1. 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

2. Ludwig Boltzmann Institute for Experimental und Clinical Traumatology Head: Heinz Redl

3. 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

4. Risk factors in Sepsis Gender Age LBI Trauma AUVA

5. Human immuno- inflammatory system Age-related differences Gender-related differences Our Starting Point LBI 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 Therapy against Sepsis

6. Incidence and mortality rate of sepsis increase with age Martin et al. Crit Care Med, 2006 LBI Trauma AUVA

7. Schroeder et al. Arch Surg, 1998 Unclear Gender Benefits ICU Mortality Sepsis subgroup LBI Trauma AUVA

8. 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

9. AUVA Establishing a Relevant Mouse Model LBI 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

10. 2 nd hit – Sepsis –Semilethal Cecal ligation and puncture (CLP) Outbred mice, 3 age groups (3, 15, 20M), analgesia, fluid resuscitation, antibiotics 1 st hit – Trauma/Hemorrhagic Shock (TH) – Femur fracture with local tissue damage – Sublethal hemorrhage Establishing a Relevant Mouse Model LBI Trauma AUVA Establishing a Relevant Mouse Model LBI Trauma AUVA Establishing a Relevant Mouse Model LBI 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

11. –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 20µl 20 parameters In total Establishing a Relevant Mouse Model LBI Trauma AUVA

12. Survival post TH-CLP: young&females have advantage Females Males A steady approx. 20-30% inter-gender survival difference Drechsler et al. Plos One 2012 LBI Trauma AUVA

13. pre-CLP Inflammatory score Organ function score DIE vs. SUR Only marginal age/gender differences after TH Drechsler et al. Plos One 2012 LBI Trauma AUVA

14. Pre-CLP: Individual cytokines in 15m ♂ 0 200 400 600 800 1000 MIP-1  * * -24h0h pg/ml 0 500 1000 1500 IL-5 * -24h0h SUR DIE pg/ml 0 2000 4000 6000 8000 IFN-  * -24h0h pg/ml SUR vs. DIE Septic outcomes can be hardly predicted based on the TH response Drechsler et al. Plos One 2012

15. Post-CLP: Gender/age did not greatly affect magnitude of pre-lethal cytokine responses Drechsler et al., PlosONE, 2012 LBI Trauma AUVA

16. Post-CLP: Gender/age did not affect magnitude of pre-lethal organ function responses AUVA LBI Trauma

17. Summary of Findings: Drechsler et al. Plos One 2012 LBI Trauma AUVA

18. n=32 n=20n=24 n=363 n=168n=242 No „menopause“ in CD-1 mice Weixelbaumer et al. almost (??) accepted in SHOCK LBI Trauma AUVA R. Palme & A. Tichy

19. 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

20. On 15.09.2011, the breeder pulled the plug… EUR 53/15M old mouse EUR 160/mouse LBI Trauma AUVA

21. -24h 0h0h 24h CLP 48h Acute sepsis (days 1-5) Anti-PAI-1siRNA (0.1 & 10uM/mouse) I.P. I.V. PAI-1 In blood PAI-1 in blood & lavage fluid protein expression Liver, kidneys, lungs, heart gene expression P=0.06 n=10 n=8 downregulation of PAI-1 in the heart/liver at 48h LBI Trauma AUVA siRNA 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

22. 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 AUVA Large Biodystribution Study: Achim Aigner Heiko Maninga Richard Hotchkiss

23. LBI Trauma AUVA Scramblin’ to stay afloat… got scooped up… ouch!

24. PAI-1 is increased in septic non-survivors Shapiro et al. Crit Care 2010 dying survivors PAI-1 (ng/ml) Septic patients LBI Trauma AUVA CLP mice Raeven et al. PLoS ONE 2013 dying survivors * * 0244872 0 1000 2000 3000 6 Hours post CLP ng/ml

25. Effects of anti-PAI-1 Co-Treatment in CLP Sepsis PAI-1 ELISA Partial Neutralization of Plasma PAI-1 -73% Fibrin Plate Assay Full Restoration of early Fibrinolysis Raeven et al. Crit Care 2012 LBI Trauma AUVA Paul Declerk

26. Inhibition of Mouse PAI-1: no benefit in all-inclusive population Co-treatment 18h post-treatment 30h post-treatment 17% 14% Raeven et al. Crit Care 2012 LBI Trauma AUVA

27. CLP 24h 30h 48h72h 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

28. Detrimental effect of PAI-1 inhibition 29% 0 20 40 60 80 100 012345677142128 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 30h post-treatment Days post CLP Percent survival Two-tailed test Retrospective stratification (3.3 ng/ml IL-6 cut-off) Raeven et al. Crit Care 2012 LBI Trauma AUVA

29. P=0.15 Only a neg. trend after prospective stratification Low 3.3 ng/ml cut-off MA-Control-treated P-SUR started dying! LBI Trauma AUVA

30. 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

31. -72h MICE ( ♀, CD-1, 3 months old) Overexpressing PAI-1: the design Hydrodynamic i.v. delivery pLIVE.PAI-1 50µg pLIVE empty 50µg -48h -24h 0h 17G cecal ligation & puncture (CLP) 24h d28 daily observation facial vein 20-30µl 1:10 dilution Complete blood count Active PAI-1 (ELISA) plasma pellet Organ function parameters LBI Trauma AUVA

32. Gene over-expression increases plasma PAI-1… p=0.02 2-fold * n=10 N=22 mean ± s.e.m. LBI Trauma AUVA n=12

33. … but does not affect CLP survival p = 0.73 07142128 0 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 prior to the onset of sepsis has no effect

34. 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

35. 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!

36. 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

37. LBI Trauma AUVA The heck of an experience, no doubt! 2008 2013

38. 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...

39. Soheyl Bahrami- IC Dept. Head and co-PI Heinz Redl – LBI Head Thank you! – questions? Paul Christina Anna Pierre Kathrin Pia Tony …and many other LBI employeesw ho helped! Georg Martijn Michaela Mohammad Suzy Ewa