The Glue Grant: Inflammation and the Host

The Glue Grant: Inflammation and the Host

Response to Injury
Response to Injury
Methods in Bioengineering
Methods in Bioengineering
Ronald G. Tompkins, M.D., Sc.D.
Ronald G. Tompkins, M.D., Sc.D.
J ohn F. Burke Professor of Surgery
Harvard Medical School
Chief of Staff
Shriners Burn Hospital Boston
Chief, Burn Service
Massachusetts General Hospital
Burden of Trauma
Burden of Trauma
Hundreds of thousands of Americans die each year and over Hundreds of thousands of Americans die each year and over
2.6 million are hospitalized from trauma, sepsis, and burns at a 2.6 million are hospitalized from trauma, sepsis, and burns at a
societal cost estimated at over $260 billion societal cost estimated at over $260 billion
Morbidity and mortality rates of those surviving initial Morbidity and mortality rates of those surviving initial
resuscitation have not improved resuscitation have not improved
Clinical trials in trauma have been industry Clinical trials in trauma have been industry- -supported supported
Benefits from traditional research approaches have been limited Benefits from traditional research approaches have been limited
SIRS and CARS in Burn Patients
SIRS and CARS in Burn Patients
Initial resuscitation followed by Initial resuscitation followed by
the pro the pro- -inflammatory response inflammatory response
( (SIRS SIRS) )
Proportional to severity of Proportional to severity of
the injury the injury
Can lead to early MODS Can lead to early MODS
After a period of relative clinical After a period of relative clinical
stability, a compensatory anti stability, a compensatory anti- -
inflammatory response ( inflammatory response (CARS CARS) )
Suppressed immunity Suppressed immunity
Diminished resistance to Diminished resistance to
infection infection
May be discharged May be discharged
uneventfully or develop late uneventfully or develop late
MODS MODS
PORC (R. Maier, D.Herndon)
Genomics (R. Davis)
PACB (L. Moldawer)
CAM (D. Schoenfeld)
DIC (R. Tibshirani, D. Donaho, W. Wong,
J . Storey, W. Xiao,)
Proteomics (R. Smith, C. Miller-Graziano)
IDDC
Administration
Our Core Structure
Our Core Structure
PATIENT-ORIENTED
RESEARCH CORE (PORC)
SOP

ACUTE
TRAUMA BURN INFLAMMATION

UT HOUSTON MGH UMDNJ - RWJMC
U COLORADO UT GALVESTON
U WASHINGTON U WASHINGTON
WASHINGTON U LOYOLA
U PITTSBURGH UT SOUTHWESTERN
NWU
U ROCHESTER
UT SOUTHWESTERN
INFLAMMATION AND THE HOST RESPONSE TO INJURY
U54 GM062119


DELIVERABLES IN RED
SAMPLES = THIN ARROWS
INFORMATION = THICK ARROWS



PROTEIN ANALYSIS AND CELL BIOLOGY CORE
SOP

SAMPLE COLLECTION AND
COORDINATION SITE
U FLORIDA
CELL SIGNALING
AND
HUMANCYTOKINE MURINE CYOKINE CYTOKINE INTRACELLULAR
ELISA ELISA PRODUCTION REGULATION

UMICHIGAN UFLORIDA UROCHESTER NWU


LC/MS HIGHTHROUGHPUT
2-DGELS PROTEOMICS

MGH PNNL

I IN NF FO OR RM MA AT TI IO ON N D DI IS SS SE EM MI IN NA AT TI IO ON N A AN ND D D DA AT TA A
C CO OO OR RD DI IN NA AT TI IO ON N C CO OR RE E
TRAUMA-RELATED DATABASE
M MG GH H
COMPUTATIONAL ANALYSIS AND MODELING CORE

BIOSTATISTICAL GENOMIC DATAMINING
ANALYSIS ANALYSIS ALGORITHMS
MGH UWASHINGTON MIT
STANFORDU, MGH
GENOMICS CORE
SOP


RNAISOLATION(WASHU)
AND
cDNAPREP (STANFORD U)
TRAUMAPORC
MICROARRAY MVC BURNPORC
SNP MICROARRAY MICROARRRAY

STANFORDU WASHINGTONU UFLORIDA


MODEL VALIDATION CORE (ANIMAL CORE)
SOP
ACUTE
TRAUMA-HEMORRHAGE BURN INFLAMMATION

UAB BWH U MICHIGAN

ADMINISTRATION CORE
MGH
CLINICAL DATA
VAILIDATION BY PORC
AND CAMCORES
U54 program at MGH - currently in 5th year
22 performance sites
Genome-wide expression profiling in humans
and partial profiling in mice
Phenotype-genotype link to clinical database of
patients with trauma, sepsis and/or burns
Program Highlights
Program Highlights
Cur r ent Ac c ompl i shment s
Biological Accomplishments
Demonstrated genome-wide changes in gene exp. after
injury (~10,000 genes) in buffy coat
Limited number of genes (~1,000) predicted a differential
outcome towards MODS
Identified over 3,500 distinct proteins in plasma with
~600 proteins over the first 7 days
Demonstrated tissue-specific genome-wide expression
with contrasts and commonalities to buffy coat
Identified novel functional modules based on initial
analysis of leukocyte subpopulations
Leukocyte RNA Isolation By Buffy Coat
Leukocyte RNA Isolation By Buffy Coat
and Lysis Techniques
and Lysis Techniques
Buffy coat Buffy coat isolation of RNA from leukocyte isolation of RNA from leukocyte- -enriched enriched
population population
Centrifuge to obtain interface between RBC and plasma Centrifuge to obtain interface between RBC and plasma
Elimination of residual RBC with lysing solution (Buffer EL, Elimination of residual RBC with lysing solution (Buffer EL,
Qiagen, Inc.) Qiagen, Inc.)
Standard RNA isolation Standard RNA isolation
Lysis method Lysis method involves mixing whole blood first with involves mixing whole blood first with
lysis buffer (ACK Buffer) to remove lysis buffer (ACK Buffer) to remove RBCs RBCs
Centrifuge to pellet Centrifuge to pellet unlysed unlysed WBCs WBCs. .
Wash Wash pelleted pelleted cells, and standard RNA isolation cells, and standard RNA isolation
S.D. From Mean
<-2.0 -1.0 0 1.0 >2.0
Buffy coat PAXgene In common with both methods
u
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943* probe sets discriminated between SEB
stimulated and unstimulated whole blood
with Buffy coat
303* probe sets discriminated between SEB
stimulated and unstimulated whole blood
with PAXgene
254 probe sets in common
Physi ol Genomi c s
19:247-54, 2004
200
500
1000
2000
4000
6000
Buffy
coat
PAXgene
Buffy coat
unstim SEB
PAXgene
unstim SEB
Blood sample was obtained from
a single subject, and total RNA isolated by
either PAXgeneor Buffy coat methods.
cRNA was generated from 10 g of starting
material using Affymetrix protocols, and
detected using a 2% agarose gel and an Agilent
2100 system.
Physi ol Genomi c s
19:247-54, 2004
#G004 GenMAPP Analysis
Physi ol Genomi c s
19:247-54, 2004
Pearson Correlation Coefficient
from cRNA hybridization (n=4)
from RNA starting material (n=4) 0.994 0.002
Leukocyte gene expression from
same healthy subject over 24 hrs
(n=4 subjects, 4-6 time points, per
subject)
0.991 0.003
Leukocyte gene expression from
individual healthy subjects (n=17)
0.955 0.017
from individual leukocyte
populations in different healthy
subjects (n=6)
T cells
0.974 0.011
Monocytes
0.968 0.010
comparing different cell types from
same healthy subjects (n=6)
MO vs T cells
0.862 0.016
T cells vs BC
0.888 0.023
MO vs BC
0.929 0.013
Leukocyte gene expression from
individual trauma patients (n=14)
0.913 0.037
0.997 0.001
Cobb, et al . PNAS
2005; 102:4501-6.
Healthy Subjects
Trauma Patients
B.
A. C.
Healthy Subjects
Trauma Patients
Pearson Correlation
Coefficient
Among Individual
Healthy Subjects
0.955 0.017
Among Individual
Trauma Patients
0.913 0.037
Between Healthy
Subjects and Trauma
Patients
0.888 0.037
Figure 4
0
500
1000
1500
2000
2500
3000
3500
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Coefficient of variation
N
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b
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r

o
f

p
r
o
b
e

s
e
t
s
Pearson
Correlation
Coefficient
Subject #1
Subject #2 0.990 0.002
Subject #3 0.993 0.002
Subject #4 0.993 0.002
0.990 0.006
Coefficient
of Variation
Replicate
Healthy
Subjects
Individual
Healthy
Subjects
Individual
Trauma
Patients
Mean S.D. 0.0930.0003 0.1820.0006 0.2070.001
Median 0.086 0.162 0.167
90% 0.134 0.275 0.359
10% 0.057 0.103 0.100
A. B.
C.
Cobb, et al . PNAS
2005; 102:4501-6.
Lysis
Monocytes T cells
Lysis
Monocytes
T cells
A.
B.
C.
Pearson Correlation
Coefficient
Lysis vs Monocytes 0.929 0.013
Lysis vs T cells 0.888 0.022
Monocytes vs T cells 0.862 0.016
Human LPS Model
Cell
LPS
Gram-negative
bacteria
LBP
TLR-4
Cytokine
expression
CR1 Blood Collection Schema
Tompk i ns, Nat ur e, 2005
0
20
40
60
80
100
0 4 8 12 16 20 24
Hours After LPS
P
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o
f

W
B
C
s
PMNs
Lymphs
Monos
Tompk i ns, Nat ur e, 2005
A network of transcriptional factors
A virtual cell of innate immunity
time point 0h
time point 2h
time point 4h
time point 6h
time point 9h
time point 24h
0. RELA genes
1. HLA-D
2. TUB-A
3. POLR-II
4. NDUFS
5. ATP-V
6. CCT
7. PSM
8. RPS/RPL
Global Innate Immunity Network
Cur r ent Ac c ompl i shment s
Infrastructure Development
Guidelines for early management of severe trauma and
burn patients
Analytical protocols for cell isolation
Web-based fully relational database
Novel computational and bioinformational tools
Novel approaches for high throughput proteomics
Microfluidics for the isolation of enriched leukocyte
populations
www.gluegrant.org
5
5

m
m
Er yt hr oc yt e Lysi s
BulkLysis
~ 15 to 20 min
Microfluidic Lysis
~ 1 sec
Lysis buffers: Ammonium Chloride, Deionized Water
Di f f er ent i al s Fol l ow i ng Mi c r of l ui di c Lysi s
Microfluidic lysis retains normal subcellular populations
Sethu et al., Analytical Chemistry (in press)
Fl ow Cyt omet r y
Granulocytes
Monocytes
Lymphocytes
Granulocytes
Monocytes
Lymphocytes
Ammonium Chloride lysis
Microfluidic lysis
0
1
2
3
4
5
6
7
8
Whole Blood
Microfluidic Lysis
BulkLysis
Total
Leukocytes
Lymphocytes Monocytes Granulocytes
C
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l
l

C
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n
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t
r
a
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(
x

1
0
6
c
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l
l
/
m
L
)
Bulk lysis results in significant cell loss
Sethu et al., Analytical Chemistry (in press)
Cel l Sur f ac e Ac t i vat i on Mar k er s
Sethu et al., Analytical Chemistry (in press)
Website: www.Gluegrant.org
Public access to educational information
Consortium member access to
Clinical, analytical, and animal model
protocols
Data
Results
Publications
Experimental methods
Participating investigator to GLIMS, GMDS,
Clinical Databases and protocols under
development
Genetics and Molecular Medicine
Genetics and Molecular Medicine
The impact of genomics and computing on healthcare will accelera The impact of genomics and computing on healthcare will accelerate te
and progress over the next 10 years. and progress over the next 10 years.
Phase I > 5 Years Phase I > 5 Years
Dramatic expansion in diagnostic tests for existing disease Dramatic expansion in diagnostic tests for existing disease
ID of discrete molecular pathologies in major diseases (right Rx ID of discrete molecular pathologies in major diseases (right Rx: :
right disease) right disease)
Phase II > 5 Phase II > 5- -10 Years 10 Years
Increasing no. of new Rx derived by rational analysis of molecul Increasing no. of new Rx derived by rational analysis of molecular ar
basis of disease basis of disease
ID of pharmacogenetic markers for patient responses to Rx ID of pharmacogenetic markers for patient responses to Rx
(pharmacogenetics) (pharmacogenetics)
New imaging probes for dynamic assessment of body functions New imaging probes for dynamic assessment of body functions