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 CenterVienna, Austria

Ludwig Boltzmann Institute for Experimental und Clinical Traumatology Head:

Heinz Redl

http://www.my-ibook.net/browser/piclins/piclins_publick/gallery.html

One Disease, Many Faces...>>Differential Host Response<<LBI Trauma AUVA

SIRS

Systemic Inflammatory Response Syndrome

MARS

Mixed Anti-inflammatory Response Syndrome

CARS

Compensatory Anti-inflammatory Response Syndrome

Pulmonary

Gastro-intestinal

Genito-urinary

Blood

skin

CNS

Risk factors in Sepsis

Gender

Age

LBI Trauma AUVA

Humanimmuno-

inflammatory system

Age-relateddifferences

Gender-relateddifferences

Our Starting PointLBI Trauma AUVA

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 Therapyagainst Sepsis

Incidence and mortality rate of sepsis increase with age

Martin et al. Crit Care Med, 2006

LBI Trauma AUVA

Schroeder et al. Arch Surg, 1998

Unclear Gender Benefits

ICU Mortality

Sepsis subgroup

LBI Trauma AUVA

LBI Trauma AUVA

Human Physiology

?Far from ideal…

Mouse Physiology

lymphocyte rich blood neutrophil rich blood LPS-induced NO release by macrophages - yes

LPS-induced NO release by macrophages - no

BALT presence - strong BALT presence - absent

different IgG and IgA isotypes/subtypes

caspase 10 - no caspase 10 - yes MHC II on T cells - no MHC II on T cells - yes

≠

Mestas & Hughes. J Immunol. 2004

AUVAEstablishing a Relevant Mouse ModelLBI Trauma

Vincent JL et al., Crit Care Med, 2006

Tien H et al., Cur Orthopedics, 2004

ICU

35% developing secondary Sepsis

27% mortality in septic patients

Trauma + Hemorrhagic Shock

• 2nd hit – Sepsis – Semilethal Cecal ligation and puncture (CLP)

• 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

-48 -24 0 24 48 72 96

TH CLP

Pre-CLP Post-CLP

blood sampling

Hours6-48 -24 0 24 48 72 96

TH CLP

Pre-CLP Post-CLP

blood sampling

Hours6

Establishing a Relevant Mouse ModelLBI Trauma AUVAEstablishing a Relevant Mouse ModelLBI Trauma AUVAEstablishing a Relevant Mouse ModelLBI Trauma

Drechsler et al. J Biomed Biotechnol 2011

Serious glitch:AUP only after 12 months!

Wichmann et al., J. Surg 1996;Gentile et al., CCM 2013

Immunosuppression

– 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

Repetitive daily low-volume blood sampling

Weixelbaumer et al. Shock 2010

20µl20

parametersIn total

Establishing a Relevant Mouse ModelLBI Trauma AUVA

Survival post TH-CLP: young&females have advantage

0

20

40

60

80

10015fem3fem

20fem

Hours

CLP

-24-48 72 96 Day 160 24 48

% s

urvi

val

0

20

40

60

80

10015male3male

20male

CLP

-48 -24 0 24 48 72 96 Day 16Hours

% s

urvi

val

A BFemales Males

A steady approx. 20-30% inter-gender survival difference

Drechsler et al. Plos One 2012

LBI Trauma AUVA

pre-CLP

A D

B E

C F

0.0

0.5

1.0345

24 3125 25

-48h (baseline)

28 283m 3m 15m 15m 20m 20m

infla

mm

ator

y sc

ore

0.0

0.5

1.0345

12 12 22314 11 7 18

-48h (baseline)

208 269

3m 3m 15m 15m 20m 20m

infla

mm

ator

y sc

ore

0

1

2

3

4

5

51 4925 25

-24h

*

27 28

3m 3m 15m 15m 20m 20m

femalemale

infla

mm

ator

y sc

ore

0

1

2

3

4

5

24 27 9 4014 11 7 18

*-24h

0.05

8 19 2 26

3m 3m 15m 15m 20m 20m

SURDIE

infla

mm

ator

y sc

ore

0

1

2

3

4

5

54 5125 25

0h

0.06

29 26

*

3m 3m 15m 15m 20m 20m

infla

mm

ator

y sc

ore

0

1

2

3

4

5

28 25 9 4214 11 7 18

0h

8 21 2 24

3m 3m 15m 15m 20m 20m

infla

mm

ator

y sc

ore

Inflammatory score Organ function score

0

1

2

3

4 -48h (baseline)

80 5233 25 32 293m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

0

1

2

3

4

12 12 22314 11 7 18

-48h (baseline)

208 269

3m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

0

1

2

3

4

79 5043 25

-24h

31 29

*

3m 3m 15m 15m 20m 20m

femalemale

orga

n fu

nctio

n sc

ore

0

1

2

3

4

24 27 9 4014 11 7 18

-24h

8 19 2 26

3m 3m 15m 15m 20m 20m

SURDIE

orga

n fu

nctio

n sc

ore

0

1

2

3

4

80 5225 25

0h

31 293m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

0

1

2

3

4

28 25 9 42 14 11 7 18

0h

8 21 2 24

3m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

A

B

C

D

E

F

DIE vs. SUR DIE vs. SUR

Only marginal age/gender differences after TH

Drechsler et al. Plos One 2012

LBI Trauma AUVA

Pre-CLP: Individual cytokines in 15m♂

0

200400

600800

1000 MIP-1a* *

-24h 0h

pg/m

l0

500

1000

1500 IL-5*

-24h 0h

SURDIE

pg/m

l

0

2000

4000

6000

8000 IFN-g*

-24h 0h

pg/m

l

SUR vs. DIESeptic outcomes can be hardly

predicted based on the TH response

Drechsler et al. Plos One 2012

Post-CLP: Gender/age did not greatly affect magnitude of pre-lethal cytokine responses

Drechsler et al., PlosONE, 2012

0

2

4

6

8

10

314224 24

24h

*

*

19 22

femalemale

3m 3m 15m 15m 20m 20m

infla

mm

ator

y sc

ore

0

2

4

6

8

10

18 13 6 3614 10 8 11

24h

* * *

** *

202816

3m 3m 15m 15m 20m 20m

DIESUR

LBI Trauma

AUVA

0

1

2

3

4

508242 23

6h

31 273m 3m 15m 15m 20m 20m

femalemale

orga

n fu

nctio

n sc

ore

0

1

2

3

4

23 19 7 16 31 51 11 39

6h

8 23 2 25

3m 3m 15m 15m 20m 20m

SURDIE

orga

n fu

nctio

n sc

ore

0

1

2

3

4

466941 25

24h

21 223m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

0

1

2

3

4

24 17 7 18 29 40 11 13

24h

202835

**

3m 3m 15m 15m 20m 20mor

gan

func

tion

scor

e

0

1

2

3

4

335437 21

48h

16 123m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

0

1

2

3

4 48h**

* * * *

24 13 7 14 25 29 11 8 102822

3m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

A

B

C

D

E

F

Post-CLP: Gender/age did not affect magnitude of pre-lethal organ function responses

0

1

2

3

4

24 13 7 14 25 29 11 8

48h

102822

*** * * *

3m 3m 15m 15m 20m 20m

orga

n fu

nctio

n sc

ore

DIESUR

AUVA

LBI Trauma

Summary of Findings:

Drechsler et al. Plos One 2012

LBI Trauma AUVA

n=32 n=20 n=24 n=363 n=168 n=242

No „menopause“ in CD-1 mice

Weixelbaumer et al. almost (??) accepted in SHOCK

LBI Trauma AUVA

R. Palme & A. Tichy

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

LBI Trauma AUVA

On 15.09.2011, the breeder pulled the plug…

EUR 53/15M old mouse EUR 160/mouse

LBI Trauma AUVA

-24h 0h 24h

CLP

48h

Acute sepsis (days 1-5)

Anti-PAI-1siRNA(0.1 & 10uM/mouse)

I.P.I.V.

PAI-1In blood

PAI-1in blood

&lavage

fluid

proteinexpression

Liver, kidneys, lungs, heartgene

expression

VEH SCRsiRNA 10siRNA0

200

400

600

800

1000

1200 PAI-1 48h post-CLPPilot 1 and 2

P=0.06

n=10 n=10n=8

downregulation of PAI-1 in the heart/liver at 48h

LBI Trauma AUVAsiRNA drama unfolding...

4 transfection reagents tested:• Invivofectamine• DOTAP• Virus Like Particle (VLP)• jetPEI• PEI-25-LMW

in heatlhy and CLP mice:• anti-PAI-1• anti-VEGF

-48h-72h

• extended treatments,• different doses,• different N/P ratios

Never a reproducible k/o effect achieved

4 transfection reagents tested:• Invivofectamine• DOTAP• Virus Like Particle (VLP)• jetPEI• PEI-25-LMW

in heatlhy and CLP mice:• anti-PAI-1• anti-VEGF

• extended treatments,• different doses,• different N/P ratios

hemorrhagic necrosis

ischemic necrosis

liver intestine

effective PEI-mediated systemic AF680 siRNA delivery

LBI Trauma AUVALarge Biodystribution Study:

Achim Aigner Heiko ManingaRichard Hotchkiss

LBI Trauma AUVAScramblin’ to stay afloat…

got scooped up… ouch!

PAI-1 is increased in septic non-survivors

Shapiro et al. Crit Care 2010

dying

survivors

PAI-1 (ng/ml)

Septic patientsLBI Trauma AUVA

CLP mice

Raeven et al. PLoS ONE 2013

dying

survivors

**

0 24 48 720

1000

2000

3000

6Hours post CLP

ng/m

l

Effects of anti-PAI-1 Co-Treatment in CLP Sepsis

PAI-1 ELISA

healthy MA-Control MA-MP6H60

100

200

300

400

CLP - + +

*

activ

e PA

I-1 (n

g/m

l)

Partial Neutralization of Plasma PAI-1

-73%

0 10 20 30 400

3

6

9

12

2 4

**

plasma dilution (fold)di

amet

er (m

m)

Fibrin Plate Assay

CLP + MA-MP6H6

healthyCLP + MA-Control

Full Restoration of early Fibrinolysis

Raeven et al. Crit Care 2012

LBI Trauma AUVA

Paul Declerk

Inhibition of Mouse PAI-1: no benefit in all-inclusive population

0 1 2 3 4 5 6 70

20

40

60

80

100

7 14 21 28

MA-MP6H6 (n=15)MA-Control (n=14)

p = 0.51

co-treatment Days post CLP

Perc

ent s

urvi

val

0 1 2 3 4 5 6 70

20

40

60

80

100

7 14 21 28

MA-MP6H6 (n=10)MA-Control (n=10)

18hpost-treatment

Days post CLP

Perc

ent s

urvi

val

0 1 2 3 4 5 6 70

20

40

60

80

100

7 14 21 28

MA-MP6H6 (n=23)MA-Control (n=23)

p = 0.34

30hpost-treatment

Days post CLP

Perc

ent s

urvi

val

Co-treatment

18h post-treatment 30h post-treatment

17%

14%

Raeven et al. Crit Care 2012

LBI Trauma AUVA

CLP

24h 30h 48h 72h 96h 28day

IL-6 as predictor

predicted to DIE

predicted to LIVE

Stratification (IL-6 cut-off 14 ng/ml)

Treatment:Anti-PAI-1 ab

(i.p., 10 mg/kg b.w.)

50% treated (T)

50%Treated (T)

50% not-treated (NT)

50%not-treated (NT)

Treatment

Targeted anti-PAI-1 treatment CLP sepsis

Raeven et al. J Throm Homeastasis 2013 - submitted

LBI Trauma AUVA

Detrimental effect of PAI-1 inhibition

29%

0

20

40

60

80

100

0 1 2 3 4 5 6 7 7 14 21 28

P-DIE + MA-MP6H6 (n=9)P-DIE + MA-Control (n=10)

P-SUR + MA-MP6H (n=14)P-SUR + MA-Control (n=13)

p = 0.02

p = 0.04

30hpost-treatment

Days post CLP

Perc

ent s

urvi

val

Two-tailed test

Retrospective stratification (3.3 ng/ml IL-6 cut-off)

Raeven et al. Crit Care 2012

LBI Trauma AUVA

P=0.15

Only a neg. trend after prospective stratification

Low 3.3 ng/ml cut-offMA-Control-treatedP-SUR started dying!

LBI Trauma AUVA

pLIVE plasmid DNA (Mirus Bio Co.)

Wooddell et al. J Gene Med, 2008

+ full-length mouse PAI-1 cDNA

Trying the other end: Overexpressing PAI-1 in the liver

M.-C. Alessi/D. Bastellica

LBI Trauma AUVA

-72h

MICE ( , CD-1, 3 months ♀old)

Overexpressing PAI-1: the design

Hydrodynamic i.v. delivery

pLIVE.PAI-150µg

pLIVE empty50µg

-48h

-24h

0h

17G cecal ligation & puncture (CLP)

24h

d28

daily observation

facial vein20-30µl

1:10 dilution

Complete blood count

Active PAI-1 (ELISA) plasma

pellet

Organ function parameters

LBI Trauma AUVA

Gene over-expression increases plasma PAI-1…

p=0.02

2-fold

*

n=10N=22

mean ± s.e.m.

LBI Trauma AUVA

n=12

… but does not affect CLP survival

p = 0.73

0 7 14 21 280

20

40

60

80

100

LBI Trauma AUVA

pLIVE Empty (n=10)pLIVE PAI-1 (n=22)Restoration of fibrinolysis in early sepsis

does not help and appears to harm

Liver specific PAI-1 over-expression priorto the onset of sepsis has no effect

WWTF Grant Summary:Major Obstacles/Mistakes

• 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!)

Our Bad Judgement:

• Delayed AUP: 12 month of burning time/resources

Bad Luck:

• The price hike on aged mice: implosion of the budget

LBI Trauma AUVA

LBI Trauma AUVA

10 direct papers (+1 review) published/under review/ in writing

WWTF Grant Summary:Output/Visibility

16 conference contributions (published)

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

Pierre won the 2012 ASS 5k Presidential Run!

WWTF Grant Summary:Other Positives

LBI Trauma AUVA

we pulled through & stayed afloat – Hail to the team!

strong international network – 7 partners

all students graduated with distinction,major career advances to participants

Paul Declerk

R. Palme & P. Schmidt

M.-C. Alessi/D. Bastellica

Achim Aigner

Heiko Maninga

Richard Hotchkiss

LBI Trauma AUVA

The heck of an experience, no doubt!

2008

2013

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...

Soheyl Bahrami-IC Dept. Head and co-PI

Heinz Redl –LBI Head

Thank you! – questions?

Paul

Christina

Anna

Pierre

Kathrin

PiaTony

…and manyotherLBI

employeeswho helped!

Georg

Martijn

Michaela

Mohammad

Suzy

Ewa