Age & gender related septic complications in trauma patients:
individually tailored treatment during posttraumatic phase
M. Osuchowski/S. Bahrami
LBI Trauma
Ludwig Boltzmann Institute for Experimental
and Clinical Traumatology
AUVA Trauma Research Center
Vienna, Austria
Ludwig Boltzmann Institute for
Experimental und Clinical Traumatology
Head:
Heinz Redl
LBI Trauma
Pulmonary
AUVA
>>Differential
Host
Response<<
One Disease,
Many
Faces...
Blood
SIRS
Systemic Inflammatory Response Syndrome
skin
Gastrointestinal
MARS
Mixed Anti-inflammatory Response Syndrome
CNS
Genitourinary
CARS
Compensatory Anti-inflammatory Response Syndrome
http://www.my-ibook.net/browser/piclins/piclins_publick/gallery.html
LBI Trauma
AUVA
Our Starting Point
LBI Trauma
AUVA
Age-related
differences
Gender-related
differences
Human
immunoinflammatory
system
plasminogen activator inhibitor 1 gene (PAI-1)
Interferon gamma-induced protein 10 gene (CXCL10/IP-10)
Olfactomedin 4 gene (OLFM4)
Secretory phospholipase A2 gene (PLA2G2A)
Characterization and Targeted Therapy
against Sepsis
LBI Trauma
Incidence and mortality rate of sepsis
increase with age
AUVA
Martin et al. Crit Care Med, 2006
LBI Trauma
Unclear Gender Benefits
Schroeder et al. Arch Surg, 1998
ICU Mortality
Sepsis subgroup
AUVA
LBI Trauma
AUVA
Human Physiology
Mouse Physiology
?
Far from ideal…
 lymphocyte rich blood
 LPS-induced NO release
by macrophages - yes
 BALT presence - strong
 caspase 10 - no
 MHC II on T cells - no
≠
 neutrophil rich blood
 LPS-induced NO release
by macrophages - no
 BALT presence - absent
 caspase 10 - yes
 MHC II on T cells - yes
 different IgG and IgA
isotypes/subtypes
Mestas & Hughes. J Immunol. 2004
LBI Trauma
Establishing a Relevant Mouse Model
AUVA
Trauma + Hemorrhagic Shock
ICU
35% developing secondary Sepsis
27% mortality in septic patients
Vincent JL et al., Crit Care Med, 2006
Tien H et al., Cur Orthopedics, 2004
LBI Trauma
Establishing a Relevant Mouse Model
AUVA
• Outbred mice, 3 age groups (3, 15, 20M), analgesia,
fluid resuscitation, antibiotics
• 1st hit – Trauma/Hemorrhagic Shock (TH)
– Femur fracture with local tissue damage
– Sublethal hemorrhage
Immunosuppression
Wichmann et al., J. Surg 1996;
Gentile et al., CCM 2013
• 2nd hit – Sepsis
– Semilethal Cecal ligation and puncture (CLP)
blood sampling
Serious glitch:
AUP only after 12 months!
Pre-CLP
TH
-48
Post-CLP
CLP
-24
06
24
48
72
96
Hours
Drechsler et al. J Biomed Biotechnol 2011
LBI Trauma
Establishing a Relevant Mouse Model
AUVA
Repetitive daily low-volume
blood sampling
20µl
20
parameters
In total
– Inflammatory Cytokines
• KC (CXCL-1), MIP-1α, TNFα, MCP-1, IFNγ, IL-1β,IL-5, IL-6, IL-10
– Complete Blood Count
• Red blood cells (RBC), hemoglobin (Hb), platelets (PLT), white blood
cells (WBC), neutrophils (NEU), lymphocytes (LYM)
– Circulating Organ Function Parameters
• ALT, AST, LDH, Glucose, Urea
Weixelbaumer et al. Shock 2010
Survival post TH-CLP:
young&females have advantage
LBI Trauma
AUVA
A steady approx. 20-30%
inter-gender survival difference
Females
% survival
100
3fem
15fem
20fem
80
60
40
20
0
-48 -24
CLP
0
24 48 72 96
Hours
Males
B
100
% survival
A
80
60
40
20
Day 16
3male
15male
20male
0
-48 -24
CLP
0
24 48 72 96
Hours
Day 16
Drechsler et al. Plos One 2012
pre-CLP
LBI Trauma
Inflammatory score
3m 3m
25
25
3m 3m
51
49
27
28
15m 15m 20m 20m
3m
5
12 12
9 23
8 20
2 26
3
2
1
0
3m 15m 15m 20m 20m
-24h
3m 3m
B
SUR
DIE
*
4
33 25
4
-24h
80
52
32
29
organ function score
7 18
3
0.05
2
1
0
14 11
3m
7 18
24 27
9 40
8 19
2 26
15m 15m 20m 20m
4
-48h (baseline)
3
2
1
0
4
9 23
14 11
7 18
3m
3m 15m 15m 20m 20m
12 12
E
female
male
0h
*
3
0.06
1
25
25
3m 3m
54
51
29
26
15m 15m 20m 20m
5
*
2
1
0
43
25
3m 3m
79
50
31 29
15m 15m 20m 20m
8 20
2 26
SUR
DIE
-24h
0h
3
2
1
14 11
3m
7 18
28 25
9 42
8 21
2 24
3m 15m 15m 20m 20m
3
2
1
0
24 27
9 40
8 19
2 26
14 11
7 18
3m
3m 15m 15m 20m 20m
F
C
4
0
3
3m 15m 15m 20m 20m
F
4
0
14 11
-48h (baseline)
4
0h
3
2
1
0 25 25
3m 3m
80
52
31
29
15m 15m 20m 20m
organ function score
inflammatory score
1
2
0.0
E
*
2
0
0.5
4
DIE vs. SUR
Only marginal age/gender differences after TH
3
C5
inflammatory score
28
female
male
-24h
4
28
15m 15m 20m 20m
B
5
31
-48h (baseline)
organ function score
24
25
D
A
organ function score
25
DIE vs. SUR
organ function score
0.0
4
3
1.0
Organ function score
organ function score
0.5
inflammatory score
-48h (baseline)
inflammatory score
5
4
3
1.0
D5
inflammatory score
inflammatory score
A
AUVA
4
0h
3
2
1
0
28 25
9 42
14 11
7 18
8 21
2 24
3m 3m 15m 15m 20m 20m
Drechsler et al. Plos One 2012
Pre-CLP: Individual cytokines in 15m♂
1000
MIP-1 a
*
*
-24h
0h
pg/ml
800
600
400
200
0
1500
IL-5
SUR
DIE
*
SUR vs. DIE
pg/ml
Septic outcomes can be hardly
predicted based on the TH response
1000
500
0
pg/ml
-24h
8000
IFN-g
6000
*
0h
4000
2000
0
-24h
0h
Drechsler et al. Plos One 2012
Post-CLP: Gender/age did not greatly affect
magnitude of pre-lethal cytokine responses
AUVA
inflammatory score
LBI Trauma
10
24h
female
male
8
*
6
24
3m 3m
42
31
19
22
15m 15m 20m 20m
0
*
*
*
*
*
2
24
DIE
SUR
*
4
2
0
24h
8
6
*
4
10
14 10
8 16
18 13
6 36
8 11
2 20
3m 3m 15m 15m 20m 20m
Drechsler et al., PlosONE, 2012
Post-CLP: Gender/age did not affect magnitude
of pre-lethal organ function responses
LBI Trauma
D
3
2
4
1
0 42 23
3m 3m
4
3
48h
82 50
*
15m 15m
31
27
20m 20m
*
*
2
24h
3
2
1
1
13 46 7 14
24 69
21 2225 29
0 41 25
3m 3m 15m 15m 20m 20m
0
C
organ function score
organ function score
female
male
4
6h
SUR
DIE
3
2
DIE
SUR
1
0
23 19
*
3m
7 16
31 51
11 39
8 23
2 25
*
3m 15m 15m 20m 20m
*
E
organ function score
6h
4 24h
3
2
*
*
1
1124 22
17
0
7 18
8 29840
2 10
8 13
11 35
2 20
3m 3m 15m 15m 20m 20m
20m 20m
3m 3m 15m 15m
F
4
48h
3
2
1
0 37 21
3m 3m
54
33
16
12
15m 15m 20m 20m
organ function score
organ function score
B
4
organ function score
organ function score
A
4 48h
*
3
*
*
7 14
25 29
*
*
*
2
1
0
24 13
11 22
8 8
2 10
3m 3m 15m 15m 20m 20m
AUVA
LBI Trauma
AUVA
Summary of Findings:
Drechsler et al. Plos One 2012
No „menopause“ in CD-1 mice
LBI Trauma
n=32
n=20
n=24
R. Palme & A. Tichy
n=363
n=168
AUVA
n=242
Weixelbaumer et al. almost (??) accepted in SHOCK
LBI Trauma
Vaginal cytology did not reflect
concentration of systemic fecal estrogens
No correlation between
cycle phases & fecal estrogen:
r=-0.117 in 3M;
r=0.026 in 15M
r=0.112 in 20M
AUVA
LBI Trauma
On 15.09.2011, the breeder pulled the plug…
EUR 53/15M old mouse
EUR 160/mouse
AUVA
siRNA drama unfolding...
LBI Trauma
Acute sepsis (days 1-5)
• extended treatments,
• different doses,
• different N/P ratios
PAI-1
in blood
&
lavage
fluid
PAI-1
In blood
Anti-PAI-1siRNA
(0.1 & 10uM/mouse)
I.P.
I.V.
-72h -48h
4 transfection reagents tested:
• Invivofectamine
• DOTAP
• Virus Like Particle (VLP)
• jetPEI
• PEI-25-LMW
-24h
CLP
0h
Liver, kidneys, lungs, heart
1200
PAI-1
48h post-CLP
Pilot 1 and 2
800
downregulation of PAI-1
in the heart/liver
at 48h
n=8
P=0.06
600
400
200
n=10
n=10
0
VEH
protein
expression
48h
24h
1000
in heatlhy and CLP mice:
• anti-PAI-1
• anti-VEGF
AUVA
SCRsiRNA
10siRNA
gene
expression
LBI Trauma
Large Biodystribution Study:
effective PEI-mediated
systemic AF680 siRNA delivery
• extended treatments,
• different doses,
• different N/P ratios
4 transfection reagents tested:
• Invivofectamine
• DOTAP
• Virus Like Particle (VLP)
• jetPEI
• PEI-25-LMW
Never a reproducible
k/o effect achieved
liver
intestine
in heatlhy and CLP mice:
• anti-PAI-1
• anti-VEGF
hemorrhagic
necrosis
Richard Hotchkiss
Achim Aigner
Heiko Maninga
ischemic
necrosis
AUVA
LBI Trauma
Scramblin’ to stay afloat…
got scooped up… ouch!
AUVA
LBI Trauma
PAI-1 is increased in septic non-survivors
Septic patients
AUVA
CLP mice
PAI-1 (ng/ml)
dying
ng/ml
3000
2000
dying
1000
*
survivors
0
Shapiro et al. Crit Care 2010
*
survivors
0 6
24
48
72
Hours post CLP
Raeven et al. PLoS ONE 2013
LBI Trauma
AUVA
Effects of anti-PAI-1 Co-Treatment in CLP Sepsis
Partial Neutralization
of Plasma PAI-1
Full Restoration
of early Fibrinolysis
12
*
300
diameter (mm)
active PAI-1 (ng/ml)
400
-73%
200
100
0
healthy
CLP
MA-Control MA-MP6H6
-
+
PAI-1 ELISA
Paul Declerk
+
9
6
3
*
healthy
CLP + MA-Control
CLP + MA-MP6H6
*
0
024
10
20
30
40
plasma dilution (fold)
Fibrin Plate Assay
Raeven et al. Crit Care 2012
Inhibition of Mouse PAI-1: no benefit
in all-inclusive population
LBI Trauma
100
AUVA
MA-MP6H6 (n=15)
MA-Control (n=14)
Percent survival
80
60
p = 0.51
40
17%
Co-treatment
20
0
0
1
2
3
6
80
60
40
20
77
14
21
28
100
MA-MP6H6 (n=10)
MA-Control (n=10)
Percent survival
Percent survival
5
Days post CLP
co-treatment
100
4
18h post-treatment
0
MA-MP6H6 (n=23)
MA-Control (n=23)
80
60
14%
p = 0.34
40
20
30h post-treatment
0
0
1
2
18h
post-treatment
3
4
5
6
77
Days post CLP
14
21
28
0
1
2
3
4
5
6
77
14
21
28
Days post CLP
30h
post-treatment
Raeven et al. Crit Care 2012
LBI Trauma
AUVA
Targeted anti-PAI-1 treatment CLP sepsis
CLP
24h 30h 48h 72h 96h
28day
IL-6 as predictor
Stratification (IL-6 cut-off 14 ng/ml)
predicted
to DIE
Treatment:
Anti-PAI-1 ab
(i.p., 10 mg/kg b.w.)
predicted
to LIVE
Treatment
50%
50%
treated (T) not-treated (NT)
50%
Treated (T)
50%
not-treated (NT)
Raeven et al. J Throm Homeastasis 2013 - submitted
Detrimental effect of PAI-1 inhibition
LBI Trauma
AUVA
Retrospective stratification (3.3 ng/ml IL-6 cut-off)
100
p = 0.04
Percent survival
80
29%
P-SUR + MA-MP6H (n=14)
P-SUR + MA-Control (n=13)
60
40
P-DIE + MA-MP6H6 (n=9)
P-DIE + MA-Control (n=10)
20
p = 0.02
0
0
1
2
3
4
5
6
77
14
21
28
Days post CLP
30h
post-treatment
Two-tailed test
Raeven et al. Crit Care 2012
LBI Trauma
AUVA
Only a neg. trend after prospective stratification
Low 3.3 ng/ml cut-off
MA-Control-treated
P-SUR started dying!
P=0.15
LBI Trauma
AUVA
Trying the other end: Overexpressing PAI-1
in the liver
pLIVE plasmid DNA (Mirus Bio Co.)
Wooddell et al. J Gene Med, 2008
+ full-length mouse PAI-1 cDNA
M.-C. Alessi/D. Bastellica
Overexpressing PAI-1: the design
LBI Trauma
AUVA
MICE (♀, CD-1, 3 months old)
Hydrodynamic i.v. delivery
pLIVE.PAI-1
pLIVE empty
50µg
50µg
Organ function parameters
Active PAI-1 (ELISA)
plasma
pellet
Complete blood count
facial vein
20-30µl
1:10
dilution
daily observation
-72h -48h -24h 0h
24h
17G cecal ligation & puncture (CLP)
d28
LBI Trauma
AUVA
Gene over-expression increases plasma PAI-1…
*
2-fold
p=0.02
n=10
n=12
N=22
mean ± s.e.m.
LBI Trauma
… but does not affect CLP survival
100
pLIVE Empty (n=10)
pLIVE PAI-1 (n=22)
80
Restoration
of fibrinolysis in early sepsis
does
not help and appears to harm
60
p = 0.73
40
Liver specific PAI-1 over-expression prior
20
to the onset of sepsis has no effect
0
0
7
14
21
28
AUVA
LBI Trauma
AUVA
WWTF Grant Summary:
Major Obstacles/Mistakes
Bad Luck:
• Delayed AUP: 12 month of burning time/resources
• The price hike on aged mice: implosion of the budget
Our Bad Judgement:
• Overloaded grant plan: almost no slack for “s..t happens”
• Overconfidence on (untested) siRNA: total failure of the in vitro to
in vivo translation (a grant on its own!)
LBI Trauma
AUVA
WWTF Grant Summary:
Output/Visibility
10 direct papers (+1 review)
published/under review/ in writing
16 conference contributions (published)
Pierre won the 2012 ASS 5k Presidential Run!
11 Travel Awards and/or Invited Oral Talks to the team
Qualified twice to New Invest. Award C. at 2011/13 ESS
Best Abstract Award at 2012 Intrl Sepsis Forum
LBI Trauma
WWTF Grant Summary:
Other Positives
AUVA
we pulled through &
stayed afloat –
Hail to the team!
Richard Hotchkiss
strong international
network – 7 partners
Heiko Maninga
all students graduated with distinction,
major career advances to participants
Paul Declerk
Achim Aigner
M.-C. Alessi/D. Bastellica
R. Palme & P. Schmidt
LBI Trauma
AUVA
The heck of an experience, no doubt!
2013
2008
Special thanks to
 M001, M002, M003, M004,
M005, M006, M007, M008
M009…, …M749, M750,
M751, M752, M753, M754,
M755, M756, M757, M758
M759, M760, M761, M762,
M763, M764, M765, M786,
M787, M788, M789, M790,
M791, M792, M793, M794,
M795, M796, M797, M798,
M799, M800, M801, M802,
M803, M804, M805, M806,
M807, M808, M809, M810,
M811, Ect...
…and many
other
LBI
employeesw
ho helped!
Christina
Soheyl BahramiIC Dept. Head and co-PI
Kathrin
Suzy
Heinz Redl –
LBI Head
Michaela
Thank you! –
questions?
Martijn
Pierre
Paul
Ewa
Mohammad
Georg
Pia
Tony
Anna