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Age & gender related septic complications in trauma patients:

LBI Trauma. Age & gender related septic complications in trauma patients: individually tailored treatment during posttraumatic phase. M. Osuchowski/S. Bahrami. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology AUVA Trauma Research Center Vienna, Austria.

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Age & gender related septic complications in trauma patients:

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  1. LBI Trauma Age & gender related septic complications in trauma patients: individually tailored treatment during posttraumatic phase M. Osuchowski/S. Bahrami 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. Pulmonary Blood SIRS Systemic Inflammatory Response Syndrome Gastro-intestinal skin MARS AUVA LBI Trauma Mixed Anti-inflammatoryResponse Syndrome Genito-urinary CNS CARS Compensatory Anti-inflammatory Response Syndrome >>Differential Host Response<< One Disease, Many Faces... http://www.my-ibook.net/browser/piclins/piclins_publick/gallery.html

  4. AUVA LBI Trauma

  5. Age-related differences Gender-related differences AUVA LBI Trauma Our Starting Point Human immuno- inflammatory system Olfactomedin 4 gene (OLFM4) plasminogenactivatorinhibitor 1 gene (PAI-1) Secretory phospholipase A2 gene (PLA2G2A) Interferon gamma-induced protein 10 gene (CXCL10/IP-10) Characterization and Targeted Therapy against Sepsis

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

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

  8. lymphocyte rich blood • neutrophil rich blood AUVA LBI Trauma • LPS-induced NO release by macrophages - yes • LPS-induced NO release by macrophages - no • BALT presence - strong • BALT presence - absent • caspase 10 - yes • caspase 10 - no • MHC II on T cells - no • MHC II on T cells - yes • different IgG and IgA isotypes/subtypes Human Physiology Mouse Physiology ? Far from ideal… ≠ Mestas & Hughes. J Immunol. 2004

  9. AUVA ICU LBI Trauma Establishing a Relevant Mouse Model Trauma + Hemorrhagic Shock 35% developing secondary Sepsis 27% mortality in septic patients Vincent JL et al., Crit Care Med, 2006 Tien H et al., Cur Orthopedics, 2004

  10. Immunosuppression Wichmann et al., J. Surg 1996; Gentile et al., CCM 2013 AUVA AUVA LBI Trauma LBI Trauma LBI Trauma Establishing a Relevant Mouse Model Establishing a Relevant Mouse Model Establishing a Relevant Mouse Model • 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 Serious glitch: AUP only after 12 months! • 2nd hit – Sepsis • Semilethal Cecal ligation and puncture (CLP) Drechsler et al. J Biomed Biotechnol 2011

  11. 20 parameters In total 20µl AUVA LBI Trauma Establishing a Relevant Mouse Model Repetitive daily low-volume blood sampling • 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

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

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

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

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

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

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

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

  19. Vaginal cytology did not reflect concentration of systemic fecal estrogens AUVA LBI Trauma No correlation between cycle phases & fecal estrogen: r=-0.117 in 3M; r=0.026 in 15M r=0.112 in 20M

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

  21. siRNA drama unfolding... • extended treatments, • different doses, • different N/P ratios PAI-1 in blood & lavage fluid protein expression -48h -72h CLP AUVA LBI Trauma gene expression Liver, kidneys, lungs, heart Acute sepsis (days 1-5) PAI-1 In blood Anti-PAI-1siRNA (0.1 & 10uM/mouse) I.P. I.V. 0h -24h 48h 24h • 4 transfection reagents tested: • Invivofectamine • DOTAP • Virus Like Particle (VLP) • jetPEI • PEI-25-LMW downregulationof PAI-1 in theheart/liverat 48h • in heatlhy and CLP mice: • anti-PAI-1 • anti-VEGF P=0.06 n=8 n=10 n=10

  22. Large Biodystribution Study: effective PEI-mediated systemic AF680 siRNA delivery Never a reproducible k/o effect achieved Richard Hotchkiss AUVA Achim Aigner LBI Trauma liver intestine Heiko Maninga hemorrhagic necrosis ischemic necrosis • extended treatments, • different doses, • different N/P ratios • 4 transfection reagents tested: • Invivofectamine • DOTAP • Virus Like Particle (VLP) • jetPEI • PEI-25-LMW • in heatlhy and CLP mice: • anti-PAI-1 • anti-VEGF

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

  24. PAI-1 is increased in septic non-survivors CLP mice 3000 2000 dying ng/ml AUVA * LBI Trauma 1000 * survivors 0 0 24 48 72 6 Hours post CLP Raeven et al. PLoS ONE 2013 Septic patients PAI-1 (ng/ml) dying survivors Shapiro et al. Crit Care 2010

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

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

  27. 28day AUVA Treatment: Anti-PAI-1 ab (i.p., 10 mg/kg b.w.) IL-6as predictor LBI Trauma Stratification(IL-6 cut-off 14 ng/ml) predicted to DIE predicted to LIVE Treatment 50% treated (T) 50% not-treated (NT) 50% not-treated (NT) 50% Treated (T) Targeted anti-PAI-1 treatment CLP sepsis CLP 24h 30h 48h 72h 96h Raeven et al. J Throm Homeastasis 2013 - submitted

  28. 100 80 60 P-SUR + MA-MP6H (n=14) AUVA P-SUR + MA-Control (n=13) 40 LBI Trauma P-DIE + MA-MP6H6 (n=9) P-DIE + MA-Control (n=10) 20 0 0 1 2 3 4 5 6 7 7 14 21 28 Detrimental effect of PAI-1 inhibition Retrospective stratification (3.3 ng/ml IL-6 cut-off) 29% p = 0.04 Percent survival p = 0.02 Days post CLP 30h Two-tailed test post-treatment Raeven et al. Crit Care 2012

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

  30. AUVA M.-C. Alessi/D. Bastellica LBI Trauma 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

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

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

  33. AUVA LBI Trauma … but does not affect CLP survival 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 100 pLIVE Empty (n=10) pLIVE PAI-1 (n=22) 80 60 p = 0.73 40 20 0 0 7 14 21 28

  34. Bad Luck: • Delayed AUP: 12 month of burning time/resources • The price hike on aged mice: implosion of the budget AUVA LBI Trauma 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!) WWTF Grant Summary:Major Obstacles/Mistakes

  35. AUVA LBI Trauma 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 WWTF Grant Summary:Output/Visibility 10 direct papers (+1 review) published/under review/ in writing 16 conference contributions (published) Pierre wonthe 2012 ASS 5k Presidential Run!

  36. we pulled through & stayed afloat – Hail to the team! Richard Hotchkiss Paul Declerk AUVA Achim Aigner strong international network – 7 partners M.-C. Alessi/D. Bastellica LBI Trauma R. Palme & P. Schmidt Heiko Maninga all students graduated with distinction, major career advances to participants WWTF Grant Summary:Other Positives

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

  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. Kathrin Christina Pierre Mohammad Georg Pia Anna …andmany other LBI employeeswhohelped! Soheyl Bahrami- IC Dept. Head and co-PI Suzy Heinz Redl – LBI Head Michaela Thankyou! – questions? Martijn Paul Ewa Tony

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