cool to room temp 28.) After time course, add 100 microliters from row 3 wells to row 4 wells. 29.) Add 900 microliters of sterile saline to row 4 wells, pipette up and down 30.) Place 1 mL from row 4 wells onto TSB plates and spread evenly using inoculating loop 31.) Incubate all samples at 37 C overnight
Day 3: Count CFUs and quantify bacteria
Count CFUs by marking each CFU with a dot using a sharpie, keep track of your dot count for each TSB plate
Create excel spreadsheet comparing CFU formation from experimental versus control groups. Include standard error, standard deviation, and averages.
Seitz, A. P., Grassme, H., Edwards, M. J., Pewzner-Jung, Y., & Gulbins, E. (2015). Ceramide and
sphingosine in pulmonary infections. Biol. Chem., 611-620.
Day 1: Plate bacteria overnight for growth
Obtain TSB plate from 4 C cold room and allow to warm to room temperature
Obtain Pseudomonas aeruginosa from -80 C freezer and use inoculating loop to gently spread one scrape of frozen bacteria onto TSB plate, place bacteria back into freezer immediately to avoid freeze-thawing
Incubate overnight at 37 C
Day 2: BAL killing assay
1.) Perform BAL, collect in 15 mL conical vial
2.) Centrifuge at 450G for 10 minutes at 21 C, collect supernatant
3.) Centrifuge at 10,000G for 10 minutes at 21 C, collect supernatant
4.) Obtain 5 vials of 8 mL BBL Trypticase Soy Broth from 4 C cold room, pre warm, and combine them in a sterile 125 mL flask, poke hole in tin foil cover
5.) Use inoculating loop to scrape plated bacteria from day 1 into flask
6.) Pipette up and down using 10 mL pipette to break up any bacteria clumps 7.) Incubate in C25 Incubator Shaker at 37 C and 125 rpm for 1 hour with tin foil cover on 8.) Centrifuge BAL MPs at 25,000G for 30 minutes at 21 C 9.) Turn on Photospectrometer to warm up (15 minutes minimum needed) 10.) Resuspend MP pellet in 5 mL of MP free saline 11.) Take 100 microliter aliquot to enumerate on NTA 12.) Calculate volume needed for 10E7 MPs 13.) Collect bacterial solution from 125 mL flask and pour into 50 mL conical vial 14.) Centrifuge at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 15.) Resuspend bacteria pellet in 40 mL PBS 16.) Centrifuge again at 2,800 rpm 9 acceleration, 9 deceleration for 10 minutes at 21 C, decant supernatant 17.) Resuspend bacteria pellet in 40 mL PBS 18.) Open plastic wrapped, sterile, round bottomed test tube, serial number 1451500, for cuvette (avoid fingerprinting cuvette by carrying at the top) and add 2 mL PBS 19.) Calibrate a blank standard on the Photospectrometer, set blank to 600 nm with 100 transmittance and 0 absorbance 20.) Dropwise, add bacteria solution to cuvette using transfer pipette and vortex, continue until an absorbance of 0.04 is reached (1 to 2 drops is typically sufficient). If surpassed 0.04, add PBS dropwise to decrease the absorbance until desirable. This absorbance corresponds to 4.80E6 CFUs per 2 mL. 21.) Obtain 24 well clear flat bottom plate and add 100 microliters of bacteria to top row of wells (each well holds 2 mL). This provides 2.4E5 CFUs per well. 22.) Add 900 microliters of sterile saline to top wells with bacteria. Also add 900 microliters of sterile saline to rows 2 and 3. 23.) Take 100 microliters from the top row of wells and add to row 2 wells directly below, pipette up and down gently.
24.) Take 100 microliters from row 2 wells and add to row 3 wells directly below, pipette up and down gently. 25.) Add 10E7 microparticle dose to row 3 wells, pipette up and down gently, for positive control, add equivalent volume of sterile saline, and for negative control add equivalent volume of K5, then begin time course killing assay 26.) Perform time course killing assay by incubating at 37 C for 5, 15, 30, and 60 minutes 27.) Once the time course begins running, remove TSB plates from 4 C cold room and allow them to cool to room temp 28.) After time course, add 100 microliters from row 3 wells to row 4 wells. 29.) Add 900 microliters of sterile saline to row 4 wells, pipette up and down 30.) Place 1 mL from row 4 wells onto TSB plates and spread evenly using inoculating loop 31.) Incubate all samples at 37 C overnight
Day 3: Count CFUs and quantify bacteria
Count CFUs by marking each CFU with a dot using a sharpie, keep track of your dot count for each TSB plate
Create excel spreadsheet comparing CFU formation from experimental versus control groups. Include standard error, standard deviation, and averages.
1. Reid, V. L., & Webster, N. R. (2012). Role of microparticles in sepsis. British Journal of
Anaesthesia, 503-513.
2. Braga, P. C., Sasso, M. D., & Zuccotti, T. (2000). Assessment of the Antioxidant Activity of the SH
Metabolite I of Erdosteine on Human Neutrophil Oxidative Bursts. Arzneimittelforschung,
739-746.
3. Seitz, A. P., Grassme, H., Edwards, M. J., Pewzner-Jung, Y., & Gulbins, E. (2015). Ceramide and
sphingosine in pulmonary infections. Biol. Chem., 611-620.
