Ch t i ti Id tifi ti d C ti P t iCharacterization, Identification, and Comparative Proteomic Kinetics of Innate Host Defense Proteins in Experimental IPA
and Pseudomonas Pneumonia
Thomas Walsh MDThomas Walsh, MDNIH/NCI
Pulmonary Aspergillosis & Pseudomonas Pneumonia3737
AVG
Subcutaneous Tunnel
Ext. max. v.
Int . max. v.
Ext. jugular v. AAVG
Subcutaneous Tunnel
Ext. max. v.
Int . max. v.
Ext. jugular v. AAVG
Subcutaneous Tunnel
Ext. max. v.
Int . max. v.
Ext. jugular v. AAVG
Subcutaneous Tunnel
Ext. max. v.
Int . max. v.
Ext. jugular v. A
Normal Pseudomonas
+Aspergillus
+++Neutropenic
++
RA
RV
AVC
RA
RV
AVC
RA
RV
AVC
RA
RV
AVC
Cytarabine M th l d i l
+ +++++Methylprednisolone
Antibiotics Intratracheal A. fumigatus
Blood SamplingBAL Sampling
Day 0 1 2 3 4 5 6 7 8 9 10 . . . . . .14
Expression of Serum and BAL GalactomannanExpression of Serum and BAL Galactomannan
Comparison of Spectral Regions of PlasmaComparison of Spectral Regions of Plasmain Aspergillus and Pseudomonas Pneumoniain Aspergillus and Pseudomonas Pneumonia
3838
in Aspergillus and Pseudomonas Pneumonia in Aspergillus and Pseudomonas Pneumonia
AspergillusPseudomonasPseudomonas
Hierarchical Clustering of SpectraHierarchical Clustering of SpectraAspergillus
3939
31 high intensity peaks
p g
SELDI-TOF 26 l i t it
pdecreased by D3
Spectra peaks > 10 x S/N - ratio, 0.3%width
Animals ordered by
26 low intensitypeaks
increased by D3
Animals ordered by time in columns.
Peak clusters with p < 0.001 shown in
Pseudomonas
19 high intensity peaks each row, ordered
by unsupervised hierarchical clustering
peaksdecreased by D5
15 low intensity peaks
increased by D3
Aspergillus Experiment Day 0 2 3 4 5 6 7 8 9
A
Figure 1 18715.74 9355.55
28101.97 27902.19 13975.89 A28798.34 21229.53 14024.14 13697.76 13835.81 9910.16 9631 53
11978.71 11775.95 11894 449631.53
8972.84 104678.25 42189.64 39977.81 93065.02 14944 80
11894.44 88782.76 11707.09 23156.16 57390.17 13215.56 51237.31
Pseudomonas Experiment Day 0 1 2 3 4 5 6 7 8 9
14944.80 14657.34 12950.65 12749.62
49376.72 53154.56 8669.27 7990.07
55793.64
Pseudomonas Experiment Day 0 1 2 3 4 5 6 7 8 9
B 9631.53 9355.55 8972.84 9910.13
28101.97 27902 19
11894.44 11775.95 11978.71 11707.09 27902.19
13975.89 28798.34 42189.64 14024.14
88782.76 57390.17 13215.56 58836.80 7990.07
16079.81 15531 5415531.54 16170.20 15489.20 15360.01 81724.33
Plasma Protein Peak (m/z)
Random Forest Analysis of Top 30 Spectral Points Random Forest Analysis of Top 30 Spectral Points Distinguish Between Distinguish Between AspergillusAspergillus and and PseudomonasPseudomonas
P iP i
4040
PneumoniaPneumonia
m/z
2
2.5
nten
sity
1
1.5
Log
Peak
I
2
0.5
0 1 2 3 4 5 6 7Post Inoculation Day
1
1.5
Log
Peak
Inte
nsity
0
0.5
0 1 2 3 4 5 6 7Post Inoculation Day
The image of 1D SDS-Page gel with coomassie blue stain. Line 1 is molecular markers; Line 2 is samplemarkers; Line 2 is sample #34 (pseudomonas) and Line 3 is sample #38 (aspergillus). On the right image, 23k, 28k, 40k and 44k are just band labels. They do not necessarily represent their actual molecular weight. The protein IDs with significant hit (p<0.05) are listed on th l ft Th i lthe left. The numeric value in the parenthesis () is Mowse score.
Reverse antibody array
This RPA image (contrast adjusted) shows an array immuno-stained with anti-rabbit HPT antibody and indicates theand indicates the layout of samples on the slide. Each unique sample has 16
t dspots composed of 8, 4, 2 and 1 deposition curves printed in quadruplicate.q p
Protein validation with reversed phase array for Haptoglobin
Asp vs Pseudo P = <0.001
Protein validation with reversed phase array of ApolipoproteinA1
Asp vs Pseudo P = <0.001
The change of relative CRP level with time course in Aspergillus and Pseudomonas animals by reverse phase array
Asp vs Pseudo P = <0.001
Protein validation with reverse phase array for transthyretin (pre-albumin)for transthyretin (pre albumin)
Asp vs Pseudo P = 0.038
ConclusionsConclusions•• Proteomic analysis of serum and BAL proteins in Proteomic analysis of serum and BAL proteins in
experimental experimental AspergillusAspergillus and and PseudomonasPseudomonas pneumonia pneumonia demonstrate spectral regions that are shared in earlydemonstrate spectral regions that are shared in earlydemonstrate spectral regions that are shared in early demonstrate spectral regions that are shared in early infection and distinguish between the two etiologies at infection and distinguish between the two etiologies at later stages of infectionlater stages of infection
ff•• These data support the concept that a pattern of These data support the concept that a pattern of proteins are expressed with different microbial proteins are expressed with different microbial infectionsinfections
•• Haptoglobin, apolipoprotein A1, and CHaptoglobin, apolipoprotein A1, and C--reactive protein reactive protein display distinct kinetic profiles and with probable roles display distinct kinetic profiles and with probable roles i th th i f i i li th th i f i i lin the pathogenesis of invasive pulmonary in the pathogenesis of invasive pulmonary aspergillosisaspergillosis
•• This approach may enhance our understanding of hostThis approach may enhance our understanding of host--pp y gpp y gpathogen interactions, detection and prognosis pathogen interactions, detection and prognosis
Acknowledgments
• Ruta Petraitiene (NCI)• Vidmantas Petraitis (NCI)Vidmantas Petraitis (NCI)• Honghui Wang, PhD (CCMD)
Ch i D MD (NCI)• Chris Devor, MD (NCI)• Densie Gonzales, MD(CCMD)• Steve Kern, MS (CCMD)• Anthony Suffredini MD (CCMD)Anthony Suffredini, MD (CCMD)
