TRANSCUTANEOUS AURICULAR VAGUS NERVE STIMULATION ATTENUATES PRO-INFLAMMATORY CYTOKINE RESPONSE IN HEALTHY VOLUNTEERS
S.S. Chavan, M. Dancho, V.A. Pavlov*, and K.J. Tracey*. Feinstein Institute for Medical Research, Manhasset, NY
The central nervous system interacts dynamically with the immune system to modulate inflammation through vagus nerve signals. Vagus nerve stimulation (VNS) modulates inflammatory responses in animal models of inflammatory diseases including endotoxemia, sepsis, and rheumatoid arthritis. Recent clinical studies demonstrated that electrical vagus nerve stimulation efficiently improves disease scores in rheumatoid arthritis patients (Koopman et al, Arthritis and Rheumatism 2012: 64(10), Abstract 451). This physiologic mechanism termed ‘the inflammatory reflex’ has major implications in therapeutics. Here we have developed a non-invasive technique to stimulate the auricular branch of the vagus nerve. Healthy subjects received transcutaneous stimulation either at the cymba concha, a region on the outer ear that is innervated by vagus nerve, or at the calf region (as control) on two different visits. Levels of TNF, IL1 and IL6 were monitored in whole blood assay before and after stimulation. Auricular vagus nerve stimulation resulted in fattenuation of inflammatory responses (e.g. IL1 1554 vs 765 pg/ml; p<0.005 Pre-VNS vs post-VNS); whereas stimulation at the calf region failed to modulate cytokine levels in response to endotoxin challenge (e.g. IL1 1787 vs 1667 pg/ml pre-VNS vs post-VNS). Importantly, in our pilot study with rheumatoid arthritis patients (n=9), auricular vagus nerve stimulation decreased disease scores significantly (DAS 4.0 decreased to 2.8 after 7 days, p<0.01). Together, these findings unravel a novel strategy for non-invasive vagus nerve stimulation that can be applied as a therapeutic modality in the context of human inflammatory diseases.
ESTRADIOL-PROVIDED CARDIAC PROTECTION FOLLOWING BURN TRAUMA IS MEDIATED THROUGH A REDUCTION IN MITOCHONDRIAL DAMPS
D. Maass, X. Yao, L. Ma, S. Wolf*, J. Minei*, J.G. Wigginton*, and Q.S. Zang*.
University of Texas Southwestern Medical Center, Dallas, TX
Objective: Mitochondria-derived danger-associated molecular patterns (DAMPs) play important roles in sterile inflammation after acute injuries. This study was designed to test the hypothesis that 17β-estradiol protects the heart via suppression of mitochondrial DAMPs in burn trauma.
Methods: SD rats were given a third-degree scald burn comprising 40% TBSA. 17β-estradiol (0.5 mg/kg) or control vehicle was administered subcutaneously 15 minutes post-injury. Heart tissues were harvested 24 hrs later and subcellular fractions were isolated. In mitochondria, structural integrity, respiratory function, lipid oxidation and antioxidant capacities were compared. In cytosol, quantities of cytochrome C (Cyto C) and mtDNA fragments were measured. In the heart tissue, levels of inflammatory cytokines (TNF-α, IL-1β and IL-6), ASC inflammasome and activation of NF-κB were examined. Serum troponin-I (Tn-I) levels were evaluated as an indication of cardiac injury.
Results: In cardiac mitochondria from burned rats, 17β-estradiol dramatically decreased oxidative stress and increased antioxidant defense. As results, mitochondrial structure was protected and respiratory function was improved. Further, the release of mitochondrial DAMPs, such as mtROS, cytochrome C and mtDNA, was significantly reduced in estradiol-treated hearts. In parallel, estradiol limited cytokine production and suppressed NF-κB- and ASC-mediated inflammation pathways in myocardium. As expected, circulation of cardiac risk factor troponin-I in these animals was significantly reduced in response to estradiol treatment.
Conclusion: Our data suggest that post-burn application of estradiol protects the heart via remedy of mitochondrial damage, leading to effective reduction in mitochondrial DAMPs and alleviation of downstream cardiac inflammation.
TLR4 ON BOTH HEPATOCYTES AND MACROPHAGES CONTRIBUTES TO TNFR1 SHEDDING IN VIVO DURING SEPSIS
M. Deng1, P. Loughran2, M.J. Scott1, S. Chanthaphavong1, S. Chhinder3, D. Hackam3, and T. Billiar*1. 1UPMC, Pittsburgh, PA, 2UPMC CBI, Pittsburgh, PA, 3UPMC Division of Pediatric Surgery, Pittsburgh, PA
Our previous work showed that LPS induces a TLR4-dependent shedding of TNFR1 from hepatocytes (HC) in vitro, and that shedding is driven by iNOS as part of a novel complex at the cell surface. Here we tested the hypothesis that TLR4 on both HC and macrophages would contribute to TNFR1-shedding in vivo. WT (C57BL/6), global TLR4KO, TLR4Flox (cell-specific KO controls), HC-specific TLR4KO (HCTLR4KO), macrophage-specific TLR4KO (MΦTLR4KO), MyD88KO and TrifKO mice were injected with LPS (5mg/kg, ip) for 8h (n=6 mice/group). As expected TNFR1-shedding was significantly upregulated in WT mice (404+/-126ng/mL vs 82+/-12 ng/mL in control, p<0.05) after LPS stimulation but not in global TLR4KO mice (122 +/-39ng/mL) (Figure 1). TNFR1-shedding in both HCTLR4KO and MΦTLR4KO mice was half of that in Flox (228+/-51ng/mL in HCTLR4KO, 258+/-56 ng/mL in MΦTLR4KO vs 540 +/- 124 ng/mL in Flox, p<0.05) indicating that LPS-induced TNFR1-shedding is dependent on TLR4 on both HC and MΦ in vivo. Inhibition of iNOS using 1400W treatment in LPS-treated mice further inhibited TNFR1-shedding only in MΦTLR4KO but not in HCTLR4KO suggesting the TLR4 upregulation of iNOS occurred specially in response to TLR4 stimulation on HC. Furthermore, TNFR1-shedding was suppressed completely in the MyD88KO mice but not in TrifKO after LPS stimulation in vivo and in vitro (Figure 1). Likewise TNFR1-shedding was MyD88-dependent (334+/-83ng/mL in WT vs 179+/-36ng/mL in MyD88KO, p<0.05) but not Trif-dependent (284+/-114ng/mL) in a polymicrobial sepsis model (CLP with 5mg/kg imipenem, 8h, n=6 mice/group). These findings establish that TNFR1-shedding in sepsis and endotoxemia is MyD88-dependent. TLR4-dependent shedding exhibits responses that are cell type specific, including the role of TLR4 on HC leading to iNOS-dependent TNFR1-shedding.
A BETTER UNDERSTANDING OF WHY MURINE MODELS OF TRAUMA DO NOT RECAPITULATE THE HUMAN SYNDROME
L.F. Gentile, D. Nacionales, M. Lopez, A. Cuenca, E. Vanzant, F.A. Moore*, L. Moldawer*, H. Baker, and P.A. Efron*. University of Florida, Gainesville, FL
Introduction: Genomic analyses have demonstrated that mouse injury poorly reflects human severe trauma at the level of the transcriptome (PNAS, in press). Some have argued that this is due to the modest severity of murine injury models, inappropriate age and timing, and dissimilar circulating leukocyte populations between the two species. We hypothesized that altering these variables might improve the similarity of the response to severe trauma between mice and humans.
Methods: Young (6-10 wk) and old (18-24 mo) B6 mice underwent 90 minutes of shock (MAP 30 mmHg) and resuscitation via femoral artery cannulation followed by laparotomy alone (TH) or with cecectomy and femur fracture+muscle tissue damage (PT). Total blood WBC samples were collected at 2h, 1d and 3d after operation. Fold expression changes in genome-wide microarray analyses between controls and young/old mice (p<0.001 (f-test)) were compared to human total blood WBC and to enriched WBC expression analyses of severe trauma patients at 0.5, 1, 4, 7, 14, and 28d after injury (Glue Grant).
Results: Increasing the severity of the murine trauma model (PT) significantly improved the Spearman correlation at all-time points (p<0.001). The highest correlations (R) in WBC expression patterns between human and mice were 0.13 (TH) and 0.31 (PT), illustrating PT better replicated human gene expression after trauma. When comparing the best murine correlation (young PT 3d) to the human response, 91-95% of the top 100 upregulated human genes were also upregulated in the mouse. Surprisingly, matching aged mice to their human age equivalent (>55 yo) did not improve the correlation. Finally, comparing expression from individual human WBC populations (PMNs, monocytes, T cells) improved the correlation, with total WBCs from aged mice at 3d vs 28d human PMNs having the best correlation (0.49).
Conclusion: Although genome-wide expression remains weakly correlated between humans and mice, the association is significantly improved when the severity of the injury is increased, and the specific leukocyte population is narrowed. However, mouse age does not appear to be an important determinant. Finally, the majority of the top up-regulated genes in humans after injury are similar in the mouse. Further investigation regarding these variables will be vital to translating murine sepsis and trauma research to the bedside.
CLINICAL GRADE HUMAN BONE MARROW DERIVED MESENCHYMAL STEM CELLS IMPROVE ARTERIAL OXYGENATION AND REDUCE LUNG WATER IN AN OVINE MODEL OF SEPSIS
S. Asmussen1, H. Ito1, D.L. Traber*1, M. Matthay*2, J.W. Lee*2, D.F. McAuley*2, R.A. Cox*1, L.D. Traber*1, D.N. Herndon*1, D.H. McKenna*3, and P. Enkhbaatar*1. 1University of Texas Medical Branch, Galveston, TX, 2University of California San Francisco, San Francisco, CA, 3University of Minnesota, Program in Assisted Cell Therapeutics, Saint Paul, MN
Introduction: Recent reports in a small animal model and an isolated perfused human lung preparation indicate that human bone marrow derived mesenchymal stem cells (hMSC) may have beneficial effects in acute lung injury (ALI). We hypothesized that iv administration of clinical grade allogeneic hMSC treatment would reduce bacterial growth and attenuate the severity of ALI in well established ovine model of Pseudomonas aeruginosa (PSA) pneumonia-induced septic shock.
Methods: Nineteen adult ewes (30–40 kg) were surgically prepared for chronic study and randomly allocated to either injured non-treated control (n=8) or injured but treated groups: single (SD, 5×106 cells/kg; n=7) and double dose (DD, 10×106 cells/kg; n=4) hMSC. Under deep anesthesia a tracheostomy was performed, smoke was insufflated into the lung and 1011 CFU PSA was instilled into the lungs via bronchoscope. Sheep were mechanically ventilated, resuscitated with lactated Ringer’s solution to maintain hematocrit within baseline levels and studied for 24h in an awake state. The treatment groups received hMSC as SD or DD as continuous i.v. infusion over 60 min, starting 1 h post injury, control group received the vehicle. Statistical analysis was performed with t-test, ANOVA and Bonferroni post hoc comparisons. Data are expressed as mean ± SEM. Significance set as p<0.05.
Results: The vehicle-treated group developed a clinically defined ALI at 24h with a PaO2/FiO2 ratio of 97±33 mmHg that was significantly improved in SD and DD (SD: 288±55; DD: 327±2). Pulmonary shunt fraction (Qs/Qt) was significantly reduced in both treatment groups (24h: Control: 0.47±0.01 vs SD 0.33±0.06 vs DD: 0.24±0.01). Lung water content was significantly lower with DD treatment compared to other groups (Control: 7.1±0.5 vs SD 6.9±0.47 vs DD 5.22±0.28). The bacteria CFU (Control:1.5±0.9 ×106 vs SD 0.27±0.08 ×106 vs DD 0.31±0.09 ×106) and neutrophil count (Control: 1.83±0.8 vs SD: 0.78±0.3 vs DD: 0.83±0.5 cells/ul) in bronchoalveolar lavage fluid showed significantly lower values with hMSC treatment groups compared to the vehicle treated group.
Conclusions: hMSC reduced the severity of lung injury as measured by improved oxygenation in an ovine pulmonary septic shock model. DD significantly reduced the quantity of lung edema. Further studies are warranted to investigate the underlying mechanistic aspects.
INHIBITION OF FATTY ACID SYNTHASE WITH C75 REDUCES ORGAN INJURY AFTER HEMORRHAGIC SHOCK
M. Kuncewitch1, W. Yang*1,2, A. Khader1, M.D. Giangola1, J. Nicastro1, G.F. Coppa1, and P. Wang*1,2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Hemorrhagic shock is an emergent condition that renders patients critically ill. Several organs, particularly the lungs, are vulnerable to the injurious effects of hemorrhage and resuscitation. Excess free fatty acids (FFA) have been implicated in triggering inflammation in several disease conditions. C75 is a small compound that inhibits fatty acid synthase, a key enzyme in the control of fatty acid production. We therefore hypothesized that C75 treatment would be protective in hemorrhagic shock.
Methods: Male SD rats were subjected to hemorrhagic shock by controlled bleeding from the femoral artery. Blood was shed to maintain a mean arterial pressure of 30 mmHg for 90 minutes, followed by resuscitation over 30 minutes with a crystalloid volume equal to twice the volume of shed blood. Immediately after resuscitation rats received intravenous infusion of C75 (1 mg/kg BW) or vehicle (20% DMSO). Blood and tissue were collected 6 hours after resuscitation for analysis.
Results: C75 blunted the rise in serum FFA following hemorrhagic shock by 48% (p<0.05 vs. vehicle) and significantly reduced the levels of organ injury markers (lactate, AST, LDH, BUN, and creatinine) in serum (Table). C75 treatment significantly improved the histologic integrity of the lungs as viewed by light microscopy. This correlated with decreased neutrophil infiltration, evidenced by decreased Gr-1-stained cells and myeloperoxidase activity in the lungs of C75-treated rats. Finally, C75 administration was associated with reduced hepatic and pulmonary tissue mRNA levels of IL-6 by 73% and 65%, respectively, as well as their mRNA levels of COX-2 by 82% and 87%, respectively (p<0.05 vs. vehicle).
Conclusions: C75 reversed the increase of serum FFA in animals subjected to hemorrhagic shock and thereby downregulated the expression of inflammatory mediators, leading to attenuation of organ injury. In addition, C75 inhibited neutrophil infiltration of the lungs and reduced lung injury. Thus, targeting fatty acid synthase may represent a promising therapeutic strategy for patients following hemorrhagic shock.
MFG-E8 REGULATES NEURAL STEM CELL PROLIFERATION AND MIGRATION VIA INTEGRIN AVB3/PPARγ/CYCLIN D2/NETRIN-1 PATHWAY
C. Cheyuo*, W. Yang*, M. Aziz, A. Jacob*, M. Zhou*, and P. Wang*. The Feinstein Institute for Medical Research, Manhasset, NY
Background: Neurogenesis is crucial to neurological recovery after brain injury. Milk fat globule-EGF factor 8 (MFG-E8) is a 66-kDa glycoprotein ligand of integrin αvβ3/αvβ5 shown to protect against acute cerebral ischemia. However, the role of MFG-E8 in neurogenesis-mediated recovery after ischemic stroke has not been investigated.
Aim: We assessed the effects of recombinant MFG-E8 on neural stem cell (NSC) proliferation and migration.
Methods: Striatal embryonic (E14) NSC were exposed to 1 μM BrdU in the presence of trace rhEGF, 1 ng/ml, and increasing doses of rmMFG-E8 (125-1000 ng/ml) or PBS as control. NSC proliferation was assessed by BrdU immunofluorescent staining after 24 h. NSC transwell migration assay was performed, with SDF-1 as the chemoattractant. The effects of MFG-E8-mediated integrin αvβ3 signaling on the gene expression of cyclin D2 and netrin-1, mediators of NSC proliferation and migration respectively, and PPARγ were assessed using an integrin αv-blocking antibody. PPARγ signaling was further assessed using the agonist, rosiglitazone in NSC culture. In additional experiments, adult male Sprague-Dawley rats underwent 90-min transient middle cerebral artery occlusion (tMCAO) followed by intracerebroventricular administration of rhMFG-E8, 0.4 μg/μl, or PBS, and daily intraperitoneal 0.1mM BrdU injections. Rats were sacrificed on day 7, and the brain slides obtained were stained for BrdU, nestin (NSC marker) and doublecortin (migration marker).
Results: Compared with PBS, MFG-E8 dose-dependently increased the fraction of proliferating NSC (BrdU+), starting at a dose of 125 ng/ml (44% vs 64%) and achieving maximal proliferation at 500 ng/ml (44% vs 77%). MFG-E8 also increased the number of migrated NSC at 24 h compared with PBS (178 vs 321) (p<0.05). MFG-E8-mediated stimulation of NSC proliferation and migration was associated with integrin αv-dependent upregulation of cyclin D2, netrin-1 and PPARγ. The PPARγ agonist, rosiglitazone, had an additive effect with MFG-E8 in the upregulation of cyclin D2 and netrin-1. Correspondingly, rhMFG-E8 increased the numbers of proliferating (BrdU+nestin+) and migrating (BrdU+doublecortin+) NSC in the striatum after tMCAO in rats.
Conclusions: MFG-E8 may promote recovery after ischemic stroke by stimulating neurogenesis via a novel integrin αvβ3/ PPARγ/cyclin D2/netrin-1 pathway.
PROBIOTICS IMPROVE THE OUTCOME IN EXPERIMENTAL PSEUDOMONAS AERUGINOSA INDUCED PNEUMONIA: ROLE OF REGULATORY T CELLS
L. Khailova, C.H. Baird*, and P.E. Wischmeyer*. University of Colorado Anschutz Medical Campus, Aurora, CO
Background: Pneumonia is a common cause of mortality in the USA with more than 50,000 deaths each year. Clinical trials demonstrate probiotics may reduce nosocomial infections, VAP and improve clinical outcome in critical illness. Recent data show enhanced T regulatory cell responses correlate with resistance to invasive bacterial pneumonia.
Objective: To evaluate the effects of probiotic treatment in a Pseudomonas aeruginosa induced pneumonia mouse model and the role of regulatory T cells (Tregs).
Methods: 6-week old FVB/N mice were treated (o.g.) with or without Lactobacillus rhamnosus GG (LGG:109CFU/ml), Bifidobacterium longum (BL:107CFU/ml) or LGG+BL (109+107CFU/ml) and intratracheally injected with 4×108CFU P. aeruginosa or saline. At 12h, blood and BAL were cultured and IL-6 analyzed in serum. Lungs were evaluated for injury and presence of Tregs (CD4, CD25 and Foxp3). In survival studies, mice were followed for 7 days and given probiotic treatment every 24h.
Results: Survival at 7 days was significantly higher in LGG (55%), BL (37%) and LGG+BL (60%) treated mice (p<0.01) compared to untreated animals (14%). Bacteremia and bacterial growth in BAL was reduced (p<0.02) in mice treated with LGG or LGG+BL compared to untreated control mice (0±0 vs. 1.4×103±604 CFU/ml; LGG:2.9×103±1.6×103,BL:1.3×104±1.3×104 vs. 2.6×105±1.2×105 CFU/ml respectively). Serum IL-6 was significantly lower in mice receiving probiotics compared to pneumonia mice (LGG 867±150 pg/ml; BL 883±105 pg/ml; LGG+BL 723±140 pg/ml vs. Pneumo 1412±121 pg/ml; p<0.05). Pulmonary pathology was improved in lungs of probiotic-treated mice. The number of MPO positive cells in lungs of all probiotic groups was significantly decreased (LGG 9±1; BL 13±1; LGG+BL 8±1) compared to pneumonia control group (43±2) (p<0.05). Lung mRNA levels of Tregs markers were significantly higher (p<0.05) in all probiotic groups compared to pneumonia control group (Table).
Conclusions: LGG, BL or LGG+BL treatments improve survival and pulmonary pathology, decrease systemic IL-6 levels in mouse model of P. aeruginosa induced pneumonia. Probiotics increased Tregs in the lungs, which may improve resistance to bacterial pneumonia. Thus, either of these probiotics may be used as novel therapy for prevention of pneumonia.
HMGB1 MEDIATES PERSISTENT SPLENOMEGALY, LEUKOCYTOSIS, AND SPLENOCYTE PRIMING IN SEPSIS SURVIVORS
S.I. Valdes-Ferrer, M. Rosas-Ballina, P.S. Olofsson, B. Lu, M. Dancho, H. Yang*, J. Li, S. Chavan, and K. Tracey*. Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Severe sepsis is a life-threatening complication of infection and injury that occurs in at least 700,000 subjects in the United States each year, being one of the top 10 causes of mortality. At least two thirds of patients with severe sepsis will survive to be discharged. Recent epidemiologic and follow-up studies have revealed high incidence of cognitive impairment, disability, and functional impairment in sepsis survivors. Mortality is 50-80% within five years. The pathophysiology of this common syndrome is largely unknown and there is no specific treatment. High-mobility group box 1 (HMGB1) is a cytokine known to be necessary and sufficient mediator of sepsis pathogenesis causing endothelial dysfunction, inflammation and organ damage. We hypothesized that persistently elevated levels of HMGB1 mediate immune dysfunction in sepsis survivors.
Methods: Sepsis was induced by cecal ligation and puncture (CLP) and survivors were followed for 12 weeks. Levels of circulating HMGB1 were analyzed by western-blot. Inflammatory cytokines were measured by ELISA. Cell phenotype was determined by flow cytometry. We used liquid chromatography tandem mass spectrometric analysis to characterize the redox state of HMGB1. Splenomegaly and leukocytosis was observed in sepsis survivors up to 4 weeks. Levels of HMGB1 were elevated for at least 8 weeks after CLP. In contrast to HMGB1, IL-6 and CXCL-1 were transiently elevated, while no increase was observed in TNF, IL-1β, IFN-γ, or other inflammatory cytokines. Splenocytes derived from sepsis survivors had an augmented response to LPS ex vivo. Administration of neutralizing anti-HMGB1 antibody to survivors reversed splenomegaly, leukocytosis and reduced splenocyte priming. Administration of recombinant HMGB1 to naïve mice induced a similar immunophenotype as observed in sepsis survivors. Interestingly analysis of circulating HMGB1 from sepsis survivors by mass spectroscopy demonstrated a stepwise increase of reduced form of HMGB1 (with known chemo-attractant properties) during the first 3 weeks, followed by disulphide form (with known inflammatory properties) 4-8 weeks after CLP.
Conclusion: Our findings suggest that prolonged increase in circulating HMGB1 plays a causative role in mice surviving severe sepsis. Agents directed against HMGB1 may provide a tool to treat immune system abnormalities in sepsis survivors.
CAN PERIPHERAL BLOOD MONONUCLEAR CELLS BE USED AS A PROXY FOR MITOCHONDRIAL DYSFUNCTION IN VITAL ORGANS DURING HEMORRHAGIC SHOCK AND RESUSCITATION?
M.A. Karamercan*1, J.P. Villarroel2, Y. Guan2, S.L. Weiss2, E.C. Werlin2, R. Figueredo2, L.B. Becker2, and C.A. Sims*2. 1Gazi University, School of Medicine, Department of Emergency Medicine, Ankara, Turkey, 2University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA
Introduction: Although mitochondrial dysfunction is thought to contribute to the development of post-traumatic organ failure, current techniques to assess mitochondrial function (MF) in tissues are invasive and clinically impractical. We hypothesized that MF in peripheral blood mononuclear cells (PBMCs) would reflect cellular respiration in other organs during hemorrhagic shock and resuscitation (HS&R).
Methods: Using a fixed pressure HS model, Long Evan’s rats were bled to a mean arterial pressure (MAP) of 40 mmHg. When blood pressure could no longer be sustained without intermittent fluid infusion (Decompensated HS), Lactated Ringer’s (LR) was incrementally infused to maintain the MAP at 40 mmHg until 40% of the shed blood volume was returned (Severe HS). Animals were then resuscitated with 4× total shed volume in LR over 60 minutes (Resuscitation). Control animals underwent same surgical procedures, but were not hemorrhaged. Animals were randomized to Control (n=6), Decompensated HS (n=6), Severe HS (n=6) or Resuscitation (n=6) groups. Kidney, liver, and heart tissues as well as PBMC’s were harvested at each time point to measure MF using high resolution respirometry. Flowcytometry was used to assess mitochondrial membrane potential (Ψm) in PBMCs. One-way ANOVA and Pearson correlations were performed (significance: p <0.05).
Results: Although mitochondrial respiration decreased in all tissues including PBMC’s, the degree of impairment varied significantly during HS&R (table). Of the tissues investigated, PBMC MF and Ψm only correlated well with kidney MF during HS (complex I:r =0.65; complex II:r=0.65; complex IV:r=0.52; p<0.05). This correlation, however, disappeared with resuscitation.
Conclusion: All tissues including PBMC’s demonstrated mitochondrial dysfunction following HS&R. While PBMC mitochondrial respiration and Ψm correlated well with kidney mitochondrial dysfunction following HS, the variability in mitochondrial response between tissues during HS&R limited the usefulness of using PBMC’s as a proxy for tissue-specific cellular respiration.
GROWTH ARREST-SPECIFIC PROTEIN 6 (GAS6) ATTENUATES NEUTROPHIL MIGRATION AND ACUTE LUNG INJURY IN SEPSIS
M.D. Giangola1, W. Yang*1,2, S.R. Rajayer1, J. Nicastro1, G.F. Coppa1, and P. Wang*1,2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Sepsis is an acute inflammatory condition associated with high morbidity and mortality in the intensive care unit. Of those that die, septic patients succumb to the ensuing septic shock and multiple organ failure, especially in the lung. Gas6 has been proposed as a broad regulator of the innate immune response and attenuates the NF-κB inflammatory pathway. Thus, we hypothesized that Gas6 could have a protective role in attenuating the severity of acute lung injury (ALI) and sepsis.
Methods: Male mice were subjected to sepsis by cecal ligation and puncture (CLP). After CLP operation, recombinant murine Gas6 (rmGas6; R&D Systems; 5 μg/mouse) or normal saline (vehicle) was injected via the jugular vein. Blood and lung tissues were collected at 20h after CLP for various measurements. In addition, human promyelocytic HL60 cells were differentiated by DMSO for studying the effect of rmGas6 on the migration of neutrophils.
Results: rmGas6 significantly reduced serum levels of organ injury markers (LDH, AST, and ALT) and proinflammatory cytokine IL-17 after CLP (Table). rmGas6 improved the microscopic structure of the lungs, judged by histological examination. The MPO activity and number of neutrophils immunostained with Gr-1 in the lungs were significantly reduced in the rmGas6 group (Table). The degradation of IκBα induced by CLP in the lungs was significantly inhibited by the rmGas6 treatment. The lung mRNA levels of TNF-α, IL-1β, IL-6, MIP-2 and IL-17 were decreased by 86%, 80%, 54%, 77% and 93%, respectively, with rmGas6 treatment as determined by real time RT-PCR (p<0.05). Finally, rmGas6 reduced the in vitro migration of differentiated HL60 cells from 452±83 to 162±9 cells/field (p<0.05).
Conclusions: Administration of rmGas6 effectively attenuated organ injury as well as ALI after CLP. rmGas6 inhibited neutrophil migration and infiltration and lowered the amounts of pro-inflammatory cytokines and chemokines in the lungs by inhibiting NF-κB activation after CLP. Thus, Gas6 clearly has a potential to be developed as a novel therapeutic agent to treat patients with sepsis.
IL-6 NEUTRALIZATION REDUCES NEUROINFLAMMATION AND BRAIN INJURY EXACERBATED BY BRIEF HYPOXIA EXPOSURE IN A MURINE MODEL OF TRAUMATIC BRAIN INJURY
S. Yang, M. Gangidine, T. Pritts, M. Goodman*, and A. Lentsch*. University of Cincinnati, Cincinnati, OH
Introduction: Secondary insults after mild traumatic brain injury (mTBI) have been linked to worse clinical outcomes. Likewise, increased expression of IL-6 after TBI has also been associated with worse outcomes. Brief episodes of hypoxia, such as those experienced during aeromedical evacuation, have not been evaluated for their effects on functional outcomes. Similarly, interventions aimed at reducing IL-6 have not been examined. In the present study, we examined the effects of brief hypoxic exposure after mTBI on neuroinflammation, brain injury, and functional outcomes and also evaluated the effects of IL-6 neutralization in this setting.
Methods: A murine model of mild traumatic brain injury (TBI) was induced by a weight drop (500 g from 1.5cm) over the cranium overlying the frontal lobes. Mice were then exposed to hypoxia (FiO2=15.1%) or normoxia (FiO2=21%) for 30 minutes. Serum and brain samples were analyzed for inflammatory cytokines 24 hours after traumatic brain injury by ELISA. Neuron specific enolase (NSE) was measured as a serum biomarker of brain injury. Furthermore, doses of monoclonal antibody to IL-6 were given intraperitoneally following TBI to neutralize systemic IL-6. Functional evaluations of motor coordination were conducted using a rotorod device for 5 days after head injury.
Results: Mice undergoing TBI had significant increases in serum and brain IL-6. When exposed to hypoxic conditions after TBI, there was a 1.5 fold increase in brain IL-6. Furthermore, hypoxia markedly exacerbated brain injury, as evidenced by a 5-fold increase in NSE levels. Neutralization of IL-6 abrogated neuroinflammation and significantly reduced brain injury. Functionally, mTBI resulted in a significant deficit in motor coordination. However, mice given IL-6 neutralizing antibody had motor coordination skills equivalent to sham animals, indicating that IL-6 may be a primary contributor to motor coordination defects after mTBI.
Conclusion: The data demonstrate that brief exposure to hypoxia after mTBI greatly increases neuroinflammation and brain injury. Furthermore, the data suggest that IL-6 is a significant contributor to brain injury and functional deficits after mTBI and may represent a viable therapeutic target.
PYR-41, A UBIQUITIN-ACTIVATING ENZYME E1 INHIBITOR, ATTENUATES ORGAN INJURY IN SEPSIS
S. Matsuo, W. Yang*, and P. Wang*. Hofstra North Shore-LIJ School of Medicine and The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Sepsis is defined as a systemic inflammatory response that can lead to multiple organ dysfunction. The proteasome pathway, consisting of ubiquitylation and degradation steps, is essential in controlling the protein turnover of IκB and subsequently the activation of NF-κB. The inhibition of proteasome degradation step has been shown to reduce inflammatory responses in sepsis. However, targeting the ubiquitylation step has not yet been addressed. PYR-41 is a small molecule compound that selectively inhibits the ubiquitin-activating enzyme E1. We hypothesized that PYR-41 attenuated organ injury caused by sepsis, especially in the lungs.
Methods: Male C57BL/6 mice (20-25g) were subjected to cecum ligation and puncture (CLP). PYR-41 (5 mg/kg BW) or 20% DMSO (vehicle) in 0.2 ml saline was injected intravenously after CLP. Blood and tissues were collected at 20 h after CLP for various measurements. An in vitro model of LPS-stimulated RAW264.7 murine macrophages was used to assess the anti-inflammatory activity of PYR-41.
Results: PYR-41 treatment significantly decreased levels of organ injury markers (AST, ALT and LDH) and proinflammatory cytokines (TNF-α, IL-1β and IL-6) in serum after CLP (Table). In the lungs, PYR-41 improved the integrity of microscopic structure, reduced the number of TUNEL-positive cells and the expression of cleaved caspase-3, and decreased MPO activity after CLP. PYR-41 also inhibited the expression of ubiquitylated proteins and degradation of IκB induced by CLP to levels similar to the shams. Consequently, PYR41 reduced lung mRNA expressions of several NF-κB-induced genes, including TNF-α, IL-1β, KC, MIP-2, iNOS and COX2, by 76%, 67%, 48%, 94%, 76%, and 53%, respectively, compared to the vehicle group (p<0.05). In addition, TNF-α release and degradation of IκB in RAW264.7 cells stimulated with LPS (1 ng/ml) were significantly inhibited by PYR-41.
Conclusions: PYR-41 effectively inhibits ubiquitylation and NF-κB activation in vivo and in vitro, leading to reduction of inflammatory responses and attenuation of organ injury in septic mice. Thus, PYR-41 may be a potential therapeutic agent for sepsis.
MOLECULAR EVIDENCE FOR UPREGULATION OF THE CHOLINERGIC ANTI-INFLAMMATORY PHENOTYPE DURING SEPSIS
D.B. Hoover, C. Bond, T.R. Ozment*, D. McDonald, and D.L. Williams*. East Tennessee State University, Johnson City, TN
Non-neuronal cholinergic mechanisms have the potential for exerting prominent anti-inflammatory effects in the spleen, but little is known about in situ expression of cholinergic markers in spleen or the response of these markers to sepsis. To address this deficiency, we evaluated mouse spleens for basal expression of specific markers required for cholinergic function and the response of these markers to sham surgery and sepsis induced by cecal ligation and puncture (CLP). Spleens were obtained from non-operated mice and operated mice at 8h after sham or CLP surgery and placed into RNA later (n=8 per group). RNA was extracted, and expression of cholinergic markers was determined by quantitative PCR. Specific markers evaluated were the high affinity choline transporter (CHT), choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT), and the α7 nicotinic acetylcholine receptor (α7nAChR) subunit, which is known to mediate the cholinergic anti-inflammatory response. Expression of cholinesterase enzymes, which degrade acetylcholine, was evaluated in separate samples using an activity assay. Each marker was expressed at a low level basally. Sepsis increased the expression of VAChT (control: 77.8+/-17.0 transcripts per 50 ng RNA; sepsis: 139.7+/-24.2; P=0.021), and immunostaining for VAChT was increased in sections of spleen from septic mice. Expression of ChAT and CHT mRNAs was not altered by sepsis. In contrast, expression of α7nAChR mRNA within the spleen decreased significantly at 8h post-CLP. Acetylcholinesterase (AChE) was the sole cholinesterase present in spleen extracts, and total AChE activity was low and unaltered by sepsis. Upregulation of VAChT within the spleen at 8h post-CLP suggests that the cholinergic anti-inflammatory pathway is activated by sepsis and might release additional ACh into the extracellular compartment. Low, unaltered levels of AChE in the septic spleen increase the probability that released ACh will avoid degradation long enough to reach and activate α7nAChRs on monocytes and thereby exert an anti-inflammatory influence. Decreased expression of α7nAChRs in septic spleen could reflect trafficking of α7nAChR-positive monocytes from the spleen at 8h post-CLP. These findings provide the first in situ evidence for expression of cholinergic genes in the spleen and regulation of the cholinergic phenotype during sepsis.
INHALATION OF HYDROGEN GAS DURING CARDIOPULMONARY RESUSCITATION IMPROVES NEUROLOGIC AND CARDIAC OUTCOME IN A RAT CARDIAC ARREST MODEL
K. Hayashida1, M. Sano2, N. Kamimura3, T. Yokota3, S. Ohta3, K. Fukuda2, and S. Hori1.
1Department of Emergency & Critical Care Medicine, School of Medicine, Keio University, Tokyo, Japan, 2Department of Regenerative Medicine and Advanced Cardiac Therapeutics, School of Medicine, Keio University, Tokyo, Japan, 3Department of Biochemistry and Cell Biology, Institute of Development and Aging Science, Graduate School of Medicine, Nippon Medical School, Kanagawa, Japan
Objective: The prognosis for cardiac arrest remains very poor despite the use of therapeutic hypothermia with cardiopulmonary resuscitation (CPR). Molecular hydrogen (H2) has potential as a novel antioxidant. Here, we investigated whether inhalation of hydrogen gas during CPR improves neurologic and cardiac outcomes in post-cardiac arrest of rats model.
Methods and Results: After 5 min of untreated cardiac arrest by ventricular fibrillation, rats were randomly assigned to 1 of 4 experimental groups at the beginning of CPR: mechanical ventilation (MV) with 2% N2 and 98% O2 under normothermia (37°C), the control group; MV with 2% H2 and 98% O2 under normothermia; MV with 2% N2 and 98% O2 under therapeutic hypothermia (TH), 33°C; and MV with 2% H2 and 98% O2 under TH. Resuscitation was performed by manual chest compression in combination with 8 μg IV epinephrine (DC shocks of 3 J, if necessary). Gas inhalation continued 2 hours after return of spontaneous circulation (ROSC). The H2 group had significantly better overall survival at 72h after ROSC compared with the control group to an extent comparable to the TH group. A neurologic deficit score (NDS) of survivors was either higher in the H2 or the TH group than the control group. H2 inhalation prevented a rise in left ventricular end-diastolic pressure and increase in serum IL-6 level after ROSC. Histological staining of cardiomyocytes showed H2 attenuated degeneration and necrosis induced by ischemic/reperfusion injury. The salutary impact of H2 was at least partially attributed to the radical-scavenging effects of H2 gas, because both 8-OHdG- and 4-HNE positive cardiomyocytes were markedly suppressed by H2 inhalation after ROSC.
Conclusion: Inhalation of hydrogen gas during and after CPR is promising strategies to reduce the neurologic and cardiac oxidative damage in patients with post cardiac arrest syndrome.
COENZYME Q10 SUPPLEMENTATION IMPROVES SURVIVAL AND BACTERIAL CLEARANCE ALONG WITH REVERSAL OF HYPERLACTATAEMIA AND NEUTROPHIL ORGAN INFILTRATION IN SEPTIC MICE
M. Fujii1,2, M. Yamada3, S. Ishikawa4, J. Lederer*4, and M. Kaneki*3. 1Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 2Hiroshima University School of Medicine, Hiroshima, Japan, 3Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Charlestown, MA, 4Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Coenzyme Q10 (CoQ10) is an essential cofactor of the mitochondrial respiration and also acts as an anti-oxidant. Recent studies by others and us have shown that circulating levels of CoQ10 are lower in patients with septic shock and in a broad range of critically ill patients as compared with healthy controls. Based on these findings, CoQ10 insufficiency has been proposed as a potential contributor to worse clinical outcome of septic patients. The effect of CoQ10 supplementation, however, has not yet been investigated in sepsis or critical illness. Hyperlactatemia is associated with mitochondrial dysfunction and has been identified as a predictor of the mortality of septic patients. CoQ10 supplementation (4, 12, 40 mg/kg/day, SC) starting at 2 h after cecum ligation and puncture (CLP) dose-dependently improved survival of septic male CD-1 mice at 7 weeks of age (n=24 per group, p<0.0001) and the maximum pro-survival effect was observed at 40 mg/kg. We evaluated the effects of CoQ10 (40 mg/kg) on bacterial clearance, plasma levels of lactate and alarmins, and neutrophil infiltration into organs at 16 h after CLP, as compared with placebo. CoQ10 significantly reduced bacterial burden in the circulation and peritoneal cavity in septic mice. CLP increased plasma lactate levels 2.5-fold compared with sham-operation, which was reversed by CoQ10. CoQ10 prevented marked increases in plasma levels of cell-free DNA and HMGB1 in septic mice. Although CoQ10 reduced bacterial burden, CoQ10 did not decrease the number of neutrophils in the peritoneal cavity of septic mice. In contrast, CoQ10 attenuated CLP-induced neutrophil infiltration, as evaluated by meyloperoxidase activity and granulocyte receptor (Gr)-1 expression, into liver, heart and kidney of septic mice. These results clearly demonstrate that CoQ10 supplementation is efficacious in improving survival and bacterial clearance, and reverses hyperlactatemia and elevated circulating alarmins. Of interest, our data suggest that CoQ10 supplementation may alter neutrophil trafficking pattern into the site of infection vs. multiple organs, which in turn contributes to the protective effects of CoQ10. Our study provides scientific rationale to develop a clinical study to evaluate the safety and efficacy of CoQ10 supplementation in septic patients.
SPECIFIC INHIBITION OF MITOCHONDRIAL ROS CONTROLS LUNG DAMAGE IN A RAT PNEUMONIA-RELATED SEPSIS MODEL
L. Ma, D. Maass, X. Yao, S. Wolf*, J. Minei*, and Q.S. Zang*. University of Texas Southwestern Medical Center, Dallas, TX
Objective: Previously, we have shown that mitochondrial-targeted vitamin E (Mito-Vit-E), an antioxidant specifically suppresses mtROS, protects hearts in a rat pneumonia-related sepsis model. The aim of this study was to evaluate whether application of Mito-Vit-E alleviates sepsis-induced lung damage using the same model.
Methods: In SD rats, sepsis was produced by intratracheal injection of S. pneumoniae (4×1,000,000 CFU/rat). Mito-Vit-E, vitamin E, both at 21.5 micro moles/kg, or control vehicle was orally administered 30 minutes post-inoculation. Lung tissues were harvested at multiple time points post-inoculation. Levels of mitochondrial oxidative stress were indirectly determined by mitochondrial aconitase activities. Cytokines in tissue lysates were quantified by ELISA. Myeloperoxidase (MPO) levels were analyzed by immunohistochemistry, and lung morphology was analyzed by hematoxylin and eosin (HE) stain.
Results: In the lung of septic rats, Mito-Vit-E and vitamin E significantly improved mitochondrial aconitase activity and suppressed the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). In all the measurements, Mito-Vit-E exhibited significantly higher efficacy compared with vitamin E. Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induce MPO accumulation. HE staining indicated that Mito-Vit-E effectively reduced pulmonary pathology, such as thickening of the alveolar wall and infiltration of leukocytes, in septic animals.
Conclusion: Our data suggest that mitochondria-targeted antioxidants may become an effective therapeutic approach to control sepsis-induced lung damage.
COMPLEMENT FACTOR B CONTRIBUTES TO ACUTE KIDNEY INJURY IN POLYMICROBIAL SEPSIS
L. Zou*1, Y. Feng1, C. Chen1, Y. Gong1, J. Cai1, L. Wang3, J.M. Thurman2, and W. Chao*1. 1Massachusetts General Hospital, Boston, MA, 2University of Colorado Health Sciences Center, Denver, CO, 3Children’s Hospital Los Angeles, Los Angeles, CA
Introduction: Acute kidney injury (AKI) is a frequent complication of sepsis and associated with a high mortality. Complement factor B (cfB) is an essential component of alternative pathway activation and involved in ischemic kidney injury. However, the role of cfB in AKI during sepsis is unknown.
Methods: Polymicrobial sepsis was created by cecum ligation and puncture (CLP). Wide type (WT) and cfB-/- mice were subjected to sham or CLP surgery. At 24 or 48 h, serum and kidneys were collected for complement and AKI assessment. cfB and C3 expression was detected by Western blot and their tissue distribution by immunohistochemistry. AKI was determined by kidney NGAL and KIM-1 gene expression. Mice subjected to CLP were monitored for mortality for up to 14 d.
Results: CLP resulted in a marked and time-dependent increase in serum and kidney cfB. Immunohistochemistry showed a basal expression of cfB and C3 in the kidney. CLP induced a significant increase in cfB and C3 expression, primarily in tubular epithelial cells and basement membranes (Fig). Septic WT mice developed AKI within 24 h as demonstrated by a marked up-regulation of kidney NGAL and KIM-1 gene expression, two sensitive biomarkers of AKI, compared with sham mice (139±36 and 20±9 fold, respectively, P<0.01, n=4-7). In comparison, septic cfB-/-mice displayed attenuated AKI with significant reduction in the kidney NGAL and KIM-1 mRNA (23±4 and 2±0.3 fold, P<0.05 and P<0.01, respectively, n=4-7). This protective effect was associated with reduced C3/iC3b/C3dg deposition in septic cfB-/- kidney (Fig). Finally, septic cfB-/- mice had significantly improved survival compared with septic WT mice (58% vs. 35% on day 14, P<0.05, n=48-49).
Conclusions: Our studies demonstrate that 1) polymicrobial sepsis induces cfB and C3 up-regulation in the kidney, 2) cfB deficiency leads to reduced kidney C3/C3 fragment deposition, attenuates AKI, and improves survival during polymicrobial sepsis. These data suggest that cfB may play a role in the pathogenesis of AKI during sepsis.
CHOLINERGIC NEURONAL SIGNALS ATTENUATE INFLAMMASOME ACTIVATION THROUGH INHIBITING MITOCHONDRIAL STRESS
B. Lu*, Y. Levine, K. Kwan, H. Yang*, P.S. Olofsson, U. Andersson, S.S. Chavan*, and K. Tracey*. The Feinstein Institute for Medical Research, Manhasset, NY
The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Immunity is highly linked with mitochondrial stress, which importantly regulates the activation inflammasome, a key component of innate immunity. Here we show that cholinergic neuronal signals attenuate inflammasome activation by inhibiting mitochondrial stress. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibited the inflammasome activation and mitochondrial stress; whereas genetic deletion of alpha 7 nicotinic acetylcholine receptor (a7 nAchR) significantly enhanced the inflammasome activation and mitochondrial stress. Immunohistochemical analysis revealed that a7 nAchR is localized in the mitochondria. We also found that extracellular acetylcholine rapidly influxes into macrophage cytoplasm upon ATP stimulation in a P2X7 receptor-dependent manner. Importantly, acetylcholine significantly attenuated calcium or hydrogen oxide-induced mitochondrial damage and the subsequent mitochondrial DNA release. Together, these findings unravel a novel neurotransmitter-mediated signaling pathway, in which acetylcholine translocates into cytoplasm of immune cells during inflammation, and inhibits inflammasome activation by preserving mitochondrial membrane integrity.
Supported in part by grant from NIH (RO1 GM57226 to K. J. T).
DELETION OF TOLL LIKE RECEPTOR SIGNALING DOES NOT AFFECT EXPANSION OF HEMATOPOIETIC STEM CELLS DURING POLYMICROBIAL SEPSIS IN NEONATES
A. Cuenca, A. Cuenca, L.F. Gentile, S. Islam, D. Kay, L. Moldawer*, and S.D. Larson. University of Florida, Gainesville, FL
Background: Neonates have increased susceptibility to infections and increased mortality. In previous studies, we have shown that neonates, unlike young adults, have an inappropriately modest early inflammatory response, and their neutrophil and macrophage functions are impaired (Cuenca et al. Infect. Immunity 2011). Since early inflammatory responses are dependent upon NFkB and type I interferon signaling, we examined whether the absence of MyD88, TRIF or MyD88/TRIF signaling altered myelopoietic response in neonates to polymicrobial sepsis.
Methods: 7d old wild-type (WT) B6 and MyD88, TRIF, or MyD88/TRIF KO mice received intraperitoneal administration of a cecal slurry (LD30), and surviving mice were sacrificed at 0, 36 hrs, or 7 days post sepsis. Blood, bone marrow and spleen cell populations were harvested and phenotyped by flow cytometry.
Results: Following neonatal polymicrobial septic challenge in WT neonates, hematopoietic stem cells (Lin-Sca-1+c-Kit+) or LSK cells in bone marrow increased from 0.34 ± 0.04 to 0.64 ± 0.20 % of total bone marrow cells at 36 hours post sepsis (p<0.05). Similar increases in LSK cells were noted in the spleen of WT neonates, 0.18 ± 0.03 to 0.74 ± 0.3 (p<0.05). In addition, common myeloid progenitors (Lin-c-Kit+Sca-1-Il-7-) were also increased in the bone marrow by 7 days 1.5 ± 0.5 to 2.9 ± 0.9 % of total cells (p<0.05). Importantly, this expansion of progenitor stem cell populations was also seen in the absence of MyD88 and/or TRIF signaling.
Conclusions: We have previously reported that neonates have a reduced capacity to expand their early bone marrow and spleen progenitor cell populations in response to polymicrobial sepsis when compared to young adults. These studies reveal that like adults, however, neonates do not rely on either MyD88 and/or TRIF signaling to induce this expansion. This data also support our findings that survival to neonatal polymicrobial sepsis does not depend on TLR signaling.
DC PLAY AN UNEXPECTED ROLE IN TLR4 DEPENDENT INFLAMMATION AND ORGAN DAMAGE FOLLOWING HEMORRHAGIC SHOCK WITH TISSUE TRAUMA
K.R. Zettel*, S. Korff, S. William, T. Lauryn, S.S. Darwiche, M.J. Scott*, A. Morelli, and T. Billiar*. University of Pittsburgh, Pittsburgh, PA
Background: Dendritic cells (DC) are widely known as antigen presenting cells, which modify T-cell responses to infectious pathogens, with less understanding of their other roles in early inflammatory responses. DC are known to respond to DAMPs, and DAMPs are thought to drive TLR4 dependent inflammation and organ injury in trauma models. Here, we tested the hypothesis that DC are important contributors to trauma induced inflammation and organ injury.
Methods: To test our hypothesis several mouse strains were subjected to hemorrhagic shock with soft tissue injury (HS+STI) with endpoints of circulating IL6 (marker of systemic inflammation) and AST levels (marker of tissue damage) measured at the 6-hr time point. Mouse strains included DC deleted mice (CD11c-DTR), cell-type selective DC TLR4 knockout mice (CD11c-cre x TLR4loxp/loxp), and TLR4-/- mice reconstituted with TLR4+/+ DC. For the reconstitution experiments, we adoptively transferred 10x10∧6 TLR4-positive or TLR4-negative DC to TLR4 KO mice 1 day prior to control or HS+STI. Conventional DC were generated by magnetic bead isolation following bone marrow culture with IL-4 and GM-CSF.
Results: Both total deletion of CD11c-pos DC and cell type selective TLR4 deletion from CD11c positive cells resulted in a significant reduction of both IL6 and AST levels following trauma (p< 0.05). Because CD11c is expressed on cells other than DC we confirmed that conventional DC were involved in the injury response by transferring purified TLR4-positive DC into TLR4 knockout mice one day prior to HS +STI. There was no difference in tissue injury in control mice given either TLR4-pos or TLR4-neg DC as demonstrated by similar transaminases (AST 153 U/I vs. 129 U/I; ALT 51 U/I vs. 53 U/I). The infusion of TLR4-pos DC increased tissue injury compared to those given TLR4-neg DC measured by AST (2105 U/I vs. 1211U/I) and ALT (5633U/I vs. 2588 U/I)(p<0.05, n= 4-6).
These results show that DC play an important role in driving TLR4-dependent inflammation and tissue damage following severe HS + STI. This represents an unexpected role for DC in the early response to sterile injury.
EFFECT OF MPLA ON IMMUNE RESPONSES TO A BURN WOUND INFECTION
J. Bohannon and E. Sherwood*. Vanderbilt University, Nashville, TN
Due to loss of the protective skin barrier and injury-induced immune alterations, opportunistic infections remain the leading cause of death in severely burned patients that survive initial injury. Prior sensitization with low doses of the immunomodulators lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA) is known to induce a state of tolerance to subsequent LPS challenge, which is associated with an enhanced ability to clear bacteria following systemic Pseudomonas aeruginosa infection. Whereas LPS is highly toxic, MPLA is relatively innocuous and would be more suited for clinical use to enhance immune responses to infection. Previous studies have shown that treatment of mice with MPLA greatly enhances bacterial clearance leading to improved survival in a model of systemic sepsis, mediated in part by increased numbers of phagocytic myeloid cells. The current study was designed to determine if MPLA treatment of burned mice would similarly alter immune functions, leading to improved bacterial clearance and survival following a clinically-relevant wound infection. Mice underwent a full thickness burn injury to ∼35% total body surface area. Systemic treatment with MPLA or lactated ringers (LR; control) was administered on days 3 and 4 post-burn. Wounds were then topically inoculated with a lethal dose of P. aeruginosa on day 5 and mice were monitored for survival, bacterial clearance and cytokine analysis. A systemic infection model (P. aeruginosa injected i.p.) was utilized to characterize immune cell populations responding to the site of infection. MPLA-treated mice showed improved survival (Fig 1). Although circulating pro-inflammatory cytokine levels were decreased during infection in MPLA-treated mice, local and systemic bacterial dissemination was decreased. MPLA increased numbers of innate immune cells responsible for clearing bacteria locally. This evidence suggests that MPLA provides protective immune-modulating potential against deadly burn wound infections, while simultaneously controlling damaging pro-inflammatory cytokine levels.
PLATELET TLR4 REGULATES COAGULOPATHY AND ORGAN INJURY AFTER HEMORRHAGIC SHOCK IN MICE
M.D. Neal, N. Ding, G. Chen, P. Loughran, and T. Billiar*. University of Pittsburgh Medical Center, Pittsburgh, PA
Objective: Platelet TLR4 is a key regulator of platelet function and contributes to the activation of other cell types in multiple inflammatory conditions. We have shown that TLR4 signaling is critical to the development of organ injury in trauma and hemorrhagic shock. However, the functional significance of platelet TLR4 in hemorrhagic shock remains unexplored.
Methods: We generated transgenic mice globally deficient in TLR4 (TLR4-/-) and specifically lacking TLR4 on platelets (TLR4-PLT). Mice were subjected to a validated model of hemorrhagic shock consisting of bilateral femoral artery cannulation and hemorrhage followed by resuscitation (HS-R). Thromboelastography (TEG) was performed on shed blood at time 0 and after hemorrhage to assess coagulopathy. Tissues were harvested for histopathology and platelets were collected and sorted by FACS for markers of activation (CD42,CD62p).
Results: Platelet function was dramatically impaired in wild-type (WT) animals subjected to HS-R, as measured by TEG. WT platelets were activated following hemorrhage as measured by increased CD42/CD62p. TLR4-/- mice were protected from HS-R and did not develop coagulopathy. Strikingly, selective deletion of TLR4 from platelets resulted in platelets that were resistant to activation and prevented coagulopathy despite HS-R (Figure). Additionally, TLR4-PLT mice were protected from lung and liver injury and exhibited a marked reduction in systemic inflammation as measured by circulating IL-6 following HS-R.
Conclusions: We demonstrate for the first time that platelet TLR4 is an essential mediator of platelet activation, coagulopathy, and inflammation in hemorrhagic shock. Modulating TLR4 signaling on platelets may represent a novel therapeutic target for preventing coagulopathy and organ injury after hemorrhage.
MITOCHONDRIAL FUNCTION DURING HEMORRHAGIC SHOCK AND RESUSCITATION: WHERE YOU COME FROM COUNTS
C.A. Sims*1, Y. Guan1, M.A. Karamercan2, M. Selak1, J.A. Baur1, and L.B. Becker1. 1University of Pennsylvania, Philadelphia, PA, 2Gazi University Hospital, Acil Tip Anabilin Dali, Besevler, Turkey
Objective: Although mitochondrial dysfunction is known to occur during hemorrhagic shock and resuscitation, we hypothesized that different tissue types would exhibit diverse mitochondrial respiratory behavior in response to acute blood loss.
Methods: Using a validated model of decompensated hemorrhagic shock, Long-Evans rats were bled to a mean arterial pressure (MAP) of 40 mmHg and maintained until the MAP could not be sustained without fluid infusion (Decompensation). When 40% of the shed blood volume (40% SVT) had been returned in Lactated Ringer’s (LR), animals were resuscitated with 4× the shed volume in LR over 60 minutes and observed for 18hrs. Animals (n=6 per group) were sacrificed prior to hemorrhage (Baseline), at Decompensation, at 40%SVT, following resuscitation (60R) or 18 hours post-resuscitation. Tissue samples (liver, kidney, heart) were taken at each time point in order to assess individual complexes (CI, CII, and CIV) using isolated mitochondria and high resolution respirometry. Data was analyzed by ANOVA (*p<0.05).
Results: At baseline, individual mitochondrial complex function varied significantly depending on tissue source (table). Tissues also demonstrated significant diversity in their response to hemorrhagic shock and resuscitation. The kidney demonstrated the greatest functional decline during shock with all complexes remaining significantly impaired at 18 hrs. All liver complexes demonstrated significantly reduced function during shock, but CI and CIV improved following resuscitation. Cardiac mitochondrial function, however, was preserved during shock. Although CI and CII activity declined significantly with resuscitation, CIV activity remained stable and all complexes returned to baseline function by 18 hrs.
Conclusions: Not all mitochondria are created equal. Depending on the tissue source, mitochondria exhibit significant differences in respiratory function both at baseline and in response to hemorrhagic shock-resuscitation.
THE COMBINED ADMINISTRATION OF INSULIN AND PROPRANOLOL BENEFITS SEVERLY BURNED CHILDREN
J. Tanksley1, D.N. Herndon*1, C. Andersen1, A. Ali1, O. Suman1, M.G. Jeschke*2, and C.C. Finnerty*2. 1University of Texas Medical Branch, Houston, TX, 2Sunnybrook Health Sciences Centre, Toronto, ON, Canada
Rationale: Hyperglycemia, insulin resistance and hypermetabolism are hallmarks of the response to burn injury, which subsequently lead to increased morbidity and mortality. Insulin, which decreases endogenous glucose production and increases glucose uptake, reduces post-burn hyperglycemia. Propranolol, a beta-adrenergic receptor blocker, reduces the hypermetabolic and catabolic post-burn states. We hypothesized that treatment with a combination of both drugs would improve endogenous glucose kinetics (EGK), reduce hypermetabolism, and improve patient outcomes.
Objective: To determine whether the co-administration of insulin to decrease hyperglycemia and propranolol to decrease the heart rate by ∼15% would have greater benefit for severely burned children.
Methods: Eighty pediatric patients with burns > 30% of total body surface area were enrolled in a prospective study. Patients were randomized to control (n=65) or insulin plus propranolol (I+P) (n=15) throughout acute admission. Insulin was administered to maintain glucose within 110-140 mg/dl in combination with 4mg/kg/d propranolol to decrease the heart rate by ∼15%. Glycemic, body composition, cytokine, acute phase proteins, resting energy expenditure (REE), and cardiac function measurements were recorded throughout hospitalization. A generalized additive mixed model and mixed models of covariance and variance were used to model the relationship between day and by treatment for all variables.
Results: Demographics were similar. Glucose measurements for daily mean, minimum and 6am values were significantly improved for the I+P group compared to control. I+P patients yielded a reduction in HR, MAP, and RPP (p<0.0001) compared to the control. Similarly, reduction was seen in the inflammatory response (p<0.001). No change was seen in REE. 35 hypoglycemic incidents occurred in patients on I+P therapy compared to 45 incidents in the control group.
Summary: Combined insulin and propranolol in pediatric burn patients improves EGK, and reduces hyperdynamic cardiac function and inflammation in severely burned children.
Conclusion: The combination of anti-hyperglycemic and beta-blocking therapies should be explored further to determine whether extended administration improves patient outcome following severe burn injury.
B-TYPE NATRIURETIC PEPTIDE AND NEUTROPHIL GELATINASE ASSOCIATED LIPOCALIN TESTING FOR PREDICTING ACUTE KIDNEY INJURY DURING BURN RESUSCITATION: A PILOT STUDY
E. Howell, S. Sen, A. Steele, T. Palmieri*, Z. Godwin, J. Bockhold, D.G. Greenhalgh*, and N.K. Tran. University of California, Davis School of Medicine, Sacramento, CA
Introduction: Adequate intravenous fluid resuscitation is vital to survival in severe burn injury. Acute kidney injury (AKI) is a common sequelae of inadequate resuscitation. Urine output (UOP) and serum creatinine have been used to assess the adequacy of resuscitation and severity of AKI. We propose using B-type natriuretic peptide (BNP) and neutrophil gelatinase associated lipocalin (NGAL) as biomarkers of AKI to optimize fluid resuscitation. Point-of-care (POC) BNP and NGAL testing enables timely treatment decisions during critical care. We hypothesize POC BNP and NGAL measurements will help predict AKI during acute resuscitation in severely burned adult patients.
Methods: We conducted a pilot observational study of 16 patients with ≥20% total body surface area (TBSA) burns. Serial BNP, NGAL and creatinine levels were measured every 4 hours (h) for the first 48 h of admission using novel POC testing devices. Area under the curve (AUC) above the reference interval was calculated for BNP, NGAL and creatinine. Age, TBSA burn, fluid rates, UOP, vitals and laboratory creatinine were also recorded. AKI was determined based on the RIFLE criteria. BNP, NGAL and creatinine levels were compared between AKI v. non-AKI patients.
Results: Patients who developed AKI (n=8) had significantly higher mean NGAL values compared to non-AKI patients (200.0±58.4 v. 122.0±52.5 ng/ml, p=0.016). The same was observed for BNP (31.9±19.7 v. 9.9±4.8 pg/ml, p=0.025). NGAL values were an independent predictor of AKI (OR 1.03, 95% CI 1.00-1.05, p=0.036). Mean NGAL results were abnormally elevated 12 h earlier than creatinine levels in AKI patients. AUC analysis of NGAL values above the reference interval during the resuscitation period was significantly higher in AKI patients (68.1±30.2 v. 26.6±34.1 ng*hr/mL, p=0.012). Age, TBSA burned, fluid rates, vitals and UOP were similar between AKI v. non-AKI patients.
Conclusions: POC BNP and NGAL measurements enable rapid results for acute resuscitation management. NGAL appears to be an early predictor of AKI compared to creatinine. BNP values were also elevated in AKI patients, which may be due to cardiorenal syndrome. Further studies are warranted to characterize the kinetics of BNP and NGAL to help determine clinical decision points for POC testing-guided acute burn resuscitation.
TRAUMATIC BRAIN INJURY POLARIZES MONOCYTES TOWARDS AN ANTI-INFLAMMATORY PHENOTYPE
S.J. Schwulst*, D.M. Trahanas, and H. Perlman. Northwestern University, Chicago, IL
Background: Traumatic brain injury (TBI) results in a rapid and profound immune suppression leaving the host susceptible to secondary infection. In fact, infection is the leading cause of death following TBI. Hypothesis: We hypothesized that circulating monocytes initiate the pathogenesis of TBI-induced immune dysfunction by creating and driving an anti-inflammatory milieu.
Methods: C57BL/6 male mice underwent closed head injury via a weight drop technique (n=34) vs. sham injury (n=18). Blood was collected at 1 day, 3 days, 7 days, 14 days, 1 month, and 2 months post injury. Monocytes and their subsets were identified via flow cytometry and counted via an automated cell counter. Data was analyzed with the statistical software program PRISM and are reported as the mean number of cells/dl ± SEM.
Results: TBI resulted in an early and sustained loss of monocytes as compared to sham-injured mice—1 day (372±22 vs. 1359±267; p<0.01), 7 days (402±63 vs. 993±112; p=0.01), 14 days (514±74 vs. 904±65; p<0.01), and 1 month (477±71 vs. 930±261; p=0.04). The overall monocyte population returned to sham levels 2 months post injury. However, when delineated into the pro-inflammatory M1 subset (Ly6C+/CD62L+) or the anti-inflammatory M2 subset (Ly6C-/CD62L-), the observed reconstitution was entirely accounted for by an increase in the anti-inflammatory M2 subset (Figure 1).
Conclusions: TBI results in an early loss of circulating monocytes followed by a late reconstitution comprised of anti-inflammatory M2 polarized cells. Future studies will determine whether these M2 polarized monocytes differentiate into regulatory macrophages, thus driving a sustained anti-inflammatory milieu leaving the brain-injured host susceptible to secondary infection.
FTY720 EFFECT ON TRAUMA/HEMORRHAGIC SHOCK INDUCED GUT INJURY
J.A. Bonitz*, B. Chandler, J.Y. Son, and E. Deitch*. UMDNJ-New Jersey Medical School, Newark, NJ
Background: Trauma/hemorrhagic shock (T/HS) is one of the major consequences of battlefield injury as well as civilian trauma. FTY720 (sphingosine-1 phosphate agonist) has the capability to decrease the activity of the innate and adaptive immune systems and, at the same time, maintain endothelial cell barrier function and vascular homeostasis during stress. For this reason, we hypothesize that FTY720, as part of resuscitation therapy, would limit T/HS induced multiple organ dysfunction syndrome (MODS) in a rodent hemorrhagic shock model.
Methods: Rats subjected to trauma/sham-shock (T/SS) or T/HS (35 mm Hg x 90 min), were administered FTY720 (1 mg/kg) during volume resuscitation. Lung injury (permeability to Evans Blue dye; EBD), polymorphonuclear leukocyte (PMN) activation (respiratory burst (RB) activity), and red blood cell (RBC) rigidity (rigidity increases reflected in increased Kei) were all tested. In the next set of experiments, lymph duct cannulated rats were used to quantify the effect of FTY720 on gut injury (permeability and morphology) and the biologic activity of T/HS vs. T/SS lymph on PMN-RB and RBC deformability.
Results: T/HS-induced increased lung permeability, PMN activation and RBC rigidity (decreased deformability) were all abrogated by FTY720 (Table). The systemic protective effect of FTY720 was only partially at the gut level, since FTY720 did not prevent T/HS-induced gut injury (morphology or permeability), however, it did abrogate T/HS lymph-induced increased respiratory burst and RBC rigidity.
Conclusion: FTY720 decreased lung injury, red cell injury, and neutrophil activation. Although FTY720 did not limit gut injury either morphologically or by permeability, it did reduce the biologic activity of the lymph.
HMGB-1-INDUCED INDIRECT LUNG INJURY
P.M. Jeziorczak1,2, S. Kaul1,2, E.R. Jacobs2, K.A. Pritchard*1,2, and J.C. Densmore*1,2. 1Children’s Research Institute, Milwaukee, WI, 2Medical College of Wisconsin, Milwaukee, WI
Purpose: No targeted treatments exist for Acute Respiratory Distress Syndrome (ARDS). The pathogenesis of this disease requires an initial increase in endothelial permeability. We have previously described the role of Endothelium Derived Microparticles (EMPs) as an initiator of lung injury. As a putative mechanism, the RAGE receptor is highly expressed in lung tissue, is activated by high-mobility group box-1 (HMGB1) protein, increases vascular permeability, and induces NF-kB transmigration when activated. We hypothesize that HMGB1 is capable of inducing indirect lung injury via RAGE activation, that EMPs contain HMGB1, and that carbenoxolone (Cbnx) will function as an HMGB1 inhibitor.
Methods: EMPs were generated from murine pancreatic endothelial cells stimulated with murine plasminogen activation inhibitor-1 (50 ng/ml). Levels of HMGB1 in EMPs were measured using ELISA. Male C3H/HeOuJ mice aged 6-8 weeks were divided into four groups [n=3/grp; PBS, HMGB1 (10 μg/kg), HMGB1 + EMP (10∧6/ml blood), HMGB1 + Cbnx (6μg/kg), and HMGB1 + Cbnx + EMP]. Permeability data were obtained via Evans Blue Albumin extravasation. Morphometric analysis of cohort lungs was completed. T-tests were performed for statistical analysis.
Results: Lung permeability, neutrophil counts, and edema were significantly increased in HMGB1, HMGB1/EMP, and HMGB1/Cbnx treated mice over PBS (p<0.05). Cbnx was an ineffective HMGB1 blocker in this model of lung injury. HMGB1 was identified in EMPs at a concentration of 2ng/10∧6.
Conclusions: This is the first description of indirect lung injury resulting from intravenously delivered HMGB1. ELISA data show that EMPs contain HMGB1 at low levels. This signal is likely amplified in vivo. The absence of additive effect of EMPs with HMGB1 may reflect maximum injury threshold or saturation of the HMGB1/RAGE pathway. Future work will focus on identifying more effective inhibitors of HMGB1, the RAGE receptor, and NF-kB activation. Intermediate amplifiers of the EMP-HMGB1 signal will be identified as therapeutic targets.
MICROPARTICLES ISOLATED FROM SEPTIC MICE SUPPRESS CARDIAC FUNCTION IN VIVO AND INDUCE CELLULAR INJURY IN MACROPHAGES
M. Gao, X. Zhang, R. Kao, T. Ha, X. Wang, C. Lu, J. Hu, J. Kalbfleisch, D.L. Williams*, and C. Li*. East Tennessee State University, Johnson City, TN
Microparticles have been demonstrated to play an important role in cell-cell communication and to serve as a novel vector for the transfer of proteins and microRNAs from one cell to another. We hypothesized that microparticles released during sepsis/septic shock play a critical role in cardiac dysfunction and systemic inflammatory responses. To evaluate this hypothesis, we induced polymicrobial sepsis by CLP in mice. Sham surgery served as sham control. Microparticles were isolated from serum of septic (septic microparticles) and sham control (sham microparticles) mice and delivered to the myocardium of normal mice through the right carotid artery (n=6/group). Cardiac function was measured with a Millar system before and after delivery of microparticles. Septic microparticles significantly suppress left ventricular pressure (LVP) in normal hearts. LVP was decreased by 45.3% at 60 min and 58.0% at 100 min after delivery of septic microparticles. Vehicle and sham microparticles did not induce changes in LVP. In a separate experiment, microparticles were added to murine J774 macrophages (n=5/group). Twenty-four hrs after treatment, we assessed LDH release, caspase-3/7 and -8 activities and TNFα and IL-6 production. In vitro data revealed that septic microparticles significantly increased LDH activity (↑105%), decreased J774 cell viability (27%), and increased caspase-3/7 and -8 activities. In addition, septic microparticles markedly increased IL-6 and TNFα production in J774 macrophages. Sham microparticles did not induce cellular injury or inflammatory cytokine production in cultured J774 macrophages. We conclude that microparticles produced in response to sepsis may contain DAMPs that induce cardiac dysfunction, cellular injury and inflammatory responses.
MICRO-RNAS FROM EXOSOMES OF SEPTIC SHOCK PATIENTS: A PRELIMINARY ANALYSIS
J. Real1, L. Azevedo1,3, J.E. Bezerra1, F.R. Machado2, and R. Salomao2. 1Hospital Sirio-Libanes, Sao Paulo, Brazil, 2Federal University of São Paulo, São Paulo, Brazil, 3State University of São Paulo, São Paulo, Brazil
Introduction: We previously demonstrated the presence of a subtype of microparticles (exosomes) in the circulation of septic shock patients. Studies showed that these microparticles carry on messenger RNA (RNAm) and micro RNA (miRNA), thus indicating that they may act as transmitters of distant cell signaling. However, the importance of this mechanism during sepsis was not evaluated. We report here preliminary data of the pattern of global miRNA expression in exosomes isolated from sepsis patients. Our objective was to compare these results with normal volunteers and during time course of sepsis.
Methods: In this preliminary study, blood (20 mL) was collected from 14 patients with septic shock in the first 48 hours of disease and, for the survivors, after 7 days. Five healthy volunteers served as controls. Exosomes were isolated by filtration (up to 0.22 mcM) and ultracentrifugation. 30 ng of the total RNA was reversely transcribed and pre-amplified using specific primers and probes. The expression profile of 754 human miRNAs was evaluated by real-time quantitative PCR (qPCR) using the Taqman Low Density Array v3.0 (TLDA - Applied Biosystems). The data were analyzed in StatMiner 3.0 software considering global expression level for normalization, the parameter t-test (Limma) for relative quantification, and the Benjamini-Hochberg multiple testing correction. Fold-change was calculated based on the estimated mean difference (2∧(-DCT)).
Results: Compared to healthy volunteers, 3 miRNAs were differentially expressed in exosomes in the first 48 hours of septic shock (miR-1290 2∧(-DCT) 2.78, p=0.02; miR-1298 2∧(-DCT) 4.02, p=0.03 and miR-146a 2∧(-DCT) -2.51, p=0.02). Additionally, 5 miRNAs presented different levels in septic exosomes seven days after disease as compared to controls (miR-1260 2∧(-DCT) 2.29, p=0.02; miR-1274A 2∧(-DCT) 2.83, p=0.02; miR-1274B 2∧(-DCT) 3.31, p=0.02; miR-192 2∧(-DCT) 1.83, p=0.02; miR-604 2∧(-DCT) -6.41, p=0.02). No different expression profile of miRNAs was observed between the different stages of sepsis.
Conclusion: In this early report, we demonstrate the differential expression of miRNA in exosomes of septic shock patients early in the disease and in the recovery phase of sepsis, as compared to normal individuals. These vesicles may represent new conveyors for genetic material exchange and signaling mechanisms in the vascular milieu during sepsis.
TNF-α STIMULATES NEUTROPHIL DERIVED MICROPARTICLE PRODUCTION THROUGH INCREASED CASPASE-8 AND NF-κB ACTIVITY
B.L. Johnson, and C.C. Caldwell*. Department of Surgery, University of Cincinnati, Cincinnati, OH
Introduction: Neutrophil-derived microparticle (NDMP) numbers are increased under inflammatory or cell death-promoting conditions that can occur during trauma or sepsis. However, the neutrophil signaling pathways activated during NDMP production are relatively unexplored. In pilot studies, we observed that peritoneal NDMPs increasingly accumulate using a murine model of sepsis. In addition, we have found that TNF-α concentrations are increased in the early stages of sepsis. In this study, we hypothesized that treatment of unactivated neutrophils with TNF-α would lead to an increase in NDMP production.
Methods: Bone marrow neutrophils were isolated from healthy mice. These neutrophils were treated with highly specific mediators that altered NDMP generation with the resultant NDMPs characterized and enumerated by flow cytometry.
Results: We observed that TNF receptor 1 (TNFr1) and TNF receptor 2 (TNFr2) activation independently and additively increased NDMP production. In addition, Caspase 8 inhibition diminished NDMPs generated through TNFr1, but not TNFr2, activation. Inhibition of NF-κB abrogates NDMPs generated after activation of either TNFr1 and TNFr2. Altogether, these data suggest that during NDMP generation: 1) NF-κB acts as a downstream regulator of Caspase 8 through TNFr1 activation, and 2) NF-κB acts in a caspase-8 independent manner after TNFR2 activation.
Conclusion: Our data continue to build a framework depicting the molecular mechanisms regulating NDMP generation. Our observations that TNFα, caspase 8, and NFκB can increase NDMP numbers agree with our earlier observation of increased accumulation of NDMPs under inflammatory conditions. This study was funded, in part, by NIH R01 GM100913.
DIETARY RESTRICTION IMPROVES AGE-RELATED INTOLERANCE TO ENDOTOXEMIA BY REDUCING THROMBOSIS
M. Starr*, B.M. Evers, and H. Saito*. University of Kentucky, Lexington, KY
Background: Despite the obvious increased risk of mortality from sepsis in older patients, the underlying mechanisms for this age-related predisposition remain largely unknown. Our previous studies, using murine endotoxemia (LPS) and peritonitis (CLP) models of sepsis, showed that augmented inflammation and thrombosis in the aged is associated with increased mortality. We recently reported that adipose tissue is a major source of pro-inflammatory (IL-6) and pro-coagulant (plasminogen activator inhibitor (PAI)-1 and -2, tissue factor (TF), and thrombospondin (Thbs)-1) factors during endotoxemia and that these factors are more abundantly produced in aged compared to young mice (Aging Cell 2013).
Objective: To test the hypothesis that loss of fat mass in middle-aged (11-12 months) and aged (23-24 months) mice would remove the major source of many pro-inflammatory and pro-coagulant factors and thus improve age-associated sensitivity to endotoxemia.
Methods: To reduce fat mass, we utilized a short-term model of dietary restriction (DR) in which mice were given 40% reduced amount of food. After 3 weeks of DR, mice were injected with LPS (2.5mg/kg initial body weight) and monitored for survival or sacrificed for tissue harvesting. RNA from epididymal fat pads and circulating plasma proteins were analyzed by qRT-PCR and ELISA, respectively.
Results: DR at middle-age effectively reduced body weight and fat mass, and significantly ameliorated the induction of circulating cytokines (IL-6 and IL-1β) and the expression of pro-inflammatory (IL-6) and pro-coagulant (TF, PAI-1, PAI-2, Thbs-1) factors in adipose tissue during endotoxemia. Survival rate of middle-aged mice during endotoxemia was increased by 74% after DR (p=0.01 compared to freely fed age-matched mice). DR in aged mice also reduced body weight and fat mass, and increased survival rate during endotoxemia by 75% (p=0.005 compared to freely fed age-matched mice). Improved survival was associated with reduced expression of pro-coagulant factors in the adipose tissue; however, the inflammatory profile of aged mice during endotoxemia did not change with DR.
Conclusions: Reducing fat mass by restricting food intake significantly increased survival rates during endotoxemia in middle-age and old-age. Improved survival in old-age is likely due to reduced thrombosis but independent of the inflammatory response.
A STROMAL CELL-DERIVED FACTOR (SDF)-1ALPHA ANALOGUE AND ENDOTHELIAL PROGENITOR CELLS SYNERGISTICALLY IMPROVE OUTCOME IN A SEVERE SEPSIS MODEL
H. Fan*1, K. Borg1, P. Halushka1, B. Zingarelli*2, and J. Cook*1. 1Medical University of South Carolina, Charleston, SC, 2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
The SDF-1α analogue CTCE-0214 (CTCE) has been shown to be safe in phase 1 clinical trials and is beneficial in experimental sepsis induced by cecal ligation and puncture (CLP). In the present study we examined the hypothesis that CTCE will enhance endothelial progenitor cell (EPC) function and improve survival in severe CLP sepsis. EPCs were isolated and cultured from human cord blood. Mice were subjected to CLP and administrated CTCE or vehicle (i.v. and s.c.) at 2, 6, 24 and 48 h after CLP. In other groups, mice were administrated i.v. EPCs or HUVEC (control cells) at 24h after CLP. In designated groups the treatment regimen of CTCE or vehicle and EPC or control cells were combined. All mice received imipenem (25mg/kg s.c.) at the same time intervals as CTCE. When treated with CTCE(10mg/kg) or EPCs (1×106 cells/mouse) alone survival to CLP-induced sepsis was improved (80% and 87%, respectively vs. 33% CLP controls, P<0.05). The beneficial survival effect of CTCE or EPC alone was lost when the dose of CTCE was reduced to 1mg/kg or the number of EPCs reduced to (1×105 cells/mouse). However when these sub-threshold doses of CTCE or EPC treatment were combined, survival from CLP-induced sepsis was synergistically improved (CTCE + EPC CLP 80% vs. 33% CLP controls, p<0.05). Subsequent in vitro studies were initiated to determine the effect of CTCE on EPC function. CTCE (1 μg/ml) enhanced EPC proliferation 1.2±0.02 fold vs. vehicle (by MTT assay) and inhibited TNFα-induced EPC senescence 48±3 % vs. vehicle (by β-galactosidase staining). CTCE also greatly augmented endothelial angiogenesis 1.7±0.5 fold vs. vehicle (by tube formation assay). Unstimulated EPCs constitutively express the microRNA miRNA34a which suppresses angiogenesis. CTCE treatment at intervals up to 24h markedly inhibited miRNA34a expression (72±4 %). These data demonstrate that sub-threshold protective doses of CTCE or EPC administration when combined, synergistically improve outcome in severe sepsis. This effect may be a consequence of CTCE enhanced pro-survival signaling in EPCs and CTCE augmented angiogenesis regulated in part by suppressed miR34a expression. (Supported by NIH GM27673, GM67202).
CARBENOXOLONE BLOCKS LPS-INDUCED HMGB1 RELEASE BY IMPAIRING PKR ACTIVATION
W. Li*1,2, M. Ashok1,2, J. Li*, S. Zhu*1,2, A.E. Sama2,1, and H. Wang*1,2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2North Shore University Hospital, Manhasset, NY
Background: The pathogenesis of sepsis is mediated by pathogen- and damage-associated molecular patterns (e.g., bacterial endotoxin and ATP), which activate the double-stranded RNA-activated protein kinase R (PKR) to trigger the inflammasome-dependent release of late proinflammatory mediators (e.g., HMGB1). ATP activates the inflammasome partly through binding to the purinergic P2X7 receptor (P2X7R), triggering the opening of the ATP-gated P2X7R cation-channel and the pannexin-1 (panx-1) hemichannel (for cations and anions up to 1000 Da).
Objectives: To determine whether P2X7R or panx-1 channel inhibitors can abrogate LPS-induced PKR activation and HMGB1 release.
Methods and Results: Here we demonstrated that a panx-1 inhibitor, carbenoxolone (CBX), dose-dependently abrogated LPS-induced HMGB1 release in macrophage cultures with estimated IC50 ≈ 5 μM. In an animal model of sepsis (induced by cecal ligation and puncture, CLP), repetitive CBX administration beginning 24 h after CLP led to a significant reduction of circulating and peritoneal HMGB1 levels, and a parallel increase in animal survival rates (from 42% to 85%, N = 26-27 mice/group, P < 0.05). Like P2X7R antagonists (e.g., oATP), CBX also effectively attenuated LPS-induced ATP-gated channel activation (as judged by the reduction of Lucifer Yellow dye uptake) and PKR phosphorylation in macrophages.
Conclusions: These results suggested that CBX blocks LPS-induced HMGB1 release possibly through impairing ATP-gated channels and PKR activation, supporting the importance of P2X7R and PKR in the regulation of HMGB1 release.
(Supported by the National Institutes of Health Grants R01GM063075 and R01AT05076).
SR-BI IS A KEY DETERMINANT AND HDL IS A CRITICAL MODULATOR OF THYMOCYTE APOPTOSIS IN SEPSIS
X. Li, L. Guo, and Z. Zhong. University of Kentucky, Lexington, KY
Background: Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood.
Methods and Results: We induced sepsis in scavenger receptor BI (SR-BI) null and wild type littermates with cecal ligation and puncture (CLP). CLP induced profound thymocyte apoptosis in SR-BI+/+ mice, but no thymocyte apoptosis in SR-BI-/- mice, indicating that SR-BI is a key determinant of thymocyte apoptosis in sepsis. SR-BI controlled inducible glucocorticoid (GC) generation during sepsis, which is required for triggering thymocyte apoptosis. Unexpectedly, supplementation with GC only partly restored thymocyte apoptosis in SR-BI-/- mice. We demonstrated that HDL is a critical modulator for thymocyte apoptosis. SR-BI+/+ HDL significantly enhanced GC-induced thymocyte apoptosis but SR-BI-/- HDL had no such activity. Further study revealed that SR-BI+/+ HDL modulates GC-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI-/- HDL loses such regulatory activity. To understand why SR-BI-/- HDL loses its regulatory activity, we analyzed HDL cholesterol contents and found that there was 3-fold enrichment of unesterified cholesterol in SR-BI-/- HDL compared with SR-BI+/+ HDL. Importantly, normalization of unesterified cholesterol in SR-BI-/- HDL by probucol administration restored GC-induced thymocyte apoptosis both in vitro and in vivo, and incorporating unesterified cholesterol into SR-BI+/+ HDL rendered SR-BI+/+ HDL dysfunctional.
Conclusions: The findings in this study reveal a previously unrecognized regulatory mechanism of thymocyte apoptosis in sepsis, involving SR-BI as a key determinant and HDL as a critical modulator.
HMGB1 BINDS TO MD-2 IN THE TLR4/MD2 COMPLEX TO ELICIT INFLAMMATORY RESPONSES
H. Yang*1, Z. Ju1, P. Lundback2, L. Ottosson2, M. Ochani*1, J. Li1, B. Lu*1, S. Chavan1, H.E. Harris2, U. Andersson2, and K. Tracey*1. 1Feinstein Institute, Manhasset, NY, 2Departments of Women’s and Children’s Health, Medicine and Rheumatology Research Laboratory, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden
High mobility group box 1 (HMGB1) is a cytokine mediator in the pathogenesis of inflammatory diseases (Yang et al, BBA, 2009). HMGB1 is released from activated immune cells during infection and also from damaged cells during sterile inflammation. Once released, HMGB1 acts through cell surface receptors on immune cells to elicit inflammatory responses. Previously, we showed that TLR4 is the primary receptor needed for promotion of macrophage activation, cytokine release and tissue damage (Yang et al, PNAS 2010). TLR4 activity and interaction with its ligands depend on the extracellular adaptor MD-2 (Visintin et al, 2006). Here we investigated the role of MD-2 in HMGB1-induced cytokine responses. Biosensor-based surface plasmon resonance (BIAcore) analysis revealed that HMGB1 binds human MD-2 with high affinity (apparent Kd = 8nM), but not to TLR4 alone. Knocking down MD-2 using specific siRNA significantly reduced HMGB1-stimulated TNF release in both murine RAW 264.7 and human monocytic THP-1 cells. Supporting these findings, macrophages from MD-2 knockout mice had complete abrogation of HMGB1-induced TNF release; whereas TLR2-dependent TNF stimulation by peptidoglycan (PGN, TLR2 agonist) was not significantly altered. Gain-of-function experiments confirmed that MD-2 is sufficient to restore HMGB1-induced IL-8 release in HEK293 cells over-expressing TLR4. Thus, our data indicate that MD-2 is critical in HMGB1-induced TLR4 signaling and subsequent inflammatory responses. (Supported by grants from NIH, NIGMS RO1GM62508 to KJT and RO1GM098446 to HY).
DAMPS AND ENDOTHELIAL DYSFUNCTION IN A MOUSE MODEL OF STX2 TOXEMIA
C. Mayer, C.S. Leibowitz, S. Kurosawa*, and D. Stearns-Kurosawa*. Boston University, Boston, MA
Introduction: Shiga-toxin producing E. coli (EHEC) is the leading cause of acute renal failure in otherwise healthy U.S. children. The bacterial Shiga-like toxins (Stx1, Stx2) induce severe endothelial dysfunction that can progress to thrombotic microangiopathy with acute kidney injury (AKI), and Stx2 is associated with more severe disease. EHEC also cause significant tissue injury, so we questioned whether damage-associated molecular patterns (DAMPs) such as histones, HMGB1, or mitochondrial DNA (mtD) might contribute to EHEC pathogenesis.
Objective: Determine whether DAMPs are released in vivo in a murine Stx2 toxemia and evaluate the impact of DAMPs on in vitro Stx-induced endothelial dysfunction. We defined endothelial dysfunction here as a shift towards a pro-thrombotic environment.
Methods: In vivo: C57BL/6J mice (n=30) were injected ip at T0 and Day3 with Stx2 (1ng/20g bw). Blood was collected on Day 0, 2, 3, 5, and at sacrifice. Plasma DAMPs (ELISA) and kidney function (BUN) were measured. In vitro: human aortic endothelial cells were incubated with Stx1 or Stx2 (100ng/ml) ± histones (50ug/ml) or mitochondrial DNA (mtD, 1ug/mL) for 24-48 hrs. Surface expression of anti-coagulant thrombomodulin (TM), endothelial protein C receptor (EPCR), and PAR1 were measured by indirect ELISA or flow cytometry.
Results: In vivo: Plasma DAMPs increased after Stx2 challenge in mice. By 48 hours HMGB1 rose 384.2±182.3% relative to T0 values. By 72 hours plasma BUN increased by 23.08±1.9% and plasma histones increased by 118.5±28.8%. In vitro: Stx1, Stx2, or histones decreased TM expression by 15.0±2.9%, 12.2±2.9% and 9.2±3.0%, respectively. They decreased EPCR by 29.6±13.1%, 18.6±0.005% and 38.05±4.1%. Stx2 +histones decreased EPCR by 44.7±5.6%. At 24 hours, Stx1 or Stx2 decreased PAR1 by 23.0±5.9% and 16.0±1.9% respectively. At 48 hours, PAR1 decreased by 15.1±5.0% (Stx1) and 15.5±1.0% (Stx2).
Conclusions: These data show for the first time that Stx induce release of DAMPs in vivo, and that HMGB1 is an early marker, rising before measurable AKI. In vitro Stx and DAMPs shifted endothelium toward a pro-coagulant surface, particularly with Stx2 and histones together as would occur during severe infection. These insights into endothelial dysfunction elucidate exciting new opportunities for therapy during EHEC infections.
SEPSIS-INDUCED CARDIAC AUTOPHAGY IS MEDIATED THROUGH MITOCHONDRIAL REACTIVE OXYGEN SPECIES
Q.S. Zang*, L. Ma, X. Yao, D. Maass, S. Wolf*, and J. Minei*. University of Texas Southwestern Medical Center, Dallas, TX
Objective: Our research has been focused on revealing the mechanisms of sepsis-induced cardiac dysfunction using both in vitro and in vivo sepsis models. This study was designed to investigate the role of mitochondrial reactive oxygen species (mtROS) in sepsis-associated autophagic responses in the heart by utilizing mitochondria-targeted vitamin E (Mito-Vit-E), an antioxidant targeting specifically at mtROS.
Methods: In vivo, SD rats were given sepsis by intratracheal injection of S. pneumoniae (4× 1,000,000 CFU/rat). Mito-Vit-E (21.5 μmoles/kg) or control vehicle was orally administered 30 minutes post-inoculation. Heart tissues were harvested day 1 and day 4 post-inoculation. Autophagy levels were determined by morphology analysis using electron microscopy (EM) and by detected changes in the expression of autophagy marker protein Beclin-1 using western blot. In vitro, neonatal cardiomyocytes overexpressing GFP-tagged LC3 were challenged by lipopolysaccharide (LPS) and treated with Mito-Vit-E. Autophagy levels were quantified by the formation of GFP-foci.
Results: Accumulation of autophagic vesicles and increase in Beclin-1 expression were significant in the heart of septic rats but not in sham and Mito-Vit-E treated rats. In neonatal cardiomyocytes, LPS induced dramatic aggregation of GFP-labeled autophagosomes. This increase was inhibited by Mito-Vit-E.
Conclusion: Our data suggest that mtROS is a critical causative factor for cardiac autophagy after sepsis. Since we have previously shown that Mito-Vit-E improves heart function in the same sepsis model, these data together suggest that sepsis-induced cardiac autophagy is potentially maladaptive, and mitochondria-targeted antioxidants may become an effective therapeutic approach to control this process.
REDUCTION OF IMMUNOCOMPETENT T CELLS FOLLOWED BY PROLONGED LYMPHOPENIA IN SEVERE SEPSIS IN THE ELDERLY
S. Inoue*, K. Suzuki-Utsunomiya, Y. Komori, and S. Inokuchi. Tokai University, Isehara, Japan
Background: Aging is known as a significant factor and is associated with a poor prognosis in sepsis; however, the mechanism of immunological changes in elderly septic patients is still unclear. The purpose of this study was to clarify the immunological changes in sepsis of elderly patients.
Materials and Methods: Seventy-nine patients with severe sepsis and 72 healthy donors (HD) were prospectively enrolled in the study. Blood samples were collected within 48 hours of diagnosis of severe sepsis, and peripheral blood mononuclear cells were purified for flow cytometric analysis.
Results: In elderly septic patients (aged 65 years and over), 3-month survival was significantly reduced compared to that for adult patients (18-64 years) (60% vs. 89%, p < .01). We found that lymphopenia was prolonged for at least 21 days in elderly non-survivors of sepsis, while the number of lymphocytes recovered in both adult and elderly survivors of sepsis. T cell levels were significantly reduced in both adult and elderly septic patients, compared to those in HD patients (56% and 57% reduction, respectively). Interestingly, the immunocompetent CD28+ subset of CD4+ T cells decreased, whereas the immunosuppressive PD-1+ T cells and the percentage of regulatory T cells (CD4+ T cells that are both Foxp3+ and CD25+) increased in elderly patients, especially non-survivors, presumably reflecting the initial signs of immunosuppression.
Conclusion: Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.
PROFOUND VARIATION OF INFLAMMATORY PROFILES ASSOCIATED WITH TWO SNPS OF THE HUMAN GLUCOCORTICOID RECEPTOR
M.V. Lasker1, T.L. Green2, D. Lim2, S.M. Leventhal2, K. Lee2, L. Yee2, K. Cho*2,1, and D.G. Greenhalgh*2,1. 1University of California, Davis, Sacramento, CA, 2Shriners Hospitals for Children, Northern California, Sacramento, CA
Glucocorticoids are indispensable therapeutic agents in diseases of inflammation, but their use in septic shock demonstrates variable outcomes. Previous studies in our laboratory have associated human glucocorticoid receptor (hGR) polymorphisms as one of the likely reasons for this variability. We examined the inflammatory properties of two single nucleotide polymorphisms (SNPs) of the hGR. We hypothesized that the hGR SNPs would elicit unique inflammatory profiles. Two hGR SNPs that were previously identified in our laboratory, T1463C and A2297G, and reference (NCBI) hGRα, were transfected into RAW 264.7 cells (mouse leukemic monocyte/macrophage cell line). Real-time PCR was then used to quantify amounts of mRNA of the following molecules: intercellular adhesion molecule-1 (ICAM), interleukin-1-beta (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and cyclooxygenase-2 (COX2). The results we obtained are graphed below with error bars demonstrating standard error of the mean. T1463C was characterized by significantly higher levels of ICAM (p<.01), iNOS (p<.01), and COX2 (p<.01) when compared to A2297G and reference hGRα. IL-6 was differentially expressed only between T1463C and A2297G (p<.01). A2297G was characterized by significantly lower levels of IL-1β (p<.01) and TNF-α (p<.01) when compared to both T1463C and hGRα. These results suggest that unique proinflammatory molecule profiles elicited by hGR SNPs help dictate the inflammatory state of the organism. This offers a possible explanation for the clinical variability seen amongst individuals in response to stress or shock.
TRAUMA HEMORRHAGIC SHOCK INDUCES CD36 MEDIATED ERYTHROCYTE ADHESION TO ENDOTHELIUM WITH SUBSEQUENT LOSS OF MONOLAYER INTEGRITY AND INCREASED ENDOTHELIAL PERMEABILITY
G. Vinluan1, L. Mason1, E. Feketeova1, M. Condon2, W.N. Duran1, Z. Spolarics1, G.W. Machiedo1, and E. Deitch*1. 1UMDNJ-New Jersey Medical School, Newark, NJ, 2VA New Jersey Healthcare System, East Orange, NJ
Introduction: Increased vascular permeability is common after trauma hemorrhagic shock (T/HS) and leads to decreased microcirculatory flow and multi-organ failure. We hypothesize that erythrocytes from T/HS rats adhere to endothelium and induce endothelial cell (EC) retraction and increase permeability.
Methods: Blood obtained from T/HS and trauma sham shock (T/SS) rats was incubated with human umbilical vein endothelial cells (HUVEC) and erythrocyte adherence, endothelial cell retraction, and endothelial permeability were investigated. Red blood cell (RBC) adherence was quantified by counting the adherent RBC per high power field. EC retraction was measured by planimetry. In vitro permeability was assessed via the Costar two-chambered Transwell System. A panel of antibodies to various RBC and endothelial receptors was used in marking and blocking experiments to determine the receptors responsible for RBC adhesion. Blood from severely injured trauma patients was tested to validate the rat model.
Results: RBC adhesion to HUVEC is increased in T/HS vs. T/SS (69±8 vs. 2.6±0.1 adherent RBC per high power field; p<0.05). This was associated with an increase in CD36 expression (28±2 vs. 2±0.2 % CD36 positivity; p<0.05). Furthermore, incubation with CD36 blocking antibodies abrogated T/HS RBC adherence. T/HS RBC-EC interaction resulted in HUVEC retraction after incubation with T/HS vs. T/SS whole blood (17±5.3 vs. 3.9±0.9 % exposed surface area; p<0.05). A similar extent of EC retraction was observed after incubation with purified RBC, but not with plasma. Incubation with T/HS RBC increased HUVEC monolayer permeability to 40Kd dextran compared to incubation with T/SS RBC (5±1.9 vs. 1.4±1.9 μl/cm2/hr; p<0.05). Erythrocytes from trauma patients also manifest increased adhesion and increased CD36 expression.
Conclusion: We report for the first time that T/HS induces an increase in erythrocyte CD36 expression which augments RBC-EC adhesion and that this results in EC retraction and increased EC monolayer permeability. This may have clinical relevance since this observation is corroborated by data from trauma patients.
EARLY, SINGLE-DOSE POST-BURN ESTROGEN SIGNIFICANTLY DECREASES RENAL PRO-INFLAMMATORY CYTOKINES FOR 45 DAYS FOLLOWING SEVERE BURN INJURY
K. DeSpain, K. Abdelfattah, D. Maass, V. Warren, P.E. Pepe, A. Idris*, Q.S. Zang*, S. Wolf*, J. Minei*, J. Gatson, and J.G. Wigginton*. University of Texas Southwestern Medical Center Dallas, Dallas, TX
Introduction: In patients with severe burns, renal ischemia and injury is thought to be a significant contributor to morbidity and mortality. We tested estrogen, a potent anti-oxidant, anti-apoptotic, and anti-inflammatory drug as a potential therapy for mitigation of kidney inflammation following severe remote thermal injury.
Methods: 177 male rats were randomized into 3 groups: Group 1 (0.5mg/kg of 17β estradiol (E2) 15 min after injury), n=84; Group 2 (placebo), n=84; and Group 3 (sham-burn control), n=9. Groups 1 & 2 were given a 3° 40% TBSA dorsal scald burn. Eight burned/treated rats per group were sequentially sacrificed at 0.5, 1, 2, 4, 6, 8, 12, 18, 24 hours and 7 days post-burn, with four rats/treatment sacrificed 45 days post-burn. Renal tissue was analyzed by ELISA for pro-inflammatory cytokines (including IL-6, TNF-α, IL-1β).
Results: Administration of a single, early dose of E2 greatly decreased all measured cytokines for 45 days, as exemplified by TNF-α (see Figure).
Conclusions: Early, single-dose estrogen administration following severe remote burn injury reduces the pro-inflammatory response in the renal tissue at all measured time points for 45 days post-burn. These findings help support consideration of clinical testing of estrogen as a post-burn resuscitation therapy in the future.
DEFICIT IN INTESTINAL TRANSIT ACCOMPANIES AN INCREASE IN GUT BACTERIAL LOAD AND GUT INFLAMMATION AFTER BURN INJURY
S. Akhtar, X. Li, R. Shankar, R.H. Kennedy, R.L. Gamelli*, and M.A. Choudhry*. Loyola University Chicago Health Sciences Division, Maywood, IL
Intestinal ileus/stasis or a decrease in intestinal transit has been documented after burn and may contribute to an increase in gut bacterial growth after burn injury. We examined the effect of burn injury on intestinal transit and determined whether any change in intestinal transit has a relationship with gut bacterial load and gut inflammation. To accomplish this, 8-10 week old male C57Bl/6 were anesthetized and subjected to ∼20% total body surface area burn injury by exposing to ∼85oC water in a bath for 7-8 sec. Sham-injured mice received identical anesthesia, but were immersed in isothermic water. Mice were sacrificed one day after injury. Intestinal (distal ileum) luminal content and intestinal tissue were harvested, homogenized and plated on tryptic soy agar and MacConkey agar for the analysis of total and Gram-negative bacterial colony forming units (CFU), respectively. Intestinal tissue homogenates were analyzed for inflammatory mediators IL-6 and KC using their respective ELISA. For intestinal transit, a group of sham and burn mice were gavaged with 0.4 ml of 22 mg/ml FITC-dextran 3 hours prior to sacrifice. Stomach, intestinal and colonic luminal contents were collected, and FITC levels were determined. As compared to sham, a significant decrease (p<0.05) in intestinal transit was noted one day after burn injury. This was accompanied with a several fold increase in total and Gram-negative bacterial CFU both in the intestinal tissue and luminal content. Furthermore, the levels of IL-6 and KC were significantly (p<0.05) elevated in the intestine one day after injury. Together, these findings suggest that the decrease in intestinal transit may contribute to increases in gut bacterial growth and inflammation after burn injury. (Support: W81XWH-10-2-0172; R01AA015731).
TLR4 MEDIATES THE INFLAMMATORY RESPONSE OF REMOTE NON-ISCHEMIC MYOCARDIUM AND CONTRIBUTES TO HEART FAILURE IN A MOUSE MODEL OF MYOCARDIAL INFARCTION
Y. Zhai, L. Ao, N. Yousif, F. Al-amran, D. Fullerton, and X. Meng*. University of Colorado Denver, Aurora, CO
Toll-like receptor 4 (TLR4) plays a role in acute myocardial inflammation associated with myocardial ischemia/reperfusion (I/R) injury. Inflammation is implicated in the mechanism underlying adverse left ventricle (LV) remodeling after myocardial infarction. However, it is unclear whether TLR4 mediates an inflammatory response in remote non-ischemic myocardium and whether TLR4 plays a role in post-infarction heart failure. We hypothesize that TLR4 mediates post-infarction inflammatory response of non-ischemic myocardium and contributes to the mechanism of heart failure. The purposes of this study were to determine: 1) the effect of TLR4 mutation on the expression of chemokines and adhesion molecules, and mononuclear cell accumulation in non-ischemic myocardium of mouse heart at day 7 and 14 after regional myocardial I/R and 2) whether monocyte depletion improves cardiac function at day 14.
Methods and Results: C3H/HeJ (TLR4 mutant, with dysfunctional TLR4) and C3H/HeN (TLR4-competent) mice were subjected to left descending coronary artery ligation (30 min) and reperfusion for 7 or 14 days. In TLR4-competent mice, levels of MCP-1 and KC were not elevated in non-ischemic myocardium. Interestingly, ICAM-1 and VCAM-1 levels were increased in non-ischemic myocardium at day 7 and 14. Increased ICAM-1 and VCAM-1 levels were associated with higher density of mononuclear cells in non-ischemic myocardium. TLR4 mutation markedly reduced the changes in adhesion molecule levels and mononuclear cell densities in non-ischemic myocardium. More importantly, LV function measured by microcatheter at day 14 was significantly improved in TLR4-mutants. Further, prior depletion of monocytes in TLR4-competent mice using liposomal clodronate reduced mononuclear cell accumulation in non-ischemic myocardium, and resulted in improved LV function at day 14 after I/R.
Conclusion: These findings provide evidence that TLR4 mediates the inflammatory response of remote, non-ischemic myocardium and contribute to post-infarction heart failure. Modulation of TLR4 signaling and mononuclear cell accumulation may have therapeutic potential for protection of heart function after myocardial infarction.
RESVERATROL PROTECTS PRIMARY HEPATOCYTES FROM THE OXIDATIVE STRESS ASSOCIATED WITH HYPOXIC INJURY
R.D. Powell*, E. Brandon-Warner, T. Huynh*, I.H. McKillop, and S.L. Evans*. Carolinas Medical Center, Charlotte, NC
Introduction: Ischemia-reperfusion after hemorrhagic shock results in hypoxic injury and oxidative stress, leading to cellular damage and multiple organ dysfunction syndrome (MODS). The liver is a key organ involved in MODS and is uniquely vulnerable to oxidative stress during hemorrhagic shock resuscitation (HSR). The aims of this study were to determine if the antioxidant resveratrol protects mitochondria (major intracellular site of ROS production), and prevents cell death in a hepatocyte model of hypoxic injury and re-oxygenation in vitro.
Methods: Primary hepatocytes were isolated from 250-300g male Sprague-Dawley rats. Hepatocyte purity was >95% with ≈97% viable (trypan-blue exclusion). Cells were cultured on collagen-coated plates (1.0×106 cells/well) to achieve 65-70% confluence. Cells were exposed to 8-Hrs hypoxia (CO2 ≥10%; HYP) with and without pretreatment with resveratrol (RES; 75μM). Cells were analyzed immediately for survival and markers of hypoxic injury or after 6-hrs hypoxia reversal. Cell survival was determined using a lactate dehydrogenase (LDH) cytotoxicity assay, and mitochondrial integrity was analyzed with a fluorescent JC-1 probe.
Results: Hypoxia (8Hrs) followed by normoxia (6Hrs) (HYP) resulted in 37±3.4% cell death (p<0.05 vs. non-hypoxic control; C). Cells treated with resveratrol (RES) prior to hypoxic injury significantly inhibited cell death to 15±2.4% (n=4, p<0.05 vs. Hyp). Analysis of culture medium LDH demonstrated hypoxia alone led to a 3-fold higher level of LDH (p<0.05 vs. C). In contrast, RES abrogated the effects of hypoxia on LDH measurement to a level not significantly different to C. Analysis of mitochondrial integrity with JC-1 probe at 560/595nm (red) demonstrated RES cells were 45-55% more likely to retain mitochondrial integrity vs. HYP (p<0.05, n=3). Conversely, analysis at 485/535nm (green) demonstrated HYP cells were 2-fold more likely to undergo mitochondrial membrane depolarization compared to C, whereas RES reversed mitochondrial membrane depolarization to a level not significantly different to C.
Conclusions: Our studies demonstrate RES treatment preserves mitochondrial integrity in primary hepatocytes and protects against hypoxia-induced oxidative stress and cell death. These data suggest antioxidant therapy may protect against cell damage that contributes to organ dysfunction and MODS related to HSR.
HYDROGEN GAS REDUCES HYPEROXIC LUNG INJURY VIA THE NRF2 PATHWAY
A. Nakao1,2, T. Kawamura2,3, M. Aoyama*1, and J. Kotani*1. 1Hyogo College of Medicine, Nishinomiya, Japan, 2University of Pittsburgh, Pittsburgh, PA, 3Osaka University, Osaka, Japan
Introduction: Highly concentrated oxygen is administered to patients who can’t breathe efficiently, however oxygen toxicity can occur over a prolonged period, causing hyperoxic lung injury. Successful abrogation of hyperoxic lung injury would have a huge impact on critical care medicine. Recently, hydrogen has been recognized as a therapeutic gas and a gaseous signaling molecule. We have demonstrated that hydrogen can reduce hyperoxic lung injury and induce heme oxygenase (HO)-1, a heme-degrading enzyme that is highly induced by oxidative stress and that protects against oxidative damage. The purpose of this study was to investigate underlying mechanisms of the HO-1 induction by hydrogen. We highlighted the critical role of the redox-dependent transcription factor, NF-E2-related factor 2 (Nrf2), in regulating expression of heme oxygenase (HO)-1.
Methods: We randomly assigned wild type (Nrf2+/+) or Nrf2-deficient (Nrf2-/-) C57BL/6J mice to four experimental groups and administered the following gas mixtures for 60 hours: 1) air with 2% nitrogen, 2) air with 2% hydrogen, 3) 98% oxygen with 2% nitrogen, 4) 98% oxygen with 2% hydrogen. We examined lung function by blood gas analysis of the arterial blood. We also examined tissue lipid peroxidation by assessing malondialdehyde (MDA) levels, histology and expression of HO-1 and Nrf2-dependent genes.
Results: When mice were exposed to hyperoxia, pO2 levels decreased, and tissue MDA levels increased. Hydrogen treatment significantly improved blood oxygenation in Nrf2+/+ mice and reduced lung MDA levels in both Nrf2+/+ and Nrf2-/- mice. However, there was no protective effect in Nrf2-/- mice. Nrf2-/- mice exposed to hyperoxia had more marked histological changes even in the presence of hydrogen. Real-time RT-PCR revealed that the Nrf2-dependent genes (HO-1, NAD(P)H dehydrogenase quinine (Nqo)-1 and glutathione S-transferase (GST) A2) were up-regulated in the Nrf2+/+ mice lungs receiving hydrogen during hyperoxic conditions. In Nrf2-/- mice, hydrogen treatment did not induce HO-1, Nqo1, or GSTA2. This result suggests that hydrogen induced HO-1 via the Nrf2 signaling pathway.
Conclusions: Our study demonstrated that hydrogen ameliorates hyperoxic lung injury by modulating the Nrf2 signaling pathway. The findings suggest a potentially novel and easily applicable solution to hyperoxic lung injury and provide new insight into our scientific knowledge of hydrogen.
GLYCOGEN SYNTHASE KINASE-3 CONTROLS AUTOPHAGY DURING RENAL ISCHEMIA/REPERFUSION
E. Esposito1, A. Ahmad1, M. Campolo1, I. Paterniti1, and S. Cuzzocrea*1,2. 1University of Messina, Messina, Italy, 2Manchester Biomedical Research Centre, Manchester Royal Infirmary, School of Medicine, University of Manchester, Manchester, United Kingdom
Autophagy occurs at basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis. In response to cellular stress, autophagy is up-regulated and can provide an adaptive strategy for cell survival, but may also directly or indirectly lead to cell demise. With the dual role in life and death, autophagy is involved in various physiological processes, and linked to the pathogenesis of a wide array of diseases. However, the defined role of autophagy is further complicated by the crosstalk and coordinated regulation between autophagy and apoptosis. In this study, we determined the contribution of autophagy and apoptosis in renal tubular cell injury using different models of renal ischemia/reperfusion (I/R) and C57BL/6 mice (6 mice in each group) were anesthetized using a ketamine (150 mg/kg) and xylazine (15 mg/kg) mixture i.p., and subjected to sham operation or 30 minutes of bilateral renal ischemia, followed by reperfusion (6 and 24 hrs). On reperfusion a significant amount of LC3-II accumulated in renal tissues in a time-dependent manner, starting at 6 hours and further increasing after 24 hours (2.1-fold over control). Moreover, I/R increased mitochondrial cytochrome C release, renal O2(-*) level and 3-nitrotirosine accumulation, Beclin-1 expression (4-fold over control), Bax/Bcl-xL ratio, caspase 3 expression and poly-(ADP-ribose)-polymerase fragments. We found that I/R-induced damage was significantly decreased by intraperitoneal injection of the inhibitor of GSK-3β SB216763 (0.6 mg/kg i.p., 5 minutes prior to reperfusion) in mouse kidney. A higher level of autophagy was also detected after SB216763 treatment. In the Human Embryonic Kidney (HEK) 293 cell line following hypoxia and reoxygenization, SB216763 (5 μM) activated autophagy and suppressed inflammatory response. These data show suppressed kidney damage by activating autophagy after ischemic kidney injury, thus offering a new target for prevention of kidney damage.
POST-TRAUMA ABERRANTLY ACTIVATED NEUTROPHILS MAY CONTRIBUTE TO ANTIGEN PRESENTING CELL DEFECTS
G. Bandyopadhyay*, P.E. Bankey*, and C.L. Miller-Graziano*. Department of Surgery, University of Rochester Medical Center, Rochester, NY
We previously showed that a cohort of trauma patients (Pt) with impaired MO to DC differentiation (Dysf-DC) has depressed immunity and increased infection risk. Elevated thrombospondin-1 (TSP-1) levels in these Pt correlates with MO differentiation dysfunction and subsequent generation of a dysfunctional CD16+DC subset with decreased T cell stimulatory capacity. CD16 expressing DC have been suggested as tolerogenic rather than immunostimulatory. An increase in circulating neutrophil (PMN) numbers, prolonged PMN activation and delayed PMN apoptosis also characterize these Pt. TSP-1 activates SHP-1 phosphatase through its receptor CD47. SHP-1 inhibition increases PMN apoptosis, suggesting TSP-1 activation could delay PMN apoptosis inducing aberrant activation. We assessed TSP-1 activated control PMNs’ and Pt PMNs’ ability to induce Dysf-DC via increased triggering of inhibitory receptor ILT-5 (trauma activated PMN express increased ILT-5 mRNA levels).
Experimental Design: Trauma patients isolated neutrophils and TSP-1 mimic treated control PMN (Tm-PMN) were assessed for co-inhibitory receptors. MO from healthy donors were co-cultured 3 days with Pts’ PMN or TSP-1-mimic treated control PMN [MO: PMN = 1:10] and then re-isolated using magnetic beads. Re-isolated MO were in vitro differentiated to DC using IL-4 + GM-CSF. DC receptor expression and T cell stimulatory capacity were compared between DC differentiated from those PMN-exposed MO and the controls.
Result: Trauma patients’ (Pt) increased neutrophil expression of ILT-5 [1.6 ± 0.2 fold in Pt with Dysf-DC vs. 0.8 ± 0.6 in DC-competent Pt] correlated with DC differentiation defects [*p < 0.05 by Spearman test]. Tm-PMN showed delayed apoptosis and increased ILT-5 expression [2.3 ± 0.9 fold increase]. Exposure to both Pts’ PMN and Tm-PMN resulted in decreased differentiated DC numbers [58.8% and 52.1% respectively compared to control PMN-treated MO]. The few differentiated DC from MO previously exposed to Pt PMN or Tm-PMN had increased CD16 expression [177% and 156% of Cnt PMN-treated MO derived DC respectively] and reduced T cell activation capacity.
Conclusion: Our data suggest that post-trauma TSP-1 induced activated PMN can negatively affect MO differentiation to immuno-stimulatory DC thus contributing to decreased T cell stimulatory capacity.
GLUTAMINE MITIGATES LUNG INJURY INDUCED BY GUT ISCHEMIA/REPERFUSION VIA INTESTINAL PPAR-GAMMA
Z. Peng, K. Ban, and R.A. Kozar*. University of Texas Medical School at Houston, Houston, TX
Objective: The mortality of multiple organ dysfunction syndrome (MODS) remains high, in part due to lack of early interventions to prevent shock-induced gut dysfunction. We have demonstrated that enteral glutamine protects the post ischemic gut and protection is associated with peroxisome proliferator activating receptor (PPAR-gamma). Since gut protection is thought to lessen MODS, we hypothesized that enteral glutamine protects the gut and mitigates MODS via intestinal PPAR-gamma.
Methods: PPAR-gamma conditional knockout mice (KO) targeted deletion of PPAR-gamma in intestinal epithelial cells (IEC) and C57BL/6J(WT) were subjected to 1 hour of intestinal ischemia through occlusion of the mesenteric artery, followed by 6 hours reperfusion (IR). The glutamine group received enteral glutamine (60 mM) at the time of reperfusion. Sham mice underwent the same operative intervention except for clamping of the superior mesenteric artery. Lungs and bronchoalveolar lavage (BAL) were analyzed with lung injury and inflammatory parameters.
Results: There were no differences between sham WT and KO mice. There was a significant increase in lung injury, edema and inflammation after IR in WT mice that were further increased in PPAR-gamma KO mice. Glutamine significantly reduced all parameters in WT but not KO mice (table).
Conclusions: Using an IEC-targeted loss-of-function approach, we have shown for the first time that enteral glutamine reduced lung injury in the post ischemic gut via intestinal IEC PPAR-gamma. These data suggest that early enteral glutamine may be a potential therapeutic modality to reduce shock-induced gut dysfunction and subsequent distant organ injury.
REDUCTION IN GUT COMMENSAL FLORA PROTECTS AGAINST VENTILATED LUNG ISCHEMIA REPERFUSION (IR) INJURY BY PREVENTING RECRUITMENT OF DIFFERENTIALLY ACTIVATED NEUTROPHILS
A. Prakash*, S.V. Sundar, and J. Hellman*. University of California San Francisco, San Francisco, CA
Background: Ischemia reperfusion (IR) injury is one type of sterile inflammatory process that can occur in routine and critical clinical scenarios, from tourniquet use in orthopedic surgical procedures to reperfusion following cardiac arrest or organ transplantation. Acute lung injury (ALI) is a major source of morbidity and mortality in severely-ill patients in part due to direct or remote IR injury. Moreover, the concurrent incidence of pulmonary infections in these patients, not unexpectedly, adds to the burden of disease. We previously validated a clinically-relevant surgical model of ventilated lung IR and demonstrated a role for TLR4 and alveolar macrophages in this process.
Methods: Mice were treated for 6-8 weeks with gut-localized antibiotics (neomycin and polymyxin B) to deplete gut commensal flora. Control mice did not receive antibiotics. The mice underwent reversible left pulmonary artery occlusion without interruption in ventilation. Lung histology and plasma cytokine levels were assayed at various times post-IR. Separately, alveolar macrophages were stimulated ex vivo and release of inflammatory mediators assessed.
Results: In the mouse ventilated lung IR model, we determined that neutrophil recruitment appeared to peak early following IR without long-term damage to the affected lungs. Additionally, the neutrophils recruited were minimally activated compared to those recruited in response to infectious stimuli. Furthermore, as part of the investigation into the endogenous TLR4 ligands that maybe involved in lung IR responses, we examined the role of gut commensal flora. Their depletion resulted in marked reduction in inflammatory cell recruitment to the lung. Alveolar macrophages from these antibiotic-treated mice were found to be less responsive ex vivo when stimulated with TLR agonists and inflammasome activators as compared to those from antibiotic-untreated mice. This suggests that circulating endotoxin from commensal flora may prime alveolar macrophages to respond to lung IR. However, the neutrophils recruited are not activated and do not appear to cause lasting injury in the affected lungs. This could be part of an evolutionary mechanism to mobilize innate immune cells to sites of IR in order to generate inflammation only when pathogen is present.
ENDOGENOUS UBIQUINOL, OXIDATIVE STRESS, AND KIDNEY INJURY FOLLOWING CARDIAC SURGERY
F.T. Billings1, J. Gamboa1, C.K. Yeung2, D.D. Shen2, J. Himmelfarb2, and A. Ikizler1. 1Vanderbilt University, Nashville, TN, 2University of Washington, Seattle, WA
Intraoperative oxidative stress independently predicts the development of acute kidney injury (AKI) following cardiac surgery, and the pattern of F2-isoprostane vs. isofuran expression might be consistent with renal mitochondrial dysfunction. We measured ubiquinol, ubiquinone, F2-isoprostane, and isofuran concentrations prior to, during, and one day following cardiac surgery in 40 patients that developed stage I AKI (defined using AKIN criteria) and 40 risk-matched control patients to test the hypothesis that endogenous ubiquinol and ubiquinol/ubiquinone redox ratios are associated with oxidative stress and kidney injury following cardiac surgery.
Baseline and intraoperative subject characteristics were similar between AKI and control subjects. Preoperative concentrations of ubiquinol and ubiquinone were 590 ng/ml (IQ range 346, 870) and 87 ng/ml (53, 137) in AKI subjects vs. 388 (284, 587) and 60 (38, 103) in control subjects (P=0.02 ubiquinol, P=0.02 ubiquinone). Ubiquinol concentrations decreased 234 +/- 262 ng/ml during surgery in all subjects but 164 ng/ml more (95% CI: 50 to 279, P=0.003) and 11.7% more (P=0.05) in AKI subjects (Figure). Each 10 unit increase in baseline ubiquinol/ubiquinone redox ratio, indicative of reduction potential, was associated with a 12.2 pg/ml (95% CI: 5.3 to 19.2, P=0.001) and a 14.7 pg/ml (95% CI: 3.2 to 26.2, P=0.01) reduction in circulating F2-isoprostanes and isofurans on postoperative day 1, adjusted for BMI and statin use. Urine isofuran/F2-isoprostane ratios were 113% higher on postoperative day 1 in AKI subjects (P=0.002), were increased in patients with low baseline ubiquinol/ubiquinone redox ratios (r = -0.26, P=0.02, Figure), and may indicate oxidative stress in presence of dysfunctional mitochondria and AKI.
Ubiquinol plasma concentrations decrease more during cardiac surgery in patients that develop AKI, and high baseline ubiquinol/ubiquinone redox ratios are associated with reduced oxidative stress during and following surgery.
INTERLEUKIN-15 AND SEPSIS: FRIEND OR FOE?
Y. Guo1, and E. Sherwood*2. 1Vanderbilt University, Nashville, TN, 2Vanderbilt University, Nashville, TN
Sepsis is a common and devastating syndrome in critically ill patients that causes prolonged hospitalization and death. However, the immunological mechanisms underlying sepsis remain poorly understood and few treatment options are available. Interleukin (IL)-15 is a non-secreted cytokine that is trans-presented with its receptor α chain by a variety of cell types to activate the β and common γ chain on lymphocyte populations, particularly natural killer (NK) and memory CD8+ T cells and plays an essential role in the development, survival and functional maturation of IL-15-dependent cells. Data generated in our lab shows that mice with deficiency in IL-15 (IL-15KO) are protected from septic shock induced by cecal ligation and puncture (CLP) when compared to wild type mice. IL-15 KO mice display less sepsis-induced hypothermia (32.7 ± 1.2 vs. 25.4 ± 0.6 °C, p< 0.001) and attenuated IL-6 production in plasma (14577 ± 6926 vs. 40519 ± 9424 pg/ml, p< 0.02) compared to wild type (WT) mice in a lethal CLP procedure, although bacterial counts in blood and peritoneal cavity were not significantly (p>0.05) affected. Treatment of mice with IL-15/IL-15Rα complex (IL-15 superagonist) at a dose of 5 μg, but not at ≤ 2 μg, amplifies sublethal CLP-induced hypothermia (28.4 ± 1.8 vs. 36.1 ± 0.5 °C, p< 0.01) and IL-6 production (43901 ± 15502 vs. 4985 ± 3054 pg/ml in plasma, p<0.05). Examination of lymphocyte populations in IL-15 KO mice shows greatly decreased NK and memory CD8+ T cell populations that can be restored by exogenous IL-15 superagonist treatment (Figure). Antibody-induced depletion of NK and CD8+ T cells causes attenuated hypothermia and IL-6 production as well as improved survival in WT mice. Therefore, IL-15 appears to be an important mediator of inflammatory responses during acute septic shock and contributes to the lethality of septic mice, possibly by activation of NK and memory CD8 T lymphocytes.
CYTOKINES INDUCE DISTINCT ACTIVATION SIGNATURES IN THE VAGUS NERVE
S.S. Chavan, S. Robbiati, P.T. Huerta, and K.J. Tracey*. Feinstein Institute for Medical Research, Manhasset, NY
The mammalian immune system and the nervous system coevolved under the influence of infection and sterile injury. In fact, stimulation of the vagus nerve inhibits cytokine release, attenuates tissue injury, and ameliorates inflammation-mediated injury in models of inflammatory diseases. This neural circuit, termed ‘the inflammatory reflex’, requires action potentials (also called “spikes”) arising from the vagus nerve. Recent developments of neurophysiological and immunological techniques have enabled the study of efferent neural circuits that maintain immunological homeostasis and are essential for health in mammals. Here, we have mapped the spike patterns of afferent fibers of the vagus nerve in response to systemic cytokines. We recorded in vivo from the cervical branch of the vagus in anesthetized, adult BALB/cJ mice. Following 10 min of recording, in which the vagus showed ∼20 Hz baseline spiking, we injected (i.p.) a bolus of endotoxin (lipopolysaccharide, LPS) TNF, or IL1. We found that LPS induced an interesting time-dependent increase in spiking, consisting of an early peak at ∼10 min and a late peak at ∼20 min post-injection. Injection of TNF and IL1 induced dose-dependent increases in vagus nerve activity. Physiologically high levels of TNF (50 microgramg/mouse) induced a doubling of spiking, from 20 Hz to 40 Hz, whereas lower TNF levels failed to produce an increase. Administration of IL1 induced a tripling of spiking, from 20 Hz to 60 Hz, with a very early peak at ∼5 min post-injection. Use of principal component analysis, auto-correlograms and cross-correlograms of the vagus spikes recorded has allowed us to identify cytokine-specific signatures, which likely belong to different axonal populations. In particular, there seems to be an IL1-specific set of fibers that respond rapidly and at high-frequency. Taken together, these studies unravel the neural mechanisms by which peripheral neural circuits sense the inflammation in the body.
TRAUMATIC BRAIN INJURY REDUCES PNEUMONIA RISKS IN PATIENTS AND MICE
S. Yang1, D.M. Stepien2, D. Hanseman1, M. Goodman1, T. Pritts*1, B. Robinson1, C.C. Caldwell*1, D.G. Remick*2, and A. Lentsch*1. 1University of Cincinnati, Cincinnati, OH, 2Boston University, Boston, MA
Traumatic brain injury (TBI) is major component of traumatic events, both in civilian and military populations, and is associated with significant morbidity and mortality. A primary contributing factor is infectious complications, particularly pneumonia, which is thought to be induced by immunosuppression after neurotrauma. Pneumonia rates for moderate and severe TBI patients (Glasgow Coma score <8) have been reported to be 20-60% and are thought to be increased compared to non-head injured patients. In the current study, we evaluated pneumonia rates in over 700,000 trauma patients using the National Trauma Data Bank. When propensity scoring was used to closely match patient cohorts, we found that ventilated patients with TBI had significantly reduced rates of pneumonia compared to their matched blunt traumatically injured controls. Supportive of these clinical findings, we found that mice subjected to mild TBI had improved survival after pneumonia or abdominal sepsis. TBI mice had increased recruitment of neutrophils and macrophages into the lung. Furthermore, there was increased bacterial clearance after pneumonia. Administration of a substance P antagonist abrogated the survival benefits observed in mice with mild TBI suggesting a mechanism for improved survival. Together, our data demonstrates that patients with TBI have reduced rates of pneumonia compared to matched blunt trauma patients without TBI. These findings are supported by our preclinical studies showing mild TBI enhances bacterial clearance and improves survival. Furthermore, these effects may be mediated by the release of substance P after TBI.
INSULIN TREATMENT DIRECTLY RESTORES NEUTROPHIL PHAGOCYTOSIS AND BACTERICIDAL ACTIVITY IN DIABETIC MICE, THEREBY IMPROVING SURGICAL SITE INFECTION WITH STAPHYLOCOCCUS AUREUS
M. Kinoshita*1, H. Yano2, S. Ono3, A. Kimura4, R. Takahata4, Y. Tanaka2, and S. Seki1. 1National Defense Medical College, Tokorozawa, Japan, 2Department of General Medicine, Tokorozawa, Japan, 3Division of Traumatology, Research Institute, Tokorozawa, Japan, 4Department of Surgery, Tokorozawa, Japan
Bacterial infections including surgical site infections (SSI) are a common and serious complication of diabetes. Staphylococcus aureus (S. aureus), which is mainly eliminated by neutrophils, is a major cause of SSI in diabetic patients. However, the precise mechanisms by which diabetes predisposes to staphylococcal infection are not fully elucidated. The effect of insulin on this infection is not also well understood. We therefore investigated the effect of insulin treatment on SSI and neutrophil function in diabetic mice. S. aureus was inoculated into the abdominal muscle in diabetic db/db and high fat diet (HFD)-fed mice with or without insulin treatment. Although the diabetic db/db mice developed SSI, insulin treatment ameliorated the infection. db/db mice had neutrophil dysfunction, such as decreased phagocytosis, superoxide production and killing activity of S. aureus; however, insulin treatment restored these functions. Ex vivo treatment (co-incubation) of neutrophils with insulin and euglycemic control by phlorizin suggest that insulin may directly activate neutrophil phagocytic and bactericidal activity independently of its euglycemic effect. However, insulin may indirectly restore superoxide production by neutrophils through its euglycemic effect. HFD mice with mild hyperglycemia also developed more severe SSI by S. aureus than control mice and had impaired neutrophil phagocytic and bactericidal activity, which was improved by insulin treatment. Unlike db/db mice, in HFD mice superoxide production was increased in neutrophils and subsequently suppressed by insulin treatment. Glycemic control by insulin also normalized the neutrophil superoxide-producing capability in HFD mice. Thus, insulin may restore neutrophil phagocytosis and bactericidal activity, thereby ameliorating SSI.
AGE-DEPENDENT CHANGES OF AMP-ACTIVATED PROTEIN KINASE PATHWAY CORRELATE WITH KIDNEY INJURY IN HEMORRAGIC SHOCK
B. Zingarelli*, G. Piraino, P.W. Hake, and M. O’Connor. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Age is an important factor for the development of multiple organ dysfunction in trauma patients. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis by modulating mitochondrial biogenesis and autophagy. Here, we investigated whether hemorrhage-induced kidney injury is age-dependent and whether it is associated with changes of AMPK activation. Hemorrhagic shock was induced in anesthetized young (2-3 months), mature (11-12 months) and old male rats (22-24 months old) by withdrawing blood to a mean arterial pressure of 50 mmHg. After 3h, rats were rapidly resuscitated by infusing the shed blood. Kidneys were harvested 3h after resuscitation. In young rats, kidney injury was characterized by mild edema and infiltration of neutrophils (myeloperoxidase activity 2.8±0.5 U/100 mg tissue). In contrast, severe tubulo-interstitial injury was observed in mature and old rats and tissue myeloperoxidase activity was significantly higher (5.1±0.8 and 7.7±0.9 U/100 mg tissue, respectively) when compared to young rats (P<0.05). At Western blot analysis, there was increase of nuclear translocation of the active catalytic subunit pAMPKα in young rats, but not in mature or old rats. Since AMPK regulates mitochondrial function through peroxisome proliferator-activated receptor γ co-activator α (PGC-1α), we also evaluated the expression of this co-factor. Young rats exhibited an increase of PGC-1α nuclear translocation at the end of resuscitation. Interestingly, in mature and old rats PGC-1α was retained in the cytosol, suggesting an impairment of nuclear translocation. We next determined the capacity to form autophagic vesicles by evaluating the expression of the light chain (LC3B) I and II isoforms. Under basal conditions LC3B-II values were similar in sham rats of all ages. At the end of resuscitation, young rats exhibited an increase of LC3B-II conversion in the kidney (3.80±0.55 relative intensity), suggesting formation of autophagic vesicles. However, LC3B-II conversion was significantly reduced in mature and old rats (1.31±0.15 and 0.49±0.06 relative intensity, respectively) when compared to the young group (P<0.05). Thus, our data suggest that during hemorrhagic shock AMPK-dependent mechanisms of autophagy and mitochondrial repair are activated. However, this metabolic recovery diminishes with age. (Supported by NIH R01 AG-0227990).
EE-3SO4 GIVEN AFTER TRAUMATIC BRAIN INJURY (TBI) ENHANCES PHYSICAL ACTIVITY AND RESTORES PLASMA ICAM-1
A. Ayub, W.J. Hubbard, C.L. Floyd, N. Day, and I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
We have shown that 17β-estradiol (E2) given 1 hr after TBI reduces brain edema 50% measured 24 hrs after iv E2. To study if ethynyl estradiol-3SO4 (EE; longer T½ than E2) has similar or enhanced physiological protective effects post-TBI, three groups of adult male rats ∼300g (n=4/group) underwent lateral fluid percussion injury (LFP) for TBI or sham operation. Groups 1 and 2: TBI induction 1 day post-craniectomy; group 3: sham animals. Group 1 was treated with EE (1mg/kg) and group 2 with saline (vehicle; Veh) 1 hr post-injury. We utilized passive infrared (PIR) motion detection to determine physical activity for 3 days. Plasma ICAM-1 levels were evaluated 24 hrs post-injury by ELISA. Six to 8 hrs post-injury PIR sensors were placed in cages. Single animals were monitored for movement activity. Motion events were recorded every 10 sec. Activity was monitored during dark cycle (6PM to 6AM). The graph below shows activity was reduced after TBI but was markedly improved with EE treatment. ICAM levels (pg/ml) on day 2 in sham, TBI-Veh and TBI-EE were 281.59±14.1, 411.1±35.2 and 237.7±3.6, respectively. Rats were also monitored for weight gain/loss; values (g %) on days 1, 2 and 3 in sham, TBI-Veh and TBI-EE were 1.611±0.49, -5.25±0.56 and -3.51±0.95; 2.78±0.65, -4.05±0.58 and -3.13±0.42; and 4.53±0.82, -2.90±0.67 and -1.45±0.37, respectively. Differences were significant (p<0.05) in normal vs. TBI-Veh. The weight loss by 3 days post-TBI was significantly less in the EE-treated group than in the vehicle-treated group. Thus, EE3-SO4 injected one hr post-TBI improves physical activity of animals, decreases their weight loss and normalizes the ICAM-1 levels in rats one day post-injury (DOD W81XWH-08-2-0153).
INCREASED MYOCARDIAL EXPRESSION OF MICRORNA-125B ATTENUATES SEPSIS INDUCED CARDIAC DYSFUNCTION
X. Wang, M. Gao, C. Lu, X. Zhang, T. Ha, J. Hu, J. Kalbfleisch, R. Kao, D.L. Williams*, and C. Li*. East Tennessee State University, Johnson City, TN
MicroRNAs (miRs) are novel endogenous regulators of gene expression via degradation or translational inhibition of their target mRNAs. MiR-125b is expressed in several organs including lung, heart, spleen, brain and skeletal muscle. NF-κB activation regulates miR-125b expression, which in turn acts as a negative feedback regulator of NF-κB activation. MiR-125b also down regulates apoptotic signaling. This study examined whether miR-125b will attenuate sepsis induced cardiac dysfunction. We constructed a lentiviral vector expressing miR-125b (LmiR-125b). Lentivirus expressing scrambled miR served as control (LmiR-control). LmiR-125b or LmiR-control was transfected into mouse hearts (n=6/group). Seven days after transfection, mice were subjected to cecal ligation and puncture (CLP). Untreated CLP mice served as sepsis controls (n=6). Sham surgical operated mice served as sham control (6/group). Cardiac function was examined by echocardiography before and 6 hrs after CLP. CLP sepsis resulted in significant cardiac dysfunction. Ejection fraction (EF) was decreased by 24.6% and fractional shortening (FS) by 31.1% respectively when compared with control. In contrast, transfection of LmiR-125b into the myocardium significantly attenuated CLP-induced cardiac dysfunction. The values for EF (60.4±2.09% vs. 49.69±1.13%) and FS (31.4±1.47% vs. 24.5±0.31%) were significantly (p<0.05) higher in LmiR-125b treated CLP mice than in the untreated CLP group. Transfection of LmiR-125b into the heart significantly suppressed the expression of Bax and Bak1 in the myocardium. Increased myocardial miR-125b expression also attenuated (p<0.05) CLP induced increases in serum TNFα and IL-1β levels. We conclude that myocardial miR-125b over expression attenuates sepsis induced cardiac dysfunction and cardiac apoptosis. Myocardial miR-125b also inhibits sepsis induced systemic inflammatory responses. These data suggest that modulation of miR-125b may be a useful approach to prevent or treat septic cardiomyopathy.
IN VIVO AND IN VITRO EVIDENCE THAT ECRG4(133-148) IS A NOVEL CHEMOTACTIC FACTOR
J. Lee, X. Lu, T.W. Costantini*, A. Baird*, R. Coimbra*, and B. Eliceiri*. University of California San Diego, San Diego, CA
Background: The c2orf40 gene encodes Ecrg4, a membrane-anchored protein that is highly expressed on the surface of resting, circulating granulocytes. Upon stimulation with lipopolysaccharide (LPS) however, peptides processed from Ecrg4 are released into conditioned media. One such peptide, the 16 amino acid Ecrg4(133-148) has been identified by mass spectrometry in human plasma, CSF and the conditioned media of Ecrg4 transfected cells. Because Ecrg4(133-148) can stimulate NF-kB signaling in mouse peritoneal macrophages, we examined its chemotactic activity in vitro and in vivo.
Methods: For an in vivo assessment of chemotactic activity, polyvinyl alcohol sponges were implanted s.c. into mice and injected with varying concentrations of Ecrg4(133-148). At different times, the cells invading the sponges were harvested and cell surface markers analyzed by flow cytometry. For in vitro assays, target cells were placed onto a matrigel-coated modified Boyden chamber transwell and the chemotactic activity of Ecrg4(133-148) quantified by quantitative microscopic analyses of the bottom surface.
Results: Maximal cell invasion in response to Ecrg4(133-148) in vivo was obtained with a 2 microgram of peptide over 3 days. The cells invading the PVA sponges were characterized by high expression levels of Gr1 and CD11b, which are markers for immature myeloid-derived cells. In vitro, Ecrg4(133-148)-induced a cell invasion response through matrigel within 18 hours of the experiment and the maximal responses were observed at 10nM concentration of peptide.
Conclusion: The results support the hypothesis that Ecrg4(133-148) is a novel chemotactic factor that is capable of mobilizing a macrophage response in vivo and in vitro. Whether the presence of membrane-anchored Ecrg4 precursor is a ‘latent’ form of Ecrg4(133-148) that serves as a reservoir on the surface of granulocytes or is active in its own is currently under investigation.
ROLE OF CARDIAC VS. MYELOID MYD88 IN ENDOTOXEMIA - A STUDY WITH TISSUE-SPECIFIC DELETION MODELS
Y. Feng*, L. Zou*, C. Chen, Y. Yao, J. Cai, and W. Chao*. Massachusetts General Hospital, Charlestown, MA
MyD88 is an adaptor molecule critical for innate immune signaling and plays an important role in host defense as well as inflammatory injury. We and others have shown that signaling via MyD88 contributes to cytokine storm, cardiac depression, and high mortality during endotoxemia. However, the role of MyD88 of different origins in endotoxemia is unknown.
Methods: Cardiac- and myeloid-MyD88 deletion models: Cre recombinase transgenic mice with α-myosin heavy chain (α-MHC) or Lysozyme M (Lyz) promoter were cross-bred with mice with loxP flanking the exon 3 (MyD88fl/fl), to generate cardiac- (α-MHC-MyD88-/-) or myeloid- (Lyz-MyD88-/-) MyD88 deletion models (Fig). MyD88fl/fl littermates were used as the control. Endotoxin shock model: Mice were subjected to 15 mg/kg LPS (ip). The following endpoints were examined: cardiac function by ECHO, body temperature, and mortality up to 96h.
Results: α-MHC-MyD88-/- mice had 61% and 87% reduction in MyD88 gene and protein expression, respectively, in cardiomyocytes, whereas Lyz-MyD88-/- had 73% and 67% decrease, respectively, in macrophages (MΦ). MΦ from Lyz-MyD88-/- mice had a markedly attenuated TNFα production in response to Pam3cys (TLR2 ligand) but a normal response to poly(I:C) (TLR3 ligand). After LPS administration, MyD88fl/fl littermates had 60% of mortality whereas both α-MHC-MyD88-/- (n=10) and Lyz-MyD88-/- (n=10) mice had marked improved survival with zero and 20% mortality, respectively. Unlike global MyD88-/- mice, both α-MHC-MyD88-/- and Lyz-MyD88-/- mice showed the signs of endotoxin shock, e.g., hypothermia and cardiac dysfunction. However, Lyz-MyD88-/- mice had higher body temperature (34±1°C vs. 30±1°C, P<0.01 at 6h; 31±1°C vs. 27±1°C, P<0.001 at 24h after LPS injection, n=10) and better cardiac function at 6h after LPS administration (FS: 43±2% vs. 31±2%, P<0.05; EF: 75±2% vs. 60±2%, P<0.05, n=10) than the controls.
Conclusions: The pilot studies demonstrate that 1) myeloid MyD88 plays a role in cardiac dysfunction during endotoxemia and 2) both cardiac- and myeloid-MyD88 contribute to the mortality.
CONTRIBUTION OF P2X7 RECEPTOR SIGNALING TO IMPROVED SURVIVAL IN SEPSIS
G. Haskó*2, B. Csóka2, B. Koscsó2, E. Kókai2, Z. Spolarics*2, L. DiFazio1, R. Rolandelli1, and Z.H. Németh1,2. 1Morristown Medical Center, Morristown, NJ, 2UMDNJ-New Jersey Medical School, Newark, NJ
Objective: The purine nucleotide adenosine triphosphate (ATP) is a biologically active signaling molecule that is released into the extracellular space during inflammation, infection, shock, and sepsis. Extracellular ATP regulates a wide variety of immunological processes by binding to its receptors. These fall into two classes: the G protein coupled P2Y (P2Y2, P2Y4, P2Y11, P2Y13) and the ionotropic P2X (P2×X1-7) receptors. P2X7 is the most highly expressed P2 receptor on cells of hematopoietic origin, such as macrophages and T cells, and has emerged as an important regulator of immunity and inflammation.
Methods: The immunological status of septic animals was assessed by measuring cytokine levels and bacterial counts from blood, peritoneal lavage fluid and vital organs 6 and 16 h after cecal ligation and puncture (CLP) of P2X7 receptor knockout (KO) and wild type (WT) mice. Markers of immune cell apoptosis were examined in the protein extract from thymus and spleen tissue using Western blotting.
Results: P2X7 KO mice showed increased mortality in comparison with WT mice. This was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum with the exception of the down regulation of IL-1β in KO mice. Pharmacological inhibition of P2×X7 receptors with oxi-ATP (40 mg/kg i.p., 30 min prior to CLP) similarly increased bacterial burden, cytokine and chemokine levels in septic shock. To confirm that P2X7 receptor signaling on hematopoietic cells was necessary for controlling inflammation, we generated P2X7 receptor bone marrow-chimeric mice by transferring P2X7 KO or WT bone marrow into irradiated WT mice. P2X7 KO→P2X7 WT chimeras that have KO bone marrow and WT parenchyma exhibited higher cytokine and chemokine levels than P2X7 WT→P2X7 WT mice with WT parenchyma and WT bone marrow. In addition, septic P2X7 KO mice had increased apoptosis in the thymus and tissue specific inflammation in vital organs, such as the heart and lung.
Conclusion: Our findings that P2X7 receptor inactivation leads to higher mortality and cell death in sepsis suggest that perhaps stimulation of this signaling pathway could convey a protective benefit.
EXTRACELLULAR SIGNAL-REGULATED KINASE 5, A CENTRAL MEDIATOR OF TOLL-LIKE RECEPTOR SIGNALING
J. Hellman*, F. Xu, A. Tran, K. Wilhelmsen. University of California, San Francisco, San Francisco, CA
Background: Sepsis is initiated by interactions between microbes and Toll-like receptors (TLRs), which leads to inflammation, coagulopathy and organ failure. We recently made the novel discovery that extracellular signal-regulated kinase 5 (ERK5, aka “Big Map Kinase 2), a member of the mitogen activated protein kinase family, mediates TLR2-dependent activation of monocytes and endothelial cells, and promotes TLR2-dependent expression of the plasminogen activator inhibitor 1 (PAI-1). We tested the hypotheses that ERK5 also regulates TLR4-dependent endothelial activation, and that ERK5 mediates TLR2- and TLR4-dependent activation of inflammation and coagulopathy in vivo in mice.
Methods: HUVEC and human lung microvascular EC (HMVEC-Lu) were treated with TLR2 agonists (FSL-1 or Pam3Cys), or with the TLR4 agonist, LPS, in the presence or absence of a highly specific ERK5 inhibitor (XMD8-92). Mice were pre-treated with ERK5 inhibitor and then challenged IV with Pam3Cys. Cytokines were quantified in HUVEC supernatants and mouse plasmas. PAI-1 was quantified in mouse plasmas.
Results: ERK5 inhibition resulted in the downregulation of TLR2- and TLR4-dependent cytokine production by HUVEC and HMVEC-Lu in vitro (p<0.05). Similarly treatment with ERK5 inhibitor reduced the induction of IL-6 and PAI-1 in plasmas of mice treated with Pam3Cys or LPS (p<0.05). Conversely, treatment with ERK5 inhibitor led to augmented plasmas levels of soluble E-selectin in mice treated with Pam3Cys or LPS (P<0.05).
Conclusions: Our results highlight critical, previously overlooked functions for ERK5 in regulating TLR-dependent inflammatory signaling, and suggest that ERK5 may be a novel drug target in endotoxemia and sepsis.
RESVERATROL REDUCES HIPPOCAMPAL INJURY DURING THE ACUTE AND CHRONIC PHASES AFTER MILD TBI IN MICE
J. Gatson*, M. Liu*, J.G. Wigginton*, J. Minei*, and S. Wolf*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Following a mild traumatic brain injury (TBI) event, the secondary brain injury consists of increased oxidant injury, inflammation, hyper-phosphorylated tau, and neuronal cell death.
Methods: In this study, using the controlled cortical impact device we produced a mild-to-moderate closed head injury in young adult C57 Bl/6 mice. At 5 minutes after injury, the mice were treated subcutaneously with either placebo or resveratrol (100 mg/kg). Mice were also treated at 12, 24, 36, and 48 hours after injury. At 72 hours (acute phase) after injury, hippocampal brain tissue was harvested, protein concentration was determined, and a Western analysis was conducted to detect the levels of total and phospho-tau. Also, at 2 weeks (chronic phase) after injury, the brains were fixed, isolated and sectioned near the injury zone. The sections were stained for apoptosis (TUNEL assay), astrocyte/microglia activation, total tau, and phospho-tau.
Results: In this study we found that treatment with resveratrol led to a decrease in phospho-tau in the hippocampus during the acute and chronic phases after injury. Resveratrol also decreased TUNEL + staining (P < 0.04), GFAP staining (P < 0.05), and Iba1 staining (P < 0.01) in the hippocampus.
Conclusions: Resveratrol may be an ideal therapy to treat mild-to-moderate TBI to reduce both acute and chronic secondary brain injury to conserve learning and memory after injury.
THE ROLE OF MLCK IN SEPSIS-INDUCED INTESTINAL HYPERPERMEABILITY
C.A. Lorentz, B.P. Yoseph, E.R. Breed, Z. Liang, R. Mittal, and C. Coopersmith*. Emory University, Atlanta, GA
Introduction: Alterations in gut permeability are associated with critical illness, which may result in part from epithelial tight junction (TJ) disruption. Myosin light chain kinase (MLCK) plays a significant role in TJ regulation and may mediate permeability changes seen in sepsis.
Objective: To determine the role of MLCK in sepsis and potential mechanisms for gut barrier dysfunction.
Methods: MLCK knockout (MLCK-/-) and C57Bl/6 (WT) mice were subjected to 2x25 cecal ligation and puncture (CLP). Permeability was measured by assessing FD-4 levels in serum 5 hrs after gavage. Western blot assessed jejunal TJ proteins, and bead array analyzed serum cytokines. T-test and ANOVA with Tukey post-test were used for two-way and multiple group analyses, respectively.
Results: MLCK-/- mice showed improved 7-day survival after CLP over WT (95% vs. 22%, n=18/group, p < 0.0001). Septic WT mice demonstrated increased permeability over non-manipulated mice 6, 12, and 24 hrs after CLP. However, permeability was attenuated in MLCK-/- mice compared to WT animals at each of these time points (Figure; p=0.02, 0.002, 0.02, respectively). MLCK-/- mice showed increased jejunal ZO-1 expression 24 hrs after CLP (p=0.03). There was no significant difference in Claudin-2 and Claudin-5 levels. Levels of IL-10 were significantly decreased in MLCK-/- animals after 24 hrs (24868 ± 10369 pg/μL vs. 1821 ± 609 pg/μL, p=0.004), while no significant difference was found in serum IL-6, IFN-γ, IL-4, or IL-13.
Conclusions: These data show that MLCK knockout mice have a survival advantage in sepsis, which is associated with decreased gut permeability, increased ZO-1 in the jejunum, and decreased systemic IL-10. Thus, MLCK appears to play a critical role in gut barrier function in sepsis.
UPREGULATION OF B7-H1 ON NEUTROPHILS, ALONG WITH ELEVATED PRO- AND ANTI-INFLAMMATORY CYTOKINES LEVEL, MAY BE BIOMARKERS OF SEPTIC PROGRESSION/ MORBIDITY
X. Huang, Y. Chen, C. Chung*, S.F. Monaghan, and A. Ayala*. Rhode Island Hospital and Brown University, Providence, RI
Identifying relevant mediators responsible for the pathological process during sepsis may lead to finding novel diagnostic and therapeutic targets. Recent studies indicate programmed cell death receptor (PD)-1 plays a significant role in the development of immune suppression associated with sepsis. However, while PD-1 is reported to interact with two cell surface ligands B7-H1 and B7-DC, it is not clear what their role to septic morbidity may be. Here we report that B7-H1 is up-regulated extensively on various immune cells during sepsis and B7-H1 gene deficiency protected mice from the lethality of sepsis. In terms of histological development of multiple organ damage and inflammatory cytokine levels in circulation or at infectious site, B7-H1 deficient mice showed a remarkable reduction in these indices when compared with WT mice. However, B7-H1 gene deficiency did not alter bacterial burden when compared to WT mice. In addition, we found that, during sepsis, while there were no marked differences affecting ex vivo macrophage cytokine productive capacity between PD-1 and B7-H1 gene deficient mice (as gene deficiency partially restores septic mouse cells function back to Sham levels); preservation of ex vivo septic mouse macrophage phagocytic function was only seen in septic PD-1 knockout mice cells. Finally, higher % B7-H1+ neutrophils in septic mouse peripheral blood was correlated not only with higher levels of blood pro- and anti-inflammatory cytokines/ chemokines (MCP-1, IL-6, MIP-2, KC, TNF-a, and IL-10) but with lethal outcome as well. Together, these results indicate not only that B7-H1 contributes to septic morbidity, but suggest B7-H1 expression on neutrophils could be used as a biomarker indicative the severity of sepsis. (Supported by NIH-GM046354).
ADIPOSE TISSUE IS ALTERED IN OBESE MICE AFTER SEPSIS
T. DeMartini, M. Nowell, P. Lahni, and J.M. Kaplan*. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
Obesity is associated with an enhanced inflammatory response. Our previous data demonstrate that high fat diet (HFD)-fed mice have more inflammation and higher mortality following sepsis. We hypothesize that sepsis alters adipose tissue inflammation and is affected by obesity. C57BL/6 mice (6 wk old) were randomized to HFD (60% kcal fat) or a control diet (CD) (16% kcal fat). After 6 wks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0 or 6h after CLP. Plasma, white adipose tissue (WAT) and brown adipose tissue (BAT) were obtained for analysis. p≤0.05 was considered significant. Following 6 wks of feeding, HFD mice had higher plasma levels of the adipose cytokines, leptin and resistin compared to CD (31 ± 5 vs. 1 ± 0.2 ng/ml and 54 ± 0.2 vs. 2.5 ± 0.7 ng/ml respectively, p≤0.05 by rank sum test). Plasma adiponectin decreased during sepsis in HFD-fed mice (850 vs. 329 μg/ml, p≤0.01 by t-test) but not in CD. HFD-fed mice had lower plasma adiponectin after CLP compared to CD-fed mice (p≤0.01 by t-test). Plasma resistin decreased in HFD-fed mice after CLP compared to time 0 and compared to CD-fed mice at 6h after CLP. Plasma leptin increased after CLP in CD mice compared with time 0 (7 vs 1 ng/ml, p≤0.05 by rank sum test). However, leptin was higher in HFD-fed mice after CLP when compared with CD mice. To understand the molecular mechanisms of this different inflammatory response, we investigated the expression of peroxisome proliferator activated receptor-γ (PPARγ), a nuclear transcription factor involved in lipid metabolism and inflammation. At Western blot analysis, PPARγ expression was lower in WAT from HFD-fed mice compared to CD-fed mice both at 0 and 6h after CLP (0.8 ± 0.2 vs. 1.4 ± 0.5 and 0.6 ± 0.2 vs 3.4 ± 0.4 relative units, p≤0.05 by t-test). PPARγ protein expression was significantly higher in BAT in CD-fed mice after CLP compared to prior to CLP (3.5 vs. 1.4 relative units, p≤0.01 by t-test). However there was no change in PPARγ expression in BAT in HFD-fed mice after CLP. The expression of uncoupling protein-1, which is usually found in BAT and has a role in thermogenesis, was significantly increased in BAT in HFD-fed mice both at 0 and 6 h after CLP compared with CD (p≤0.05 by t-test). Our data demonstrate that both WAT, BAT and plasma adipokines are altered in sepsis and affected by obesity. Supported by NIH K08GM093135.
A COMPUTERIZED ICU SYSTEM FOR RECOGNITION AND MANAGEMENT OF SURGICAL SEPSIS
C.A. Croft1, F.A. Moore*1, P.A. Efron*1, P.S. Marker2, A. Gabrielli1, L.J. Caruso1, F.J. Casimir1, J. Jordan1, L. Lottenberg1, L.S. Westhoff2, V. Klink1, R.M. Sailors1, and B.A. McKinley*1. 1University of Florida, Gainesville, FL, 2Shands at University of Florida, Gainesville, FL
Purpose: To describe a sepsis management system used by a surgical ICU. Method: A rule based, data driven system is operated using computerized medical record and protocol components designed using literature and guideline evidence, and local expert consensus. Rules were devised and organized to create a care process that provides early recognition and guides patient specific management of sepsis including: 1. modified early warning signs - sepsis recognition score (MEWS-SRS; summative point score of ranges of selected vital signs, level of consciousness and WBC; Q4hr) by bedside RN used the electronic medical record system; 2. suspected site assessment (vascular access, lung, abdomen, urinary tract, soft tissue, other) by MD extender used a paper form; 3. sepsis management protocol (clinical interactive computerized protocol) by RN, MD and extender used patient bedside workstations. Sepsis severity was determined using standard criteria. Result: In 25 weeks (Jun-Nov 2012), the system was used to manage 111 consecutive sepsis encounters (4.5±0.4 cases/wk) in 103 patients (64% m; age 58±2). Positive MEWS-SRS elicited 358 suspected site assessments and 118 (32%) were thought due to an infectious source. On further review by intensivist, 7 were considered false positive and 111 had sepsis diagnoses-sepsis management protocol orders. Incidence and outcome are tabulated. (X2, ANOVA)
Hospital mortality rate was 13% for all surgical sepsis patients and 20% for combined severe sepsis-septic shock (compare other published mortality rate >30%). 24 (50%) septic shock patients were transferred to ICU from elsewhere. ICU LOS and hospital mortality rate increased with sepsis severity, but MEWS-SRS did not. 33% of patients who survived hospital stay went home.
Conclusion: Sepsis management for acute care surgery patients is frequent and effective with a computerized system that facilitates early recognition and directs evidence and consensus based care. Vital signs or WBC derangement is non specific for sepsis severity, indicating need for protocol directed, patient specific intervention. Hospital sepsis survival is associated with disposition to ongoing care.
CIRCULATING BIOMARKER NETWORKS DIFFERENTIATE SUSCEPTIBILITY TO INFECTION FOLLOWING TRAUMA IN HUMANS
R.A. Namas*1,2, K.W. Almahmoud1, A. Zaaqoq3, J. Yin1, N. Azhar1, R. Zamora*1,2, Y. Vodovotz*1,2, and T. Billiar*1,2. 1University of Pittsburgh, Pittsburgh, PA, 2Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 3Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA
Introduction: We hypothesized that an exaggerated early inflammatory response is associated with nosocomial infection following trauma in humans. To test this hypothesis, we analyzed an extensive time course of circulating levels of 24 inflammatory biomarkers in patients presenting following blunt injury.
Methods: From a cohort of 484 blunt trauma survivors studied, 48 patients with nosocomial infection post-trauma (30 males and 18 females; age: 48±2.9; Injury Severity Score [ISS]: 26.2±1.6) were matched with 48 trauma patients with no infection i.e. controls (29 males and 19 females; age: 49.2±2.2; ISS: 25.3±1). None of the patients had received transfusions or underwent emergency surgical procedures to avoid the confounding impact of these interventions. Plasma (3 samples within the first 24 h and then from days 1 to 7 post-injury) was assayed for 23 inflammatory cytokines and NO2-/NO3-. These data were compared by 2-Way ANOVA and Dynamic Bayesian Network (DBN) inference. Human genomic DNA was isolated and amplified for known TNF-α, IL-6, IL-10, TGF-β1, and TLR4 (Asp299Gly and Thr399Ile) single nucleotide polymorphisms (SNPs).
Results: Statistically significant differences in ICU length of stay (LOS), total LOS, and ventilator days were observed in the nosocomial infection patients vs. control patients. Plasma IL-1β, IL-1Ra, IL-2, sIL-2Rα, IL-4, IL-5, IL-6, IL-7, IL-8, IL-13, IL-15, IL-17, GM-CSF, IFN-γ, MIP-1α and MCP-1 were significantly elevated even within the first 24 hours in patients that developed nosocomial infection vs. controls. Further analysis of the dramatic and unique differences in the biomarker patterns between groups by DBN suggested that MIG and MCP-1 induce each other through forward-feedback loops that drive IL-6 production in patients destined to develop nosocomial infections, whereas MIG alone drives IL-6 and IL-8 in the uninfected controls. SNPs did not differentiate between patients with or without infection.
Conclusion: These data show that even upon admission and over the initial 24 hours post-injury, unique inflammatory biomarker patterns emerge in patients prone to develop infections. By using stringently matched cohorts, we show that these patterns are independent of ISS, age, inflammatory SNPs or gender in the patients with moderately severe injuries. Network analysis points to MIG and MCP-1 as key drivers of this process.
DETRIMENTAL EFFECTS OF ERK1/2 INHIBITION ON THE INTESTINE DURING ISCHEMIA/REPERFUSION
K. Ban, Z. Peng, and R.A. Kozar*. University of Texas Medical School at Houston, Houston, TX
Introduction: The extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway has recently been associated with protection of the gut after ischemia/reperfusion (IR). To further investigate the potential role of this pathway, a specific ERK1/2 inhibitor, U0126, was used to assess intestinal damage and inflammation after gut IR.
Methods: Mice were randomly divided into sham, sham+U0126, IR alone and IR+U0126 groups (6 animals/group). U0126 groups were injected with U0126 (1mg/kg) and sham and IR groups were injected with vehicle intraperitoneally. After 30 minutes, IR groups were subjected to 1 hour ischemia via clamping of the superior mesenteric artery and 6 hours of reperfusion. The sham groups underwent identical procedures without IR. The small bowel was harvested for phosphorylation of ERK1/2 analysis by Western blot, histological damage using the Chiu score, neutrophil infiltration and accumulation by leukocyte staining and myeloperoxidase (MPO) activity. Blood was collected for cytokines by ELISA. Data is presented as mean ±SEM, ANOVA, Tukey post hoc.
Results: ERK1/2 activity was significantly inhibited by U0126 after I/R (Fig. 1A). There was minimal injury in the sham and sham+ U0126 groups (no difference between groups). Histologic injury significantly increased after IR but was further increased after IR+ U0126 (Fig.1B and C). Leukocyte staining demonstrated no difference between shams (6.0±5.8 vs 8.6±5.0) but a significant increase after IR (88.2±26.6) that was further increased by U0126 (214.5±38.4). Similar findings were shown for MPO (0.15±0.06 shams, 0.13±0.05 sham U0126, 1.53±0.21 IR, and 2.72±0.15 IR+U0126).
Discussion: These results demonstrate the detrimental role of ERK1/2 inhibition on the intestine in a rodent model of gut IR, further supporting the protective role of ERK1/2 in the intestine. Further investigation into the precise mechanism for this protection is warranted.
ADMINISTRATION OF ANTI-INTERLEUKIN-6 ANTIBODY DECREASED INFLAMMATORY CASCADE AND ATTENUATED IMMUNODEPRESSION IN MICE WITH PSEUDO-FRACTURE AND HEMORRHAGE
Y. Zhang, W. Xiang, J. Zhang, S. Korff Sebastian, K. Zettel, Kent R*, G. Chen, and T. Billiar*. University of Pittsburgh Medical Center, Pittsburgh, PA
Introduction: Our recent studies indicated that IL-6 is one of central nodes in inflammatory cascade induced by T/HS utilized multiplexing cytokine analysis coupled with data-driven modeling. However, it remains unclear whether blockade of massive IL-6 will attenuate trauma/hemorrhage induced immunosuppression. The goal of this study was to investigate the effect of neutralizing anti-IL-6 monoclonal antibody on immune responses in mice subjected to trauma and hemorrhage.
Methods: Male C57/BL6 mice underwent pseudo-fracture combined with hemorrhage, and isotype IgG (3mg/kg) or anti-IL-6 mAb (3mg/kg) was administered intraperitoneally before resuscitation. Mice were sacrificed at 6h, 24h or 48h. Liver and lung injury were assessed by H&E histopathology and serum ALT levels. Serum IL-6, IL-10, KC, MCP-1, IP-10 and MIP-1α, Th1 cytokines (IFN-γ and TNF-α) and Th2 cytokines (IL-10) release of splenocyte were measured by ELISA; splenocyte proliferation was determined by 3H-thymidine incorporation assays.
Results: Administration of the anti-IL-6 antibody effectively suppressed the elevation of Plasma IL-6, IL-10, KC, MCP-1and MIP-1α levels at 6h and 24h following PF and HS, while the serum IP-10 concentration was indistinguishable between both HS+PF mice. Anti-IL-6mAb treatment significant attenuated the lung and lung injury, confirmed by lower lung injury scores (p < 0.05) and circulating ALT level (p < 0.05) compare to IgG treated HS+PF mice. PF and HS resulted in a significantly depressed cytokine production capacity of splenocytes. However, the decrease in Th1 (IFN-γ and TNF-α) cytokines were more significantly in IgG treated group (p < 0.05), while the production of Th2 (IL-10) cytokine were significantly lower in anti-IL-6 antibody group (p < 0.05). Furthermore, Interleukin-6 neutralizing antibody markedly attenuated the suppression of ConA-induced splenocyte proliferation following PF and HS at 24h and 48h (p < 0.05).
Conclusions: These results show that IL-6 blockade after PF and HS decreased inflammatory cascade and attenuated the organ damage. Treatment with anti-IL-6 neutralizing antibody markedly restored splenocyte proliferative capacity and Th1/Th2 cytokine balance. It suggests that excessive IL-6 may be a potential target for therapeutic interventions for post-traumatic immune dysfunction.
NLRP3 INFLAMMASOME RECOVERY IMPROVES MONOCYTE FUNCTION AFTER MAJOR TRAUMA
J.P. Horstmann, K. Wilhelm, I. Marzi*, and B. Relja. University Hospital of the Goethe-University Frankfurt, Frankfurt am Main, Germany
Objectives: Suppressed function of monocytes in the early posttraumatic response plays a key role in the initiation of the inflammatory response by inflammasome activation and subsequent IL-1beta release. NLRP3 inflammasome deficiency is associated with several inflammatory diseases. We analyzed the monocyte function in a 5-day time course after trauma and their capacity to recover by NLRP3 gene insertion in isolated cells from trauma patients.
Methods: Twenty seven severely injured trauma patients [ISS≥16] and ten healthy volunteers were enrolled. Monocytes were isolated by CD14+ microbeads daily up to day 5 after trauma and assayed for their ex vivo in vitro IL-1beta production after LPS-stimulation (10μg/ml, 24h) by ELISA. Fresh monocyte RNA was analyzed for IL-1beta daily. In parallel, isolated monocytes were transfected with an expression plasmid encoding NLRP3 and 48 h later they were stimulated with LPS to uncover their functionality.
Results: LPS-stimulation increased markedly the IL-1beta release in healthy volunteers compared to unstimulated controls (p<0.05) and the LPS-response early after trauma. IL-1beta mRNA in samples from trauma patients was significantly enhanced compared to healthy volunteers early after trauma declining on day 3 and 4. Transfecting monocytes obtained from trauma patients after admission and on day 3 after trauma with NLRP3 significantly increased IL-1beta release after LPS-stimulation compared to corresponding unstimulated samples. The IL-1beta release in NLRP3-transfected and LPS-stimulated monocytes from patients was comparable to IL-1beta release in healthy volunteers.
Conclusions: Reduced IL-1beta release from monocytes early after trauma is paralleled by enhanced IL-1beta gene expression. This monocyte dysbalance can be partly counterbalanced by NLRP3 gene insertion, suggesting potential new targets and strategies for immunorecovery after trauma.
THE ROLE OF HSP25 IN MEDIATING MORTALITY IN SEPSIS
E.R. Breed1, B.P. Yoseph1, Z. Liang1, A. Hadley1, R. Mittal1, C. Hunt2, and C. Coopersmith*1. 1Emory University, Atlanta, GA, 2University of Texas Southwestern, Dallas, TX
Background: Heat shock proteins (HSPs) play an important role in the host response to pathophysiological stresses, including infection, oxidative damage, and thermal stress. HSP25 is one of the major stress inducible HSPs, however its role in human disease is not well characterized.
Objective: To determine the role of HSP25 in mediating the host response to sepsis.
Methods: Young (6-12 week old) HSP25-/- and wild type (WT) mice were subjected to 2x25 cecal ligation and puncture (CLP) and followed seven days for survival (n=22-23/group). Additional mice were sacrificed at 24 hours following CLP for mechanistic studies. Inflammatory cytokine response in peritoneal fluid and serum was measured using a cytometric bead array (n= 8-12/group). Splenocyte cell populations were assayed by flow cytometry and lymphocyte apoptosis was quantified by PI and Annexin V expression (n=5-10/group). Gut epithelial apoptosis was quantified using H&E and active caspase-3 staining (n=7-12/group).
Results: Mortality was higher in HSP25-/- mice than in WT mice subjected to CLP (65% vs. 35%, respectively; p=0.02). Compared to WT mice, HSP25-/- mice had an increase in the local IFN-γ response (155±62 vs. 894±273 pg/mL; p=0.04) and decreased systemic levels of IL-6 (40337±7555 vs. 11139±4091 pg/mL; p=0.01) and CXCL1 (65563±5940 vs. 43053±6909 pg/mL; p=0.01). Frequencies of CD4+ and CD8+ lymphocyte populations were decreased in HSP25-/- mice compared to WT mice (21% vs. 30% and 11% vs. 14%; p=0.005 and p<0.05, respectively). In addition, CD4+ and CD8+ lymphocyte apoptosis was increased in HSP25-/- mice compared to WT mice (13% vs. 9% and 40% vs. 30%; p=0.01 and p=0.02, respectively). There were no differences in gut epithelial apoptosis between HSP25-/- mice and WT mice.
Conclusion: HSP25 is an important determinant of survival in septic mice. The difference in mortality between HSP25-/- and WT mice indicates that HSP25 may play a protective role in the host response to sepsis by altering the host cytokine response and preventing lymphocyte apoptosis.
ENTERAL TRANEXAMIC ACID ATTENUATES LUNG ENDOTHELIAL PROTEIN DAMAGE FOLLOWING HEMORRHAGIC SHOCK
A.E. Altshuler, D. Li, J. Chou, A.H. Penn, and G.W. Schmid-Schonbein*. University of California, San Diego, La Jolla, CA
Objective: Hemorrhagic shock (HS) causes damage to peripheral organs, such as the intestine and lung. The reduced perfusion to the intestine leads to increased intestinal mucosal permeability with junctional protein degradation and leakage of luminal contents (e.g. pancreatic digestive enzymes, degradation products) into the circulation. In addition, the hypotension may also cause systemic matrix metalloproteinase (MMP) activation. We examined whether inhibition of protease activity in the lumen of the intestine or peripheral MMP inhibition may attenuate tissue and endothelial protein breakdown in the lungs.
Methods: Male Sprague Dawley rats were grouped into no shock, sham HS and HS with and without an MMP inhibitor, (doxycycline, IV, 5 mg/kg; IP, 10 mg/kg) or serine protease inhibitor (tranexamic acid, 200 mM, lumen of intestine) (N=7/group). Mean arterial blood pressure (MAP) in HS animals was reduced to 30 mmHg (90 min) followed by reperfusion of shed blood. Post-HS plasma, intestine, and lungs were collected at 3 hr after reperfusion. Myeloperoxidase (MPO) activity was assessed in intestine and lung homogenates. Bronchoalveolar lavage fluid (BALF) was collected and analyzed for MPO activity. Protease activity in plasma was measured by plate zymography (MMP-9 specific substrates for plasma) or by gel zymography (plasma and lung). MMP-9 and TIMP-1 levels were measured. We also determined VEGFR-2 receptor levels in the lung and circulating VEGF.
Results: After reperfusion, no significant differences were noted in the MAP among HS groups. Macroscopic hemorrhages in the intestine in untreated HS were reduced with both MMP and serine protease inhibition. None of the treatments reduced MPO activities in the intestine or lung compared to untreated HS. MMP-9 activities and levels were elevated in plasma and lung tissues after HS with enteral tranexamic acid treatment but not doxycycline (p<0.05). Plasma TIMP-1 levels remained unchanged in all groups. Plasma VEGF levels were elevated following HS (p<0.05). VEGFR-2 levels were significantly reduced in lung tissue and restored with tranexamic acid treatment but not doxycycline.
Conclusion: Enteral treatment with tranexamic acid but not systemic doxycycline treatment may prevent progression of VEGFR-2 protein breakdown in the lung and reduce the progression of organ failure. Supported by GM85072.
THE ESTROGEN RELATED RECEPTOR (ERR)-β AND -γ AGONIST DY131-DERIVED CARDIOPROTECTION IN TRAUMA-HEMORRHAGIC SHOCK (T-H) IS THROUGH PREVENTION OF T-H-INDUCED INCREASE IN CARDIAC TRANSCRIPTION FACTOR NF-κB
S. Yang, S. Hu, R. Raju, K.I. Bland, and I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
Our studies have shown that DY131-derived cardiac protection is closely related to DY131-induced restoration of cardiac ERRs and PGC-1α. We have also shown that cardiac depression after T-H is via T-H-induced upregulation in cardiac NF-κB and administration of estrogen (E2) after T-H prevented T-H-induced increase in cardiac NF-κB. However, it is unclear if DY131 can also prevent T-H-induced increase in cardiac NF-κB. To study this, DY131 was administered after T-H in male adult Sprague-Dawley rats; seven groups were established (n=6/group): sham; sham + DY131; T-H + vehicle; T-H + E2 (1 mg/kg); T-H + ICI 182,780 (ICI; 3 mg/kg) + E2; T-H + DY131 (40 μg/kg); and T-H + ICI + DY131. DY131 or E2 was delivered IV at the onset of resuscitation and the ER antagonist ICI was given IP 30 min before DY131 or E2 administration. Two hours after T-H and resuscitation, left ventricular (LV) performance was measured, then blood and heart tissue were harvested. Cardiac nuclear NF-κB and Nrf2, and cardiac IκB-α were determined by Western blot. Plasma and heart tissue cytokines including TNF-α, IL-6 and IL-10 were measured by ELISA. Our results indicate that like E2, DY131 prevented T-H-induced increase in cardiac NF-κB and phospho-IκB-α (Figure). Furthermore, DY131 restored cardiac Nrf2. DY131 also attenuated the increase in plasma and cardiac TNF-α and IL-6. Interestingly, ICI did not alter the effect of DY131 on LV performance and all measured signaling pathways and cytokines. Thus, it can be concluded that cardioprotection by DY131 following T-H is via DY131-derived restoration of cardiac NF-κB, Nrf2 and IκB-α (NIH R01 GM 39519).
MITOCHONDRIAL DNA COPY NUMBER VARIATION IN BURN PATIENTS
R.M. Huebinger*1, N. Phillips2, R.K. Roby2, A. Burris1, G. Purdue1, B. Arnoldo1, J. Hunt1, R. Barber2, and S. Wolf*1. 1University of Texas Southwestern Medical Center, Dallas, TX, 2University of North Texas Health Science Center, Fort Worth, TX
Recovery from trauma requires vast amounts of energy which is primarily generated through mitochondrial metabolism. Variation in the number of mitochondria per cell may affect the amounts of cellular energy that can be generated. We hypothesize that mitochondrial copy number variation in burn patients is associated with differences in clinical outcomes. Mitochondrial DNA copy number per cell was calculated by TaqMan quantification assays in peripheral white blood cells from burn patients. A total of 179 patients were examined for mitochondrial copy number upon admission to the Parkland Burn ICU with TBSA≥15. The number of mitochondria varied from 30 to 1687 copies per cell. Median cellular copy number in this group was 104.3 mitochondria (IQR 78.55-149.7). Copy number was not significantly correlated with age (p=0.36), burn size (p=0.63), or Baux score (p=0.28). Results from logistic regression analysis was significantly associated with sepsis (aOR for each additional mtDNA copy 1.004; p=0.006) but not for mortality (aOR 0.995; p=0.16). For the ROC analysis mtDNA copy number was converted to a dichotomous variable (0=below mean, 1= above the mean). ROC analysis of sepsis yielded 0.544 AUC for mtDNA copy number, 0.724 AUC for Baux score and 0.739 AUC for Baux+mtDNA copy number. Mitochondrial DNA copy number in the peripheral blood in burn patients varies greatly between individuals. The clinical relevance of these associations is presently undetermined. A portion of these analyses were presented at the Society for Critical Care Medicine in San Juan, Puerto Rico (January 2013).
ANTI-HMGB1 MONOCLONAL ANTIBODY AMELIORATES IMMUNE DYSFUNCTION AFTER PERIPHERAL TISSUE TRAUMA
S.S. Darwiche1, X. Ruan3, 1, K.R. Zettel1, C. Cai1, H. Pape*2, 1, and T. Billiar*1. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2University of Aachen Medical Center, Aachen, Germany, 3First Municipal People’s Hospital of Guangzhou, Affiliated Hospital of Guangzhou Medical College, Guangzhou, China
HMGB1 is a key damage-associated molecular pattern (DAMP) molecule released after injury (Andersson Ann Rev Immunol 2011). The immuno-inflammatory response to injury is comprised of both the upregulation of innate and suppression of adaptive immune responses (Flohe Innate Immun 2008). HMGB1 serves as an early trigger of sterile inflammation after severe trauma (Scaffidi Nature 2002) however the role of HMGB1 in the suppression of adaptive lymphocyte dysfunction after injury remains unclear. We chose to test the hypothesis that HMGB1 plays a role as a mediator in the injury-induced immune suppression.
Through the in-vivo use of neutralizing anti-HMGB1 monoclonal antibodies in a murine model of peripheral tissue injury, we evaluated the post-traumatic immune response. Systemic inflammation and end organ dysfunction were measured at 6h after injury through circulating IL-6 levels and serum transaminase levels respectively. Post-traumatic immune dysfunction was assessed at 48h after injury by ex-vivo splenocyte proliferation, Th1 cytokine release and at 24h after injury by ARG1 induction within splenic myeloid-derived suppressor cells (MDSC).
Plasma HMGB1 levels increased in a time-dependent manner to peak at 24h after injury. HMGB1 neutralization did not improve the early systemic response after injury as evidenced by persistent and significant elevation in IL-6 and ALT levels in injured mice administered the anti-HMGB1 mAb. In contrast to the early response, neutralization of HMGB1 did attenuate the depressed lymphocyte responses at 48hr after injury as shown by preservation of splenocyte proliferation and cytokine release capacity. Interestingly, the anti-HMGB1 mAb also reduced the splenic MDSC expansion after trauma. However, MDSC upregulation of immunosuppressive ARG1 remained present after injury even with the administration of the anti-HMGB1 mAb.
This study confirms the hypothesis that HMGB1 plays a role as a mediator in the immune dysfunction after peripheral tissue trauma. These findings demonstrate an HMGB1-dependent MDSC-mediated T lymphocyte dysfunction that occurs in an ARG1-independent manner. Interestingly, the use of anti-HMGB1 mAb did not improve the early systemic response after peripheral tissue injury in this murine model. This study suggests potential for therapeutics that could limit the delayed immune dysfunction following injury.
SUBSTANCE P INHIBITION IMPROVES SURVIVAL IN MURINE POLYMICROBIAL SEPSIS BY ENHANCED CARDIOVASCULAR FUNCTION
J. Mella1, E.L. Chiswick2, E.G. King1, E. Duffy2, A. Stucchi1, and D.G. Remick*2. 1Boston University Medical Center, Department of General Surgery, Boston, MA, 2Boston University Medical Center, Department of Pathology, Boston, MA
Introduction: The Rivers protocol demonstrated that maintaining cardiovascular function and end organ perfusion during the golden hours of sepsis are critical for survival. While prior trials modulating the inflammatory response have been unsuccessful at demonstrating survival benefit, there has not been much of an emphasis on modulating the vascular response. Substance P binding to neurokinin-1 receptor (NK1R), mediates neurogenic inflammation in sepsis by promoting vasodilation, vascular permeability and plasma extravasation. We hypothesize that inhibiting substance P will improve cardiovascular function and survival following murine polymicrobial sepsis.
Methods: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in aged neurokinin-1 receptor knockout (NK1R KO) and wild type Balb-c mice. Plasma cytokines IL-6, MIP-2, and IL-1RA were evaluated by ELISA. Physiologic parameters were monitored closely following CLP. The same experimental design was utilized in out bred ICR mice randomized to IP treatment of CJ-12255, a neurokinin-1 receptor antagonist (NK1RA), at the time of CLP and administered for 5 additional doses every 12 hours, or the vehicle, normal saline, following CLP to corroborate the findings.
Results: Inhibiting the effects of substance P in NK1R KO mice or using NK1RA in out bred mice improved survival. Better survival correlated with significantly decreased plasma cytokine (IL-6, MIP-2, IL-1RA) values. Interestingly, there were dramatic differences in physiologic parameters. The experimental mice experienced a dramatic improvement in cardiovascular function, as determined by heart rate and pulse distension (a surrogate for stroke volume), during the early hours following the septic insult.
Conclusion: Blocking the substance P receptor in either NK1R KO mice or NK1RA treated mice attenuated neurogenic mediated inflammation by minimizing plasma extravasation, preventing capillary leakage and maintaining intravascular volume. This resulted in improved cardiovascular function with enhanced organ perfusion without the administration of extra resuscitation during the golden hour of sepsis improving survival.
BURN-INDUCED MUSCLE INSULIN RESISTANCE AND ITS REVERSAL BY FARNESYLTRANSFERASE INHIBITOR (FTI) ARE ASSOCIATED WITH ALTERATIONS IN CIRCULATING ALARMINS
M. Kaneki*, and M. Yamada. Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Charlestown, MA
Metabolic derangements are a major complication of burn injury and affect the clinical outcome of burn patients. These metabolic aberrations include hypermetabolism, muscle wasting and hyperlactatemia, in all of which muscle insulin resistance plays a role. Protein farnesylation is a lipid modification of cysteine residues and leads to membrane trafficking and functional alterations of many proteins. We have previously shown in mice that the maximum levels of burn-induced insulin resistance and increased farnesyltransferase (FTase) expression are observed at 3 days post-burn, and that FTase inhibitor, FTI-277, reverses burn-induced muscle insulin resistance. However, relation between burn-induced muscle insulin resistance and systemic inflammatory response remains to be clarified. Circulating alarmins (e.g., HMGB1) have emerged as a key player in inflammatory response. Recently, cell-free DNA has been proposed as a predictor of the mortality of septic patients. We studied, therefore, the effects of burn on circulating alarmins and cell-free DNA at 0, 6, 24, 72 h and 7 days post-burn. A full-thickness third degree burn injury (30% total body surface area) was produced in 8-week old male C57BL/6 mice under anesthesia by exposing the trunk to 80°C water. In parallel with the development of insulin resistance, burn significantly increased plasma levels of HMGB1, histone H3, cell-free DNA and lactate in a time-dependent manner and the maximum levels were observed at 3 days after burn compared with naïve and sham mice. Treatment of burned mice with FTI-277 (5 mg/kg/day) starting at 2 h post-burn reversed increased levels of HMGB1, cell-free DNA and lactate at 3 days after burn, as compared with vehicle. FTI-277 also prevented burn-induced infiltration of macrophages and neutrophils into skeletal muscle. In contrast, FTI-277 didn’t alter these parameters in sham mice. These results clearly demonstrate that alterations in plasma levels of alarmins and cell-free DNA in burned mice were associated with development of muscle insulin resistance and its reversal by FTI-277. Our data indicate a close biological link between insulin resistance, metabolic dysfunction and systemic inflammatory response. Overall, our study identifies FTase as a novel potential target to improve the clinical outcome of burn patients.
FIBROCYTES IMPROVE OUTCOME AND PROMOTE A TH1 RESPONSE IN SEPTIC MICE
C. Fry and J. Nemzek*. University of Michigan, Ann Arbor, MI
Numerous studies correlate mortality with loss of T cells during sepsis. Consequently, treatments that preserve T cells have proven beneficial. We hypothesized the adoptive transfer of tissue derived fibrocytes would improve sepsis outcomes by increasing T cell proliferation and Th1 cytokines. Following CLP in male mice, adoptive transfer of fibrocytes, fibroblasts, or saline was performed. At one week, control animals receiving saline or fibroblasts had 40% and 20% survival respectively. Animals receiving fibrocytes had a significantly improved survival of 80%. To identify mechanisms for survival, the study was repeated and animals euthanized at various time points. Mice that received fibrocytes had lower plasma IL-6 and less organ injury than those given saline. Animals receiving fibrocytes had significant increases of splenic CD4 T-cells from 12 to 168hrs after CLP compared to fibroblasts or saline. Treatment with fibrocytes also resulted in significant increases of CD8 T-cells at 12 and 24hrs compared to the other groups. Then splenocytes were harvested at 24hrs post CLP and stimulated with anti CD3/CD28. After 18hrs, intracellular IFN-γ increased significantly in CD4 and CD8 T-cells from animals treated with fibrocytes compared to fibroblasts or saline. Therefore, treatment of mice with fibrocytes prior to CLP improved survivability and promoted a localized Th1 response. A potential mechanism for T cell proliferation was demonstrated by a significant increase of IL-2 at 24hrs in fibrocyte treated animals compared to controls. To identify molecular mechanisms; T-cells were cultured alone, with fibroblasts or fibrocytes. The results showed increased proliferation of CD4 T-cells when fibrocytes were co-cultured with T-cells compared to fibroblasts or T-cells alone. The increase in T-cell proliferation occurred without addition of a defined antigen. Fibrocytes were then derived from wild-type (WT) or MHC Class II knockout mice and co-cultured with splenic T-cells for one week. There were no significant differences between T-cells incubated with WT or KO fibrocytes. Collectively, these data suggest that adoptive transfer of fibrocytes increase survivability of mice after CLP by reducing systemic inflammation, enhancing T-cell proliferation, and promoting MHC class II independent Th1 responses. Fibrocytes may prove to be an effective therapy for the treatment of sepsis.
EXPRESSION OF COSTIMULATORY MOLECULE EXPRESSION ON PBMCS DURING SEPSIS CORRELATES WITH OBSERVED IMMUNE FUNCTION
C. Valentine*, A. Bergerat, and D.G. Remick*. Boston University Medical Center, Boston, MA
Objectives: Costimulatory molecules (CSM) play a key regulatory role during interactions between the innate and adaptive response by providing positive or inhibitory signals to lymphocytes, resulting in activation and proliferation, or anergy. Our goal was to examine the expression of CSM on peripheral blood monocytes (PBMCs) during the acute and chronic phases of sepsis to determine if there were changes that would reflect altered immune function observed in septic patients.
Methods: CD-1 mice underwent cecal ligation and puncture (CLP) to induce sepsis. Serial blood sampling was done via facial vein bleed at 24h, 72h, day 7 and day 14 post CLP. CSM expression was measured by flow cytometry on PBMCs. Spleens were isolated from some mice at 24 hours post CLP, and dendritic cell sub populations analyzed by flow cytometry.
Results: Mice that survived sepsis developed marked monocytopenia within 24 post-CLP, which began to recover by day 7 post CLP. This decrease in the peripheral monocyte population corresponded with a marked increase in the number of monocyte-derived dendritic cells (moDCs) found in the spleens 24h post CLP. CD86 expression on peripheral monocytes peaked by day 7, and then declined. Interestingly, by day 14, mice that survived sepsis demonstrated a distinct subpopulation of peripheral monocytes with extremely high levels of PD-L1 expression. Peripheral CD4+ lymphocytes showed a significant increase in CD28 expression within 24hours of CLP; however the expression of CLTA-4 which is upregulated after T cell activation providing a negative signal to limit proliferation was unchanged during the 14 day period.
Conclusions: The sharp increase in CD28 expression without a counterbalance in CTLA-4 expression may provide the opportunity for unregulated immune activation in the acute phase. While the monocytic expression of both positive and negative signaling CSM are increased in the acute phase of sepsis, PD-L1, an inhibitory CSM, predominates in the chronic phase. The moDCs observed in spleens are the predominant population and are immature. These findings correlate with the dysregulated immune function observed in septic patients, which is characterized by an early highly inflammatory state, followed by a period of immunoparalysis, marked by decreased antigen presentation and a poor lymphoproliferative response.
RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) SERVES A PROTECTIVE ROLE DURING KLEBSIELLA PNEUMONIAE-INDUCED PNEUMONIA
M. van Zoelen*1, 2, A. Achouiti1, A.F. de Vos1, C. van ’t Veer1, S. Florquin1, P. Nawroth3, A. Bierhaus3, and T. van der Poll1. 1Academic Medical Center, Amsterdam, Netherlands, 2University Medical Center Utrechtt, Utrecht, Netherlands, 3University of Heidelberg, Heidelberg, Germany
Background: Sepsis is the most common cause of death in non-coronary critical care units in the United States with > 750,000 cases per year. Gram-negative pneumonia is a common cause of sepsis with Klebsiella (K.) pneumoniae as one of the major pathogens involved. The receptor for advanced glycation end products (RAGE) plays a key role in diverse inflammatory responses. High mobility group box 1 (HMGB1) is a high-affinity ligand of RAGE.
Aim: To investigate the role of RAGE in the host response during K. pneumoniae pneumonia.
Methods: Normal wild-type (Wt) and rage gene deficient (RAGE-/-) mice were intranasally inoculated with 1 ×x 104 CFU of viable Klebsiella bacteria. In a separate experiment, Wt and RAGE-/- mice intranasally received highly pure lipopolysaccharide (LPS) from Klebsiella.
Results: K. pneumoniae pneumonia resulted in an increased pulmonary expression of RAGE and its high-affinity ligand HMGB1 compared to healthy, uninfected mice. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE deficient mice. RAGE-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. RAGE-/- mice displayed an unaltered response to intranasally instilled Klebsiella LPS.
Conclusion: The current study is the first to establish that RAGE is important for antibacterial defense during pneumosepsis by a common gram-negative causative pathogen, K. pneumoniae. We here show that RAGE plays a protective role during Klebsiella pneumonia, by improving antibacterial defense in lungs and reducing bacterial dissemination. This could at least in part be explained by a better phagocytosis capacity of neutrophils in the presence of RAGE.
RESUSCITATION WITH ARGININE VASOPRESSIN REDUCES OXIDATIVE DAMAGE IN KIDNEY MITOCHONDRIA FOLLOWING HEMORRHAGIC SHOCK
E.C. Werlin, Y. Guan, B. Agarwal, J.A. Baur, J.P. Villarroel, R. Figueredo, M.A. Karamercan, L.B. Becker*, and C.A. Sims*. University of Pennsylvania, Philadelphia, PA
Objectives: To determine if AVP use in resuscitation of HS improves mitochondrial function & reduces oxidative damage (OD) in kidney.
Methods: L-E rats were bled to MAP 40mmHg & maintained until the MAP could not be sustained w/o fluid. Once 40% of the shed volume (40%SVT) was returned in LR, animals were resuscitated over 60min w/4x the shed volume in LR or the same fluids w/AVP (0.5 U/kg+2 U/kg/hr). Animals (n=5/group) were sacrificed before hemorrhage (Sham), at 40%SVT, following resuscitation (60R, 60R w/AVP) or 18hrs post-resuscitation (18hr, 18hr w/AVP). Kidney samples were taken to assess histology, mitochondrial OD by measuring HNE and nitrotyrosine, & mitochondrial respiratory capacity (RC).
Results: Resuscitation w/LR+AVP led to higher MAP at 60R (125±3 vs 77±7mmHg;p<0.01) & 18hr (82±6 vs 69±5mmHg;p<0.05), & demonstrated normal renal histology. AVP supplementation significantly reduced mitochondrial lipid peroxidation as measured by HNE at 60R w/AVP (0.9±0.2 vs 1.7±0.1;p<0.01), but had minimal effect at 18hr (1.1±0.1 vs 0.9±0.1). Resuscitation w/AVP also decreased NO damage w/decreased nitrotyrosine seen at 60R (0.9±0.1 vs 1.4±0.2;p<0.01), but minimal influence at 18hr (0.9±0.1 vs 1.0±0.2). AVP significantly improved complex I RC at 60R (103.1±10.2 vs 84.7±12.1nmolesO2/min/mg;p<0.05) & at 18hrs (97.9 ±17.6 vs 80.3 ± 17.4nmolesO2/min/mg;p<0.05), but had no impact on complex II.
Conclusions: Following HS, resuscitation w/AVP results in improved hemodynamics, preserved renal architecture, decreased OD, & improved complex I RC.
ANALYSIS OF GENE EXPRESSION OF ENZYMES CONTRIBUTING TO PAIN VIA FORMATION OF OXIDIZED LIPIDS IN TRAUMA PATIENTS
H.M. Brandfellner*, S. Ruparel, D. Green, J. Gelfond, and K.M. Hargreaves. 1University of Texas Health Science Center San Antonio, San Antonio, TX
Adequate pain management in traumatically injured patients is difficult and often complicated by many adverse effects of the medications being prescribed. In response to injury and inflammation linoleic acid is metabolized to form oxidized-linoleic-acid-metabolites(OLAMs), which are direct agonists for the pain receptor, Transient Receptor Potential Vanilloid Subtype-1(TRPV1). Thus, OLAM production after trauma likely contributes to pain. The list of possible enzyme candidates involved in metabolizing linoleic acid is large and before additional studies can begin to target those enzymes involved in OLAM formation, a narrower focus must be established. Here, we tested the hypothesis that traumatic injury triggers a sustained upregulation in the expression of mRNA transcripts encoding oxidative enzymes that may be involved in OLAM synthesis.
The Glue Grant Trauma-Related Database (TRDB) was used to identify changes in expression patterns of transcripts from peripheral circulating leukocytes (PCL) after traumatic injury. The Affymetrix HG-U133_Plus_2 human microarray was used to analyze a total of 105 genes, including 86 genes known to oxidize poly unsaturated fatty acids (the general class of lipids that includes linoleic acid). 187 traumatically injured subjects had an associated 785 microarrays from PCL samples. The injured subjects were compared against 95 controls. Data was analyzed by log2 expression differences from control.
As compared to controls, traumatic injury in humans triggered significant upregulation of specific (Cytochrome P450(CYP) and Lipoxygenase (LOX)) gene transcripts in peripheral circulating leukocytes samples. The most significant upregulated and down regulated genes with a p<0.001 were well conserved from Day 0 to 28 post injury. Analysis of the relationship between the genes and Injury Severity, Max Denver 2, and APACHE II scores demonstrated common overlap amongst the genes with p<0.001.
Traumatic injury triggers a massive, selective and sustained alteration in the expression pattern of enzymes capable of forming OLAMs in the blood. The CYP family appears to play a critical role in OLAM formation, thus identifying and targeting specific CYP enzymes responsible for OLAM synthesis after injury could provide potential strategies for development of novel analgesics.
GLUTAMINE IS PROTECTIVE IN THE INTESTINE VIA DIFFERENT PROTECTIVE PATHWAYS IN HEAT AND OXIDANT INJURY MODELS: ROLE OF NETRIN-1
S. Niederlechner*, C. Aherne, A. Riegel, H. Eltzschig, and P.E. Wischmeyer*. University of Colorado, Aurora, CO
Background: Glutamine (GLN) protects intestinal cells from both heat and oxidant injury and decreases mortality in critically ill patients. The laminin-related, neuronal guidance molecule Netrin-1 (Ntn-1) has been implicated as a survival factor in the intestine. The purpose of our study was to determine whether GLN protects via Ntn-1 expression during both heat and oxidant injury.
Methods: IEC-6 cells were treated (15 min) with 0 mM GLN or 2 mM GLN and exposed to either non-lethal injury (43°C for 45 min or 600 μM H(2)O(2) for 30 min) or lethal injury (44°C for 50 min or 4 mM H(2)O(2) for 30 min). Survival was determined via MTS assay. Ntn-1 was evaluated via Western blot following a 3 h recovery after heat and oxidant stress. Small interfering RNA (siRNA) was used to block Ntn-1 expression. Cells transfected +/- 20 nM Ntn-1 siRNA (Ntn-1si) or non-coding control oligos (NC) (48h) were treated (15 min) with 0 mM (CT) or 2 mM GLN, and subjected to lethal HS (44°C / 50 min) for cell survival or non-lethal HS (43°C / 45 min) for protein expression. Ntn-1 and cleaved caspase-3 (CC-3) levels, a key mediator for apoptosis, were measured via Western blot.
Results: MTS assays showed that GLN increased cell survival significantly in both heat and oxidant injury (P<0.05). Ntn-1 levels were decreased by 80 % after HS in cells exposed to 0 mM GLN (P<0.05). 2 mM GLN prevented this decrease in Ntn-1 expression after HS (P<0.05). However, no attenuations in Ntn-1 levels in 0 mM GLN groups were seen after H(2)O(2) treatment. Ntn-1si transfection experiments showed that CC-3 levels were decreased in GLN-treated cells after HS (P<0.05). Ntn-1si (70 % Ntn-1 knockdown, p<0.05 vs. non silenced cells), but not NC oligos, attenuated the reduction in CC-3 in the HS GLN groups (P<0.05). MTS assays revealed that GLN increased survival 2-3 fold after lethal HS (P<0.05). Ntn-1si attenuated GLN’s protection by 91 % after HS (P<0.05).
Conclusion: GLN protects intestinal cells from both heat and oxidant injury. However, this is the first data showing that Ntn-1 expression seems to be essential in GLN’s protective mechanism after heat but not oxidant injury, suggesting GLN’s mechanism of protection may vary in different models of injury.
LFA-1 BLOCKADE IMPROVES SURVIVAL IN A MURINE MODEL OF SEPSIS VIA A LYMPHOCYTE-DEPENDENT MECHANISM
M. Serbanescu, A. Hadley, B.P. Yoseph, C. Coopersmith*, and K. McConnell*. Emory University School of Medicine, Atlanta, GA
Introduction: Activation and trafficking of lymphocytes are essential components of the immune response. The integrin, Leukocyte Function Antigen 1 (LFA-1), is expressed on the surface of all leukocytes and plays a crucial role in leukocyte trafficking and firm adhesion to endothelium. LFA-1 stabilizes T cell:APC interactions allowing for T cell activation, proliferation, and cytokine production.
Objective: To determine whether LFA-1 blockade is beneficial in a preclinical model of sepsis.
Methods: C57Bl/6 and Rag-/- mice were made septic by 2x25 cecal ligation and puncture and then given either saline or αLFA-1 antibody. Animals were either followed seven days for survival or sacrificed at 24 hours for flow cytometry at which time blood was assayed for frequency and absolute number of immune cell subsets. Activation status of T cells was evaluated by surface expression of CD44.
Results: Administration of αLFA-1 antibody improved survival from 14% to 62% (n=21/group, p=.003, figure 1). There was no survival benefit seen in Rag-/- mice treated with αLFA-1. Flow cytometry revealed that sepsis results in a decrease of circulating CD4 and CD8 cells, and administration of αLFA-1 antibody results in a lesser reduction of both lymphocyte subsets: (CD4 8.8% vs. 3.6%, CD8 4.58% vs. 1.0%). LFA-1 blockade resulted in a lower frequency and absolute number of CD44 (vs. CD44LO) CD4 cells in blood compared to controls (15.6% CD4 CD44HI vs 67.8%).
Conclusion: LFA-1 blockade improves survival in sepsis in a lymphocyte-dependent manner. This is associated with a mitigation of the sepsis-induced decrease in circulating lymphocytes and a differential effect on naïve cells.
TOO ZEALOUS TOO SOON: THE SHIFT FROM HYPER TO HYPO REACTIVE PHAGOCYTES IN POLYMICROBIAL SEPSIS
E.L. Chiswick, J. Mella, D.R. Beal, and D.G. Remick*. Boston University, Boston, MA
Objective: Six hours after cecal ligation and puncture (CLP) mice predicted to live (Live-P) or die (Die-P) have similar numbers of peritoneal phagocytes and bacterial CFUs. By 24 hours post-CLP (T-24), Die-P mice have significantly more peritoneal phagocytes and bacterial CFUs than Live-P. This suggests a cellular defect of phagocytes in Die-P mice. Preliminary work determined that peritoneal phagocytes from Die-P mice have decreased phagocytosis and reactive oxygen species (ROS) generation compared to Live-P mice. We investigated whether these deficiencies extend to cell compartments distal to the site of infection. Furthermore, we examined whether these defects translate into impaired bacterial killing.
Methods: Sepsis was induced by CLP in female outbred mice. Peritoneal Cavity (PC) and Bone Marrow (BM) cells were obtained at T-6 or T-24. Mice were stratified into Live-P and Die-P groups by plasma IL-6. Bacterial killing was determined with streptomycin resistant E. coli and PC cells. Phagocytosis and ROS was measured with pHrodo-E.coli and Dihydrorhodamine-123 via flow cytometry. Results were normalized to unstimulated controls. Comparisons of BM and PC activity were measured in the same animal.
Results: In conjunction with decreased ROS & phagocytosis in PC cells, Die-P PC exhibit less bacterial killing compared to Live-P mice at both T-24 (21% vs. 85% p<.0001) and T-6 (29% vs 92% p<.0001).
Die-P BM phagocytes show more phagocytosis and ROS compared to Live-P at T-24. Conversely, Die-P PC phagocytes display less phagocytosis and ROS compared to Live-P. Both Live-P and Die-P mice show more phagocytosis in PC compared to BM. In contrast, augmented phagocytosis-mediated ROS is found in PC vs BM in Live-P mice, while there is diminution in PC vs BM in Die-P.
Conclusion: Die-P PC phagocytes are depressed in effector functions as compared to Live-P mice as expected. However, Die-P BM cells are actually enhanced. Taken together with the similar bacterial CFUs awaiting both Live-P and Die-P BM phagocytes in the peritoneum, this suggests that these hyper responsive phagocytes are detrimental to the host.
EFFECTS OF POLYTRAUMA, HEMORRHAGE AND RESULTANT COAGULOPATHY ON INFLAMMATORY AND OXIDANT STRESS MARKERS IN RAT TISSUES
M.A. Dubick*1,2, D.N. Darlington*1, J.J. Barr1, D.L. Grubbs1, M.G. Schwacha*2, 1, and A.P. Cap1,2. 1US Army Institute of Surgical Research, San Antonio, TX, 2University of Texas Health Science Center San Antonio, San Antonio, TX
A polytrauma rat hemorrhage model with coagulopathy defined as prolonged PT and aPTT was developed and select markers of tissue inflammation and oxidant stress were investigated to explore potential mechanisms related to the coagulopathy. Anesthetized rats were subjected to polytrauma via intestinal crush, left and medial liver lobe crush and right leg femur fracture and skeletal muscle crush. They were then bled to and held at 40 mmHg until 40% of the blood volume was removed. No fluid resuscitation was given. Groups of rats were euthanized at times 0 (before trauma and hemorrhage; BTH) and 30, 60, 120, 180 and 240 min later. Tissue samples were collected at each time and assayed for cytokines, thiobarbituric acid reactive substances (TBARS), total antioxidant status, glutathione concentrations and select antioxidant enzyme activities. TNF-α peaked at 1 to 2 hr in liver and lung after start of trauma/hemorrhage, whereas IL-6 was highest at 3 and 4 hr. These cytokines were elevated 30 min sooner in jejunum. Liver IL-17 rose slowly to levels that were 1.8 fold higher than BTH at 3 and 4 hr. No significant changes were observed in TBARS or total antioxidant status in any tissue over time. Liver glutathione (GSH) levels were about 60% lower than BTH at 3 and 4 hr and Mn superoxide dismutase activity was nearly 60% lower than BTH at 2 and 3 hr. Jejunal GSH levels fell 25% from 2-4 hr compared to BTH. Lung myeloperoxidase activity began to rise after 30 min and peaked at 4 hr, whereas its activity in jejunum was only higher than BTH at 4 hr. Plasma PT peaked at 60 min and remained elevated through 4 hr. These data indicate that this model is associated with an inflammatory response and oxidative stress in the tissues examined that coincides with the rise in PT. Cytokine responses occurred prior to or at the time of changes in oxidative stress indices. The relationship of these observations to the development of coagulopathy remains to be explored. Funded by the US Army Medical Research Materiel Command.
UTILITY OF END-TIDAL AND TRANSCUTANEOUS CARBON DIOXIDE MONITORING DURING LETHAL HEMORRHAGIC SHOCK
S. Belenkiy1, 2, J. Berry1, 2, A. Batchinsky1, C. Kendrick1, 2, C. Necsoiu1, J. Salinas1, and L. Cancio*1. 1US Army Institute of Surgical Research, Ft Sam Houston, TX, 2San Antonio Military Medical Center, Ft Sam Houston, TX
Objectives: We evaluated utility of capnography and transcutaneous CO2 monitoring in setting of fatal hemorrhage. Noninvasive results were correlated with arterial carbon dioxide (PaCO2) at baseline, after 25%, 44%, 62% total blood volume hemorrhage (TBVH), and cardiac arrest in a porcine model of exsanguination-induced cardiac arrest.
Methods: Ten consciously-sedated female Yorkshire pigs were subjected to a fatal hemorrhage of 80% TBV over 20 min. End-tidal CO2 (etCO2) was monitored with Capnostream 20. Transcutaneous CO2 (tPCO2) was recorded with SenTec digital system. PaCO2 was measured with i-STAT. Statistical analysis included linear regression, Bland Altman method and one-way RM ANOVA with post-hoc Dunnett’s tests. Data are presented in mm Hg as means ± SD.
Results: See table 1.
Conclusion: In a swine model of lethal hemorrhage, neither transcutaneous nor end-tidal CO2 monitoring, when used alone, were reliable surrogates for PaCO2. However, tPCO2-etCO2 gap demonstrated statistically significant progressive increases with each subsequent hemorrhage step above 25% TVBH, suggesting possible utility of this noninvasive measurement in monitoring severity of hemorrhagic shock.
MUSCLE OXYGENATION PREDICTS SEVERITY IN AN ANIMAL MODEL OF HEMORRHAGIC SHOCK
K.A. Schenkman, D.M. McMullan, W.A. Ciesielski, and L.S. Arakaki. University of Washington, Seattle, WA
Introduction: In shock, oxygenation in muscle may decrease before brain and central organs due to preferential shunting of blood to maintain central perfusion. We hypothesize that muscle oxygenation (Mox) may serve as an important early indicator of shock in bleeding patients.
Methods: A novel noninvasive optical monitor was developed to measure Mox from the hind limb in the rabbit. A bifurcated optical probe was designed to illuminate the muscle and return light to a cooled CCD array detector. Optical spectra were analyzed by multiwavelength analysis. Animals were anesthetized and instrumented for mechanical ventilation and blood pressure monitoring. Up to 40% of the animal’s blood volume was withdrawn in three aliquots to produce shock. After an hour of shock, shed blood was reinfused in three aliquots for resuscitation. Blood gas, lactate and hematocrits were monitored every 10 minutes.
Results: Severity of shock was stratified based on the highest lactate recorded. Mox decreased almost immediately with blood loss, remained low during shock, and recovered quickly with reinfusion of blood. Lactate values lagged Mox significantly during the development of shock and resuscitation. Mean Mox was 72+/-14% at baseline in room air (n=14), 86+/-13% on 100% oxygen (n=16), and dropped to 38+/-14% with mild (n=6), 11+/-4% with moderate (n=4) and 4+/-3% with severe (n=6) shock.
Conclusions: Noninvasive measurements of Mox from the hind limb are sensitive to mild, moderate, and severe shock, as well as to resuscitation. Furthermore, Mox correlates with MAP and appears to be an earlier indicator of reduced end-organ oxygen delivery than pulse oximetry or arterial lactate.
MICROGLIAL CD14 ACTIVATED BY INOS CONTRIBUTES TO NEUROINFLAMMATION IN CEREBRAL ISCHEMIA
M. Zhou*, W. Yang*, and P. Wang*. Hofstra North Shore-LIJ School of Medicine and The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Stroke is one of the most frequent causes of death and disability worldwide. Cerebral ischemia induces acute inflammation, leading to exacerbation of primary brain damage in stroke. Toll-like receptor (TLR)- and nitric oxide-mediated signaling pathways are activated under the ischemic stress. However, the interaction between these two pathways in controlling proinflammatory cytokines has not been well addressed in stroke. Here, we used an animal model and cell culture system to demonstrate the cross-talk between these two pathways.
Methods: Adult male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) to induce stroke. The MCAO procedure resulted in a 30% infarct area in the center section of the brain after 24 h. Murine microglial BV2 cells were cultured in normoxia or hypoxia (1% O2) for 20 h with or without L-canavanine (CAN, 50 μM). Brain tissue and BV2 cell lysate were collected for RT-PCR and Western blotting.
Results: MCAO induced a 12.1- and 1.4-fold increase of TNF-α gene and protein expression, respectively, in the brain (p<.05 vs. sham). The protein expression of CD14 was elevated 2.1-fold, while TLR4 levels remained unchanged after MCAO. The levels of inducible nitric oxide synthase (iNOS) and nitrotyrosine in the infarcted brain were also increased by 1.3-and 1.9-fold, respectively (p<.05). Exposing BV2 cells to hypoxia resulted in an increased protein expression of TNF-α, CD14, iNOS, and nitrotyrosine by 2.3-, 5.1-, 3.9-, and 5.0-fold, respectively (p<.05). When BV2 cells were treated with CAN, an iNOS selective inhibitor, the elevation of TNF-α and CD14 induced by hypoxia was significantly inhibited (Figure). After hypoxia, IκB levels were decreased by 71% in BV2 cells, while their levels partially recovered by CAN (p<.05 vs. hypoxia).
Conclusions: Upregulation of TNF-α production in ischemic stroke is partially mediated by the increased iNOS and then CD14 expression, leading to the activation of the NF-κB pathway in microglia.
THERAPEUTIC POTENTIAL OF ADIPOSE-DERIVED STEM CELLS AFTER HEMORRHAGE AND TRAUMA IN RATS
C. Penzenstadler*, M. Ashmwe, A. Bahrami, A. Klotz, M. Jafarmadar, A. Banerjee, S. Wolbank, H. Redl*, and S. Bahrami*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology & AUVA Research Centre, Vienna, Austria
Background & Aim: Hemorrhagic traumatic shock (HTS) and reperfusion is often associated with general inflammatory response and organ dysfunction. Contemporary literature indicates that mesenchymal stem cells may have a cytoprotective and/or anti-inflammatory impact. We evaluated the therapeutic potential of rat adipose-derived stem cells (ASCs) in a HTS model in rats.
Methods: Rats were subjected to HTS (mean arterial pressure 30-35 mmHg till decompensation) and a resuscitation protocol, that includes prehospital restrictive reperfusion (30 ml/kg/h, MAP maintained at 50-55 mmHg for 40 min) followed by an adequate reperfusion phase (75ml/kg/h over 60 min, MAP to baseline). 20 minutes after the onset of reperfusion, animals received either 2x10∧6 ASCs (n=7) or vehicle (n=6) intravenously. Blood samples were obtained at baseline, end of resuscitation (EOR), 24 and 48 hrs after shock.
Results: Cell injury markers creatin kinase and lactate dehydrogenase peaked at 24 hrs with no difference between ASCs and vehicle groups (41724 vs 17375 U/L and 22280 vs 18352 U/L, respectively) and returned to baseline at 48 hrs. HTS-induced liver dysfunction determined by plasma alanine aminotransferases (ALT) was significantly ameliorated at EOR by ASC treatment (1194±176 vs 2458±400 U/L). Plasma ALT peak at 24 hrs did not differ between groups. Similarly, HTS-induced increase in plasma creatinine levels did not differ between groups. Inflammatory response reflected in IL-6 was less pronounced in AMSC treated group at 48 hrs (77.1±46.1 vs 422.3±184.9 pg/ml, p<0.061). Changes in IL-10 and MCP-1 were not different between groups.
Conclusion: Systemic administration of ASCs during reperfusion may ameliorate the HTS-related early liver dysfunction and/or late inflammation in rats.
EXAGGERATED CARDIAC CONTRACTILE DEPRESSION IN AGING ENDOTOXEMIC MICE IS ASSOCIATED WITH ENHANCED INFLAMMATORY RESPONSES
H. Slimani, N. Yousif, Y. Zhai, L. Ao, D. Fullerton, and X. Meng*. University of Colorado Denver, Aurora, CO
Introduction: Trauma and stress associated with major surgery can cause gut bacteria translocation, leading to endotoxemia and the systemic inflammatory response. Major surgeries performed in the elderly are increasing with the increase in life expectancy. While the general immunological function is decreased with aging, cytokine response to endotoxin is reported to be enhanced in old animals. Understanding of the impact of endotoxemia on vital organ function is helpful for improving post-surgery outcomes in the elderly. We tested the hypothesis that the vulnerability to endotoxemic cardiac depression is increased with aging due to age-related augmentation of the inflammatory response.
Methods: Adult (4-6 months) and aging (18-20 months) C57BL/6 mice were treated with a low dose of endotoxin (0.5 mg/kg, iv) for 6 h. Left ventricle (LV) function was assessed using a microcatheter. Chemokines (MCP-1, KC and MIP-1) and cytokines (TNF-α, IL-1β and IL-6) in plasma and the myocardium, as well as cardiac troponin I in plasma were analyzed by ELISA. Neutrophils and mononuclear cells in the myocardium were examined using immunofluorescence staining.
Results: Endotoxemia caused cardiac contractile depression in both adult and aging mice. However, aging endotoxemic mice exhibited worse LV function (cardiac output 1.5±2.3 ml/min vs 5.6±3.6 ml/min in adult mice). In addition, cardiac troponin I levels in plasma were higher in aging endotoxemic mice. Aging endotoxemic mice had markedly higher levels of MCP-1 and KC, but lower levels of MIP-1, in both plasma and myocardium in comparison with adult endotoxemic mice. The exaggerated cardiac contractile depression in aging mice is associated with greater densities of neutrophils and mononuclear cells in the myocardium, and higher levels of TNF-α, IL-1β and IL-6 in the circulation and myocardium.
Conclusion: Endotoxemia causes exaggerated cardiac contractile depression in aging mice. The increased vulnerability to endotoxemic cardiac depression in aging mice is associated with enhanced systemic and cardiac inflammatory responses. Our findings suggest that special attention is needed to suppress the inflammatory responses and to protect the heart in the elderly with endotoxemia.
PRE-EXISTING MALIGNANCY ALTERS INNATE AND ADAPTIVE IMMUNE RESPONSES IN A MURINE MODEL OF INFECTION
R. Mittal, M. Wagener, E.R. Breed, Z. Liang, B.P. Yoseph, C. Coopersmith*, and M. Ford. Emory University, Atlanta, GA
Objectives: To quantify and characterize the immune response to Listeria monocytogenes in the setting of malignancy as pre-existing malignancy is the second leading predisposing factor to bacterial infection.
Methods: B6 mice were injected with a pancreatic adenocarcinoma cell line (Pan02) and allowed to develop tumors for 3 weeks. The cancer group and a group of non-cancer control mice underwent adoptive transfer of 104 TCR transgenic OVA-specific Thy1.1+ CD8+ T cells. After 24 hours, all mice were infected with 104 CFU of an OVA-expressing Listeria. Blood samples at days 0, 2, 7, and 14 post-infection were collected and assayed for immune subset frequency and activation by flow cytometry (n=9-10). Spleens (n=4-5) were collected at day 14 and were assayed for frequency of immune cell subsets, T-regulatory (Treg) cells, and intracellular cytokine production following ex vivo restimulation with cognate antigen.
Results: The absolute number of NK1.1+ cells was lower in spleens of cancer mice (16541±1757 vs. 11010±410; p=0.01), and these cells contained fewer Ly49D+ and CD127+ cells (8751±806 vs. 6139±208 and 1762±135 vs. 1241±130, respectively; p<0.05). Spleens in cancer mice had reduced frequencies of total CD4+ and CD8+ T cells (18% vs. 11% and 17% vs. 12%, respectively; p<0.05), and reduced frequencies of antigen-specific Thy1.1+ CD8+ T-cells (3.6% vs 1.3%; p<0.05). In cancer mice, these cells had a lower number of cells expressing high levels of KLRG-1 (4969±870 vs. 1507±513; p=0.03), a surface maker induced on highly activated effector T cells. Restimulation resulted in lower numbers of antigen-specific IFN-γ producing CD8+ T cells (591±179 vs. 126±45; p=0.02) in the cancer group. While cancer alone causes an increase in Tregs, the combination of cancer and infection results in lower numbers of CD4+ Foxp3+ Tregs in the cancer group (5529±880 vs. 2035±512; p=0.02). The number of Helios+ thymic-derived Tregs was reduced in the cancer group (3354±596 vs. 1211±334; p=0.03), while the number of Helios- peripherally derived Tregs was not different.
Conclusion: Pre-existing malignancy alters the innate and adaptive immune responses to infection, characterized by impaired Listeria-specific CD8+ T cell expansion and effector function, diminished NK cell accumulation, and reduced counter-regulatory Treg responses.
A FAILURE TO RESOLVE INFLAMMATION RATHER THAN HYPER-INFLAMMATION CHARACTERIZES THE AGED RESPONSE TO SEVERE TRAUMA
D. Nacionales, L.F. Gentile, R. Ungaro, M. Lopez, J. Jyot, E. Vanzant, A. Cuenca, S. Gabrilovich, A. Bihorac, F.A. Moore, A. Joseph, C. Leeuwenburgh, H. Baker, L. Moldawer*, and P.A. Efron*. University of Florida, Gainesville, FL
Objectives: It is well established that the elderly have worse outcomes after trauma, including increased infections. Using a recently described murine polytrauma (PT) model (CCM in press, 2013), we compared the leukocyte responses of old and young mice after injury, as well as their survival to subsequent Pseudomonas pneumonia (Pp).
Methods: 6-10 wo young and 18-24 mo old B6 mice underwent 90 minutes of shock (MAP 30 mmHg) and resuscitation via femoral artery cannulation, followed by laparotomy with cecetomy and femur fracture + muscle tissue damage. Mice were euthanized 2h, 1d and 3d after PT and their spleens, bone marrow, blood and serum were collected; leukocyte phenotypic and functional analysis were performed. Genome-wide expression was performed on total blood leukocytes. Expression patterns were compared between healthy and young/old PT mice at p<0.001 (F test). Intranasal Pseudomonas was instilled to induce Pp 1d post PT.
Results: Serum cytokine/chemokine concentrations were not different between young and old mice and there were few differences in the numbers and activation/functional status of bone marrow, spleen and blood leukocytes. However, the blood WBC transcriptomic response to PT differed markedly in young and aged mice (p<0.05). Old and young mouse showed similar overall transcriptomic changes at 2h, but old mice did not show early increased up-regulation of genes related to PMN-mediated immunity, chemokine/chemokine receptor binding and responses to pathogen-associated molecular patterns. At 1d, the transcriptome from young mice started to return to baseline, whereas a similar return was not seen in aged mice even at 3d. Finally, aged PT mice given Pp at 1d had a significantly increased mortality (55%; p<0.02 vs naïve, Pp, young PT+Pp).
Conclusion: Although ‘inflamma-aging’ exists, it’s role in increased infections and mortality after trauma in the aged is modest. Rather, a failure of leukocytes from the aged to initiate an early innate immune response, and a subsequent inability to effectively resolve their inflammatory response to severe injury may leave them at risk to subsequent infection. A proper understanding of this phenomenon is critical to improving elderly patient outcomes in the future.
EVALUATION OF OXIDATIVE STRESS IN THE CEREBROSPINAL FLUID OF PATIENTS WITH SEVERE TRAUMATIC BRAIN INJURY
K. Borg*, D. Townsend, S. Hutchens, P. Halushka*, E. Jauch, K. Tew, and Y. Manevich. Medical University of South Carolina, Charleston, SC
Traumatic brain injury (TBI) is a significant cause of morbidity and mortality in trauma patients. Oxidative stress through reactive oxygen species (ROS) induced damage is a significant component of the secondary injury cascade that leads to lifelong disability and contributes to a leading cause of mortality in patients with TBI. In our studies of human TBI, we enrolled patients with severe TBI and evaluated oxidative stress through examination of cerebrospinal fluid (CSF) from patients who have had ventriculostomies placed for management of intracranial pressure (ICP) following blunt trauma. We serially sampled CSF and plasma of patients after injury. Protein thiols (cysteines, Cys) are known to be extremely sensitive to oxidative damage. Thus, their redox status is expected to correlate with the extent of oxidative stress. We analyzed protein thiol status in CSF and plasma samples from control and TBI patients using ThioGlo-1 sulfhydryl-specific fluorescent probe. Our data show a correlation between CSF protein-thiol oxidation and severity of TBI outcome. Similar analysis of plasma from the same patients did not show any correlation. Western blot analysis of CSF from control and TBI patients revealed high levels of peroxiredoxin VI (Prdx6) - a novel cytosolic dual-functioning antioxidant enzyme with glutathione peroxidase and PLA2 activities to protect the cellular membranes against lipid peroxidation. Prdx6 was reduced in the CSF of healthy volunteers, but substantially oxidized in the CSF of TBI patients. Its level of oxidation correlated with the severity of TBI outcome. The results suggest a potential physiological role of Prdx6 in mitigating oxidative stress damage in the CSF of TBI patients. We also measured 8-isoprostane (8-IP) in the CSF as a measure of the oxidative stress-mediated damage of phospholipids. 8-IP was elevated in patients with severe TBI as compared to that in control patients. The levels of 8-IP were elevated in patients with worsening outcome and low in patients with the best outcomes. Our studies provide potential markers, targets and insights into the role of oxidative stress following TBI in humans.
FARNESYLTRANSFERASE INHIBITOR (FTI) AS A NOVEL POTENTIAL THERAPEUTIC STRATEGY TO IMPROVE SEPSIS SURVIVAL
M. Yamada1, S. Ishikawa2, J. Lederer*2, and M. Kaneki*1. 1Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Charlestown, MA, 2Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
The lipid lowering-independent beneficial effects of statins, HMG-CoA reductase inhibitors, in septic patients have been tested in clinical trials. The mechanisms of the pleiotropic effects of statins, however, remain to be determined. Statins decrease biosynthesis of farnesyl pyrophosphate, which is a substrate of protein farnesylation (a lipid modification of cysteine residues) as well as a precursor of cholesterol. Farnesylation leads to membrane trafficking and functional alterations of many proteins. To test the hypothesis that the inhibition of protein farnesylation plays an important role in the cholesterol-independent protective effects of statins, we studied the effects of tipifarnib, a clinically applicable farnesyltransferase (FTase) inhibitor (FTI), in septic CD-1 mice. Cecum ligation and puncture (CLP) increased protein farnesylation two-fold in splenocytes, including neutrophils and NK cells. CLP also significantly increased protein farnesylation and FTase expression in liver (2-fold), heart (3-fold) and kidney (5-fold) compared with sham mice. A single injection of tipifarnib (1, 3, 10 mg/kg, SC) at 2 h after CLP dose-dependently improved survival of septic mice compared with vehicle (n=24 per group, p<0.0001) and the maximum effects were observed at 3 and 10 mg/kg. Similarly, tipifarnib (3 mg/kg) reduced the mortality after pulmonary infection induced by intranasal challenge of P. aeruginosa (4.8 x 10∧8 CFU bacteria) in mice (n=16 per group, p<0.001). Tipifarnib (3 mg/kg) significantly reduced: (1) bacterial burden in the circulation and peritoneal cavity; (2) plasma levels of cell-free DNA, HMGB1, IL-1β, IL-6 and IL-10; and (3) neutrophil infiltration in organs (i.e., liver, heart and kidney) as judged by myeloperoxidase expression at 16 h after CLP in mice. These results clearly demonstrate that sepsis increases protein farnesylation in immune cells and organs and that tipifarnib improves survival of septic mice. Our data suggest that improved bacterial clearance along with reversal of increased circulating alarmins and neutrophils infiltration into organs may contribute in concert to the pro-survival effects of tipifarnib. Our study identifies FTase as a novel potential target to improve the clinical outcome of septic patients. These data warrant a clinical study to evaluate the safety and efficacy of tipifarnib in septic patients.
DIFFERENTIAL INFLAMMATORY PROPERTIES OF GAG GENES OF TWO BURN INJURY-ASSOCIATED HUMAN ENDOGENOUS RETROVIRUSES
K. Lee1, H. Rah1, T.L. Green1, Y. Lee1, D. Lim1, J. Nemzek*2, W. Wahl3, D.G. Greenhalgh*1 and K. Cho*1. 1Shriners Hospitals for Children and University of California, Davis, Sacramento, CA, 2Unit for Laboratory Animal Medicine and Department of Pathology, University of Michigan, Ann Arbor, MI, 3Department of Surgery, Saint Joseph Mercy Health System, Ann Arbor, MI
Common polymorphisms found in gene functions have been the centerpiece of studies investigating post-burn systemic pathogenesis. However, the sum of all known conventional genes consists of only ∼3 % of the human genome, whereas ∼8% is occupied by human endogenous retroviruses (HERVs), and it is anticipated that genomic HERV profile is individual-specific. We recently reported that certain gene products of murine endogenous retroviruses can modulate the production of inflammatory cytokines in macrophages. In this study, we identified burn-associated HERVs by examining the changes in the expression of various HERV families in buffy coat cells of burn patients. Subsequently, the gag genes from two burn-associated HERVs, which were isolated from the genome of one patient (Pt1), were characterized in regard to their potential roles in inflammatory processes. The two gag genes of Pt1 were presumed to be derived from the HERV-K109 and HERV-K115 loci on the NCBI reference chromosomes 6 and 8, respectively. The putative amino acid sequences of both gag-HERV-K109Pt1 (666 amino acids) and gag-HERV-K115Pt1 (545 amino acids), which were cloned from Pt1’s genome, were not identical to the corresponding reference sequences. Overexpression of the gag-HERV-K109Pt1 markedly induced the expression of IL-6, IL-1β, COX-2, and iNOS in macrophages while only the expression of IL-1β was increased by gag-HERV-K115Pt1. The expression of TNF-α was not affected by overexpression of either HERV gag gene. These findings suggest that the inherent HERV gene polymorphisms function as uncommon genetic factors which contribute to individual-specific burn as well as other disease phenotypes.
PANCREATIC PROTEASES ARE TRANSPORTED THROUGH MESENTERIC LYMPH TO THE SYSTEMIC CIRCULATION
A.E. Altshuler, M. Richter, A. Modestino, M. Heller, and G.W. Schmid-Schonbein*. University of California, San Diego, La Jolla, CA
Objective: The lymphatics play a key role in the pathogenesis of multiple organ dysfunction following circulatory shock. Current results show that biologically active molecules produced in the gut enter the circulation via the mesenteric lymph duct, and induce neutrophil activation, increased vascular permeability, and acute lung injury. Despite several attempts to determine the protein composition of mesenteric lymph, the factors responsible for this biological activity and specifically the organ damage are unknown. The aim of this study was to investigate the transport of pancreatic digestive proteases and their activities in post-shock lymph to the central circulation.
Methods: Male Wistar rats were subjected to laparotomy, followed by mesenteric lymph duct cannulation and one hour of lymph fluid collection before shock. Five animals then underwent one hour of splanchnic artery occlusion followed by reperfusion, while sham shock animals remained perfused. Post-shock lymph was collected for three hours, with the samples aliquoted every hour. Protease activity in the lymph was analyzed using charge-changing fluorescently quenched peptide substrates specific to pancreatic digestive proteases (trypsin, chymotrypsin) and by Western blot. To confirm presence of pancreatic enzymes in the plasma following hemorrhagic shock (1.5 hr ischemia [30 mmHg], 3 hr reperfusion, N=5), pre- and post-shock plasma was collected and analyzed by gel zymography and Western blot.
Results: Zymographic measurements showed the presence of trypsin and chymotrypsin activity in the lymph (in the nanomolar range). The presence of pancreatic digestive proteases was confirmed by Western analysis. Baseline levels of protease activity in the mesenteric lymph increased following gut ischemia during reperfusion. Trypsin and chymotrypsin plasma protein levels were detected before and after hemorrhagic shock, but their activities were only elevated after shock.
Conclusions: These results indicate that digestive protease escape from the intestine and their activities are elevated following intestinal ischemia, raising the possibility that they are involved in the systemic injury following shock.
Supported by NIH grant GM-85072.
CRITICAL ROLE OF JAK/STAT1 PATHWAY IN HMGB1 RELEASE
B. Lu*, K. Kwan, D.J. Antoine, P. Lundback, H. Wähämaa, H. Yang*, J. Li, S.S. Chavan*, U. Andersson, and K. Tracey*. The Feinstein Institute for Medical Research, Manhasset, NY
Extracellular high-mobility group box 1 (HMGB1) is a necessary and sufficient mediator of inflammation during sterile and infectious injury. Activated immune cells mobilize HMGB1 from nucleus to cytoplasm, which is a critical step for HMGB1 release into extracellular space during inflammation. Previous studies establish that canonical or non-canonical inflammasome mediates pyroposis and subsequent release of cytoplasmic HMGB1 in activated immune cells. However, the mechanisms by which immune cells regulate HMGB1 translocation from nucleus to cytoplasm are poorly understood. Here we show that JAK/STAT1 pathway is essential for LPS-induced nuclear translocation and release of HMGB1 in macrophages. Pharmacological inhibition JAK/STAT1 pathway blocked LPS-induced HMGB1 nuclear translocation and release. Conversely, activation of JAK/STAT1 pathway by type 1 interferon induced robust HMGB1 nucleus to cytoplasm translocation. In an agreement with early studies that hyperacetylation and phosphorylation at the nuclear location sequences (NLSs) of HMGB1 importantly regulates HMGB1 nuclear translocation, increasing HMGB1 acetylation by pharmacological inhibition of histone deacetylases (HDAC) significantly enhanced HMGB1 translocation and release. Importantly, mass spectrum analysis revealed that pharmacological inhibition of JAK/STAT1 pathway abrogated LPS- or type 1 interferon-induced HMGB1 hyperacetylation and phosphorylation in the NLSs. Together, these results identify a critical role of JAK/STAT1 pathway in mediating HMGB1 nuclear translocation, and thus provide potential drug targets for the treatment of HMGB1-releated inflammatory diseases.
Supported in part by grant from NIH (RO1 GM57226 to K. J. T).
PROGRAMMED CELL DEATH RECEPTOR LIGAND-1 (PD-L1): A ROLE IN ANGIOPOIETIN-MEDIATED ENDOTHELIAL CELL ACTIVATION IN THE DEVELOPMENT OF ACUTE LUNG INJURY
J. Lomas-Neira*, S.F. Monaghan*, X. Huang*, Y. Chen, and A. Ayala*. Alpert School of Medicine at Brown University/Rhode Island Hospital, Providence, RI
Our laboratory has recently reported that deficiency of the co-inhibitory receptor, Programmed cell death receptor (PD)-1, provides a survival benefit in our murine shock/sepsis model for the development of acute lung injury (ALI). While PD-1 expression and its regulatory role have been associated with mainly T-cell responses, its ligand PD-L1, is broadly expressed on other non-immune cells, specifically, lung endothelial cells (ECs). Loss of endothelial barrier function is a defining factor in the development of ALI. To investigate the impact of PD-1/PD-L1 expression on lung ECs, we assessed changes in the expression of EC growth factors Angiopoietin (Ang)-1/2 and their cognate receptor, Tie2. Ang-1/Tie2 binding has been shown to promote vessel integrity, and pro-survival and anti-inflammatory signaling [Tie2 phosphorylation (pTie2)], while Ang-2/Tie2 is associated with increased vessel permeability and inflammation. Expression of these proteins was assessed (Western Blot analysis) in lung tissue lysates from C57BL/6 background, PD-1 or PD-L1 knockout (KO) mice in our model of hemorrhagic shock (Hem) or Sham Hem (Sham), followed by subsequent septic challenge (CLP), which when combined (Hem/CLP) culminates in the development of ALI unlike Sham/CLP.
Lung tissue receptor Tie2 expression increased in all mouse strains following Hem/CLP compared to respective Sham/CLP. Despite this increase, pTie2 expression decreased significantly in C56BL/6 as well as both KO mouse strains. pTie2 is associated with Ang-1/Tie2 binding and the promotion of EC barrier stability and this data is consistent with decreased Ang-1 and increased Ang-2 expression observed in the lung tissue from C56BL/6 and PD-1 KO mice. This trend, however, was not observed in the PD-L1 KO. This distinction may reflect a broader impact on inflammation associated with PD-1+ leukocyte selective interactions with PD-L1 expressing non-immune cells such as lung ECs and/or lung epithelial cells. (Supported by NIH HL-073525)
HEME OXYGENASE-2 REGULATES HYPOXIA INDUCED MITOCHONDRIAL SIGNALING AND RESPONSES IN HEPATOCYTES
B. Kautza1, S. Whelan1, S. Stratmirovics1,2, and B. Zuckerbraun*1,2. 1University of Pittsburgh, Pittsburgh, PA, 2VA Pittsburgh Healthcare System, Pittsburgh, PA
Introduction: Cellular hypoxia is part of many pathological processes including shock. Adaptive cell signaling responses allow cells to compensate and survive the insult of hypoxia. Mitochondria responses are important to prevent bioenergetic failure and in initiating cellular responses. Furthermore, heme oxygenase-2 has been implicated in sensing hypoxia. The purpose of these experiments is to test the hypothesis that heme oxygenase-2 (HO-2) regulates mitochondrial induced cell signaling and responses in hypoxia in hepatocytes.
Methods: Primary hepatocytes were harvested and cultured from C57BL/6 or gp91phox-/- mice. Cells were exposed to standard culture conditions (5%CO2, and 21% O2) or hypoxia (5%CO2, 1%O2) for 0-12 hours. In some experiments hepatocytes were pretreated with control siRNA, HO-1 siRNA, or HO-2 siRNA. Additionally, tin protoporphyrin (SnPP) was utilized as a pharmacological inhibitor of HO. Western blots or immunohistochemistry for HO-1, HO-2, hypoxic signaling proteins, mitochondrial fusion pathways, and oxidative phosphorylation proteins were performed. Reactive oxygen species were measured by DCF fluorescence.
Results: Hypoxia increased cellular ROS levels in C57BL/6 mice but not from mice with altered NADPH oxidase (gp91phox-/- mice). Mitochondrial targeted antioxidants decreased ROS levels. HO-2 but not HO-1 was expressed in hepatocytes at baseline and was localized within mitochondria. SnPP or HO-2 siRNA, but not HO-1 siRNA inhibited hypoxia induced ROS. Furthermore, hypoxia induced phosphorylation of JNK and increased HIF1a protein levels, and this was inhibited by HO-2 siRNA or SnPP. Furthermore, hypoxia induced mitochondrial fusion signaling and this was inhibited by HO-2 siRNA. Moreover, 12 hours of hypoxia exposure led to increased expression of proteins of oxidative phosphorylation and this was inhibited by HO-2 siRNA.
Conclusions: HO-2 regulates mitochondrial responses to hypoxia in hepatocytes. These results suggest that HO-2 is important molecule in initiating adaptive responses to hypoxia. Further understanding of these responses may help to guide the development of therapeutics in shock.
MIDDLE-AGED ANIMALS FAIL TO CLEAR PULMONARY BACTERIAL INFECTIONS AFTER SEVERE INJURY DESPITE INCREASED EXPRESSION OF TLR-4 ON MONOCYTES AND INCREASED SYSTEMIC CYTOKINE RESPONSE
R.E. Southard*, S. Ghosh, C. Davis, J. Hilliard, K. Chang, and I. Turnbull. Washington University in St Louis, St Louis, MO
Introduction: Nosocomial pneumonia is a common problem in critically injured patients. Mortality due to nosocomial pneumonia is increased in the elderly. Changes in the innate immune response following trauma are thought to play a key role in the development of pneumonia. In a murine model of injury and pneumonia, we have demonstrated increased susceptibility to Pseudomonas aeruginosa pneumonia in middle-aged animals. We hypothesized that aging leads to differential expression of toll-like receptor 4 (TLR-4) in blood monocytes and this plays an important role in the ability of injured animals to clear infections.
Methods: C57BL/6J retired breeder (6-8 months of age) and young (8-10 weeks) male mice were subjected to multisystem injury with partial liver laceration, bilateral hind limb crush injury, and withdrawal of 15% of total blood volume via cardiac puncture. Mice were sacrificed at 24 hours after the injury and the percentage of whole blood monocytes expressing TLR-4 was measured by flow cytometry. Mice were subjected to a pulmonary infection with intratracheal Pseudomonas aeruginosa. Cytokines were assayed using a Luminex assay before and after infection. Bacterial clearance was determined by bacterial culture of BAL fluid. Uninjured animals served as controls in all studies.
Results: Middle-aged mice subjected to severe injury showed increased expression of TLR-4 on circulating monocytes (18%) vs. uninjured mice (4%, p=0.04.) While monocytes in young mice did not show a similar response (24% injured, 20% uninjured), the baseline level of expression was higher than that of older mice (p=0.02.) Cytokine values, such as IL-6, showed that injured middle aged mice had an enhanced expression of cytokines after infection (2400 pg/mL) compared to young mice (20 pg/mL, p<0.001.) Middle-aged mice had significantly higher (1x10∧6 CFUs) bacterial counts compared to injured young mice (1000 CFUs, p<0.05.)
Conclusion: Despite an increase in monocyte TLR4 expression and enhanced cytokine production in response to injury, middle-aged animals demonstrate a markedly decreased ability to clear a pulmonary infection due to Pseudomonas aeruginosa. The deficiency in the ability to clear infection in older animals is not due to an inability to mount a systemic inflammatory response after injury.
IGF-1 REDUCES THE HEPATIC UNFOLDED PROTEIN RESPONSE IN THERMALLY INJURED ANIMALS
A. El Ayadi1, 2, A.E. Offordile1, 2, D.N. Herndon*2, S. Hegde2, A. Prasai1, 2, E.C. Diaz1, 2, L.E. Sousse*1, 2, M.G. Jeschke*3, and C.C. Finnerty*1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX, 3Sunnybrook Health Sciences Center, Toronto, ON, Canada
Objectives: We have previously shown that endoplasmic reticulum(ER) stress and the unfolded protein response (UPR) pathways are activated in the liver following burn injury. The administration of insulin following burn injury protects hepatic cells, thereby reducing ER stress and apoptosis, which leads to improved liver function. Insulin-like growth factor-1(IGF-1) mediates the effects of growth factors following prolonged stress such as burn injuries. Here we determined whether IGF-1 administration impacts the hepatic ER stress response in thermally injured rats.
Methods: A 60% total body surface area scald burn rat model was used. Subcutaneous administration of IGF-1 by continuous mini-pump infusion was started one day before the burn and continued for 2, 7, 14 or 28 days post burn. Liver protein and RNA levels of the ER stress markers were measured.
Results: IGF-1 infusion significantly decreased the post-burn elevation of the ER stress marker calnexin at 2 weeks post burn. The luminal ER stress marker calreticulin, which is increased at 2 days, 2 weeks and 4 weeks post burn, was not affected by IGF-1. IGF-1 also inhibited the increase in the GRP78 (BIP) protein levels seen at 2 days post burn. The protein disulfide isomerase (PDI), a member of the thioredoxin super family of proteins mediating the redox- dependent folding and unfolding of proteins within the ER, was not altered by IGF-1.
Summary: Burn injury activates different arms of the UPR pathways. IGF-1 prevents activation of the UPR and ER stress.
Conclusion: Supplementation of growth factors or hormones that are decreased following severe burn injury may reduce hepatic ER stress, thereby improving post-burn outcomes.
TYPE OF RESUSCITATION FLUID ALTERS BBB INFLAMMATION A DAY AFTER SHOCK: AN IN-VIVO CEREBROVASCULAR STUDY
W. Gong, S. Li, K. Kumasaka, R. Eisenstadt, K. Browne, S. Douglas, M.A. Murcy, C.A. Sims*, S.R. Allen*, and J.L. Pascual. University of Pennsylvania, Philadelphia, PA
Introduction: Neutrophil (PMN) activation in the cerebrovascular circulation (BBB) may be responsible for delirium, common in ICU patients. Hypertonic saline (HTS) resuscitation of shock blunts systemic microvascular endothelial (EC)-PMN interactions, but it is unknown if HTS also reduce the interactions in the BBB beyond the immediate post-resuscitation period. We hypothesized that HTS would decrease BBB EC-PMN interactions 18 hours following hemorrhagic shock resuscitation and sepsis.
Methods: Wistar rats were hemorrhaged (35mmHg ×X60 min) and received IV lipopolysaccharide (LPS, 1mg/kg). Resuscitation was with shed blood + either 5% HTS (6cc/kg, n=7) or RL (2×X shed blood, n=6). Sham animals underwent vascular cannulation but no shock, resuscitation or LPS (n=6). 18 hours later, rats underwent craniotomy to visualize in vivo EC-PMN interactions in BBB vessels using intravital microscopy (IVM). EC-PMN interactions were analyzed offline and post-mortem brain edema was assessed by wet-to-dry ratios. The Mann-U-Whitney test determined significance (p <0.05).
Results: Resuscitated animals displayed similar hemodynamics and acid base status. HTS animals had a lower all-cause mortality ( 30.8% vs. 70.% for RL, p=0.04). Brain edema was similar in all groups. However, in vivo PMN adhesion was greatest in HTS animals (Figure, p<0.05).
Conclusion: Type of resuscitation fluid can alter BBB microvascular PMN activation up to 18 hours post-resuscitation. HTS may paradoxically increase PMN activation in the cerebrovascular circulation. How this translates to brain tissue injury and development of delirium in ICU patients requires further study.
ROLE OF LIPID IN ANTIBIOTIC RESISTANCE OF BACTERIAL BIOFILMS
D.J. Hess*, M.J. Henry-Stanley*, and C.L. Wells*. University of Minnesota, Minneapolis, MN
Bacterial biofilms contaminate catheters, sutures, and other indwelling devices, and cause sepsis in the critically ill. Biofilms are surface-associated microbial communities in a liquid. Biofilm bacteria are often antibiotic resistant, but if removed from the biofilm, antibiotic susceptibility is similar to planktonic (free-living) bacteria. Biofilm bacteria are typically embedded in an extracellular polymeric substance (EPS) containing proteins, glycoproteins, glycolipids, polysaccharides, and extracellular DNA. There is evidence that the EPS contributes to antibiotic resistance by restricting antibiotic diffusion and/or by inhibiting antibiotic penetration of bacterial cells. Here, much attention has focused on the polysaccharide in the matrix, but we have hypothesized that EPS lipid plays a key role in mediating antibiotic resistance. We recently reported that novel lipid rich structures appear early after inoculation of Staphylococcus aureus onto clinically relevant abiotic surfaces, and similar structures were observed in catheter vegetations from critically ill patients with S. aureus sepsis. The present study was undertaken as a “proof of principle” to investigate the ability of a detergent, specifically sodium dodecyl sulfate (SDS), to modulate the activity of gentamicin in S. aureus biofilms. Suture-associated biofilms were cultivated overnight in biofilm growth medium, which was then replaced with fresh medium supplemented with 20 μg/ml gentamicin and varying concentrations of SDS. (The minimum inhibitory concentration of planktonic S. aureus is 1 μg/ml and these experimental biofilms were resistant to 20 μg/ml gentamicin.) After 16 hr incubation of intact biofilms with SDS and gentamicin, viable bacteria were quantified from sonicated biofilms. SDS alone did not affect biofilm viability, but SDS potentiated the activity of gentamicin, and biofilms normally resistant to 20 μg/ml had no detectable bacteria after incubation with 20 μg/ml gentamicin plus 0.5 mM SDS (P<0.01). Thus, a detergent which solubilizes lipid, augmented the bactericidal effect of gentamicin. These data provide proof of principle that lipid disruption facilitates antibiotic activity within an intact biofilm. Future experiments will use normally occurring human surfactants, such as phophatidylserine or glyceryl laurate, to test their effects on the antimicrobial susceptibility of biofilm bacteria.
SEPTIC CONDITION PROMOTES CANCER METASTASES IN A MURINE MODEL
Y. Matsumoto1, S. Ono2, H. Tsujimoto1, R. Takahata1, K. Yoshida1, S. Hiraki1, H. Miyazaki2, M. Kinoshita3, T. Yamori4, D. Saitoh1, J. Yamamoto1, and K. Hase1. 1Department of Surgery, National Defense Medical College, Tokorozawa, Japan, 2Division of Traumatology, National Defense Medical College, Tokorozawa, Japan, 3Department of Immunology and Microbiology, National Defense Medical College, Tokorozawa, Japan, 4Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Japanese Foundation for Cancer Research, Koto-ku, Japan
Background: We previously demonstrated that septic conditions due to postoperative infectious complications were unfavorable prognostic factors after potentially curative resection for gastric cancer (Ann Surg Oncol 2009), but the precise mechanism remains unclear. The present study was designed to evaluate the relationship between the host immune response and the progression of neoplastic cells in a septic murine model.
Method: BALB/c (8-10w old, ♀) were trans-splenic treated with NL17 colon cancer cells, which is high potential stock for liver metastases. Thereafter, treated mice were subjected to septic model induced by cecal ligation and puncture (sepsis) or sham treatment (sham). Blood and liver were harvested on day 1, day 3, day 7, and day 14. 1) Volume of liver metastases, 2) Serum and liver cytokines levels, 3) Percentage of regulatory T cells (Treg), and an expression of the MHC Class II antigen in the liver mononuclear cells (MNCs), 4) Cytotoxity of liver MNCs against neoplastic cells, 5) Expression of HGF receptor (HGFR) in NL17 cells, were examined.
Result: Liver metastases were confirmed since day 7 in septic mice, but not in sham mice. And the volume of liver metastases on day 14 was significantly higher in septic mice than in sham. Serum IL-6 levels on day 1, and serum IL-10 levels on day 7 were significantly higher in septic mice than those in sham. In contrast, concentrations of IFN-γ on day 3 and IL-12 p70 on day 7 in the liver were significantly lower in septic mice. A percentage of Tregs in CD4+ T cells on day 7 was significantly higher, and the expression of MHC class II antigen on day 7 was significantly lower in septic mice than in sham. Cytotoxity of liver MNCs against NL17 significantly decreased on day 12 in septic mice compared to sham mice. As for the NL17 stock, HGFR developed in 85% of cells, and HGF levels in serum were significantly higher in septic mice than in sham (755 vs. 1610 pg/ml).
Conclusions: Septic conditions promote cancer metastases in the liver. Not only the suppression of cellular immunity and cytotoxity in the liver but also enhanced activity of neoplastic cells through the HGF/HGFR signal transmission were confirmed in septic conditions, which may contribute to the progression of liver metastases during sepsis.
INHIBITION OF ENDOCANNABINOID DEGRADATION REDUCES LEUKOCYTE ADHESION AND IMPROVES CAPILLARY PERFUSION IN EXPERIMENTAL ENDOTOXEMIA
C. Lehmann*, M. Kianian, N. Al-Banna, J. Zhou, and M. Kelly. Dalhousie University, Halifax, NS, Canada
Background: Protection of the intestinal microcirculation represents a pivotal therapeutic target in systemic inflammation and sepsis. The endocannabinoid system (ECS) has been associated with anti-inflammatory responses. Our study aimed to examine intestinal leukocyte adhesion and capillary perfusion following selective inhibition of the endocannabinoid degradation enzyme, fatty acid amide hydrolase (FAAH), in experimental sepsis (endotoxemia).
Methods: Five groups of rats were studied: controls, endotoxemia [lipopolysaccharide (LPS)], FAAH inhibitor URB597 (0.3 mg/kg)+LPS, URB597 (0.6 mg/kg)+LPS, and URB597 (0.6 mg/kg)+cannabinoid 2 receptor (CB2R) antagonist (AM630)+LPS. After 2 h, intravital microscopy to quantify intestinal leukocyte recruitment and functional capillary density (FCD), as well as macrohemodynamic monitoring and histological examinations were performed.
Results: LPS induced a significant increase in leukocyte adhesion in collecting and postcapillary submucosal venules and a decrease in intestinal FCD. URB597 pre-treatment prevented the LPS-induced increase in leukocyte adhesion in intestinal venules and a decrease in intestinal FCD. The administration of the CB2R inhibitor, AM630, with URB597 reversed the protective effects of URB597 on the LPS-induced increase in leukocyte adhesion in intestinal venules, but not URB597’s effect on the intestinal FCD.
Conclusions: FAAH inhibition prevents the LPS-induced increase in leukocyte adhesion and improves the capillary perfusion of the gut. This might be mediated in part by CB2R activation. Our study encourages further investigation into the therapeutic potential of drugs targeting the ECS in sepsis.
DYNAMIC REPROGRAMMING OF INFLAMMATION ASSOCIATED WITH REMOVAL OF INFLAMMATORY ASCITES PREVENTS MULTIPLE ORGAN DYSFUNCTION
G. Nieman*1, N. Azhar2, L. Gatto3, G. An*4, and Y. Vodovotz*2. 1SUNY Upstate, Syracuse, NY, 2University of Pittsburgh, Pittsburgh, PA, 3SUNY Cortland, Cortland, NY, 4University of Chicago, Chicago, IL
Introduction: Sepsis-induced multiple organ dysfunction syndrome (MODS) arises from disordered systemic inflammation. We have shown that disordered inflammation in the gut/peritoneal compartment occurs early and plays a critical role in the pathogenesis of MODS (Kubiak et al, Shock. 2010. 5:525). Here, we hypothesized that inflammatory ascites drive MODS pathogenesis, and that removal of ascites with an abdominal wound vacuum prevents MODS via time- and compartment-dependent changes in inflammation that can be discerned using Dynamic Bayesian Network (DBN) inference.
Methods: Two groups of anesthetized, ventilated pigs were subjected to intestinal ischemia/reperfusion by clamping the superior mesenteric artery plus placement of a peritoneal fecal clot. All animals were given appropriate fluid resuscitation and antibiotics, and followed for 48 hrs. The Control Group (n=6) had the abdomen opened at 12 hrs post injury (T12) with attachment of a passive drain. The Peritoneal Suction Treatment (PST) Group (n=6) was treated in an identical fashion except that a vacuum was applied to the peritoneal cavity at T12 to remove ascites and maintained until T48. Multiple inflammatory mediators (Endotoxin; Interleukins-1β,-6,-8,-10,-12; tumor necrosis factor [TNF]-α; Von Willebrand factor [vWF]; C-reactive protein [CRP]; Prostaglandin E2 [PGE2]; and Transforming Growth Factor [TGF]-β1] were measured in ascites and plasma and compared to lung function (PaO2/FiO2 ratio [PF]) using DBN inference.
Results: PST prevented MODS based on intestine, lung, liver, and kidney histopathology, whereas Control animals developed MODS. DBN suggested that Endotoxin drives the inflammatory response in the ascites, interplaying with PF/lung dysfunction in a feed-forward loop that exacerbates inflammation characterized by greater production of IL-1β and IL-6 in Control vs. PST animals. In the plasma, and in agreement with prior studies, DBN inferred the induction of TGF-β1 and CRP by IL-6 in both Control and PST groups. IL-8 was an output of this process in the Control group, whereas IL-10 was the output in the PST group.
Conclusion: Combined in vivo and in silico studies suggest that dynamic reprogramming of networks controlling inflammatory and physiologic (dys) function during the process of ascites removal is a possible mechanism of MODS prevention.
INCREASED EXPRESSION OF LEUKOCYTES IMMUNOGLOBULIN-LIKE RECEPTOR B2 (LILRB2) RNA PREDICTS SURVIVAL AFTER SEPTIC SHOCK
M. Cazalis1, E. Cerrato1, L. Cavé1, F. Frager1, V. Barbalat1, A. Friggeri1, 3, M. Paye4, M. Tournoud4, A. Pachot1, G. Monneret1, 2, F. Venet1, 2, and A. Lepape1, 3. 1Joint Unit (Sepsis) Hospices Civils de Lyon - bioMérieux, Lyon, France, 2Immunology Laboratory, Hôpital Edouard Herriot, Lyon, France, 3Intensive Care Units, Centre Hospitalier Lyon-Sud, Pierre Beniete, France, 4Data and Knowledge Lab, bioMérieux, Marcy, France
Background: Septic syndromes remain the leading cause of mortality in intensive care units. Patients rapidly develop immune dysfunctions which intensity and duration have been linked with deleterious outcome.
Objectives: In this study, we investigated whether early evaluation of septic patients’ severity and risk of death through genomic biomarker measurement could predict mortality in septic shock patients.
Patients and Methods: Fifty one septic shock patients (including 17 non-survivors at day 28) and 22 healthy donors were enrolled. PAXgene® tubes were collected at day 1 after the onset of shock. Genome wide expression analysis was performed using Affymetrix U133plus 2 GeneChip. Validation of gene expression was done using qRT-PCR in 262 septic shock patients (172 survivors and 90 non survivors).
Results: LILRB2 was one of the most differentially expressed genes between survivors and non-survivors (Area Under the Curve= 0.84, p-value= 0.0002, Mann-Whitney test). Interestingly, LILRB1 mRNA level, another inhibitory LILR family member, showed good predictive value (AUC= 0.85, p-value=0.0009). The two mRNA markers were well correlated (R=0.86, p-value<0.0001, Spearman correlation test). Importantly, the differential expression of LILRB2 between survivors and non-survivors was validated by qRT-PCR in a larger cohort of patients (Odds Ratio= 0.47, p=0.0028). Survival curves confirmed that increased LILRB2 mRNA level was associated with decreased mortality after septic shock.
Conclusion: Increased LILRB2 mRNA expression significantly predicts survival after septic shock. LILRB2 is an inhibitor of the immune response though its preferential binding to HLA-G. Interestingly, increased soluble HLA-G concentrations has been described to be associated with survival in septic shock patients. This suggests that increased LILRB2 level may play an important role in the negative feedback mechanisms that limit the process of inflammation during septic shock. This needs to be confirmed at the protein level with functional experiments. Thus, LILRB2 may be a reliable marker of appropriate regulation of the immune response and a robust predictor of good outcome in septic patients.
THE ELIMINATION OF ABDOMINAL COMPARTMENT SYNDROME IN TRAUMA PATIENTS
B. Zangbar Sabegh, B. Joseph, G. Vercruysse, R.S. Friese, V. Pandit, T. O’Keeffe, A. Tang, L. Gries, J. Wynne, N. Kulvatunyou, and P.M. Rhee. University of Arizona, Tucson, AZ
Introduction: Anticipation of abdominal compartment syndrome (ACS) is a factor for performing damage control laparotomy (DCL). There have been changes in resuscitation patterns and there is a decline in use of DCL. We hypothesized that reductions in both crystalloid resuscitation and DCL have reduced the rate of primary abdominal compartment syndrome in trauma patients.
Methods: A retrospective study was performed on all patients undergoing trauma laparotomies at our level 1 trauma center over a 6 year period (2006-2011). DCL was defined as trauma laparotomy that left the fascia open at the first operation. ACS was confirmed by elevated bladder pressures and end organ dysfunction. Primary outcome was development of ACS; secondary outcome was mortality.
Results: A total of 799 patients were included. The number of DCL (39% vs. 8%, p<0.001) and amount of crystalloid use per patient (12.86±7.8 L vs. 6.62±4.2 L, p<0.001) significantly decreased from 2006 to 2011. ACS decreased significantly and was virtually eliminated during this time period (7.4% vs. 0%, p<0.001). There was a significant decrease in mortality rate (22.7% vs. 10.6%, p<0.001). The overall laparotomy rate (136 vs. 113, p=0.24), Injury Severity Score (22[13-38] vs. 18[10-30], p=0.09), Abdominal Abbreviated Injury Scale (3[2-4] vs. 3[2-4], p=0.17), and blood product requirement per patient (2.94±4 L vs. 2.17±4.8 L, p=0.79) did not change during the study period.
Conclusion: Minimizing the use of crystalloids and damage control laparotomy was associated with better outcome and has virtually eliminated primary abdominal compartment syndrome. With the adaption of damage control resuscitation, goals for a trauma laparotomy should be definitive surgical care with abdominal closure. Abdominal compartment syndrome is no longer a major factor in trauma care and may have been iatrogenic.
ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE (AMPK) EXERTS BENEFICIAL EFFECTS IN SEPSIS IN AGED MICE
R. Samraj, P.W. Hake, G. Piraino, M. O’Connor, and B. Zingarelli*. Cincinnati Children’s Hospital, Cincinnati, OH
The development of multiple organ failure and the outcome of sepsis are significantly impacted by the age of the patient. AMP-activated protein kinase (AMPK) is an important homeostasis regulator, which is activated in response to low energy levels during stress, mitochondrial dysfunction and infections. Here, we investigated whether AMPK modulates the age-related inflammatory response during polymicrobial sepsis in vivo. Sepsis was induced in aged male mice (11-12 months old) by cecal ligation and puncture. Treatment with the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside-1-β-D-ribofuranoside, 500 mg/kg i.p.) reduced tissue injury and neutrophil infiltration (as evaluated by myeloperoxidase activity) in lung and liver (280.9±51.7 and 1.4±0.5 U/100 mg tissue, respectively) when compared with vehicle-treated mice (453.6±55.4 and 4.1±1.3U/100 mg tissue, respectively; P<0.05) at 6 hrs after CLP. Transmission electron microscopy of hepatocytes of vehicle-treated septic mice demonstrated marked alterations in the mitochondrial morphology, characterized by the presence of swollen organelles with distorted cristae and translucent matrix (Figure). In AICAR-treated septic mice, most mitochondria presented normal structure with transversely orientated cristae; several autophagosomes were also observed. At molecular analysis, the hepato-protective effect of AICAR treatment was associated with increased nuclear translocation of the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1 α), a key component of mitochondrial function. In parallel survival studies, when given as single acute dose at 1h after CLP, AICAR provided an early survival advantage at 24h: in the vehicle group (n=15) 60% of animals were dead, whereas in the AICAR group only 12.5% of mice were dead (n=8; P=0.037 at Fisher’s Test). Thus, our data suggest that AMPK activation may be a potential therapeutic approach in sepsis in elderly patients. The beneficial effect of AICAR may be secondary to metabolic recovery. (Supported by NIH R01GM067202).
COLD INDUCIBLE RNA-BINDING PROTEIN (CIRP): AN IMPORTANT MEDIATOR OF ACUTE ALCOHOL INTOXICATION INDUCED BRAIN INJURY
S.R. Rajayer, A. Jacob*, W. Yang*, M. Zhou*, W. Chaung, and P. Wang*. Hofstra North Shore-LIJ School of Medicine and The Feinstein Institute for Medical Research, Manhasset, NY
Acute alcohol intoxication is associated with cerebral dysfunction. Studies have shown that ethanol-induced microglial activation sets off an inflammatory process upregulating cytokines and eventually causing neuronal damage. However, its molecular mechanism is not fully understood. We postulated that CIRP, which was found to be a novel pro-inflammatory molecule, may be a mediator in this process. To prove this, male wild-type (WT) and CIRP knockout (CIRP-/-) mice were exposed to ethanol at a dose of 1.75 g/kg intravenously followed by a continuous infusion at a rate of 200-300 mg/kg/h. After 15 h, blood and total brain tissue were collected to measure various parameters. In addition, we also cultured BV2 cells (immortalized mouse microglia cells) and incubated them in 100 mM ethanol to study the expression of CIRP. We found a 274% and 400% increase in CIRP mRNA levels at 24 and 48 h, respectively (Figure 1A). After in vivo alcohol exposure, a 75% increase in the total brain protein levels of CIRP was noted at 15 h in the WT mice (Figure 1B). Serum levels of organ injury markers, AST and LDH were increased in the alcohol exposed WT mice by 159% and 169% respectively, which was abrogated in the CIRP-/- mice. Brain cytokine levels in the WT showed 93% and 44% elevation in IL-6 and IL-1β respectively, that was attenuated in the CIRP-/- mice. Total brain protein levels of nitric oxide synthase (iNOS) were also significantly upregulated in the WT by 150% which was not observed in the CIRP-/- mice. We then investigated if PPARγ, a known neuroprotectant, plays any role in alcohol induced CIRP upregulation. In the WT group, there was a significant downregulation of PPARγ levels by 52%, whereas PPARγ was restored in CIRP-/- mice with alcohol. We conclude that alcohol exposure causes increase in brain CIRP level which subsequently results in cerebral dysfunction via the downregulation of PPARγ.
COMBINATION OF ANTITHROMBIN AND RECOMBINANT THROMBOMODULIN ATTENUATES THE CELL DEATH THROUGH THE MAINTENANCE OF MICROCIRCULATION IN ENDOTOXEMIC RAT
T. Iba*. Juntendo University, Tokyo, Japan
Introduction: Both antithrombin and thrombomodulin are the key players in physiological anticoagulant systems. Since both factors are known to decrease significantly during severe sepsis, we hypothesized that the combination therapy would be effective.
Methods: Sepsis model was made by the intravenous infusion of lipopolysaccharide (LPS). Either 125 IU/kg of antithrombin, 0.25 mg/kg of recombinant thrombomodulin or combination of both agents was injected immediately after LPS infusion (n= 7, each). The intravital observation of the mesenteric microcirculation was performed and the leukocyte adhesion and the blood flow was calculated at 3 h after LPS infusion. At 8h, circulating levels of nucleosome and high-mobility group box 1 (HMGB1) were measured.
Results: Microscopic findings revealed suppression in leukocyte adhesion, thrombus formation and endothelial damage in the combination groups. The count of adhesive leukocyte on endothelium was significantly suppressed (P< 0.01) and the blood flow was better maintained in the combination group (P< 0.05) compared to the placebo control. Organ damage markers were significantly improved in the combination group. Circulating levels of nucleosome and HMGB1 were both decreased in the combination group (P< 0.05).
Conclusion: The coadministration of antithrombin and thrombomodulin is effective for the maintenance of microcirculation which leads to the decrease of cell death during sepsis.
FED STATE CHANGES METABOLIC RESPONSE TO HEMORRHAGIC SHOCK IN LIVER
C. Determan, E.R. Lusczek, N. Witowski, K. Pokorney, K. Mulier, and G. Beilman. University of Minnesota, Minneapolis, MN
Hemorrhagic shock(HS) with injury results in changes in metabolic state and contributes to organ dysfunction and death. Prior studies of HS in murine models have shown a protective effect of carbohydrate pre-feeding (CPF). This finding has relevance for military personnel participating in hazardous missions. We performed CPF in pigs undergoing polytrauma and HS with two goals: 1) To determine if CPF or fasted (FS) state improve outcomes, and 2) To evaluate metabolomic changes in this model of HS. Eighty Yorkshire pigs were divided into groups: no HS (10), HS no resuscitation (6), FS (32) and CPF (32). Each animal was subjected to polytrauma and HS. To measure metabolic changes in HS, liver biopsies were taken at set timepoints. Metabolites were extracted and quantified using 1H NMR and profiled using Chenomx. Fifty-one metabolites were profiled. Partial-Least Squared Discriminate Analysis (PLSDA) was used to examine clustering of data with respect to outcome and fed state. Contrary to prior literature, this experiment reveals increased mortality with CPF (47% vs. 28% mortality). Cross-validated models discriminated CPF from FS. PLSDA models within groups revealed different metabolites associated with outcome suggesting an alternate response to shock. These metabolites have known relationships to energy pathways and oxidative stress. CPF tends to increase mortality and that the metabolic response to HS is different between fed states. In conclusion, we validated the use of metabolomics to identify the metabolic profile associated with fed state.
BROMOVALERYLUREA IMPROVES SURVIVAL IN RAT SEPSIS OF CECAL LIGATION AND PUNCTURE MODEL
S. Kikuchi1, T. Nishihara2, S. Ootubo1, K. Umakoshi1, T. Nishiyama1, M. Aibiki*1, and J. Tanaka2. 1Ehime University Emergency Medicine, Toon, Japan, 2Ehime University Molecular and Cellular Physiology, Toon, Japan
Introduction: Bromovalerylurea (BU) is an old hypnotic drug that is generally no longer used. Recently, we have found that the agent suppresses the production of nitric oxide (NO) from lipopolysaccharide (LPS) activated macrophages. Additionally, it reduces the release of inflammatory mediators. In this study, we evaluate therapeutic effects of the agent on rat sepsis of a cecal ligation and puncture (CLP) model.
Methods: In vitro study: Rat peritoneal macrophages were isolated by peritoneal lavage using 20ml cold phosphate buffered saline. Following a centrifugation, the pellet was resuspended. The cells were poured into polystyrene dishes for suspension cultures for one night and then LPS was added. In experiments, BU was applied to the medium containing LPS at two different concentrations, 0.3μg/ml (BU30) and 1μg/ml (BU100). Conditioned media of all dishes were collected for analyses of NO, IL-1beta and IL-6.
In vivo study: Adult male rats were divided into sham, sepsis and BU groups. Sepsis was induced by the CLP. 10ml maintenance fluid was injected to sham and sepsis groups, and 10ml fluid containing 5mg of dissolved BU was administered to BU group every 12 hours after CLP. We evaluated mortality rate every 24 hours for 7 days. We assayed serum levels of creatinine, IL-1beta and IL-6 at 24hours after CLP.
Results: The mortality rate of sham group was 0%, sepsis group 85% and BU group 50%, respectively (BU versus sepsis group, p<0.05). BU significantly decreased NO, IL-1beta and IL-6 levels in supernatants in a concentration dependent manner. Serum levels of creatinine, IL-1beta and IL-6 were significantly lower in BU group than those in sepsis group (p<0.05).
Conclusions: BU improved survival rates in rat sepsis of CLP model, which was associated with improvements of renal function and attenuations of inflammatory mediator. Thus, there is a need to clarify the cellular mechanisms of BU in rat sepsis and to test a hypothesis whether this agent could also have beneficial effects in human sepsis.
SPECIFIC INHIBITION OF MITOCHONDRIAL ROS SUPPRESSES INFLAMMASOME ACTIVATION IN THE HEART AFTER SEPSIS
X. Yao, L. Ma, D. Maass, S. Wolf*, J. Minei*, and Q.S. Zang*. University of Texas Southwestern Medical Center, Dallas, TX
Objective: We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), an antioxidant targeting specifically at mitochondrial reactive oxygen species (mtROS), limited cytokine production in the heart of a rat pneumonia-related sepsis model. This study was designed to utilize Mito-Vit-E to investigate the mechanism underlying the significance of mtROS in the induction of cardiac inflammation after sepsis.
Methods: In SD rats, sepsis was produced by intratracheal injection of S. pneumoniae (4x1,000,000 CFU/rat). Mito-Vit-E (21.5 μmoles/kg) or control vehicle was orally administered 30 minutes post-inoculation. In the heart tissue harvested day 4 post-inoculation, levels of inflammasome component ASC, activated form caspase 1 and IL-1β were measured by immunohistochemistry, Western blot, and ELISA assay respectively.
Results: In the heart, significant increases in ASC, active caspase 1 and IL-1β were evident in septic rats but not in sham and Mito-Vit-E treated rats.
Conclusion: Our data suggest that sepsis-induced overproduction of mtROS promotes inflammasome formation via up-regulation of ASC, leading to downstream activation of caspase 1 and production of IL-1β in myocardium. The results also suggest that mitochondria-targeted antioxidants may become an effective therapeutic approach to alleviate cardiac damage during sepsis.
TWO-COMPARTMENT METABOLIC NETWORKS IN PORCINE HEMORRHAGIC SHOCK AND INJURY DEMONSTRATE BCAA METABOLISM AND UTILIZATION
E.R. Lusczek, N. Witowski, C. Determan, K. Pokorney, K. Mulier, and G. Beilman*. University of Minnesota, Minneapolis, MN
Objective: We constructed muscle and serum metabolic networks using metabolomics data from a porcine model of trauma and hemorrhagic shock to examine relationships among metabolites for animals that were pre-fed carbohydrate (CPF) or fasted prior to injury, and animals that were not injured or hemorrhaged (NH).
Method: Metabolic profiles were constructed from Nuclear Magnetic Resonance spectra of muscle and serum samples collected at set timepoints in a porcine model of hemorrhagic shock that included a pulmonary contusion, a 35% controlled bleed, and a liver crush injury. Metabolites were identified and quantified with Chenomx software. Networks were constructed from these metabolic profiles using the WGCNA (Weighted Correlation Network Analysis) package for R software.
Results: Branched-chain amino acids (BCAAs) were important hubs of metabolism in muscle and serum at all experimental timepoints for fasted and CPF animals, though BCAA levels were higher for fasted pigs relative to CPF pigs (p≤0.03 in serum; p≤3.5×X10-5 in muscle). Serum urea was higher in fasted pigs relative to CPF pigs as well (p=0.0002). Serum arginine and urea were functionally related more often in fasted than CPF pigs. Muscle osmolytes betaine, trimethylamine, and trimethylamine N-oxide were important hubs during shock in fasted pigs and early resuscitation in CPF pigs. In contrast, networks of NH pigs did not identify osmolytes or BCAAs as hub metabolites and did not show a relationship between serum arginine and urea.
Conclusion: Injured pigs showed an increased reliance upon BCAAs compared to NH pigs as evidenced by their prominence as hubs in serum and muscle. Observed relationships between serum arginine and urea suggest an increased need to maintain nitrogen balance in injured pigs relative to non-injured pigs, and in fasted pigs relative to CPF pigs. When taken with increased levels of BCAAs and serum urea seen in fasted pigs, these results suggest fasted pigs rely on increased protein catabolism for energy relative to CPF pigs. The importance of muscle osmolytes in injured pigs may be related to known mechanisms that protect proteins against the denaturing effects of urea. These osmolytes appear earlier in the experimental process for fasted pigs than for CPF pigs, and further demonstrate differing responses to injury between fed and fasted pigs.
ASSOCIATION BETWEEN INSERTION/DELETION POLYMORPHISM IN ANGIOTENSIN-CONVERTING ENZYME GENE AND ALI/ARDS: A META-ANALYSIS
A. Matsuda*1, 2, M. Miyashita*2, A. Jacob*1, M. Aziz1, T. Matsutani*3, T. Hayakawa2, K. Yokoi2, E. Uchida3, and P. Wang*1. 1Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2Department of Surgery, Nippon Medical School Chiba-Hokuso Hospital, Inzai, Japan, 3Department of Surgery, Nippon Medical School, Bunkyo-ku, Japan
Rationale: Several genetic association studies reported an association between insertion/deletion (I/D) polymorphism in angiotensin-converting enzyme (ACE) gene and the risk and mortality of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Here, we conducted a systematic review and a meta-analysis between I/D polymorphism in ACE gene and the susceptibility and the mortality of ALI/ARDS.
Methods: We searched electronic databases for the terms “angiotensin-converting enzyme gene”, “acute lung injury”, and “acute respiratory distress syndrome,” and reviewed all studies that reported the relationship of the I/D polymorphism in ACE with ALI/ARDS in humans. Seven studies met the inclusion criteria, comprising 532 ALI/ARDS patients, 3032 healthy controls, and 1432 patients without ALI/ARDS. We used three genetic models: the allele, dominant, and recessive models.
Results: The ACE I/D polymorphism was not associated with susceptibility to ALI/ARDS for any genetic model. However, the ACE I/D polymorphism was associated with the mortality risk of ALI/ARDS in Asian subjects (Pallele < 0.0001, Pdominant = 0.001, P recessive = 0.002). This finding remained significant after correction for multiple comparisons.
Conclusions: There is a possible association between the ACE I/D polymorphism genotype and the mortality risk of ALI/ARDS in Asians.
HUMANIZED TRANSGENIC MICE AS A MODEL FOR FUNCTIONAL STUDIES OF HUMAN SURFACTANT PROTEIN GENETIC VARIANTS IN VIVO
G. Wang*, L. Ge, X. Liu*, O. Abdel Razek, and S. Javidiparsijani*. SUNY Upstate Medical University, Syracuse, NY
Pulmonary surfactant proteins A and B (SP-A and SP-B) are essential for normal respiratory function and host defense. Genetic variations of SP-A and SP-B are associated with multiple lung diseases including pneumonia. Humanized transgenic mouse (hTG) model has helped overcome severe limitation in the study of human biology in vivo. Utilizing the hTG mice, the physiologically and pathologically functional differences of human gene/genetic variants can be investigated in complex biological processes.
Objective: Investigate functional effects of human surfactant protein genetic variants in vivo in bacterial pneumonia model.
Methods: The hTG SP-A and SP-B mice were generated by using direct DNA microinjection into fertilized oocytes from wild-type (WT) mice. Mouse SP-A or SP-B gene was eliminated by backcrossing with SP-A or SP-B KO mice. To established pneumonia model, hTG SP-A and SP-B mice were infected intratracheally with 50μl of Klebsilla pneumonia (450 CFU) and Pseudomonas aeruginosa (1X107 CFU), respectively. The mice were sacrificed 24 hours post-infection, then histological, cellular and molecular analyses of the lungs were performed. BAL proteome and signaling pathways were studied. Differences were considered significant when p<0.05 by t-test or ANOVA.
Results: 1) The hTG SP-A and SP-B mice show normal phenotypes as WT mice. The levels of hSP-A or hSP-B protein expression in the lung of hTG mice are comparable with those in the human lung. 2) In the K. pneumonia model of hTG SP-A mice, differential effects of human SP-A genetic variants on BAL proteomic patterns and signaling pathway association were observed, indicating functional difference of human SP-A genetic variants in response to K. pneumonia infection. 3) In the P. aeruginosa model of hTG SP-B mice, hTG SP-B mice with either the C or T allele exhibited the characteristics of pneumonia pathology, but showed differences between the C and T alleles in the degree of lung inflammatory cell infiltration and surfactant protein decrease of BALF, suggesting the functional difference of human SP-B genetic variants to P. aeruginosa infection.
Conclusion: hTG mouse model is an effective tool to study the pathophysiological function of human SP-A and SP-B gene/genetic variants; even subtle differences in phenotypes caused by the transgenic gene/genetic variant can be elucidated.
ROLE OF HYPOXIA INDUCIBLE FACTOR (HIF 1α) ALPHA FOLLOWING LUNG CONTUSION
S.V. Madathilparambil, D.A. Machado, Y. Bi, N. Talarico, E. Anderson, Y. Shah, and K. Raghavendran*. University of Michigan, Ann Arbor, MI
Objective: Lung contusion (LC) is a major risk factor for the development of acute respiratory distress syndrome (ARDS). Hypoxia-inducible factor (HIF) 1α is the primary transcription factor responsible for regulating cellular response to changes in oxygen tension. We set to determine if HIF1α plays a role in the pathogenesis of acute inflammatory response and injury in LC.
Method: Non-lethal closed-chest unilateral LC was induced in C57 Bl6 mice where the Oxygen dependent domain (ODD) of HIF1α is linked with luciferase. The animals along with uninjured controls were then subjected to IVSS imaging to measure the degree of hypoxia. The explanted organs (lungs, liver and spleen) from similar animals induced with LC, were also subjected to IVSS. In separate experiments, LC was induced in alveolar type II cell specific HIF1α conditional knockout (cKO) [triple transgenic mice (SP-C-rtTA-/tg/ (tetO)7-CMV-Cretg/tg/HIF1aflox/flox) and control (CTL) mice]. In these mice feeding doxycycline induces a knockout of HIF-1α from type 2 cells. The mice were sacrificed at 5, 24, 48, and 72h time points and the extent of injury and inflammation were assessed by measuring bronchoalveolar lavage (BAL) cells (flow cytometry and cytospin), albumin (permeability injury) and cytokines (inflammation). Determination of pulmonary respiratory mechanics was performed by a flexivent. The confirmation of HIF 1α knockdown in the type 2 cells was assessed by confocal microscopy using specific antibodies against HIFα and surfactant protein C.
Results: The mice with ODD-linked luciferase showed profound hypoxia especially in the liver and spleen following LC. The extent of lung injury following LC was significantly reduced in the HIF1α cKO mice at all the time points, when compared to the CTL mice. Release of pro-inflammatory cytokines such as IL-1β, IL-6, MCP1/5, MIP-2, and KC were significantly lower, whereas that of anti-inflammatory IL-10 was significantly higher at 24 and 48h time point in the cKO mice. The histologic lesions in the CTL mice were significantly more severe than the cKO mice. Recruitment of alveolar macrophages and neutrophil infiltration after LC was significantly lower in the cKO mice. HIF1α cKO mice showed pronounced reduction in HIF1α expression in the Clara cells and type II cells when compared to the CTL mice.
Conclusion: Activation of HIF1α is a major driver of acute inflammation following LC.
PERFORMANCE OF TISSUE HEMOGLOBIN OXYGEN SATURATION AS MEASURED BY RESONANCE RAMAN SPECTROSCOPY AND NEAR INFRARED SPECTROSCOPY COMPARED TO MIXED VENOUS HEMOGLOBIN OXYGEN SATURATION AND SYSTEMIC LACTATE LEVELS DURING HEMORRHAGE
M.H. Tiba*1, 2, G.T. Draucker1, 2, R.W. Barbee*3, J. Terner3, I.P. Torres Filho*4, and K.R. Ward*1, 2. 1University of Michigan, Ann Arbor, MI, 2Michigan Center for Integrative Research in Critical Care, Ann Arbor, MI, 3Virginia Commonwealth University, Richmond, VA, 4US Army Institute of Surgical Research, Fort Sam Houston, TX
Objective: We hypothesized that regional monitoring of tissue hemoglobin oxygenation (StO2) using resonance Raman spectroscopy (Raman-StO2) is more sensitive than Near Infrared Spectroscopy (NIRS) measured StO2 in reflecting the level of mixed (SmvO2), and central (ScvO2) venous hemoglobin oxygen saturation and elevated systemic lactate levels during hemorrhage in a swine model.
Methods: Five male swine were anesthetized, instrumented, and hemorrhaged at a rate of 30 mL/min for 60 min. SmvO2, ScvO2, lactate and hemoglobin were measured every 5 minutes. Regional Raman-StO2 of the buccal mucosa and NIRS of skeletal muscle (quadriceps), were monitored continuously. Values were compared using GraphPad Prism5.
Results: Raman-StO2 changes tracked changes in SmvO2 (r= 0.918, CI: 0.8683-0.9492) and ScvO2 (r= 0.901, CI: 0.8276-0.9438) during hemorrhage, while NIRS-StO2 failed to do so for SmvO2 (r= 0.290, CI: 0.04919-0.4984) and ScvO2 (r= 0.142, CI: -0.1514-0.4124). An unpaired t test showed no significant difference between SmvO2 and Raman-StO2 (p > 0.05) and between ScvO2 and Raman-StO2 (p > 0.05) while there was a significant difference between NIRS-StO2 and both SmvO2 and ScvO2 (p < 0.0001 for both). When compared to lactate levels during hemorrhage, a non-linear regression of falling SmvO2 and ScvO2 showed a strong fit with rising lactate levels (r2 = 0.851 and 0.831 respectively). Raman-StO2 changes demonstrated a similar fit (r2 = 0.824) while NIRS-StO2 demonstrated poor fit (r2 = 0.031) to rising lactate levels.
Conclusion: Regional measurement of StO2 using Raman spectroscopy correlated better to changes in SmvO2, ScVO2, and lactate during hemorrhage than NIRS in this model.
PHOSPHORYLATED MYOCYTE ENHANCING FACTOR 2 (MEF2) IS INCREASED IN RESPONSE TO SEVERE BURN
J. Song*, J. de Libero, K. Despain, J. Hunt, J. Minei*, and S. Wolf*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Skeletal muscle impairment accompanies severe burn; in this hyper-catabolic state, muscle cells undergo damage through protein degradation and disuse. Skeletal myogenesis including satellite cell activation, proliferation and differentiation is undoubtedly affected by injury. Myocyte enhancing factor 2 (MEF2) participates in myogenic transcription genes expression, however, effects of burn on myogenesis and related muscle homeostasis are not clear. The aim of the study was to investigate mechanisms of skeletal myogenesis in response to severe burn.
Methods: Twenty-eight C57BL6 mice received 25% TBSA scald burn under general anesthesia. Mice were euthanized at day 1, 3, 7 and 14, and gluteus muscles were harvested. Muscle tissue was weighed and then halved into either in 10% neutral buffered formalin for histological process or snap frozen storage in liquid nitrogen. Tissue RNA and protein were examined by qPCR and western blot. Six animals without burn served as controls. One-Way ANOVA with Bonferroni or Kruskal-Wallis tests were applied, with significance accepted at p < 0.05.
Results: Muscle tissue wet weight and protein content decreased significantly at day 3 and 7 (p < 0.05). Cleaved caspase 3 levels increased at day 3 after burn, which was confirmed with increased TUNEL positive stained nuclei. qPCR showed increased Pax7 and myogenin at days 3 and 7, indicating increased signaling for myogenetic activation and differentiation in response to injury; Desmin mRNA expression decreased at day 7 after burn. Western blots showed that MEF2 had increased phosphorylation at day 3 after burn (p = 0.032), suggesting a mechanism for stimulation of myogenesis in response to injury.
Conclusion: Muscle changes after injury were associated with increased activation of skeletal myogenesis associated with increased myogenic enhancer factor 2 phosphorylation. These findings must be correlated to ongoing catabolic responses, and may be a counter-response to the direct effects of injury.
BURN INCREASES CIRCULATING LEVELS OF DAMAGE-ASSOCIATED MOLECULAR PATTERNS (DAMPS)
Q. Zhang1, M. Rani*1, A.P. Cap2, 1, D.N. Darlington*2, and M.G. Schwacha*1, 2. 1University of Texas Health Science Center, San Antonio, TX, 2US Army Institute of Surgical Research, San Antonio, TX
Burn is associated with profound inflammation and activation of the innate immune system that contributes to multiple complications. Recent findings suggest that DAMPs released after tissue injury play a critical role in the activation of the innate immune response. This response appears to be mediated via Toll-like receptors (TLRs) and previous finding have shown that TLRs and TLR-mediated responses are up-regulated after burn. Nonetheless, it is unclear what impact burn has on circulating levels of DAMPs. To study this, male C57BL/6 mice (n=5-7/group) were subjected to a major burn injury (3rd degree, 25% TBSA) or left untreated (i.e., naïve). Three hrs to 7 days thereafter, plasma was collected and assayed for the representative DAMPs, HMGB1, S100A and cytochrome C by ELISA, as well as DNA levels. Plasma HMGB1 levels were elevated ∼4-fold at 24 hrs after injury, as compared with naïve mice. HMGB1 levels were not different from that of naïve mice by 3 days post-injury. In contrast, cytochrome C levels, a marker of mitochondrial damage, were elevated (∼8-fold) as early as 3 hrs post-injury and remained elevated at 24 hrs. Cytochrome C levels returned to normal by 3 days post-injury. Circulating DNA levels were not significantly different between naïve mice or burn mice at any time points assessed. The calcium binding protein, S100A was not measurable in any samples. In conclusion, burn induces a significant increase in circulating levels of selected DAMPs during the first 24 hrs post injury. These findings suggest that burn-induced elevation in circulating DAMPs may be causative in innate immune activation and contribute to the development of post-burn inflammatory complications.
THE EFFECT OF BLOOD PRESSURE-CONTROLLED STEPWISE REPERFUSION IN HEMORRHAGIC SHOCK MODEL OF RATS
H. Chung1, J. Lee1, K. Kim1, Y. Jo1, K. Kang2, J. Kim1, C. Park1, C. Kang1, S. Lee1, and M. Lee1. 1Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea, 2Jeju National University Hospital, Jeju-si, Republic of Korea
Introduction: Hemorrhagic shock causes global ischemia-reperfusion injury. Current guidelines for resuscitation of hemorrhagic shock are bleeding control and achievement of adequate tissue perfusion with fluid and blood products. Acidosis produced by cellular ischemia may have some protective effects and rapid washout by reperfusion may attenuate the protective effects of acidosis. Thus, we tested the effect of slow correction of tissue perfusion by blood pressure-controlled stepwise reperfusion (SR) on survival and organ injury in pressure controlled hemorrhagic shock model of rats. We hypothesized that rapid reperfusion (RR) to normalize blood pressure would be harmful and SR may be beneficial on survival and organ injury in hemorrhagic shock.
Methods: Thirty male Sprague-Dawley rats were subjected to pressure-controlled hemorrhagic shock (MAP=38 ±1mmHg) for 60 minutes and then randomly allocated into two groups: RR (N=15) vs. SR (N=15). RR group was treated by transfusion of shed blood to reach MAP of 70mmHg immediately and maintain until the whole shed blood was transfused. SR group was treated by transfusion according to transfusion protocol (MAP was increased to 45mmHg and maintained for 3 minutes, 50mmHg for 3 minutes, 55mmHg for 3 minutes, 60mmHg for 3 minutes, 65 mmHg for 3 minutes and 70mmHg until the whole shed blood was transfused). All experiments were performed under room air condition. Survival time and MAP were recorded for 6 hours. Liver tissue and blood were collected after 6 hours and measured the histological liver injury and plasma ALT. Statistical analysis was performed using student t-test or Mann-Whitney U test, repeated measures ANOVA and Kaplan-Meier survival analysis.
Results: Baseline characteristics were not different between groups. For survival rates, SR group showed significantly higher survival rates than RR group (SR: 11/15 (73.3%) vs. RR: 5/15 (33.3%), log rank p = 0.032) and MAP was significantly different between groups (p <0.001). Although liver histological injury score was not significantly different between groups (p=0.152), it showed a tendency to be decreased in SR group compared with RR group (p=0.152) and plasma ALT was significantly lower in SR group than in RR group (p=0.032).
Conclusion: Blood pressure-controlled stepwise reperfusion increased survival rates and decreased liver injury in hemorrhagic shock.
EFFECTS OF THROMBOMODULIN TREATMENT IN EXPERIMENTAL HEAT STROKE
T. Kawasaki*1, C. Kawasaki2, K. Okamoto1, and T. Sata1. 1University of Occupational and Environmental Health, Kitakyushu, Japan, 2Sugioka Memorial Hospital, Fukuoka, Japan
Objective: Previously, we reported that the contribution of HMGB1 to liver dysfunction after heat stress. Thrombomodulin (TM) is well known as an endothelial anticoagulant cofactor that plays an important role in the regulation of intravascular coagulation. In this study, we investigated the effect of TM on heat stress induced inflammatory responses and liver dysfunction in experimental heat stroke.
Methods: Male C3H/HeN (8-10 weeks) mice were randomly assigned to TM treated group (TG) or non-treated control group (CG). In TG, mice were treated with TM (1 mg/kg, i.p.) before heat stress. In some experiments, TM (1 mg/kg, i.p.) was administrated during heat exposure. Heat stroke was induced by exposure to ambient temperature of 38°C for 4 hours. After heat stress, the levels of cytokines (TNF-alpha and IL-6), plasma HMGB1 concentrations, liver dysfunction; plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations, and immunohistochemical and histopathological characteristics of liver were determined.
Results: In CG, plasma TNF-alpha and IL-6 increased significantly after heat stress (p<0.05). Plasma HMGB1 concentration also increased in CG after heat stress (p<0.05). Plasma AST and ALT concentrations increased after heat stress (p<0.05). In CG, strong and extensive immunostaining for HMGB1 was observed. In addition, there was extensive hepatocellular necrosis observed in CG. In TG, plasma cytokines and HMGB1 concentrations suppressed after heat stress compared to CG (p<0.05). Liver injury also improved in TM. Furthermore, TM’s protective effects were shown even with delayed treatment.
Conclusion: The results of this study demonstrate that TM suppresses inflammatory responses and liver dysfunction after heat stress. TM may be a beneficial treatment for heat stroke patients.
COMPUTATIONAL IDENTIFICATION OF PRINCIPAL DRIVERS AND NETWORKS OF SYSTEMIC INFLAMMATION IN HYPOTENSIVE VS. NORMOTENSIVE BLUNT TRAUMA PATIENTS
K.W. Almahmoud1, R.A. Namas*1, 2, A. Zaaqoq3, N. Azhar1, R. Zamora*1, 2, T. Billiar*1, 2, and Y. Vodovotz*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 3Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA
Background: Hypotension-induced inflammation is frequently implicated in morbidity following traumatic injury. We sought to compare the dynamic, systemic acute inflammatory response to blunt trauma - with or without hypotension - in humans.
Methods: From a cohort of 484 blunt trauma survivors studied following IRB approval, 22 hypotensive patients (15 males and 7 females; age: 45±3.8; Injury Severity Score [ISS]: 20.5±1.8) were matched with 22 normotensive patients (15 males and 7 females; age: 45.5±3.3; ISS: 21.4±1.8). Serial blood samples were obtained from all patients (3 samples within the first 24 h and then from days 1 to 7 post-injury). Twenty-three plasma inflammatory cytokines were assayed using Luminex™. NO2-/NO3- was measured using the nitrate reductase/Griess assay. Two-way Analysis of Variance (ANOVA) was used to compare both groups. In silico modeling using Principal Component Analysis (PCA) and Dynamic Bayesian Network (DBN) inference were utilized to suggest group-specific principal inflammatory drivers and to infer causal relationships based on probabilistic measures, respectively.
Results: Shock markers (systolic blood pressure, Shock Index, pH, and lactic acid) were significantly different ( P < 0.05 by Student’s t-test) between hypotensive and normotensive patients. However, there was no statistical difference in clinical outcomes (ICU length of stay [LOS], Total LOS, Marshall Score, and prevalence of nosocomial infection) between the two groups. Plasma circulating levels of IL-10, MIP-1α, IL-8, IL-17, MCP-1, IL-15, GM-CSF, IL-1Ra, IL-7, sIL-2Rα, MIG were significantly elevated in hypotensive patients when compared to normotensive patients. PCA suggested that the inflammatory response in hypotensive patients was primarily driven by MIG, IL-1Ra, IL-8, IL-10 and IL-13, whereas the response in normotensive patients was primarily driven by IL-10, IL-6, MCP-1 and IL-8. In addition, DBN suggested a role for MCP-1 in driving IL-6 production in hypotensive patients.
Conclusion: In silico studies suggest differential inflammatory responses in hypotensive patients primarily driven by MIG and MCP-1. Our findings also suggest that, in the setting of moderate blunt trauma, hypotension leads to a reprogramming of inflammation that does not result in increased morbidity.
ANIMAL-BASED RESEARCH REPORTING IN SHOCK: IMPROVING REPORTING QUALITY
P. Wall*2 and P. Reynolds*1. 1Virginia Commonwealth University Medical Center, Richmond, VA, 2Iowa Methodist Medical Center, Des Moines, IA
Objectives: ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines were developed in response to serious and frequent deficiencies in experimental conduct and reporting revealed from systematic research reviews. The Shock Society endorsed the ARRIVE guidelines in 2012. We assessed opportunities for reporting improvements in Shock according to ARRIVE guidelines, specifically to determine (1) reporting characteristics; (2) if or how reporting changed over 5 years before specific guidelines were endorsed; and (3) risk of bias in 5 Cochrane guideline domains.
Methods: We surveyed all animal-based research articles published in Shock in 2005 (n=115) and 2010 (n = 114).
Results: The major experimental categories were: sepsis (44%), ischemia/reperfusion (25%), trauma/hemorrhage (21%), burn/heat shock (8%), and anaphylaxis (2%). Most animals were male (78%) rodents (83%). Twenty-eight studies reported fasting small mammals overnight or longer (generally not recommended). Reports frequently did not include information regarding vendor or animal origin (37%), age (72%), weight (22%), basic husbandry (71%), study design (>99%), power calculations (>99%), or number of animals (22%). Risk of bias was high. Random allocation of animals to experimental groups was stated for only 71 studies; only 2 provided randomization details. Only 26 studies reported blinding of any outcome assessments. Most studies reported large numbers of associations between interventions and outcomes, contributing to false positives. Many studies were too small to detect clinically-relevant effect sizes.
Conclusions: In common with other journals, Shock shares in system-wide deficiencies of reporting animal-based research. This creates a serious risk for less than maximally useful, biased, or even misleading research literature. Within Shock, the problem has not improved without systematic attempts to create change. Significant opportunities exist for combined editor, reviewer, and author use of ARRIVE guidelines to improve reporting quality. Creating reporting improvements should increase research process transparency. This is desirable for assessing results, comparing study outcomes, planning future studies, and translating animal model-based findings to clinically useful strategies.
AGE DISCRIMINATION: DOES IT MATTER FOR RED BLOOD CELLS?
J.F. Rappold*1, M.E. Lloyd1, K.A. Hollenbach2, Y. Li1, S.O. Lars1, J. Dujon1, A.S. Pathak1, T.A. Santora1, and A.J. Goldberg1. 1Temple University School of Medicine, Philadelphia, PA, 2UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences, San Diego, CA
Background: The age of stored packed red blood cells (pRBC) and its relationship to patient outcomes in those requiring a massive transfusion (≥10 units pRBC/24 hrs) remains controversial. Laboratory evidence has revealed evidence of the accumulation of storage lesions and decreased oxygen carrying capacity in pRBCs stored for greater than 14 days but the clinical significance of these findings remains unknown.
Methods: We conducted a retrospective cohort study on all patients receiving a massive transfusion (MTP) at an urban Level I Trauma Center from 2008 to 2012 (n=137). Patient outcome, amounts of blood products given, age of pRBCs transfused, injury severity, mechanism of injury and standard demographic data were collected. Percent of blood less than or equal to 14 days of age was calculated. Logistic regression was used to examine the relationship between age of blood and survival among the 137 patients receiving an MTP during the study period (58 survived; 79 died). A similar analysis was conducted for patients that survived their initial emergency or operating room procedures only to succumb later in their hospitalization (n = 33).
Results: The percent of blood less than 14 days of age was lower among patients who survived than among those who died or those who died after surviving their initial OR/ER experience (see Table 1). There was no significant difference in odds of death associated with percent of blood less than 14 days of age or less (OR = 1.01; 95% CI = 1.00,1.02) after adjusting for the competing effect of the total number of units of pRBCs received among MTP patients. Similar results were seen when the analysis was restricted to MTP patients who survived past their initial ER or OR procedures (OR = 1.00; 95% CI = 0.99, 1.02).
Conclusion: The percentage of pRBCs less than 14 days of age was higher in patients who died than survived among the study population. Results from this study indicate that the amount of blood transfused was a more significant predictor of death and is in contradistinction to literature supporting younger pRBCs for transfusion in critically injured patients requiring an MTP. A prospective randomized trial is warranted as are additional basic science studies to ascertain the exact effect of age on pRBC performance in this patient population.
DIFFERENTIAL REGULATION OF TNF RECEPTOR 1 AND RECEPTOR 2 IN ADIPONECTIN EXPRESSION
T. Christopher*, Y. Wang, W. Bond Lau, and X. Ma. Thomas Jefferson University, Philadelphia, PA
In vitro experiments demonstrate that adiponectin (APN), a cardioprotective cytokine, is inhibited by TNFα. However, the role of TNFα in post-myocardial infarction (post-MI) APN reduction remains unclear. More importantly, the receptor type responsible (TNFR1 vs. TNFR2) for TNFα-mediated suppression of APN production has not been identified. The current study investigated whether TNFα plays a causative role in post-MI APN reduction, and identified the receptor type responsible for TNFα-mediated suppression of APN production. Adult male TNFα-/-, TNFR1-/-, TNFR2-/- mice or their wild type littermates (WT) were subjected to MI via coronary artery occlusion. Histological and biochemical analyses were performed at days 3 and 7 post-MI. In WT mice, MI significantly increased plasma TNFα, reduced adipocyte APN mRNA, and decreased plasma APN levels. A significant negative correlation between plasma TNFα and APN levels was observed. TNFα deletion had no significant effect upon basal APN levels, and significantly restored APN expression/production post-MI (P<0.01 vs. WT). Basal APN levels were significantly increased in TNFR1-/- (P<0.05 vs. WT), and unchanged in TNFR2-/- mice. Importantly, suppressed APN expression/production by MI was markedly attenuated by TNFR1 deletion (P<0.01 vs. WT), but exacerbated by TNFR2 deletion (P<0.05 vs. WT). To obtain more evidence to support the opposite role of TNFR1 and TNFR2 in post-MI APN regulation, recombinant TNFα was administered to WT or TNFR knockout mice at a dose increasing plasma TNFα to comparable levels as that found in WT after MI. Experimental results demonstrated that TNFR1 deletion abolished, whereas TNFR2 deletion further enhanced, the inhibitory effect of TNFα upon APN expression/production. Mechanistically, TNFR1 knockout significantly inhibited, whereas TNFR2 knockout further enhanced, TNFα-induced mRNA and protein expression of ATF3, a transcriptional factor known to significantly inhibit APN expression. Collectively, our study provides the definitive evidence demonstrating TNFα overproduction is responsible for reduced APN expression/production following MI. More importantly, we have demonstrated that TNFR1/TNFR2 has opposite effects upon APN expression/production through differential regulation of ATF3.
DOES THE COOLING PHASE OF THERAPEUTIC HYPOTHERMIA RESULT IN HYPERGLYCEMIA AND HIGHER INSULIN REQUIREMENT?
D. Arumugam, J. McCoy, M. Tinti, V. Gracias, and T. Davidov. UMDNJ-Robert Wood Johnson University Hospital, New Brunswick, NJ
Background: Therapeutic hypothermia (TH) provides neurologic protection in patients after cardiac arrest by reducing metabolic rate. We sought to determine whether blood glucose and insulin requirements rise during the cooling phase of therapeutic hypothermia.
Methods: A retrospective review of patients who underwent TH after cardiac arrest from 2010 until 2012 was conducted. Age, sex, history of diabetes, chronic steroid use, and reason for admission were recorded. The control group consisted of patients admitted to the coronary care unit (CCU) for cardiac-related reasons other than TH induction. Controls were matched by age, history of diabetes, and chronic steroid use. Therapeutic hypothermia consisted of a 24 hour period of cooling to a temperature of 32-34C followed by a 24 hour period of rewarming to a temperature of 37C, per hospital protocol. Temperature, blood glucose, and insulin dosage were recorded over a five day period.
Results: A total of 54 patients were included in the study of which 27 underwent TH and 27 were matched controls. The TH group showed significantly higher mean blood glucose and insulin requirements during the cooling phase (day 1) compared to the rewarming phase (day 2) and the normothermic phase (days 3-5; p<.05 for all). Blood glucose was significantly higher in the TH group compared to controls during the cooling phase (185.5 vs.139.6, p=.01), but not during the remaining study days. There was a higher insulin requirement in TH patients compared to controls during the cooling phase (78.6 vs. 9.1, p=.00003) and rewarming phase (31.4 vs. 8.6, p=.04), but not the normothermic phase.
Conclusions: The cooling phase of therapeutic hypothermia after cardiac arrest is associated with hyperglycemia and higher insulin requirement. These changes are not seen in other phases of therapeutic hypothermia or in critically ill patients who do not undergo cooling.
INJURY-INDUCED INCREASES IN HUMAN APOLIPOPROTEIN A-1 DECREASES PLATELET ACTIVATION RESPONSES AND PROTECTS MICE FROM ARTERIAL AND VENOUS THROMBOEMBOLISM
C.R. Ramos1, W. Jones1, E.E. Moore*1, B. Branchford2, J. Di Paola2, C.C. Silliman*1, 3, A. Banerjee*1. 1University of Colorado Trauma Research Center, Aurora, CO, 2Department of Pediatrics: Hematology/Oncology/BMT, University of Colorado Denver, Aurora, CO, 3Bonfils Blood Center, Aurora, CO
Introduction: Increased levels of high density lipoprotein (HDL) have been associated with decreased occurrence of thromboembolism. Recently, proteomic studies showed that apolipoprotein A-1 (apoA-1), the active component of HDL, increased 3.5 fold following trauma, thus being a potential contributor to trauma induced coagulopathy in the severely injured. We explored the effects of ApoA-1 on platelet activation responses using functional assays and murine induced-thrombosis models.
Methods: All experiments were performed in samples from healthy volunteers at a concentration of 300 μg/mL of apoA-1. Thromboelastography (TEG) and microfluidic analysis were carried out in whole blood. Light transmission platelet aggregometry was conducted using apheresis platelet concentrates. Murine thrombosis models included ferric chloride (FeCl3)-induced carotid artery thrombosis and collagen/epinephrine-induced pulmonary embolism.
Results: ApoA-1 treated platelets decreased collagen induced platelet aggregation compared to controls. (23.9 +/- 5.8% vs. 80.7 +/- 3.3%, p<0.001). ApoA-1 induced decreased strength and increased lysis at 30 minutes (4.9 +/- 0.3 kd/cm2 and 11.8 +/- 3.1%), compared to controls (7.9 kd/cm2 and 0.8 +/- 0.6 %, p<0.01) employing kaolin-TEG. ApoA-1 also elicited a greater proportion of single or double platelets in microfluidic analyses under shear versus the large aggregates seen in controls. P-selectin surface expression was inhibited by apoA-1 compared to controls (10.4 +/-1.1 MFI vs. 17 +/- 2.3 MFI, p<0.01). Injection of collagen and epinephrine induced rapid fatality (presumably by pulmonary embolism) at 3.3 +/- 1 min in controls (n=5). ApoA-1 prevented lethality in treated mice with 100% survival at 30 minutes (n=5, p<0.001). Lastly, application of 6% FeCl3 to the murine carotid artery induced initial occlusion within 5.7 +/-0.7 min in controls treated with vehicle (n=5) vs. 7.7 +/-1.8 min in apoA1 treated mice (n=5, p<0.05).
Conclusion: We have shown that exogenous apoA-1 inhibited human platelet activation responses, protects mice from arterial and venous thrombosis, and may represent a protective compensatory mechanism initiated by injury.
S100A12 AND SOLUBLE RAGE LEVELS IN PATIENTS WITH SEVERE INFECTION
A. Achouiti1, M. van Zoelen*1, 2, D. Foell3, V. Thomas3, J.W. van Till1, P. Laterre4, T. Dugernier5, X. Wittebole4, M.A. Boermeester1, J. Roth3, T. van der Poll1. 1Academic Medical Center (AMC), Amsterdam, Netherlands, 2University Medical Center Utrecht (UMCU), Utrecht, Netherlands, 3University of Muenster, Muenster, Germany, 4St. Luc University Hospital, Brussels, Belgium, 5Cliniques St. Pierre, Ottignies, Belgium
Background: Sepsis remains a serious problem in clinical medicine today. S100A12 is highly expressed and serum levels correlate with individual disease activity in patients with inflammatory diseases.
Aim: To determine the extent of S100A12 and its soluble high-affinity receptor RAGE (sRAGE) release in patients with severe sepsis stratified to the three most common primary sources of infection (lungs, abdomen and urinary tract) and in human endotoxemia; and to determine S100A12 and sRAGE levels locally at the site of infection.
Methods: Two patient populations were studied: 1) patients with sepsis due to pneumonia (n=29), peritonitis (n=12) or urinary tract infection (n=10); 2) patients with peritonitis. In addition, healthy humans were studied after intravenous LPS injection.
Results: Patients with severe sepsis displayed increased circulating S100A12 concentrations. All severe sepsis subgroups (sepsis due to pneumonia, peritonitis and urinary tract infection) had elevated serum S100A12 concentrations. LPS injection in healthy humans elevated systemic S100A12 levels. In contrast to S100A12, sRAGE concentrations did not change during severe sepsis or human endotoxemia. During peritonitis, S100A12 concentrations in abdominal fluid were more than 100-fold higher than in concurrently obtained plasma, while sRAGE levels in abdominal fluid were lower than in plasma and did not increase.
Conclusion: In severe sepsis, S100A12 is released systemically irrespective of the primary source of infection. During abdominal sepsis, S100A12 release likely predominantly occurs at the site of infection. Concentrations of its high-affinity soluble receptor sRAGE do not change during infection or human endotoxemia.
DETECTION OF SERUM LEVELS OF NEUROFILAMENT-H AND BRAIN-DERIVED NEUROTROPHIC FACTOR IN MILD TBI PATIENTS WITH INTRACRANIAL HEMORRHAGE
J. Barillas, C. Stebbins, C. Madden, J.G. Wigginton*, H. Phelan, S. Wolf*, J. Minei*, J. Gatson*. University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Each year, in the United States, approximately 1.7 million people sustain a traumatic brain injury (TBI). Of these TBI events, about 75 percent are characterized as being mild brain injuries. Immediately following TBI, a secondary brain damage persists for hours, days, and even months. This secondary injury is comprised of oxidant injury, inflammation, diminished cognition, and even cell death. Previously, detection of neural markers of injury has proven to be useful to predict neurological outcome. Here, we hypothesized that both neurofilament-H (NFL-H) and brain-derived neurotrophic factor (BDNF) are detectable in the serum of mild TBI patients and the levels of these biomarkers of neural injury correlate with injury severity.
Methods: Blood (8 cc) was collected from subjects (n=27) suffering from mild TBI (Glasgow coma scale [GCS] of 13-15) at Parkland Hospital, Dallas, Texas, on days 1, 3, and 7 after injury. The serum levels of NFL-H and BDNF were measured using the enzyme-linked immunoassay (ELISA). Clinical assessments such as GCS and findings of hemorrhage from the computed tomography (CT) scan were utilized to determine injury severity.
Results: The mild TBI patients exhibited a modest increase in the serum levels of NFL-H (p < 0.08) on days 1 and 3 compared to non-injured controls. The NFL-H levels (p < 0.05) were exacerbated in mild TBI patients with intracranial hemorrhaging. In addition, plasma levels of BDNF increased (3-fold) in the mild TBI patients with negative CT findings compared to healthy controls. No increase was observed in the mild TBI patients with episodes of intracranial hemorrhaging.
Conclusion: This study describes the serum profile of NFL-H in patients suffering from mild TBI with both positive and negative CT findings and suggests that BDNF may mediate repair in the negative CT group, but is not increased in mild TBI patients with extensive neural injury. Detection of NFL-H and BDNF in the plasma after injury may prove to be a useful clinical tool for determining injury severity and outcomes in mild TBI patients.
LACTATE CLEARANCE WITHIN 12 HOURS PREDICTS MORTALITY AFTER MAJOR INJURY
S.C. Gale*1, 2, D. Arumugam1, V.Y. Dombrovskiy1, S. Coyle1, S.F. Lowry*1, V. Gracias1. 1Robert Wood Johnson Medical School/UMDNJ, New Brunswick, NJ, 2East Texas Medical Center, Tyler, TX
Backgroud: Early goal-directed resuscitation using simple markers such a central venous pressure (CVP), urine output and lactate clearance is effective in treating sepsis. Results of goal-directed resuscitation after injury are mixed. While serial lactate measurements are common during trauma resuscitation, no data are available for early (<12-hour) lactate clearance (ELC). We hypothesized that in severely injured patients, ELC would be highly predictive of mortality and would correlate with aggressive resuscitation.
Methods: Patients were selected from the Glue Grant Trauma Related Database with initial (0-6hr) serum lactate >4mmol/L. Early lactate clearance was calculated [(Lact0-6h-Lact6-12h)/Lact0-6h]x100% using the subsequent (6-12hr) measurement. Demographic, injury, and resuscitation data were compiled. Patients were grouped according to ELC quartiles for comparison. Chi-square, logistic regression analysis, point binary correlation, and Students’ t-test were used.
Results: A total of 614 patients met inclusion criteria; 119 patients (19.4%) died. Mean ISS and mean initial lactate were 37.1±13.6 and 7.3±2.8 respectively. ELC means by quartiles were -24.3%, 16.9%, 40.5% and 67.0%; corresponding mortality was 34.0%, 19.5%, 16.9%, and 7.19%. Logistic regression demonstrated that poor ELC predicts mortality when adjusted for ISS, age, race, gender, and BMI (p<0.0001). Initial 12-hour blood product volumes were 11386 ± 7940ml for non-survivors and 4815 ± 3120ml for survivors (p<0.0001). Total 12-hour resuscitation volumes were 27346 ± 15966ml for non-survivors and 17695 ± 9188ml for survivors (p<0.0001). Mean CVP ranges (nonsurvivors:10.38-13.88 vs. survivors:11.18-14.80) were not different (p=0.11). Survival was correlated positively with successful ELC (p<0.0001) and negatively with 12-hr transfusion volume (p<0.0001) and 12-hr total resuscitation volumes (p<0.0001).
Conclusions: Failure to achieve effective lactate clearance in the first hours of resuscitation after severe injury is highly predictive of mortality. Paradoxically, 12-hr blood product and 12-hr resuscitation volumes were negatively correlated with survival indicating that volume or CVP alone may be inadequate goals. Failure to achieve early lactate clearance, despite aggressive resuscitation, is an independent predictor of mortality and may herald a need to alter management approach. Prospective validation is indicated.
COMPARATIVE PHARMACOKINETICS OF HIGH- AND LOW-DOSE RECOMBINANT HUMAN SOLUBLE THROMBOMODULIN IN PATIENTS WITH SEPTIC DISSEMINATED INTRAVASCULAR COAGULATION DURING CONTINUOUS HEMODIAFILTRATION
E. Watanabe*, D. Setoguchi, T. Sadahiro, M. Nakamura, S. Oda*. Chiba University, Chiba, Japan
Background: Despite widespread use of recombinant human soluble thrombomodulin (rTM) for management of coagulopathy in patients with severe sepsis in Japan, little is known about the pharmacokinetic properties and dosing in the critically ill with coagulopathy during continuous hemodiafiltration (CHDF). rTM is reported to be excreted by kidney, hence the dose for patients with renal impairment is suggested to be reduced to one third of the normal dose.
Patients and Methods: We performed a single-center, randomized, prospective study between low-dose (0.02 mg/kg, n=4) and high-dose (0.06 mg/kg, n=4) rTM administration. We evaluated the effect of the high-dose compared with the low-dose on the pharmacokinetics of rTM for the 8 septic disseminated intravascular coagulation (DIC) patients during CHDF. Patients were given rTM during a 30 minute infusion for consecutive 6 days. All patients received CHDF at the time of inclusion. Most of parameters of severity, e.g., APACHE II, DIC score, HMGB1, and interleukin-6, etc., were not different between the two groups. The pharmacokinetic parameters were analyzed by one compartment model.
Results: The elimination half-life, clearance, and distribution volume of rTM were similar between the low- and high-dose (29.1 vs 24.1 hr, 1.92 vs 2.83 mL/hr/kg, and 77.1 vs 86.4 mL/kg, respectively), while the peak plasma concentration (Cmax) of rTM, and the AUC of rTM, were approximately 2.7 times higher with the high-dose daily infusions compared to the low-dose drip infusions (579 vs 1590 ng/mL and 66300 vs 175100 ng/mL per hr). The time when the concentration of rTM was >500 ng/mL, i.e., holding time, was significantly longer with the high-dose infusions than with the low-dose (129.0 versus 21.1 hr, p=0.032). Side effects were not observed in all the patients, but one patient in each group deceased because of multiple organ failure.
Conclusions: rTM displayed dose-dependent pharmacokinetic behavior with the range of doses used in the clinical setting. As the results, effective blood concentration was achieved under both low- and high-dose administration during CHDF, and also rTM was found not to be bioaccumulative. Therefore, it might be unnecessary to reduce the dose during CHDF even if DIC patients are complicated with renal failure.
THE FEASIBILITY AND METHODS OF AN EXCEPTION FROM INFORMED CONSENT PILOT STUDY OF ESTROGEN IN SEVERE TRAUMATIC BRAIN INJURY PATIENTS IN THE EMERGENCY DEPARTMENT (ED)
V. Warren1, K. AbdelFattah1, J. Gatson*1, A. Idris1, P.E. Pepe1, J. Simpkins2, D. Maass1, J. Minei*1, S. Wolf*1, J.G. Wigginton*1. 1University of Texas Southwestern Medical Center, Dallas, TX, 2West Virginia University, Morgantown, WV
Introduction: Despite significant supporting evidence to conduct clinical trials of estrogens as an acute trauma therapy, there are none to date. This is in part due to the need for rapid drug administration for optimal effect, causing logistical and consent issues. We recently completed the first-ever pilot traumatic brain injury study of estrogen for acute injury, RESCUE Traumatic Brain Injury (TBI).
Methods: 50 patients were enrolled in RESCUE TBI (GCS between 3 and 8) at 2 Level I trauma centers. Preclinical data led to ED drug dosing within 2 hours of injury. The mental status of severely injured patients and absence of an early Legally Authorized Representative (LAR) left informed consent generally unfeasible, so an exception from informed consent FDA IND was required.
Results: To date, several important findings have emerged: 1) Investigator-initiated exception from informed consent studies are feasible, with our FDA IND approval obtained in 19 months, IRB 1 approval in 25 days, and IRB 2 approval in 43 days. 2) Community consultation/notification was successfully accomplished, with no one opting out. 3) 50/50 enrolled patients (or LARs) were notified of participation. Six LARS received letters after the patient’s death and one participant was notified by letter of his participation (he was unable to be contacted otherwise) after he was prematurely discharged from the hospital prior to consent. The average number of days to verbal notification of the remaining patients was 7.21 days (range 0-47 days), as the study team began notification only after the patient or family was able to reasonably understand information about the study. No one decided against continued follow-up. Overall, both male and female patients and their families were very enthusiastic about participation. 4) The DSMB has no safety concerns after reviewing study data three times to date.
Conclusions: Exception from informed consent for novel, time-sensitive ED traumatic brain injury studies is crucial, feasible, and well accepted by patients and communities. Delayed notice of participation is justified for many reasons.
CENTRAL VENOUS PRESSURE GUIDED RESUSCITATION REDUCES MORTALITY IN OLDER AND IN MORE SEVERELY INJURED PATIENTS
D. Arumugam2, S.C. Gale*1, 2, V.Y. Dombrovskiy2, V. Gracias2. 1East Texas Medical Center, Tyler, TX, 2Robert Wood Johnson Medical School/UMDNJ, New Brunswick, NJ
Objectives: Early goal-directed resuscitation (EGDR) using central venous pressure (CVP) reduces mortality in septic patients. While supernormal and pulmonary artery catheter guided resuscitation strategies have been abandoned, the role of EGDR after injury remains unclear. The role of CVP specifically, to guide injury resuscitation, is also unknown. We sought to identify specific populations who might benefit most from early CVP-guided resuscitation using a large database of injured patients.
Methods: Data were obtained from the National Trauma Databank (NTDB) for adult patients admitted directly to the ICU (after ER/OR) from 2002 to 2009. Injured patients with documented CVP monitoring (ICD-9 89.62) within 48 hours of admission comprised the study group; patients without documented monitoring were included in the control group. Demographics, injury severity scores (ISS) and mortality rates were tabulated. Study group patients were matched 1:1 with controls based on propensity scores and compared with Chi-square analysis.
Results: During the study period, we identified 3,818 patients who had CVP monitoring reported during the initial 48hrs. Mean age was 46.3 ± 20.8 years; 70.0% were male. Mortality was 15.9%. Patients with severe injury (ISS ≥25) were most likely to have early CVP monitoring (1.55% vs. 0.45%;p<0.0001) and comprised 58.5% of the study group. Compared to matched controls, CVP monitoring was associated with significantly reduced mortality in older (> 60 years) patients (20.7% vs. 24.1%;p<0.008) and in more severely injured (ISS ≥25) patients 20.7% vs. 25.1%;p<0.0005). Infectious complications were not different between groups. Non-CVP guided patients had a higher rate of post-injury shock (p<0.02); CVP guided patients had more cardiac complications (p<0.05).
Conclusion: Early CVP monitoring to guide resuscitation is associated with lower mortality in patients > 60 years of age and in the most severely injured patients (ISS ≥25). Additional prospective studies are needed to further define which patients can most benefit from CVP-guided resuscitation early after injury.
INTERLEUKIN-10 AND INTERLEUKIN-12 PREDICT CHEMOTHERAPY ASSOCIATED TOXICITY IN TERMS OF PAINFUL GRADE 3 MUCOSITIS
E. Boelke*1, W.T. Knoefel1, M. van Griensven*2, C. Matuschek1, M. Peiper1, M. Schauer1. 1Heinrich Heine University, Dusseldorf, Germany, 2Technical University Munich, Munich, Germany
Backgound: Chemotherapy associated mucositis often prevents completion of an entire chemotherapy cycle. Severe mucositis may cause strong pain, dysphagia, malnutrition, dehydration, and severe diarrhoea. The underlying pathophysiology of chemotherapy associated mucositis has not been well established. The individual immunological predisposition of patients appears to play an important role.
Methods: 100 patients with locally advanced esophageal adenocarcinoma received neoadjuvant chemotherapy with cisplatin, 5 fluorouracil and leucovorin (PLF) followed by resection. Prior to the neoadjuvant therapy monocytes were isolated from blood samples and were stimulated with lipopolysaccharide (LPS) and interferon (IFN). An ELISA was used to measure interleukin-10 (IL-10) and interleukin-12 (IL-12) levels and correlated with patients’ clinical course.
Results: Pretherapeutic low IL-10 (<24.1pg/ml) and high IL-12 (>5500pg/ml) levels were significantly associated with grade III-IV mucositis causing a therapy interruption or even cessation. Interleukin production was not correlated to age, sex, tumor characteristics or response rates. Long term survival was not correlated with cytokine levels.
Conclusions: Chemotherapy associated high grade mucositis mainly depends on the individual immune response, and thus varies greatly from patient to patient. The proinflammatory effect of IL-12 and the anti-inflammatory effect of IL-10 seem to play an important role in the development of severe mucositis. As known from chronic inflammatory bowel disease, these cytokines are closely related to the activity of the mucositis and correspondingly to the rate of chemotherapy-interruption. A prediction seems to be possible by analyzing the monocyte function and their IL-10 and -12 production. With the characterization of patients at risk for severe mucositis the appropriate therapy can be started early and a severe painful mucositis sometimes even with impending electrolyte imbalances, dehydration and consecutive complications can be prevented.
THE USE OF TEMPERATURE PATTERNS TO PREDICT SEPSIS IN AFEBRILE ADULT INTENSIVE CARE UNIT PATIENTS
A.M. Drewry*, B. Fuller*, R.S. Hotchkiss*. Washington University in St. Louis, St. Louis, MO
Introduction: Sepsis is recognized as a time-sensitive emergency, yet early diagnosis remains challenging. In intensive care unit (ICU) patients, the presence of fever is a common trigger for the evaluation of infection. Evidence suggests that temperature pattern variability may be an earlier indicator of sepsis than the appearance of fever, but data is limited. The purpose of this study was to examine whether abnormal temperature patterns could predict the subsequent diagnosis of sepsis prior to the onset of fever.
Methods: Retrospective case-control study of 32 septic and 29 non-septic patients in a medical and surgical ICU. Temperature curves were plotted for the period starting 72 hours and ending 8 hours prior to the clinical suspicion of sepsis (for the septic group) and for the 72-hour period prior to discharge from the ICU (for the non-septic patients). The timestamp for the clinical suspicion of sepsis was determined by the first fever >38.3°C, the first order for cultures, or the first order for antibiotics, whichever came first. Seven blinded intensivists independently reviewed each temperature curve and rated the pattern as normal or abnormal.
Results: Baseline characteristics of the two groups were similar, except the septic group had more trauma patients (31.3% vs. 6.9%, P = 0.02) and patients requiring mechanical ventilation (75.0% vs. 41.4%, P = 0.008). An abnormal temperature pattern was noted by a majority of the observers in 22 (68.8%) septic patients and 7 (24.1%) control patients. Multivariate logistic regression analysis to control for baseline differences showed that an abnormal temperature pattern was significantly associated with the eventual diagnosis of sepsis (OR 5.53 [IQR 1.14, 26.71], P = 0.033). Abnormalities included increased maximum fluctuations in temperature (1.53°C vs. 1.20°C, P = .032) and a trend toward higher peak temperatures (37.75°C vs. 37.42°C, P = 0.087). The median time from the end of the last plotted temperature to the first dose of antibiotics was 16.90 hours (IQR 8.35, 34.20).
Conclusions: Temperature patterns, rather than absolute values, may aid in the earlier diagnosis of sepsis in adult ICU patients. Analysis of temperature variability may also decrease time to antimicrobial therapy. Future prospective trials should examine if temperature variation is a valid diagnostic marker for early sepsis detection.
PERFUSIONAL PARAMETERS IN EARLY FLUID RESUSCITATION OF MASSIVE BURN PATIENTS
L. Azevedo1, 2, E. Campos1, M. Park1, 2. 1Intensive Care Unit, Hospital das Clinicas, University of São Paulo School of Medicine, São Paulo, Brazil, 2Hospital Sirio-Libanes, Sao Paulo, Brazil
Introduction: Tissue hypoperfusion plays an important role in the development of multiple organ dysfunction and death in general critically ill patients. However, there are no clear recommendations regarding monitoring of perfusion parameters during early resuscitation of burn patients. Moreover, it is uncertain if the evolution of these parameters may distinguish between survivors and non-survivors. Thus, our aim was to analyze tissue perfusion markers (lactate, base excess - BE and SvO2) in massive burn patients during the initial phase of fluid resuscitation (24 hours).
Methods: This is a retrospective cohort study using data collected from May 2005 to April 2010 at an ICU specialized in burn patients from the Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. All patients admitted during this period were included. Data are expressed in median and interquartile ranges and Mann-Whitney test was used to calculate the differences between the groups. A p value below 0.05 was considered significant.
Results: One hundred and sixty three consecutive patients were studied (male 71%), with median age of 34 [25,47] years and a median hospital stay of 29 [11,50] days. Incidence of inhalation injury was 45% and median total burn surface area was 29 [18,43] %. In-hospital mortality rate was 42% (69 patients). Lactate levels at admission in survivors were 2.7 [2.3,4.3] mmol/L, while non-survivors had 4.1 [2.7,5.1] mmol/L (p<0.001). Twenty-four hours later, lactate levels in survivors were 2.4 [1.8,3.2] mmol/L, whereas non-survivors had 3.3 [2.2,4.8] mmol/L (p<0.001). BE at admission in survivors was -4.5 [-8,-2] and in non-survivors -8.5 [-12,-6] (p<0.001). After 24 hours, survivors had -4 [-6,-2] and non-survivors -8.6 [-12,-4] (p<0.001). Also, SvO2 at admission was 73 [64,82]% in survivors while in non-survivors it was 60 [46,76]% (p=0.006). After 24 hours, SvO2 increased in both groups, but it was higher in survivors than in non-survivors (79 [75,85]% vs 70 [61, 77]%; p<0.001).
Conclusions: Non-survivors of severe burns evolve with elevated lactate as well as reduced BE and SvO2 in the first 24 hours of fluid resuscitation as compared to survivors. Evaluation of perfusional parameters in this time course of resuscitation may help to distinguish patients more prone to unfavourable outcomes.
ANTIPYRETIC USE IN GRAM-NEGATIVE SEPTIC SHOCK DOES NOT DECREASE THE DURATION OF VASOPRESSOR THERAPY
N.M. Mohr*1, B.M. Fuller*2, L. Skrupky3, H. Moy4, R. Alunday2, S.T. Micek3, R.E. Fagley5. 1University of Iowa Carver College of Medicine, Iowa City, IA, 2Washington University School of Medicine, St. Louis, MO, 3Barnes-Jewish Hospital, St. Louis, MO, 4University of North Carolina School of Medicine, Chapel Hill, NC, 5Virginia Mason Hospital and Medical Center, Seattle, WA
Introduction: Fever is common in septic shock, and antipyretic therapy is a commonly used therapy. Human trials conflict on the role of antipyretic therapy in sepsis, but a recent study suggests that fever control may shorten the duration of shock and decrease 14-day mortality. The present study was designed to determine the impact of antipyretic drug administration on the duration of vasopressor therapy in patients with gram-negative septic shock.
Methods: Single-center, retrospective cohort study of 96 febrile adult patients with gram-negative septic shock at a 1,111-bed academic center from January 2002 through February 2008. Antipyretic therapy was defined as administration of acetaminophen or ibuprofen within 24 hours of a positive blood culture in a patient with septic shock. Patients were excluded for history of cirrhosis, elevated liver enzymes, acute traumatic brain injury, acute stroke, or acetaminophen allergy.
Results: Fifty-four (56%) patients were treated with an antipyretic agent, and 14 (26%) of those treated received 3 or more doses in the first 24 hours after onset of septic shock (positive blood culture). More patients in the antipyretic group were male (69% vs. 44%, p = 0.02), but the groups were otherwise well-matched. Using multivariable linear regression to account for potentially confounding factors, antipyretic therapy was not associated with duration of vasopressor therapy (beta 0.14, -0.32 - 2.34, p = 0.14). Independent predictors of the duration of vasopressor therapy were APACHE-II (p < 0.01) and initial mean arterial pressure (p = 0.02). Antipyretic use was not associated with 14-day mortality (41% vs. 39%, p = 0.88).
Conclusions: Antipyretic drug therapy was not independently associated with either vasopressor duration or 14-day mortality. These findings are hypothesis-generating for future clinical trials, as the role of fever modulation on clinical outcomes in septic shock remains unclear. (Grant UL1 RR024992, NIH-NCRR).
CLINICAL RELEVANCE OF CYTOKINES GENE POLYMORPHISM (CGP) AND LEVELS OF THESE CYTOKINES IN RECOVERY OF HEMORRHAGIC SHOCK PATIENTS
D.L. Gupta, D.N. Rao, S.K. Bhoi. AII India Institute of Medical Sciences, New Delhi, India
Introduction: Severe trauma complicated with multiple organ dysfunction syndromes (MODS) are among the leading causes of deaths in intensive care units. Alarmingly, the mortality rate, owing to multiple causes e.g. multiple organ dysfunctions (MOD) with or without sepsis, is now reported to cross the value of 50% and thus need an immediate insight to overwhelm it. The cytokine levels and their associated gene polymorphisms are now contemplated as underlying cause for the severity of trauma.
Objective: This aims to examine if there exists any link between cytokine gene polymorphisms and outburst of pro-inflammatory/anti-inflammatory cytokines in the patients with various complications (sepsis/septic shock, MOF etc.) after traumatic injury.
Methodology: A total of seventy trauma patients and fifty healthy controls were recruited in the study. The allele, genotype, and haplotype frequences of 13 cytokines genes have been evaluated using PCR-SSP. Cytokine concentrations in the individual patient’s sera were determined by sandwich ELISA in a serum level dynamics up to 14 days.
Results and Conclusion: In septic patients, changes in the alleles and genotype frequencies in cytokines TGF-β (codon10), IL-10(-1082) and IL-6(-174) have emerged as major cause of sepsis and MOF related mortality. Thus, immune paralysis (IL-10 over expression) and Th17 (enhanced TGF-β/IL-6) concluded as putative cause of death in this study.
GENERATION OF A LYOPHILIZED EXTRACT OF THE LARVA OF THE GREENBOTTLE FLY LUCILIA SERICATA: A NOVEL STRATEGY FOR THE MANAGEMENT OF CHRONIC WOUNDS
E. Boelke*1, C. Matuschek1, B. Homey1, H. Mehlhorn1, M. van Griensven*2, N. Hoff1, F. Gestmann1, P. Gerber1. 1Heinrich Heine University, Dusseldorf, Germany, 2Technical University Munich, Munich, Germany
Background: Chronic wounds, like venous, diabetic or decubital ulcers, are a common problem of the growing elderly population. These wounds may eventually result in amputations and are associated with a significantly reduced quality of life. Finally chronic wounds are associated with considerable high socio-economic costs. The so-called bio-debridement or maggot-therapy with the use of living larvae of the greenbottle fly Lucilia sericata is an effective therapy to debride chronic wounds and stimulate the generation of fresh granulation tissue, finally leading to wound closure. However, maggot-therapy is associated with various problems, including the requirements for the transport of living larvae as well as low patient compliance due to disgust, putrid smell or pain/discomfort.
The aim of this study was to generate a lyophilized extract of the larva of the greenbottle fly Lucilia sericata and analyze its effects on chronic wounds.
Methods and Results: Larvae of Lucilia sericata were shock-frozen in liquid nitrogen and subsequently homogenized. This emulsion was sterile-filtered and the resulting solution was freeze-dried to generate a lyophilized extract. Finally, the extracts were sterilized using gamma-irradiation resulting in the final product (Larveel®, Alpha-Biocare GmBH, Duesseldorf, Germany). The safety of Larveel® was tested using various assays, including the hens egg test (HET-CAM), tests for sensibilisation of the skin as well as tests for cytotoxicity (neutral red uptake) and tests for endotoxins (LAL, MAT and rabbit).
Conclusions: We successfully generated a lyophilized extract of Lucilia sericata larvae (Larveel®). In a next step, we will now select patients with chronic leg ulcers that did not respond well to standard wound therapy and will analyze the effects of Larveel® on these lesions. We feel, that Larveel® is a promising new option for the treatment of chronic wounds.
COMPARISON OF TISSUE HEMOGLOBIN OXYGEN DESATURATION USING RESONANCE RAMAN SPECTROSCOPY VERSUS NEAR INFRARED SPECTROSCOPY
K.R. Ward*1, 2, M.H. Tiba*1, 2, G.T. Draucker1, 2. 1University of Michigan, Ann Arbor, MI, 2Michigan Center for Integrative Research in Critical Care, Ann Arbor, MI
Objective: We hypothesized that monitoring of regional tissue hemoglobin oxygenation (StO2) using resonance Raman spectroscopy (Raman-StO2) is more sensitive than near infrared spectroscopy (NIRS) for detecting StO2 changes due to ischemia.
Methods: Data was gathered using an Institutional Review Board approved protocol. Ten healthy, normotensive, nonsmoking subjects (4 males, 6 females) were consented and tested. Raman-StO2 utilizes light at a wavelength of 405 nm, which results in tissue penetration of < 1 mm and thus does not penetrate into muscle tissue from the skin. NIRS utilizes light at wavelengths between 650-1000 nm and is able to penetrate several centimeters below the skin to muscle tissue. StO2 was monitored using both Raman spectroscopy of the thenar eminence and NIRS of the mid volar forearm at the same time. A cuff pressure was placed on the arm of the same extremity. Following a baseline period of 5 minutes, cuff pressure was inflated rapidly to 200 mmHg and held for 120 seconds. Cuff pressure was then deflated and tissue oxygenation was monitored and recorded for another 3 minutes.
Results: There was no significant difference between baseline StO2 values between Raman-StO2 as measured at the thenar eminence versus NIRS-StO2 of the forearm. Following cuff inflation, Raman-StO2 decreased from a baseline of 64(5)% to 12(3.6)% representing an 81% change. During this same period, however, NIRS-StO2 decreased from a baseline of 64(7)% to 55(11)% representing a 15% change. An unpaired t test showed a significant difference between Raman-StO2 and NIRS-StO2 at the 120 seconds (p < 0.0001). One minute post cuff deflation, both Raman-StO2 and NIRS-StO2 were back at baseline level 69(6)% and 68(8)% respectively with NIRS-StO2 reaching that level faster than Raman-StO2 at an average of 30 seconds and 78 seconds respectively.
Conclusion: Raman-StO2 appears to be more sensitive in detecting levels of ischemic blood flow than NIRS-StO2. Reasons for this may be due to myoglobin contamination of the NIRS-StO2 signal as myoglobin has a significantly lower P50 than hemoglobin.
RESUSCITATION VOLUME EXPANDER:HBOC COCKTAILS DO NOT OUTPERFORM STANDARDS OF CARE (BLOOD, HEXTEND) IN A RABBIT HEMORRHAGIC SHOCK MODEL
P. Reynolds*, A. Morris, B.D. Spiess. Virginia Commonwealth University Medical Center, Richmond, VA
Objective: Following severe hemorrhagic shock, correction of tissue hypoperfusion is a primary resuscitation goal. Current thinking emphasizes use of relatively small volumes of hypertonic or hyperosmotic fluids. Because these fluids are plasma expanders with limited oxygen-carrying capacity, it has been suggested that augmentation with a hemoglobin-based oxygen carrier (HBOC) would improve oxygen delivery to the tissues. We hypothesized that synergism between components would provide benefit above and beyond that afforded by any individual component. We predicted that combined resuscitation therapy with a multi-component “cocktail” composed of a volume expander and an oxygen-delivery adjunct would outperform current standards of care in a pre-clinical model of battlefield trauma. The objective was to identify the optimum combination of selected resuscitation agents that best promotes survival to two hours post-resuscitation, restores and supports hemodynamics, and promotes lactate clearance.
Methods: We tested different combinations of volume expanders, platelet supplement, and oxygen therapeutic adjuncts in a rabbit model of acute hemorrhagic shock. Anesthetized rabbits (n=35) were subjected to a controlled acute 40% total blood volume (TBV) hemorrhage over 60 minutes. Fresh whole blood (FWB; “gold standard”) was compared to two volume expanders, Hextend (HEX; combat casualty standard of care) and Resusix (RES; solvent detergent-treated and spray-dried human plasma), with or without Stasix (STA; freeze-dried human platelets) and/or oxygen therapeutic adjuncts (reconstituted OxyVita Powder [HBOC] or Efaproxiral [RSR13; allosteric hemoglobin modifier]). Animals were randomized to treatment according to a D-optimal design, testing all possible combinations.
Results: FWB, HEX, and HEX combinations provided superior survival, mean arterial pressure (MAP) support, and lactate clearance compared to RES combinations (p < 0.001). The triad of RES, STA, and HBOC was lethal within 60 minutes; animals showed evidence of cardiac dysfunction and renal damage. Hemodynamics and acid-base status of animals given RES and STA slightly improved with RSR13 augmentation (p < 0.01).
Conclusions: Adverse effects of current alternatives to FWB or HEX may offset any benefit from increased MAP support or oxygen delivery.
STORED BLOOD MODULATES THE INTERFERON REGULATORY FACTOR 1 RESPONSE OF MACROPHAGES DURING STERILE INFLAMMATION IN VITRO AND IN VIVO
J.R. Klune1, L. McDaniel1, K. Hart1, J. Raval2, M.D. Neal1, A. Tsung*1. 1University of Pittsburgh, Pittsburgh, PA, 2University of North Carolina, Chapel Hill, NC
Background: Blood transfusions are common in surgical, trauma, and critically ill patients and have been correlated with worse outcomes. Receiving blood transfusions is known to lead to increased infectious complications and increased morbidity and mortality. However, little is known about the specific pathways by which blood transfusions lead to these complications. Interferon regulatory factor 1 (IRF1) is a nuclear transcription factor known to be a central coordinator of the immune response following injury. Therefore, we hypothesized that stored blood may modulate the IRF1 response of macrophages leading to an altered inflammatory response.
Methods: Stored human red blood cell units (RBCs) were obtained with IRB approval. Cellular components were separated and the acellular serum portion was obtained. This portion was used in vitro with RAW 264.7 cultured macrophages or injected in vivo into wild-type mice prior to hepatic ischemia reperfusion (I/R) injury.
Results: In vitro, RAW cells pretreated with stored blood had decreased activation of the pro-inflammatory transcription factor Interferon regulatory factor 1 (IRF1) in response to cytomix stimulation with TNFα, IFNγ, and IL-1β. This effect was more pronounced in blood stored for increased storage periods. Additionally, these macrophages demonstrate decreased production of IRF1-dependent genes iNOS and IFNβ. In vivo, when given at the time of hepatic I/R, exposure to stored blood significantly decreased production of iNOS and IFNβ which suggests decreased IRF1 activity. This occurred independently from hepatocellular injury. Additionally, mice receiving pretreatment with stored blood had decreased inflammatory cytokine production of TNFα and IL-6. These differences were seen in both the ischemic and non-ischemic lobes, suggesting this was due to immune cell rather than hepatocyte differences.
Conclusions: Exposure to stored blood modulates the inflammatory response to sterile inflammation such as I/R injury. This appears to occur through suppression of IRF1 activity in immune cells such as macrophages. This decreased inflammatory response may be responsible for making the recipient more susceptible to infectious complications. Further research may focus on eliminating this effect in order to decrease morbidity and mortality.
INHIBITION OF HYDROGEN SULFIDE PRODUCTION DECREASES HEPATIC LEUKOCYTE ACCUMULATION INDEPENDENT OF ITS EFFECTS ON INFLAMMATORY RESPONSE
N. Feilen, E.J. Norris*, M. Clemens*. University of North Carolina at Charlotte, Charlotte, NC
The gaseous mediators NO and CO are well recognized modulators of injury in shock and sepsis. More recently, endogenously produced H2S has been shown to be upregulated in these conditions, but its effects are controversial. While H2S appears to be protective in ischemia, it may be deleterious in sepsis and endotoxemia but the mechanisms are not well understood. Leukocyte accumulation exacerbates injury after LPS and pretreatment with propargylglycine (PAG) prior to injury reduces leukocytes concomitant with decreased inflammation. We have recently shown that treatment with PAG 5.5 hours after LPS ameliorates hepatic microvascular dysfunction (increased sinusoid constriction) at 6 hours; therefore in this study we tested whether PAG post treatment might also reduce leukocyte sticking in the sinusoids. Rats were treated with LPS (1 mg / kg ip) and then 5.5 hours later given PAG (50 mg/kg) or vehicle ip. 30 minutes later they were prepared for intravital microscopy. Leukocytes were stained with acridine orange and leukocytes stationary in sinusoids or adherent or rolling in central venules were quantified via offline analysis. Liver samples were collected at the end of the experiment for measurement of cysthionine γ lyase (CSE, the enzyme that synthesizes H2S) activity to ensure the efficacy of PAG. LPS treatment upregulated CSE activity, but this was inhibited by 69% by PAG treatment in vivo 30 minutes prior to intravital microscopy. Contrary to our expectation, PAG pretreatment even after the inflammatory response had fully developed, significantly reduced sinusoidal leukocytes (13.4 +/- 4.4 vs 1.8 +/- .03 per 20x field, P=0.009). Adherent and rolling leukocytes in central venules were variable and not different between groups. These results show that inhibition of CSE in vivo even after the inflammatory response has developed effectively reduces leukocyte sticking in hepatic sinusoids but not in the larger venules. Since endotoxemia is associated with sinusoid constriction, we propose that the effect of PAG results from inhibition of H2S-dependent narrowing of sinusoids resulting in trapping of leukocytes. The effectiveness of post-treatment suggests possible therapeutic potential for limiting leukocyte-dependent liver injury as well has sinusoid hyperconstriction in sepsis.
BURN INJURY INDUCES THE FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS (NET) IN LUNG TISSUE
F.A. Rivera-Chavez, J. Song*, M. Liu, J. Minei*, S. Wolf*. University of Texas Southwestern Medical Center, Dallas, TX
Background: Pulmonary complications such as acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common among burn injured patients. Excessive recruitment of NETs has been implicated in infectious ALI/ARDS. NETs are composed of DNA, histones, elastase (NE) and myeloperoxidase (MPO). The formation of NETs in lungs after burn injury is unknown.
Aim: To investigate lung NET formation after thermal injury.
Methods: 12 Male Sprague-Dawley rats were subject to either a sham burn or a 40% total body surface area burn, followed by fluid resuscitation. Lung tissue was harvested, fixed in PFA 4%, and assessed for NET formation by immunofluorescence staining.
Results: NETs were not expressed in sham tissue. In comparison, NETs were abundantly expressed in response to acute burn injury. Figure below is representative of various experiments. Staining reveals fibrous extracellular material that stains for histone and DNA (A Column), neutrophil elastase (B column) and myeloperoxidase (C column). Image shows, overlay of the images A) DNA + histone, B) DNA + elastase, and C) DNA + MPO. The images are projections of confocal z stacks (40x) generated from lung sections of 5 to 6 _m thickness.
Discussion: Although NETs are important in trapping and killing bacteria and other pathogens, their prolonged presence in sterile inflammation such as in burns, may lead to tissue damage. NET formation has been implicated in the pathology of TRALI, which is another form of non-infectious ALI. Also, elastase and myeloperoxidase play a role in ALI.
Conclusions: We provide evidence for the formation of neutrophil extracellular traps (NETs after burn injury). However, how this impact the development of ALI/ARDS is unknown.
ELECTRICAL STIMULATION OF A BRAIN CHOLINERGIC REGION REGULATES PERIPHERAL INFLAMMATORY RESPONSES
V.A. Pavlov*1, H. Silverman1, M. Dancho1, A. Regnier-Golanov2, M. Ochani*1, Y.A. Levine1, 3, W. Hanes1, J. Johnson2, S.S. Chavan1, E. Golanov2, K.J. Tracey*1. 1Laboratory of Biomedical Science, Center for Biomedical Science, The Feinstein Institute for Medical Research, Manhasset, NY, 2Department of Neurosurgery, North Shore University Hospital, Manhasset, NY, 3SetPoint Medical Corporation, Valencia, CA
We have previously indicated a role for brain cholinergic muscarinic receptor-dependent signaling, functionally associated with a vagus nerve-mediated anti-inflammatory circuit in the regulation of peripheral inflammation (Proc Natl Acad Sci USA, 2006, 5219; Brain Behav Immun, 2009, 23:41). Here we studied whether electrical stimulation of the medial septum (MS), an important constituent of the basal forebrain cholinergic system, alters the peripheral inflammatory response during endotoxemia. MS was stimulated in isoflurane anesthetized C57Bl/6 mice using a stereotaxically placed coaxial electrode 1h prior to endotoxin (8mg/kg, i.p.) administration. Serum cytokine levels were determined 1.5h following LPS administration. Three consecutive trains (30sec each) of stimulation (bipolar rectangular pulses, 250micros, 150microA, 30Hz, 75Hz, 120Hz, respectively) followed by 10min of continuous stimulation (250micros, 150microA, 75Hz) significantly reduced serum TNF, IL-6, CXCL1, IFN-gamma, and IL-12p70 levels as compared to sham-stimulated controls. MS stimulation was also associated with increased regional cerebral blood flow (rCBF, parietal cortex, laser Doppler flowmetry) - a previously unreported physiological effect. Decreasing the stimulation intensity to 75microA and duration to 3min resulted in lower magnitude of serum TNF and other cytokine suppression and rCBF increase. Ongoing studies explore the role of MS within brain anti-inflammatory circuitry and its relation to autonomic regulation. These current results demonstrate a previously unrecognized anti-inflammatory effect of MS stimulation. Our findings are of interest for further unveiling the central mechanisms of regulation of inflammation and development of stimulation of brain cholinergic pathways as a novel anti-inflammatory approach. This study was funded in part by NIH/NIGMS.
DEVELOPMENT OF A RODENT MODEL OF REPEATED IMMUNEMODULATING RESUSCITATION FOR SEPSIS ALLOWS DELAYED IN VIVO EVALUATION OF MICROCIRCULATION
R.G. Eisenstadt1, P. Sanati-Mehrizy2, W. Gong1, M. Murcy1, K. Kumasaka1, C.A. Sims*1, S.R. Allen*1, L.B. Becker1, J.L. Pascual1. 1University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 2Icahn School of Medicine at Mount Sinai, New York, NY
Introduction: Single dose HTS resuscitation in animals blunts neutrophil (PMN) activation and interactions with endothelium (EC), reducing microvascular leakage. It is unknown if repeated HTS administration (as occurs in the ICU) has persistent effects in the microcirculation. We hypothesized that by using chronic implanted jugular catheters, repeated daily resuscitation with HTS could be tolerated by mice allowing in vivo microcirculatory evaluation 3 days later.
Methods: 29 male CD1 mice (30 g) underwent tunneled jugular catheterization 3 days prior to cecal ligation and puncture (CLP). Animals then received repeated infusions of 5% HTS (6cc/kg) or Lactated Ringer’s (30 cc/kg) every 12 hours for 3 days and were returned to their cages between infusions. Prior to sacrifice, animals underwent cremaster intravital microscopy (IVM) to assess PMN/EC interactions, and vascular permeability (leakage of FITC-albumin). Tissue water content was determined by wet:dry analysis. Parametric tests determined significance (p<0.05).
Results: 62.1% of septic animals survived repeated resuscitation boluses, yielding IVM footage visualizing PMNs interacting with EC 3 days after CLP (Figure 1). Rolling and adherent PMN to EC could be counted reliably. Vascular permeability was quantified with standard imaging software. Wet:dry ratios demonstrated reduced lung water content in HTS animals (p<0.05).
Conclusions: Repeated fluid resuscitation in septic mice using an indwelling jugular catheter is well tolerated for several days. This model appears to be more clinically relevant, simulating resuscitation as would occur in septic patients in the ICU.
GENDER AS A DETERMINANT OF MUSCLE PROTEIN SYNTHESIS IN SEVERE PEDIATRIC BURNS
E. Diaz1, 2, A. Ali1, 2, C. Porter1, 2, J. Lee1, D.N. Herndon*1, 2, M. Cotter1, 2, W.J. Meyer1, 2, L. Sidossis1, 2, E. Borsheim1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX
Introduction: Skeletal muscle catabolism, a hallmark of the burn induced stress response, is associated with increased morbidity and mortality. We have previously reported that muscle loss following a major burn is more prominent in males than in females. However, it is unknown if this difference is associated with variations between gender in the rate of muscle protein synthesis. The aim of this study was to determine if sexual dimorphism exists with regards to skeletal muscle fractional synthetic rate (FSR) in severely burned children.
Methods: A total of 44 pediatric patients (29 males and 15 females) with ≥30% Total Body Surface Area (TBSA) burns were included in this study. These patients were participants of larger prospective studies and were randomized to receive placebo. Plasma and muscle samples were collected during stable isotope infusion studies once burn wounds were 95% healed (30±10 days post burn) in order to determine skeletal muscle FSR. The Wilcoxon-Mann-Whitney test was used for comparison purposes. Significance was accepted when P<0.05.
Results: Males (age 10±1.0 years) and females (age 7±1.0 years) had 59±11% and 57±11%TBSA burns, respectively (P=NS). Flame injuries were seen in 76% of males and 80% of females (P=NS). The proportion of patients (35% vs. 36%) with inhalation injury was not different between groups (P=NS). FSR was significantly greater in females when compared to males (0.29±0.05%.h-1 vs. 0.15±0.02 %.h-1; P<0.05).
Conclusion: The current data demonstrates sexual dimorphism with regards to skeletal muscle FSR following burn injury. Whether a lower skeletal muscle FSR in males contributes to greater muscle catabolism as reported in previous studies warrants further investigation. However, our current findings suggest that the response of skeletal muscle to severe burns is gender specific in children. As such, gender specificity may be a pertinent consideration in future clinical care of patients with severe burns.
Supported by the National Institutes of Health grant P50-GM60338 and Shriners Hospitals for Children grants 84090, 84085 and 71006.
MECHANISMS OF BOWEL-MEDIATED HYPOTENSION FROM PANCREATIC-DERIVED PEPTIDES
E.B. Kistler*1, T. Alsaigh2, T. Hugli*3, G.W. Schmid-Schonbein*2. 1VA San Diego Healthcare System/University of California, San Diego, San Diego, CA, 2University of California, San Diego, Dept of Bioengineering, La Jolla, CA, 3Torrey Pines Institute for Molecular Studies, La Jolla, CA
As part of normal digestion, pancreatic proenzymes released into the small intestine lumen are themselves degraded into peptide fractions, some of which may be inflammatory. In ischemia, increased intestinal wall permeability may allow these factors normally sequestered in the bowel to breach the mucosal barrier and reach the lymph and systemic circulation, with subsequent cardiovascular collapse. We have identified 12 peptide factors from end-hydrolysis of porcine pancreas homogenate with hemodynamic activity that may contribute to the initial hypotensive state seen in shock associated with gut malperfusion. Results demonstrate that these factors cause hypotension if injected systemically (10-50 μM range) into anesthetized non-shocked rats and activate TNF-α in isolated epithelial (IEC-18) and endothelial cell (HUVEC) cultures but do not directly activate neutrophils via pseudopod assay or promote histamine release in isolated human mast cell cultures via β-hexosaminidase measurements. Major mechanisms of action by these peptides appear to be as angiotensin converting enzyme inhibitors (ACEI), which may explain in part the early hypotension seen in circulatory shock. Although it is known that endogenous ACEI can be formed in the bowel, this is the first evidence to our knowledge that the production and subsequent egress of these mediators may contribute to bowel-mediated shock. Supported by Career Development Award (CDA2) 1IK2BX001277-01A1 from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, HL67825, and the ASCCA/FAER/Hospira Physician Scientist Award.
HEMORRHAGIC SHOCK-INDUCED MITOCHONDRIAL DYSFUNCTION IS LIMITED BY INHALED CARBON MONOXIDE OR NEBULIZED SODIUM NITRITE AS ADJUNCTS TO STANDARD RESUSCITATION
D.A. Escobar, H. Gomez, B. Ataya, L. Gordon, O. Ogundele, D. Severyn, M.R. Pinsky, S. Shiva, B. Zuckerbraun*. University of Pittsburgh Medical Center, Pittsburgh, PA
Introduction: Hemorrhagic shock (HS) results in hypoperfusion and inflammation, which induces mitochondrial injury and tissue damage. Adjuncts targeted at limiting mitochondrial dysfunction may enhance the effect of standard resuscitation and decrease tissue injury. Accordingly, we investigated if inhaled Carbon Monoxide (CO) or nebulized Sodium Nitrite (NaNO2) could protect the mitochondria and thus, reduce HS-induced tissue injury.
Methods: Twenty anesthetized female Yorkshire pigs subjected to a protocol of severe hemorrhage until unable to compensate for 90 min, and resuscitation with volume/pressors. Animals were randomized to 1. Standard of care (HSR, n=5); 2. HSR+CO (CO; 250ppm x 30 min, n=6); or 3. HSR+NaNO2 (NaNO2;11mg in PBS x 30 min, n=6), and sham (n=3). CO or NaNO2 were initiated ∼ 30 min before resuscitation. Primary endpoints were changes in platelet and muscle mitochondrial respiration between baseline (BL) and 2 hrs after resuscitation (EndObs), quantified by change in proton leak respiration (PLR) and mitochondrial reserve capacity, and muscle respiratory control ratio (RCR). Secondary endpoints were kidney injury measured by the change in urine N-GAL from 60 minutes after hemorrhage (H60) to EndObs, and development of cardiac arrest after resuscitation.
Results: Platelet mitochondrial dysfunction occurred in the HSR group, manifested by an increase in PLR and decreased reserve capacity. This group also manifested a reduced RCR (p=0.04) primarily due to decreased ADP-dependent respiration, with no change in state 4 respiration. CO and NaNO2 prevented these changes in mitochondrial respiration. This benefit was associated with a negative change in N-GAL in CO and NaNO2 (-5.36 and -3.46), and a lower rate of post-resuscitation cardiac arrest (33 and 25%). In comparison, HSR had a positive change in N-GAL (10.9) and 60% post-resuscitation cardiac arrest rate.
Conclusions: CO and NaNO2 limited platelet and muscle mitochondrial injury in this model of HS. This suggests that CO and NaNO2 may protect mitochondrial function by maintaining reserve capacity and ATP-coupled respiration. Whether this translates in less renal injury and lower rates of cardiac dysfunction remains speculative despite the trends we report, and thus merit further investigations.
ESTROGEN REDUCES PANCREATIC INFLAMMATION FOR 45 DAYS FOLLOWING SEVERE BURNS
J.G. Wigginton*, J. Gatson*, V. Warren, Q.S. Zang*, A. Idris*, J. Minei*, S. Wolf*, P.E. Pepe, D. Maass. University of Texas Southwestern Medical Center, Dallas, TX
Background: Multisystem and remote organ failure following burn injuries remain a significant cause of morbidity and mortality. The mechanisms of organ failure following burns are currently not fully understood, however, it is known that the remote and systemic effects of severe burns are at least in part driven by increased inflammation. Clinical observations note that the pancreas and its function are negatively affected following remote burn injuries. More specifically, post-burn beta-cell dysfunction function and inflammation have been correlated with mortality. Therefore, we tested estrogen, an anti-oxidant, anti-apoptotic, and anti-inflammatory drug as a potential therapy for mitigation of pancreatic inflammation following burns.
Methods: 177 male rats were randomized into 3 groups: Group 1 (0.5mg/kg of 17β estradiol (E2) 15 min after injury), n=84; Group 2 (placebo), n=84; and Group 3 (sham-burn control), n=9. Groups 1 & 2 were given a 3° 40% TBSA dorsal scald burn. Eight burned/treated rats per group were sequentially sacrificed at 0.5, 1, 2, 4, 6, 8, 12, 18, 24 hours and 7 days post-burn, with four rats/treatment sacrificed 45 days post-burn. Renal tissue was analyzed by ELISA for pro-inflammatory cytokines (including IL-6, TNF-α, IL-1β).
Results: Administration of estradiol significantly decreased the IL-6 levels in the pancreas to a fraction of that found in the placebo/burn group at multiple time points over 45 days.
Conclusions: Acute administration of estrogen decreases inflammation in the pancreas following remote severe burns. Results from these studies will help further our understanding of how estrogens may protect the pancreas and decrease patient morbidity and mortality following burns.
INHIBITION OF AUTOPHAGY REDUCES INTESTINAL INFLAMMATION AFTER ACUTE ETHANOL AND BURN INJURY
M. Sigman, X. Li, R.L. Gamelli*, M.A. Choudhry*. Loyola University Chicago, Maywood, IL
We have shown that acute ethanol intoxication exacerbates intestinal immune and barrier dysfunction after burn injury. Disturbances in autophagy, or lysosomal catabolism of cellular components, have been implicated in intestinal inflammation and immune deficiency. We examined whether combined ethanol and burn injury influences autophagy and whether this relates to intestinal inflammation. To accomplish this, Male C57BL/6 mice (22-25 g BW) were gavaged with ethanol (2.9 g/kg) or water (vehicle control) prior to a 12.5% total body surface area burn. Groups of sham and injured mice were then treated with 3-methyladenine, (3MA; 10mg/kg) an inhibitor of autophagy via class III phosphatidylinositol 3-kinase (PI3K). Mice were sacrificed one day following injury. Small intestine (ileum) was harvested and processed for the measurements of inflammatory cytokines IL-6, IL-18, MIP-2 and KC by ELISA and for PCR expression of LC3B, a genetic marker for the formation of autophagosomes. LC3B expression in ethanol burn injured animals was increased 21.97±7.01 times to 1±0.19 (Relative expression p=0.02) compared to sham mice. This was accompanied by increases in IL-6 (154±60.1 to 40±14.2 pg/ml, p=0.02), IL-18 (139±85.1 to 41±25 pg/ml, p=0.01), MIP-2 (202.4±74 to 29.5±11, p=0.03) and KC (69.5±35 to 2.75±0.8 pg/ml p=0.03). Levels of inflammatory cytokines in burned mice treated with 3-MA normalized to sham levels. In conclusion, these findings suggest that increases in autophagy following ethanol and burn injury may play a causative role in intestinal inflammation seen after these injuries. Support: R01AA015731, Dr. Ralph and Marian C. Falk Medical Research Trust.
LONG-TERM ACTIVATION OF THE ENDOPLASMIC RETICULUM STRESS FOLLOWING BURN INJURY
A.E. Offordile1, 2, A. El Ayadi1, 2, D.N. Herndon*2, S. Hegde2, A. Prasai1, 2, E.C. Diaz1, 2, L.E. Sousse1, 2, M.G. Jeschke*3, C.C. Finnerty*1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospitals for Children, Galveston, TX, 3Sunnybrook Health Sciences Center, Toronto, ON, Canada
Objectives: We have previously shown that endoplasmic reticulum(ER) stress and the unfolded protein response (UPR) pathways are activated in the liver following burn injury. Burn injury induces apoptosis in the liver leading to hepatic dysfunction. Depending on the magnitude of the injury, the UPR pathway can be activated in a time dependent manner leading to nuclear transcription of ER chaperones. In this study, we determined the time course of activation of various markers of UPR following burn injury.
Methods: Rats received 60% of total body surface area scald burn and were sacrificed at 2, 7, 14 or 28 days post burn. Liver ER stress markers were measured at the protein and mRNA levels.
Results: Burn injury increased the luminal ER stress marker calreticulin at 2 days, 2 weeks and 4 weeks post burn. Calnexin levels were significantly increased at 2 weeks post burn. The glucose regulated protein 78 (GRP78 or BIP) is one of the best characterized ER chaperones that interacts with the hydrophobic domains of a wide range of proteins. The GRP78 levels significantly increased 2 days post burn before returning to baseline levels at 28 days post burn. The protein disulfide isomerase (PDI), involved in folding and unfolding of proteins within the ER, only increased 28 days following burn. The effects of burn injury on other markers of ER stress (the pro-apoptotic protein CHOP, the transcription factor XBP1 (Xbox binding protein) and the ER stress gene Dnajb) occurred soon after injury. Expression of the 3 markers was decreased only at one week post burn. Summary: Overall, our data suggest that burn injury activates different arms of the UPR pathway in a time dependent manner. This activation persists longer than previously thought and is characterized by increasing the levels of various ER chaperones as an adaptive mechanism under high stress conditions.
Conclusion: Establishing the time course for burn-induced ER stress will enable a better understanding of post-burn hepatic dysfunction and insulin resistance.
CXCR4 ANTAGONISM PLERIXAFOR IMPROVES SURVIVAL AFTER POLYMICROBIAL SEPSIS
A. Hadley, M. Serbanescu, B.P. Yoseph, R. Mittal, C. Coopersmith*, K. McConnell*. Emory University School of Medicine, Atlanta, GA
Introduction: The chemokine CXCL12 (Stromal cell derived factor 1a, SDF-1a) regulates retention of hematopoietic progenitor cells (HPCs) to the bone marrow via its interaction with receptor CXCR4. Inflammation causes alterations of the CXCL12-CXCR4 gradient, and increased secretion of SDF1a from inflamed tissues and subsequent release of HPCs and neutrophils from bone marrow into the peripheral circulation and shifts on the cytokine milieu. The CXCR4 antagonist, Plerixafor (AMD3100), is approved for mobilization of stem cells to peripheral blood autologous stem-cell transplantation, but its effect in the setting of inflammation is controversial.
Methods: C57BL/6 mice underwent 2X25 cecal ligation and puncture and received either intraperitoneal injection of Plerixafor or normal saline following injury. Mice were followed for seven days for survival or sacrificed at 6 hours after CLP for cytokine analysis using a 23-plex assay. Data were compared using Log Rank and Mann-Whitney tests respectively.
Results: Plerixafor decreased 7-day mortality from 86% to 43% (n=21/group, p=0.004). There were no differences between the two groups in the cytokine levels of: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL12(p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α (n=6-8/group).
Conclusions: Plerixafor improved survival following polymicrobial sepsis. Surprisingly, this was not associated with any changes in cytokine levels despite the marked survival benefit conferred by Plerixafor. We speculate that the benefit may be due to immune cell trafficking although further mechanistic studies are required to determine why antagonizing CXCR4 improves outcome in a model of murine sepsis.
A NOVEL P2RX7 SNP HAPLOTYPE EXPLAINS ATP-INDUCIBLE ANTI-APOPTOSIS IN ANTIGEN-PRESENTING CELLS OF AUTOINFLAMMATION AND SEPTIC SHOCK
S. Dagenbach1, A. Altmann1, H. Hohmann1, K. Foehr1, M. Weiss2, U. Brueckner*3, E. Schneider1. 1Sektion Experimentelle Anaesthesiologie, University of Ulm, Ulm, Germany, 2Dept Anesthesiology, University of Ulm, Ulm, Germany, 3University of Heidelberg, Heidelberg, Germany
The purinergic receptor P2RX7 is an important element of the inflammasome, triggered by extracellular ATP. Methods: We enriched in vivo activated antigen presenting cells (APC) by cell culture from patients with autoinflammatory diseases and at the onset of septic shock, and tested P2RX7 expression, cytokine release and ion flux after pyrosequencing of functional polymorphisms (SNPs). Results: Phenotypically, the P2RX7 ion channel activity is >10 times more active in such cultured APC than in monocyte derived immature or mature dendritic cells. Haplotypes bearing the functional SNP489 C>T and SNP 1513 A>C were more frequent in the patients’ population as compared to controls. ATP stimulated IL-1β secretion was less in the mutated genotype than in wildtype (wt) and the same was true for the ATP-induced ion flux signal (p=0.0004). When ATP was co-administered with cyclosporin A (CsA), the ion flux of SNP489/1513 mutated cells was stimulated (p=0.03) but never reached the activity of wt cells. Remarkably, ATP plus CsA led to increased IL-1RA secretion, not seen in wt cells. Although the ATP response gave rise to cell blebbing, caspase-3/7 activity was not upregulated but rather decreased when compared to baseline. Beyond that, ATP and CsA showed a synergistic anti-apoptotic effect in the presence of staurosporine (STS) and STS-induced caspase-3/7 was significantly less in mutated isolates (p<0.002). Summary: The precursor frequencies of in vivo activated APC in patients with autoinflammatory diseases including septic shock is high. A novel P2RX7 SNP haplotype found more frequently in patients, displays a reduced ion channel activity, a reduced capacity to secrete IL-1β and a higher anti-apoptotic effect by exogenous ATP. Conclusion: P2RX7 SNP combinations play a major role in immune control and apoptosis of in vivo activated APC in autoinflammation.
NEUTROPHIL EXTRACELLULAR TRAPS (NETS) AS A DIAGNOSTIC MARKER FOR MAJOR POSTBURN MESENTERIC INFLAMMATION: INHIBITION BY MELATONIN AND SIMVASTATIN
M. Kim, A. Ali, N. Fazal, A. Azim, W.M. Al-Ghoul*. Chicago State University, Chicago, IL
NETs are thought to leak from inflamed tissues to other body compartments and are therefore potential systemic inflammation diagnostic marker (Shock 30(4):352-8, 2008). Here, we used parallel microscopy, flow cytometry, and in vitro methods to compare picogreen, DHR123 as novel NETosis markers to traditional tissue inflammation markers in a murine model of major thermal injury (TI). The methods were then applied to assess the novel anti-inflammatory actions of melatonin (Mel) and simvastatin (SMV). Specifically, we tested 4 groups of 8-week-old male Balb/c mice: sham, TI, TI+Mel, TI+SMV. Thermal injury was 9-sec hot water dorsal scald (30% TBSA; 95 deg C) and Mel and SMV were injected twice at 1.86 or 0.5 mg/kg, IP doses, respectively, based on our IACUC-approved protocols. Injections were made at 0- and 18-hrs and sacrifice was at 24-hrs postburn. Inflammation assessment was performed on systemic plasma, peritoneal lavage, ileum and colon homogenates, and cryostat sections. The figure shows NETosis levels in ileum which typifies all the body fluids we tested, thus confirming that NETosis significantly pervades all body fluids compartments and that melatonin and simvastatin significantly suppress postburn inflammation in all these compartments (p<0.05). (Supported by NIGMS-MBRS-SCORE grant# 1SC3GM099632-01, to WMA).
UBIQUITIN ACTS CHEMOTACTIC ON EPC BUT PROBABLY NOT ON MSC PRECURSORS
J. Marzi, D. Henrich, J. Schultheiss, I. Marzi*. Department of Trauma Surgery, University of Frankfurt, Frankfurt, Germany
Introduction/Aim: After trauma, Ubiquitin is being released from necrotic cells and it’s serum concentration is significantly increased. It has been shown recently that this highly conserved protein binds to the chemokine receptor CXCR4 and triggers a chemotactic response. The latter, however is about 50% of the natural CXCR4-ligand SDF-1a. CXCR4 is expressed on cells involved in regenerative processes such as endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC). We assume that extracellularly released Ubiquitin provides an early chemotactic signal for EPC and MSC which helps to increase their concentration in the injured tissues. Therefore, the aim of this study was to evaluate the chemotactic effect of increasing concentrations of Ubiquitin on EPC and MSC in vitro.
Methods: Ubiqitin-binding to CXCR4 was qualitatively demonstrated by the co-incubation of EPC with FITC conjugated Ubiquitin and the specific CXCR4 blocker AMD3100 followed by a FACS-analysis. Chemotaxis-assay: EPC (5x10∧4/well) differentiated from peripheral blood mononucleated cells, respectively bone marrow mononucleated cells (2x10∧5/well) were placed in the upper chamber of a modified Boyden Chamber (pore size: 8 μm). The lower chamber contained either Ubiqitin [1, 10, 100, 1000 ng/mL], SDF-1 [10 ng/mL] or medium [control]. The number of migrated EPC was assessed by counting five randomized high power fields/well [100 x], the number of migrated MSC precursor was evaluated by a subsequent CFU-F-assay. Results were presented as median values, a p< 0.05 is significant (Kruskal-Wallis).
Results: A CXCR4 specific binding of Ubiquitin-FITC on EPC was observed. Coherently, we found a dose dependent, bell shaped chemotactic effect of Ubiqitin on EPC with an optimum at 100 ng/mL [95 cells/field 100 ng/mL vs 60 cell/field control]. The chemotactic response of Ubiquitin was slightly decreased in comparison to an equal concentration of SDF-1a [Ubiquitin 10ng/mL 80 cells/field vs SDF-1a 10ng/mL 77 cells/field]. Neither Ubiquitin nor SDF-1 exert a significant chemotactic effect on MSC precursors.
Conclusion: Our study demonstrated the binding of Ubiquitin to the CXCR4 and a significant chemotactic effect of Ubiquitin on EPC. The latter suggest that Ubiquitin might serve as early chemotactic signal for EPC. However, its significance in vivo must be proofed in further studies.
EARLY ALTERATIONS IN IMMUNE-RELATED GENE EXPRESSION IN TRAUMA PATIENTS THAT DEVELOP GRAM-NEGATIVE BACTEREMIA
C.M. Thompson1, C.H. Park2, R.V. Maier*1, G.E. O’Keefe1. 1University of Washington, Seattle, WA, 2Brookdale University and Medical Center, Brooklyn, NY
Study Objective: Using clinical and genomic data from the “Inflammation and the Host Response to Injury Large-Scale Collaborative Research Program”, we sought to determine if there were differences in early post-injury immune-related gene expression in subjects that developed gram negative bacteremia (GNB) compared to those subjects that did not (non-GNB).
Methods: After Institutional Review Board approval, we accessed the “Inflammation and the Host Response to Injury Large-Scale Collaborative Research Program” database and downloaded clinical and genomic data for all trauma patients with total leukocyte (buffy coat) microarray data at 12 hours (hrs) and 96 hrs after injury. We then normalized the data and corrected for batch effect. To reduce the heterogeneity of the non-GNB subjects, we derived a propensity score for the development of GNB based on each subject’s presenting clinical characteristics and matched each GNB subject to 2-3 non-GNB subjects. We then analyzed the GNB subjects for differential gene expression compared to non-GNB subjects using GenePattern (Broad Institute, MIT). Gene expression values are reported as mean± standard deviation.
Results: One hundred and twenty one subjects had total leukocyte microarray data at 12 and 96 hrs after injury. Ten of those subjects developed GNB during their hospital course; they were matched to 26 non-GNB subjects. At 12hrs after injury, 64 probes had differential gene expression of ≥ 1.5 fold between the two groups. At that time point, none of the 64 probes represented genes that are directly known to be related to either the innate or adaptive immune system. At 96hrs, 102 probes had differential expression of ≥1.5 fold. From that group, 21 probes representing 18 different innate or adaptive immunity genes were differentially expressed. We saw a downregulation of pro-inflammatory innate immune system genes in the GNBs vs. non-GNBs including interleukin-1 beta (785.8±439 vs. 1362.7±532; P=0.004). We also saw downregulation of genes related to the adaptive immune system in the GNB subjects including Major Histocompatibility Complex, class II, DR beta (859.4±414 vs.1459.5±626; P=0.006).
Conclusion: Based on differential gene expression, there is evidence of early suppression of both the innate and adaptive immune system in trauma patients who go on to develop gram-negative bacteremia.
LIPOXIN A4 PROMOTES MORE COMPLETE INFLAMMATION RESOLUTION IN SEPSIS COMPARED TO STABLE LIPOXIN A4 ANALOG
B. Wu, J. Walker, B. Spur, A. Rodriguez, D. Temmermand, K. Mian, P. Stein, P. Banerjee, K. Yin*. UMDNJ School of Medicine, Stratford, NJ
In sepsis, an appropriate inflammatory response is essential to inhibit bacterial spread. A sustained inflammatory response however, can lead to tissue injury or cause immunosuppression with increased susceptibility to infection. Resolution of inflammation ensures that excessive inflammation or bacterial spread does not occur. We have previously shown that Lipoxin A4 (LXA4), one of a family of endogenously produced compounds with novel pro-resolution activity, reduced systemic inflammation and bacterial load in sepsis. In this study we examined if LXA4 stable analog could provide greater benefit in sepsis. CLP rats were given either vehicle saline, LXA4 (7 μg/kg, i.v) or 16 (parafluoromethoxy)-lipoxin A4 (LXA4 analog; 7 μg/kg, i.v.) 1h after surgery. Only LXA4 increased 8 day survival. Using LC-MS/MS, levels of LXA4 analog were detected in plasma 24h after CLP. Levels of LXA4 could not consistently be detected and were similar in rats given LXA4 and those given saline vehicle. Both LXA4 compounds reduced plasma TNFα and IL-6. Neither treatment altered plasma IL-10 compared to CLP alone, but LXA4 analog, increased IL-10 by 100% compared to rats given LXA4 (P < 0.05). LXA4 reduced blood bacterial load but LXA4 analog did not. To examine the possible mechanisms for the differences, we investigated macrophage phagocytic ability and leukocyte gene expression of inflammatory mediators of cells taken from CLP rats given saline, LXA4 or LXA4 analog as described above. Only LXA4 increased phagocytic rate of peritoneal macrophages. Using relative qPCR, we found that LXA4 reduced neutrophil gene expression of iNOS by 5-fold compared to CLP rats given vehicle (P < 0.05), while LXA4 analog did not. Although both LXA4 compounds reduced macrophage gene expression of iNOS by 24h, LXA4 analog transiently increased iNOS expression at 3h. Our results suggest that at doses that reduced systemic inflammation, only the shorter lived LXA4 could inhibit bacterial spread and increase survival. This difference may be due to the shorter-lived compound being able to increase macrophage phagoyctic activity and reduce neutrophil iNOS expression.
WHOLE BREATH ANALYSIS: A NOVEL APPROACH TO PROTECTIVE MECHANICAL VENTILATION
B. Emr1, L. Gatto2,1, S. Roy1, A. Ghosh1, J. Satalin1, K. Snyder1, B. Sadowitz1, M. Kollisch-Singule1, D. Wang1, D. Huang1, P. Andrews3, N. Habashi*3, W. Marx1, G. Nieman*1. 1SUNY Upstate, Syracuse, NY, 2SUNY Cortland, Cortland, NY, 3Shock Trauma, Baltimore, MD
Introduction: It has been demonstrated that inappropriate tidal volume (Vt), plateau pressure, or end expiratory pressure (PEEP) can cause a ventilator induced lung injury (VILI). However, there are many more components in the mechanical breath that may exacerbate VILI. We have identified 11-components that comprise the Mechanical Breath Profile (MBp) and hypothesize that a complex relationship exists within the MBp that plays a critical role in VILI development. In this study we analyzed all 11-breath components delivered with three different ventilator strategies in a Rat VILI model.
Methods: Rats (n=9) were anesthetized, surgically prepared, placed on mechanical ventilation and separated into groups: 1) HVt LP (Vt 10 cc/kg, PEEP 0.5 cmH2O), 2) LVt HP (Vt 6, PEEP 5), and 3) APRV (Airway Pressure Release Ventilation). All of the parameters comprising the MBp were measured for 6h: Time at Inspiration (TI), Pressure at Inspiration (PI), Time at Expiration (TE), Pressure at Expiration (PE), Transition Time from PE to PI (ΔTI), Transition Time from PI to PE (ΔTE), Respiratory Rate (RR), Vt, Functional Residual Capacity (FRC), Inspiratory Flow (Qi), and Expiratory Flow (QE). Longitudinal data analysis was done to correlate each component with PaO2/FiO2 ratio (P/F), which was used as marker of lung injury.
Results: APRV prevented lung injury (P/F: APRV 428.3±14.3*; LVtHP 291.0±12.2; HVtLP 228.8±72.6, *= p<0.05 vs. HVtLP). Six breath components correlated with improved P/F and lung protection (Table). The APRV breath had optimal values of ΔTI, QI, TI, TE, and Vt that correlated strongly with lung protection (Table). APRV also reduced histopathologic lung damage.
Conclusion: Preemptive application of APRV can prevent the development of lung injury. The mechanism of this protection was optimization of multiple MBp components (Table). These data demonstrate that the Time of airway pressure application (i.e. High TI and Low TE) is critical in lung protection.
EXOGENOUS T3 ADMINISTRATION PROVIDES NEUROPROTECTION IN A MURINE MODEL OF TRAUMATIC BRAIN INJURY
I. Paterniti*, R. Crupi*, M. Campolo, R. Di Paola, S. Cuzzocrea*, E. Esposito*. University of Messina, Messina, Italy
Traumatic brain injury (TBI) induces primary and secondary damage in both the endothelium and the brain parenchyma. While neurons die quickly by necrosis, a vicious cycle of secondary injury in endothelial cells exacerbates the initial injury. Thyroid hormones are reported to be decreased in patients with brain injury. Controlled cortical impact injury (CCI) is a widely used, clinically relevant model of TBI. Here, using CCI in adult male mice, we set to determine whether 3,5,3-triiodothyronine (T3) attenuates posttraumatic neurodegeneration and neuroinflammation in an experimental model of TBI. Treatment with T3 (1.2 μg/100 g body weight, i.p.) 1 h after TBI resulted in a significant improvement in motor and cognitive recovery after CCI, as well as in marked reduction of lesion volumes. Mouse model for brain injury showed reactive astrocytes with increased glial fibrillary acidic protein, and formation of inducible nitric oxide synthase (iNOS). Western blot analysis revealed the ability of T3 to reduce brain trauma through modulation of cytoplasmic-nuclear shuttling of nuclear factor-κB (NF-κB). Twenty-four hours after brain trauma, T3-treated mice also showed significantly lower number of TUNEL(+) apoptotic neurons and curtailed induction of Bax, compared to vehicle control. In addition, T3 significantly enhanced the post-TBI expression of the neuroprotective neurotrophins (BDNF and GDNF) compared to vehicle. Our data provide an additional mechanism for the anti-inflammatory effects of thyroid hormone with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.
MOLECULAR EVIDENCE FOR THE INVOLVEMENT OF PPAR-δ AND PPAR-γ IN ANTI-INFLAMMATORY AND NEUROPROTECTIVE ACTIVITIES OF PALMITOYLETHANOLAMIDE AFTER SPINAL CORD TRAUMA
S. Cuzzocrea*, I. Paterniti*, R. Crupi*, R. Morabito*, M. Campolo, E. Esposito*. University of Messina, Messina, Italy
Introduction: Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. Moreover, several data suggested that PEA reduced inflammation and tissue injury associated with spinal cord trauma and showed a regulatory role for Peroxisome Proliferator-Activated Receptor (PPAR)-α signaling in the neuroprotective effect of PEA. However, several other mechanisms could explain the anti-inflammatory and anti-hyperalgesic effects of PEA, including the activation of PPAR-δ and PPAR-γ. The aim of the present study was to carefully investigate the exact contribution of PPAR-δ and PPAR-γ in addition to the PPAR-α, in the protective effect of PEA on secondary inflammatory damage associated with an experimental model of spinal cord injury (SCI).
SCI was induced in mice through a spinal cord compression by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy, and PEA (10 mg/kg, i.p., 1 and 6 h after SCI) was injected to WT mice and to mice lacking PPAR-α (PPAR-α KO). In order to deepen the ability of specific PPAR-δ and PPAR-γ antagonists to reverse the effect of PEA, mice were administered GSK0660 or GW9662, 30 min before PEA injection.
Results: Genetic ablation of PPAR-α in mice exacerbated spinal cord damage, while PEA-induced neuroprotection seemed be abolished in PPARα KO mice. Twenty-four hours after spinal cord damage, immunohistological and biochemical studies were performed on spinal cord tissue. Our results indicated that PPAR-δ and PPAR-γ also mediated the protection induced by PEA. In particular, PEA was less effective in PPAR-α KO, in GSK- or GW9662-pretreated mice, as evaluated by degree of spinal cord inflammation and tissue injury, neutrophil infiltration, pro-inflammmatory cytokine, iNOS expression and motor function.
Conclusion: This study indicates that PPAR-δ and PPAR-γ can also contribute to the anti-inflammatory activity of PEA in SCI.
PI3K/NRF2/HO-1 SIGNALLING BY ASCORBIC ACID INCREASES SURVIVAL RATE IN ENDOTOXIC MICE BY REDUCING HMGB1
K. Chang. Gyeongsang National University, Jinju, Republic of Korea
Previously we reported that HO-1 inducers negatively regulates HMGB1 release in septic conditions. We hypothesized that ascorbic acid (AA) may induces HO-1 through PI3K/Nrf-2 signaling which inhibits HMGB1 in LPS-activated macrophage cells and increases survival of endotoxic mice. Results indicate that AA increased HO-1 protein in a concentration- and time-dependent manner in RAW264.7 cells. AA-induced HO-1 expression was significantly diminished by LY294002 or Nrf-2 si-RNA-transfected cells. Mutation of keap1 of cystein to serine at 151, 273 and 283 significantly reduced HO-1 expression by AA. AA-induced reduction of HMGB1 release in LPS-activated RAW 264.7 cells was also significantly reversed by either LY294002 or transfection with mutated Keap1 or HbO2. In addition, CORM-2 significantly reduced HMGB1 release in LPS-activated cells, indicating CO, one of HO-1 products, may be responsible for this action. AA reduced LPS-activated NF-kB luciferase activity. Importantly, injection of AA improved survival rate in endotoxic mice which was significantly inhibited by the presence of ZnPPIX. Plasma level of HMGB1 was significantly elevated in endotoxic mice but AA reduced it in a ZnPPIX-sensitive manner. Significant increase of HO-1 protein was observed in heart, lung and liver tissues of endotoxic mice injected with AA compared to AA+ZnPPIX+LPS animals. Taken together, we concluded that PI3k/Nrf-2/HO-1 signaling pathway by AA plays a critical role for reduction of HMGB1 and increase of survival in endotoxic mice.
ANTICOAGULANT-FREE RENAL REPLACEMENT THERAPY IN AKI PATIENTS WITH BLEEDING COMPLICATIONS
N. Kuriyama1, O. Nishida1, Y. Hara1, T. Nakamura1, M. Suga1, Y. Shimomura1, M. Akiyama1, M. Noda1, S. Komatsu1, T. Miyasho2. 1Department of Anesthesiology & Critical Care Medicine, Fujita Health University School of Medicine, Toyoake Aichi, Japan, 2Department of Veterinary Science, Rakuno Gakuen University, Ebetsu, Japan
Introduction: In recent years, renal replacement therapy (RRT) has become an important tool in ICU. However, for bleeding patients, anticoagulant-free RRT seems to be essential. Toraylight NV (NV) (Toray, Japan) is a newly developed hydrophilic polysulfone polymer with higher mobility to absorbed water. Due to the antithrombogenicity, it is expected to reduce anticoagulant use during RRT.
Hypothesis: For AKI patients with bleeding complications, NV enables anticoagulant-free or reduced RRT.
Methods: AKI patients with bleeding episodes or concerns who had started RRT with NV were enrolled between January and July 2012. This study was approved by the Ethics Committee. The hemorrhagic volume, anticoagulant dosage, achievement rate of the duration, results of coagulation tests and JAAM (The Japanese Association for Acute Medicine) DIC score were recorded. Cytokine production when singly passing through a filter was evaluated.
Results: Ninety-three sessions (SLEDD 37, CHF 56) were conducted in 16 patients (9 with bleeding episodes and 7 with a bleeding risk). Sixty-six sessions (71%) were safely performed with no anticoagulant. Twenty-seven sessions (29%) required nafamostat mesilate (Torii Pharmaceutical, Japan), a serine protease inhibitor widely used in Japan, at a rate of 8.3±9.0 mg/hr whereas the normal usage is 30.0 mg/hr. The achievement rate of the scheduled duration was 7.5±3.0 hours (92% accounted for the target duration) in the SLEDD group and 20.1±5.6 hours (82%) in the CHF. Eighty sessions (86%) continued successfully with no clotting of the filter. At baseline, there were no significant differences in the JAAM DIC score, platelet counts, TAT and FDP between bleeding episode and bleeding risk groups. There were no significant changes in these parameters before and 24 hours after the initiation in each group. Additionally, there were no significant changes in the blood cytokine level before and after the filter. No new episode or exacerbation of bleeding was observed in any patient.
Conclusions: NV was concluded to be useful in patients with AKI with bleeding complications. Moreover excellent biocompatibility, in terms of cytokine activation, was observed even under the condition of anticoagulant-free RRT.
RECOVERY FROM SEPSIS-INDUCED IMMUNOSUPPRESSION USING HEMOPERFUSION WITH POLYMYXIN B-IMMOBILIZED FIBERS
A. Kimura1, S. Ono1, 2, M. Kinoshita1, 3, H. Tsujimoto1, S. Hiraki1, R. Takahata1, S. Aosasa1, D. Saitoh1, 2, K. Hase1, J. Yamamoto1. 1National Defense Medical College, Tokorozawa, Japan, 2Division of Traumatology, National Defense Medical College, Tokorozawa, Japan, 3Department of Immunology and Microbiology, National Defense Medical College, Tokorozawa, Japan
Background: Although sepsis-induced immunosuppression has long been considered to be a factor in the late mortality of patients with sepsis, little is known about cellular immunity and the effect of polymyxin B-immobilized fiber hemoperfusion (PMX-F) therapy on sepsis-induced immunosuppression. The present study was designed to evaluate the effect of PMX-F therapy on the recovery from sepsis-induced immunosuppression.
Methods: Septic patients who had an identified focus of infection were enrolled in this study. 1. Peripheral blood mononuclear cells (PBMC) in the septic patients were examined to evaluate the inflammatory cytokine production before, immediately after and 24 hours after PMX-F therapy. Obtained PBMC were stimulated with anti-CD3 antibody or with interleukin (IL)-2 and IL-12 or with IL-2 and IL-12 and IL-15 for 24 hours. The culture supernatants were measured for IFN-γ, TNFα production by ELISA. 2. Whole blood obtained from septic patients were incubated with polymyxin B-immobilized filter for small animals for 2 hours (PMX group), or were treated with 200μg of polymyxin B for 2 hours (PLB group), or were not treated (non-treated group). IFN-γ production of PBMC was compared among groups.
Results: 1. IFN-γ and TNFα production of PBMC were significantly lower in septic patients compared to healthy volunteers. There was a tendency to improve IFN-γ and TNFα production 24 hours after PMX-F therapy. Additional IL-15 stimulation to PBMC improved IFN-γ production in septic patients. 2. IFN-γ production of PBMC in septic patients was significantly improved both in PMX and PLB group compared to non-treated group.
Conclusions: PMX-F therapy improved IFN-γ and TNFα production of PBMC in sepsis. Polymyxin B may up-regulate immune response by induction of IFN-γ production in septic conditions.
THE INTERACTION OF STAGE OF ESTROUS AND BUPRENORPHINE TREATMENT IMPACTS SEPSIS SURVIVAL IN FEMALE MICE
L. Kennedy, H. Hwang, J. Nemzek-Hamlin*. University of Michigan, Ann Arbor, MI
The impact of opioids on the immunopathology of sepsis models in mice has been controversial. In previous work, our laboratory showed no differences in mortality or inflammatory parameters between groups of female mice given saline or buprenorphine. However, the estrous cycle was not considered. To further investigate, we hypothesized that buprenorphine use would not impact sepsis outcomes at any stage of estrous. Vaginal cytology was performed and females were placed into four groups based on stage of estrous. Each group then underwent CLP and received either buprenorphine or saline. Three-week survival studies were performed (n=20 per stage of estrous). Survival did not differ between buprenorphine and saline treated female mice overall. When stratified by the stage of estrous, the survival among the saline groups did not vary. Among the buprenorphine treated mice, those in metestrus had a significantly lower survival than mice in estrus or proestrus. To investigate inflammation as a potential mechanism for survival, mice were euthanized at 12 or 24 hours after CLP. Cell counts and cytokines were measured in the peripheral blood and peritoneal lavage fluid. Mice in proestrus that received buprenorphine had significantly more circulating neutrophils and monocytes than mice in proestrus that did not receive buprenorphine. No other differences between groups based on analgesic use or stage of estrous were seen. The results of this study suggest that the effects of buprenorphine on a 50% survival model of sepsis in BALB/c female may appear minimal overall, however, there are differential effects on the subjects based on stage of estrous. Investigators should consider the effects of buprenorphine and estrous cycle when using female mice in sepsis research.
INCREASED CIRCULATING MYELOID DERIVED SUPPRESSOR CELLS AND PICS IN SEVERE TRAUMA AND SEPSIS
E. Vanzant, L.F. Gentile, J. Lanz, R. Davis, D. Nacionales, R. Ungaro, A. Cuenca, L. Moldawer*, A. Bihorac*, B.A. McKinley, F.A. Moore*, P.A. Efron*. University of Florida, Gainesville, FL
Introduction: Many patients who survive severe trauma (ST) or sepsis (SS) develop chronic, critical illness that we have termed PICS (persistent inflammatory/immunosuppressive catabolic syndrome) (Gentile et al. J. Trauma, 2012). We hypothesize that PICS is associated with increased circulating myeloid derived suppressor cells (MDSCs) and that this increase contributes to inflammation, immunosuppression and catabolism. Yet, MDSCs have never been identified in the blood of SS or ST patients.
Methods: Using standard definitions of SS and septic shock, 38 SS patients were enrolled within 12 h of recognition. 10 Non-TBI trauma patients with hemorrhagic shock (ST) defined as a base deficit > 6 or SBP < 90 mmHg with transfusion requirements within 6 h of injury were studied within 12 h of admission. Blood samples were drawn <12h and 1, 2, 4, 7, 14, 21 and 28 d after onset of sepsis or injury. Blood from 4 controls were used for comparison. MDSCs were identified as CD33+CD11b+HLA-DR-/lowCD124+ leukocytes. The distribution of monocytic (CD14+) MDSCs (M-MDSCs), and granulocytic (CD15+) (G-MDSCs) were determined by flow cytometry.
Results: In both ST and SS, the absolute and relative numbers of total MDSCs increased at all-time points (p<0.05), except day 21 in sepsis, peaking in the 1st 24 hours. Surprisingly, the ratio of G-MDSCs to M-MDSCs also markedly increased in both populations, and remained elevated through day 28. Both SS and ST patients had persistent inflammation throughout their hospitalization, indicated by elevated CRP concentrations (SS 148 + 87; ST 143 ± 101 mg/L) and elevated total white blood cell counts. They also exhibited lymphopenia and decreased pre albumin concentrations (SS 10.5 + 6; ST 11 ± 6 mg/dl). 15% of the SS patients and 80% of the trauma patients were discharged home.
Conclusions: Both SS and ST are associated with an early and comparable expansion of immature circulating myeloid cells consistent with MDSCs(CD33+CD124+). These patients also exhibit persistent inflammation, immunosuppression and protein depletion. Findings suggest that expansion of MDSC populations is a common feature of severe stress, whether produced by microbial invasion or tissue injury. It appears that this expansion is geared toward increased immunosuppressive phenotypes indicated by increased G/M-MDSC ratio. We believe this expansion contributes to the development of PICS.
VENTILATION STRATEGY ALTERS GAS DISTRIBUTION AND STRAIN IN THE PULMONARY MICRO-ANATOMY
A. Ghosh1, B. Emr1, L. Gatto2, S. Roy1, P. Andrews3, N. Habashi*3, K. Snyder1, J. Satalin1, S. Landas1, W. Marx1, G. Nieman*1. 1SUNY Upstate, Syracuse, NY, 2SUNY Cortland, Cortland, NY, 3Shock Trauma Center, Baltimore, MD
Introduction: Mechanical ventilation can exacerbate lung injury caused by Acute Respiratory Distress Syndrome (ARDS) leading to a secondary Ventilator Induced Lung Injury (VILI). Whole-lung strain has been used as an indicator of VILI (Gattinoni L et al Crit Care Med 2010). In this study we used a novel method of measuring the change in size of alveoli (Alv%), respiratory bronchioles (RB%) and parenchymal tissue (PT%), expressed as a percent of the microscopic field, and calculated the Micro-Strain (Micro-S) on these anatomical structures in a Rat ARDS model during different ventilation strategies.
Methods: ARDS was induced in Rats by Tween lavage and separated into 3 groups: 1) Airway Pressure Release Ventilation (APRV) 2) Controlled Mechanical Ventilation (CMV) with a tidal volume (Vt) of 6mL/kg plus PEEP of 5 cmH2O (CMV+5PEEP); and 3) (CMV+16PEEP). The Control group were spontaneously breathing normal rats (SB). Lungs were fixed at peak inspiration (PI) and end expiration (EE). Alv%, RB%, and PT% were measured using digital image analysis. Micro-S was calculated using the definition: strain (e) is equal to the change in length (dL) divided by the original length (L), such that: e = dL/L.
Results: There was no statistical difference in Alv% at PI or EE between the APRV and SBV groups, while Alv% was significantly lower in both CMV groups. There was no significant difference in RB% area between groups. The PT% at EE was significantly lower with APRV. In addition, the pattern of Micro-S was similar between the APRV and SB groups (i.e. less strain in the alveolus, more strain in the ducts), which was reversed in both CMV groups (Table).
Conclusion: Our results demonstrate that gas volume is distributed differently to the alveoli and respiratory bronchioles depending on the ventilator strategy, resulting in different levels of Micro-S in each anatomical component. We postulate that APRV would cause less tissue damage since the Micro-S in both compartments is similar to that in a normal spontaneously breathing lung.
REACTIVE OXYGEN SPECIES RELEASED FROM MITOCHONDRIA REGULATE INFLAMMATORY RESPONSE IN HEPATOCYTES
A.V. Kozlov*1, J. Paier-Pourani1, J. Duvigneau*2, A. Weidinger1, H. Redl*1. 1L. Boltzmann Institute for Traumatology in AUVA Research Center, Vienna, Austria, 2University of Veterinary Medicine, Vienna, Austria
Mitochondrial reactive oxygen species (mROS) considered for a long time as byproducts of mitochondrial respiration have recently been discovered as an important component of signal transduction pathways, particularly those involved in inflammation. These pathways not only regulate innate immunity, but may also cause cellular dysfunction and organ failure upon overwhelming inflammatory response. In this study we determined mROS by means of electron spin resonance and laser scan microscopy in models of systemic inflammatory response (SIR). We show that SIR not only elevates mROS generation but also enabled mROS to diffuse out of mitochondria. This was shown in mitochondria isolated from livers of rats treated with LPS and in hepatocyte culture treated with inflammatory mediators synthesized by white blood cells incubated with LPS. These data were obtained using a combination of permeable versus non-permeable ROS sensitive spin probes and mitochondria targeted versus not targeted ROS sensitive fluorescent dyes. mROS diffusion modulated transcription of inflammatory specific genes, especially iNOS and IL-6. This was confirmed by testing effects of mitoTEMPO, a mitochondria-targeted ROS scavenger. These results show that the diffusion of ROS out of mitochondria may be an important step in the development of SIR.
A PROCESSED CHEMOTACTIC PEPTIDE FROM THE ECRG4 PRECURSOR POLYPEPTIDE INTERACTS WITH AND INTERNALIZES THROUGH THE INNATE IMMUNITY RECEPTOR
S. Podvin, X. Dang, T.W. Costantini*, B. Eliceiri*, A. Baird*, R. Coimbra*. University of California San Diego, San Diego, CA
Background: The c2orf40 gene encodes Ecrg4, a 17,000 Dalton precursor protein that modulates the course of response to infection and injury when over expressed in epithelial cells. Because of its presence on the cell surface of quiescent leukocytes, and its processing, release and ability to activate the pro-inflammatory transcription factor NF-κB after stimulation, we hypothesized that a 16 amino acid cytokine-like peptide derived by thrombin processing (Ecrg4(133-148)) could interact with the TLR innate immunity receptor complex (IIR-C).
Methods: Confocal microscopy, FACS and co-immunoprecipitation were used to show a physical association between Ecrg4 and IIR-C, specifically TLR4, CD14 and MD2, on the cell surface of leukocytes. A functional association between IIR-C and Ecrg4 was established using a quantitative ligand-targeted phage assay that demonstrated internalization of the peptide Ecrg4(133-148) through HEK cells when transfected with IIR-C but not in wild type or TNFR/IL-1βR expressing HEK cells. An NF-κB-dependent IIR-C reporter system was used to measure activation of by the Ecrg4(133-148) peptide.
Results: FACS analyses and confocal microscopy co-localize three protein components of IIR-C (TLR4, CD14, and MD2) with Ecrg4 immunoreactivity on the cell surface of leukocytes. Co-immunoprecipitation of Ecrg4 using antibodies to TLR4, CD14 and MD2 suggests that peptides containing the Ecrg4(133-148) epitope interact directly with the IIR-C. Finally, when Ecrg4(133-148) is displayed on phage, the particles acquire the ability to internalize into HEK cells that are engineered to overexpress IIR-C. This same particle fails to internalize into parental or TNFR/IL-1βR over-expressing control HEK cells. Remarkably, the internalizing Ecrg4(133-148) peptide does not activate the NF-κB-dependent reporter gene in these IIR-C signaling cells or modulate their response to LPS.
Conclusion: Biochemical and ligand phage targeting point to a functional interaction between Ecrg4 and IIR-C. The inability of Ecrg4(133-148) to activate IIR-C reporter HEK cells suggests that it may modulate NF-κB by the activation of a non-canonical inflammatory pathway involving RelB. The identification of these targets will help elucidate the molecular mechanisms through which Ecrg4 modulates the inflammatory response to injury and infection.
EXTRACORPOREAL TREATMENT FOR SEPSIS
R. Tullis*, R. Lefkowitz, R.P. Duffin, R. Lucas. Aethlon Medical, San Diego, CA
Aethlon is developing an extracorporeal treatment for sepsis in wounded Warfighters. We have developed a multivalent affinity plasmapheresis system to capture sepsis factors, such as sepsis-associated viruses (e.g. CMV). In clinical studies, HIV and HCV have been removed from the blood using Galanthus nivalis agglutinin (GNA) coupled to diatmite and placed in a plasma separator. Infected blood is pumped over the cartridge to remove the toxins and returned to the patient. GNA is selective for alpha-1,3 linked dimannosides and used for capture of many enveloped viruses including: Dengue, Orthopox viruses and other bioweapons threats. GNA also captures immunosuppressive exosomes expressed by solid tumors and in sepsis. In sepsis, our clearance efforts concern mainly INOS-containing, platelet-derived exosomes. For other targets we use specific antibodies for capture. Targets here include complement component C5 (KD∼10-10 M), and E. coli DNA (KD ∼10-7). For capture of proteolytic enzymes we have used protease inhibitors such as alpha-1-antitrypsin all with a KD less than 1 μM. As a separate project Aethlon is develop a blood handling system that can be used without the addition of anticoagulants for patients with severe bleeding who cannot tolerate anticoagulants. In conclusion, we have demonstrated successful in vitro capture of numerous sepsis factors moving toward an animal trial for the utility of this novel, multivalent treatment for sepsis.
A NOVEL RESVERATROL ANALOGUE ATTENUATES RENAL INJURY AFTER ISCHEMIA-REPERFUSION
A. Khader1, W. Yang*1, 2, M. Kuncewitch1, J. Prince1, 2, P. Marambaud2, J. Nicastro1, G.F. Coppa1, P. Wang*1, 2. 1Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY
Introduction: Renal injury as a result of ischemia-reperfusion (I/R) is a clinical problem with no specific treatment. Resveratrol, a known antioxidant, is also a stimulator of AMP-activated protein kinase (AMPK), which is a key modulator of energy metabolism and mitochondrial biogenesis. Recently, we screened a library of synthetic resveratrol analogues and identified RSVA405 as a highly potent activator of AMPK in vitro. Thus, we hypothesized that RSVA405 could be renoprotective against I/R-induced damage.
Methods: Adult male rats were subjected to renal I/R through bilateral clamping of the renal pedicles for 60 min, followed by reperfusion. RSVA405 (3 mg/kg BW) or vehicle (10% DMSO and 33% Solutol in PBS) was administered by intraperitoneal injection 1 h prior to ischemia. Blood and renal tissues were collected 24 h after I/R for histological evaluation and various measurements. Mitochondrial mass was determined by mitochondrial DNA copy number using real time PCR. Protein and mRNA levels were determined by Western blotting and RT-PCR, respectively.
Results: Administration of RSVA405 significantly reduced serum levels of renal injury markers (AST and LDH) as well as improved renal function by lowering serum BUN and creatinine, compared to the vehicle group (Table). The histologic injury score was remarkably improved in the treatment group, in comparison to the vehicles (Table). I/R resulted in 46% reduction of both renal mitochondrial mass and ATP levels, while RSVA405 treatment restored mitochondrial mass and ATP to levels comparable to those in the sham group. RSVA405 also significantly reduced the production of nitrotyrosine protein by 28% as well as mRNA levels of TNF-α, IL-6 and IL-1β by 50%, 21% and 59% respectively, in the kidney after I/R.
Conclusions: RSVA405 increased mitochondrial biogenesis and enhanced energy metabolism as well as reduced the production of reactive nitrogen species and proinflammatory cytokines. We have identified RSVA 405 as a highly promising therapeutic agent against I/R induced kidney damage.
ANTI-INFLAMMATORY EFFECTS OF ETHYL PYRUVATE AND ALCOHOL IN A TIME COURSE
B. Relja, N. Omid, K. Wilhelm, A. Schaible, K. Jurida, M. Lehnert, I. Marzi*. University Hospital of the Goethe-University Frankfurt, Frankfurt am Main, Germany
Objective: Exaggerated inflammatory response after oxidative stress results in end organ injury. Previously, we demonstrated the anti-inflammatory and anti-oxidant effects of acute alcohol (EtOH) gavage upon H/R in vivo, Moreover, the “beneficial” effects of alcohol and mimicking effects of ethyl pyruvate (EtP) applied before oxidative stress were showed in our in vitro studies. Here, we compared the “therapeutic” potential of EtP and effects of EtOH application before and after inflammatory “hit” using an in vitro experimental model of acute inflammation.
Methods: Human pulmonary epithelial cell line A549 was stimulated with IL-6. Either before or after IL-6 stimulation the cells were incubated with various doses of EtP (2.5 and 10mM), sodium pyruvate (NaP, 10mM) or EtOH (85 and 170mM) for 1, 24 or 72h. IL-8 release, neutrophil adhesion to A549 and CD54 expression were evaluated for each time point.
Results: IL-6 stimulation induces a time and dose-dependent IL-8 release with a peak at 12 and 24h after stimulation with 100 ng/ml. Pre-treatment with EtOH or EtP for 1h before the inflammatory stimuli does not influence the IL-8 release significantly, whereas pre-incubation for 24 or 72h with EtOH or EtP significantly reduces the IL-8 release. Post-treatment of A549 with EtOH or EtP reduced IL-8 release at all time points. NaP exerts similar effects to EtP. Cytokine-induced adhesion of neutrophils to monolayers is significantly reduced after both, pre- and post-treatment with EtOH or EtP. Likewise, pre-treatment with EtOH or EtP reduced CD54 expression during the complete time course, whereas the post-treatment correlated with reduced CD54 expression only after 1 h incubation.
Conclusions: Exposure of pulmonary epithelial cells to EtOH, EtP or NaP inhibited the release of inflammation-regulating IL-8. Reduced CD54 expression was paralleled by reduced adhesiveness of neutrophils to pulmonary epithelial cells. These results confirm the anti-inflammatory potential of EtOH but also EtP applied before inflammatory stimuli. Moreover, due to beneficial effects gained also by treatment after the inflammatory “hit”, these findings suggest EtP as promising treatment option for acute pulmonary conditions.
ORGAN SPECIFIC MITOCHONDRIAL PROFILE IN HEMORRHAGIC SHOCK AND RESUSCITATION (DIPSTICK ASSAYS - A RAPID AND SIMPLE APPROACH)
P. Sharma, B. Benford, V. Makler, M. Bodo. Uniformed Services University of the Health Sciences, Bethesda, MD
Introduction: Hemorrhagic shock (HS) due to traumatic injury is the third leading cause of death and disabilities in the United States, and it accounts for almost 50% of the combat fatalities. Most of the potentially preventable deaths are caused by the progression of secondary cell injury cascades, deficient cellular energy metabolism due to damaged mitochondria, increased cell death and multiple organ failure (MOF). When and how mitochondria in vital organs get damaged in HS, and cause MOF is also entirely unknown due to the lack of non-invasive blood based biomarkers of mitochondrial damage. Our goal of this study is to determine the utility of a blood based dipstick test of mitochondrial damage as a biomarker of organ damage.
Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS-30 min, followed by resuscitation (60 min) and infusion of shed blood (60 min). Control groups were (1) sham, (2) HS without resuscitation. Animals were continuously monitored for physiological, hemodynamic, biochemical parameters, and organ dysfunctions. For dipstick tests PDH (pyruvate dehydrogenase complex) and Complex I (first enzyme of the mitochondrial electron transport chain) were performed in blood collected at 0, 30, 90 and 150 min. Lysates from liver, brain and kidneys were also subjected to mitochondrial dipstick test for PDH and Complex I according to our published method.
Results: 1). Hemorrhage alone did not affect mitochondrial PDH or complex I enzyme contents. Most of the changes in these enzymes were caused by resuscitation and/or by blood reperfusion, 2). Serum deficiency of PDH and Complex I may indicate compromised mitochondrial functions in brain and 3). Mild PDH (approximately 40% of baseline) deficiency in blood may indicate compromised mitochondrial functions in liver.
Conclusions: Mitochondrial blood based dipstick test can be used as a biomarker of early metabolic failure in vital organs.
TRAUMA-HEMORRHAGE-INDUCED DECLINE IN SIRT1 ACTIVITY AND AUGMENTATION OF PDK1 LEVEL ARE REVERSED BY POST-TREATMENT WITH RESVERATROL
B. Jian, S. Yang, I.H. Chaudry*, R. Raju*. University of Alabama at Birmingham, Birmingham, AL
Severe hemorrhage leads to mitochondrial functional decline and multiple organ dysfunction. Previous studies have demonstrated a significantly decreased level of SIRT1 (silent mating type information regulation 2 homolog 1) in the heart following trauma-hemorrhage (T-H). SIRT1 is a deacetylase with cytoplasmic and nuclear distribution. SIRT1 is known to deacetylate a number of proteins involved in critical metabolic processes. It was also demonstrated that when rats subjected to T-H procedure were treated with resveratrol (RSV), a plant polyphenol and known activator of SIRT1, SIRT1 protein level in the heart was restored and left ventricular function improved. However it remained unclear if SIRT1 enzyme activity following T-H was altered and whether RSV treatment had any salutary effect on SIRT1 activity. In this study we determined the alteration of enzyme activity of SIRT1 and nuclear protein levels of SIRT1 and PGC-1α in the heart at 2 hours following T-H and resuscitation. In order to assess the shift in mitochondrial-glycolytic balance following T-H and SIRT1 regulation, PDK1 was quantified. Rats were subjected to T-H and resuscitation and a subset of the animals were administered either RSV or RSV and sirtinol during resuscitation. The activity of SIRT1 was determined by a fluorimetric assay and protein quantifications were done by Western blot. We found a significantly (p<0.05) decreased SIRT1 activity following T-H. The nuclear protein level of SIRT1 (p<0.05) and PGC-1α (<0.05) were also significantly decreased following T-H. PDK1 level was significantly elevated (p<0.05) in T-H group indicating suppression of mitochondrial oxidation. RSV treatment restored SIRT1 activity as well as the level of PDK1 and nuclear SIRT1 and PGC-1α. Sirtinol, an inhibitor of SIRT1, abolished the salutary effect of RSV. Based upon the data, we conclude that T-H not only resulted in decreased SIRT1 protein level, but also in decreased activity. RSV treatment enhanced SIRT1 activity and normalized PDK1 level possibly enhancing mitochondrial oxidation. Therefore, modulators of SIRT1 expression or activity may be important in devising therapeutic strategies for T-H induced injury.
TRAUMA PATIENT PLASMA INDUCES ENDOTHELIAL BARRIER INJURY AND IS ATTENUATED BY ACTIVATED PROTEIN C
B. Miyazawa, M.J. Cohen*. University of California San Francisco, San Francisco, CA
Background: In addition to its well-described anti-coagulant effect, activated Protein C (aPC) also has several cytoprotective functions that are still not well understood. Several studies have shown a beneficial effect of aPC on infection and organ injury, which can be attributed to the attenuation of endothelial barrier breakdown. Loss in endothelial barrier integrity is associated with the development of pulmonary edema, ALI, and infection; conditions frequently seen in trauma patients. We hypothesized that proteins associated with endothelial barrier breakdown would increase in patient plasma upon traumatic injury, and aPC will provide protection against this breakdown.
Methods: An electrical cell-substrate impedence sensing system (ECIS) was used to assess the permeability of monolayers of Human Umbilical Vein EC (HUVEC). A decrease in transendothelial electrical resistance indicates a loss in cell junction adherence, reflecting an increase in permeability. Plasma samples were prospectively collected from trauma patients upon arrival to an urban Level I trauma intensive care unit.
Results: Treatment of HUVEC monolayers with trauma patient plasma resulted in a maximal decrease in resistance of 43.3%. One hour pretreatment with aPC attenuated this decrease in resistance, resulting in a maximal decrease of only 7.5% (Fig 1). One hour pretreatment with the thrombin inhibitor hirudin also attenuated the decrease in resistance, resulting in a maximal decrease of 22% (Fig 1).
Conclusions: Trauma patient plasma does indeed disrupt endothelial barrier integrity, possibly in a thrombin dependent manner, which can be nearly completely protected by aPC. Further experiments are warranted and underway to determine what other proteins are responsible for this barrier breakdown, and to explore by which mechanism aPC attenuates this breakdown.
UPREGULATION OF GRAIL (GENE RELATED TO ANERGY IN LYMPHOCYTES) IS ASSOCIATED WITH UNRESPONSIVENESS OF CD4 T-CELLS IN SEPSIS
M. Aziz, W. Yang*, A. Jacob*, S. Matsuo, M. Zhou*, P. Wang*. Hofstra North Shore-LIJ School of Medicine and The Feinstein Institute For Medical Research, Manhasset, NY
Introduction: CD4 T-cells play an essential role in host defense against pathogens. The loss of CD4 T-cell populations and their functionality is frequently observed in sepsis; however, the mechanism remains elusive. GRAIL is a novel E3 ubiquitin ligase and is essential for the induction of T cell tolerance. We therefore aimed to examine the role of GRAIL in CD4 T-cells during sepsis.
Methods: Sepsis was initiated in 10-wk-old male C57BL/6 mice by cecal ligation and puncture (CLP). Thymocytes and splenocytes were isolated at different time points after CLP and subjected to flow cytometry to determine CD4 T-cell percentages. Apoptosis was determined by annexinV staining and caspase-3 activity was measured by fluorometric assay. CD4 T-cell proliferation assay was measured by carboxyfluorescein succinimidyl ester staining. GRAIL expression in splenocytes was measured by flow cytometry and Western blotting. Gene expression of Th1, Th2, and Th17 cytokines was determined by RT-PCR.
Results: As compared to shams, CD4 T-cells were significantly reduced in the thymus and spleen by 35% and 33%, respectively, at 20h after CLP (p<0.05). Apoptotic CD4 T-cells in the thymus and spleen at 20h after CLP were significantly higher than the sham by 18% and 6%, respectively, through the upregulation of caspase-3. CD4 T-cells isolated from septic mice were less responsive to anti-CD3/CD28- and IL-2-induced cell proliferation in comparison to the sham (proliferation index: 2.14 ± 0.04 vs. 1.42 ± 0.03; p<0.05). The induction of T-cell differentiation as determined by the expression levels of Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-10), and Th17 (IL-17A) cytokines was significantly reduced in splenocytes isolated from septic mice, compared to the sham, after 5 h-LPS stimulation. Correspondingly, GRAIL expression in splenocytic CD4 T-cells was significantly higher in septic mice than sham animals (mean fluorescence intensity: 532 ± 12.1 vs. 295 ± 7.5; p<0.05). Stimulation of splenocytic T-cells isolated from the sham mice with LPS significantly increased mRNA and protein levels of GRAIL.
Conclusion: Our findings reveal a novel association of GRAIL expression with CD4 T-cell unresponsiveness, thus implicating an emerging therapeutic target in sepsis.
THE INTRACELLULAR ROLE OF HEPATOCYTE HMGB1 IN STERILE INFLAMMATORY LIVER INJURY
H. Huang, K. McDonald, G.W. Nace, J.R. Klune, A. Tsung. Department of Surgery University of Pittsburgh Medical Center, Pittsburgh, PA
Purpose: High mobility group box-1 (HMGB1) is a chromatin associated nuclear protein. As damage associated molecular pattern (DAMP) molecule, it is secreted into the extracellular milieu as a signaling molecule during liver ischemia/reperfusion (I/R) and initiates pro-inflammatory cascades that lead to organ injury and dysfunction. Although the extracellular function of HMGB1 has been previously described, the intracellular role of HMGB1 during oxidative stress is not understood. We sought to determine the function of intracellular HMGB1 in hepatocytes, the major cellular source of HMGB1, following ischemic conditions.
Methods: Hepatocyte specific HMGB1 knockout (HMGB1-HC KO) and Flox control mice were subjected to a segmental (70%) warm hepatic I/R. Liver damage was assessed by ALT levels and histology. Protein, cytokines, mRNA, and innate immune cell population was evaluated.
Results: HMGB1-HC KO mice have markedly elevated serum transaminases as well as more necrosis in ischemia lobes compared to controls after warm I/R injury, indicating a greater degree of liver injury when HMGB1 in hepatocytes is absent. Hepatocytes from the ischemic lobe of HMGB1-HC KO mice demonstrate significantly more oxidative stress as measured by 4-hydroxynonenal (4-HNE) staining. HMGB1-HC KO mice also express lower levels of superoxide dismutases (SOD) 1 and SOD2 than controls after liver I/R. In vitro, after stimulation with hypoxia, loss of HMGB1 in hepatocytes leads to more mitochondrial instability compared to controls by exhibiting more mitochondria ROS production and damaged mitochondria. In addition, HMGB1-HC KO mice have less activation of autophagy, as demonstrated by decreased light chain 3B (LC3B) staining and protein levels and more p62, as compared to controls. These data imply that intracellular HMGB1 in hepatocytes can also contribute to the activation of autophagy.
Conclusion: Although released HMGB1 may function as a DAMP in ischemic liver injury, our study demonstrates the importance of intracellular HMGB1 in the response of hepatocytes to oxidative stress. Hepatocyte specific HMGB1 KO mice are more injured after liver I/R compared to controls through increased oxidative stress. The absence of HMGB1 in hepatocytes leads to diminished reactive oxygen species scavengers and autophagy which may exacerbate organ damage after liver I/R due to their cell survival roles under stress.
THE ESTROGEN RECEPTOR (ER)-β AGONIST DPN IS MORE EFFECTIVE THAN ER-α AGONIST PPT IN IMPROVING CARDIAC FUNCTION, CARDIAC ERS AND ESTROGEN RELATED RECEPTORS (ERRS) FOLLOWING TRAUMA-HEMORRHAGIC SHOCK (T-H)
S. Hu, S. Yang, R. Raju, K.I. Bland, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL
Our studies have shown that T-H induces a decrease in cardiac ERs and ERRs, which correspond with T-H-induced cardiac depression. We have also demonstrated that estrogen (E2), DPN, or the ERRβ and γ agonist DY131 administration after T-H restored cardiac function. Although our results indicated that the salutary effect of DY131 on the heart was not through alteration in cardiac ERs, it remained unclear if the ER agonist DPN or PPT affect cardiac ERRs after T-H. To study this and compare with the effect of E2, male adult Sprague-Dawley rats were divided into six groups (n=6/group): sham, T-H+vehicle, T-H+E2, T-H+ICI 182,780 (ICI)+E2, T-H+DPN, T-H+PPT. DPN (10μg/kg), PPT (10μg/kg), or E2 (1mg/kg) was given at the onset of resuscitation, IV, and ER antagonist ICI (3mg/kg) was given 30 min before E2, IP. Two hrs after resuscitation, left ventricular (LV) performance was determined; blood and heart tissue were harvested and cardiac nuclei were extracted. Cardiac ERs, ERRs and PGC-1α were determined by Western blot. Plasma and heart tissue cytokines were measured by ELISA. Our results indicated that DPN and E2 restored LV performance as well as cardiac ERs, ERRs and PGC-1α after T-H; however, PPT only partially improved LV performance and only restored cardiac ERα and ERRα. DPN and E2 prevented T-H-induced increase in plasma and heart TNF-α and IL-6 levels, but induced a further increase in plasma and heart IL-10. Therefore, it may be concluded that the ER-β agonist DPN is more effective in improving cardiac function, cardiac ERs and ERRs than the ER-α agonist PPT following T-H (NIH R01 GM39519).
ANALYSIS OF REGULATORY MARKERS FOR PREDICTING SUSCEPTIBILITY TO SEPSIS IN TRAUMA PATIENTS
M. Krencicki1, C. Mold2, O. Myers3, A. Ziegler1, S.D. West*1. 1University of New Mexico School of Medicine Department of Surgery, Albuquerque, NM, 2University of New Mexico School of Medicine Department of Molecular Genetics and Microbiology, Albuquerque, NM, 3University of New Mexico Health Sciences Center Clinical and Translational Science Center, Albuquerque, NM
Injured patients who survive the initial trauma and resuscitation remain at risk for late mortality secondary to septic complications. This has been attributed in part to dysregulation of the immune system. We hypothesized that a panel of inflammatory markers could be used to predict susceptibility to sepsis in trauma patients. The Human Research Review Committee approved all protocols prior to sample collection. Severely injured trauma patients defined by an Injury Severity Score of 16 or greater or requiring ICU admission were consented and blood samples were collected at enrollment and again at 48 and 72 hrs. Patients were followed through their hospital stay and any septic events were recorded, as defined by the presence of SIRS and a documented infection as defined by the CDC guidelines. We measured a panel of monocyte markers and cytokines in 50 severely injured trauma patients for 3 days following admission. The incidence of sepsis was 44%. We found that day 1 levels of both anti-inflammatory and pro-inflammatory cytokines were predictive of sepsis. IL-10 levels elevated above the median for patients had a 3.19 odds ratio for sepsis. Similarly, IL-1RA had an odds ratio of 6.73, IL-6 and IL-8 each had odds ratios of 4.57, TGF-β had a ratio of 3.78 and MCP-1 had an odds ratio of 3.19. We noted that day 2 levels of HLA-DR were inversely correlated with septic complications in trauma patients, as previously shown.
PLASMA IL-6 AND KC LEVELS DURING THE EARLY PHASE OF ACUTE PANCREATITIS CAN PREDICT LATER PROGRESSION TO A SEVERE CONDITION WITH MULTIPLE ORGAN DYSFUNCTION SYNDROME
D. Okamura, M. Starr*, B.M. Evers, H. Saito*. University of Kentucky, Lexington, KY
Introduction: Approximately 20% of acute pancreatitis (AP) patients develop a severe life-threatening condition with necrotizing pancreas, multiple organ dysfunction syndrome (MODS), and high mortality. While mortality rates in young AP patients (<40-years) are less than 1%, older patients exhibit dramatically increased mortality because disease progression to MODS is more common in the elderly. The establishment of early phase AP biomarkers, which will accurately predict later progression to MODS, is desired as such markers would enable early management decisions to reduce AP mortality. The purpose of this study was to identify such biomarkers by using an AP model with aged mice (Aging Cell 2012, 11:760).
Methods: To characterize the progression of AP to severe AP, young (4-5 months) and aged (23-25 months) C57BL/6 mice were subjected to experimental AP (caerulein 50 μg/kg, i.p., 9 times at hourly intervals) and sacrificed at multiple time points. The severity of AP was evaluated by plasma protein analyses and histological scoring. Plasma protein analyses were repeated in a separate experiment in which a small blood sample was collected from young and aged mice during the early phase of AP (1h); these same mice were sacrificed at a later phase (24h) and AP severity of each mouse was individually compared to the early phase plasma protein data.
Results: By 24h after the final caerulein injection, approximately 50% of aged mice developed renal dysfunction (plasma creatinine >0.6 mg/dL), whereas the remaining aged mice and all young mice showed no such symptoms. Aged mice with renal dysfunction also exhibited significantly increased pancreatic damage (severe AP group) compared to those without renal dysfunction (mild AP group). During the early phase (1h time point), these differences in AP severity were not yet evident. We found that only those mice with high plasma levels of IL-6 (>300 pg/ml) and KC (1000 pg/ml) during the early phase would exclusively fall into the severe AP group at the later phase (24h point). Other plasma factors including amylase, d-dimer, PAI-1, MIP-2, TNFα, and IL-1β, did not show such correlations.
Conclusions: High levels of plasma IL-6 and KC during the early phase of AP can accurately predict later development of severe AP with MODS and high risk of mortality.
COMBINED HEAD INJURY AND EXTRACRANIAL TRAUMA ENHANCES THE INFLAMMATORY BEHAVIOR OF THE IMMUNE SYSTEM
V.M. Stoecklein1, 2, S. Ishikawa1, D.R. Baker1, K. Shimizu*1, J. Lederer*1. 1Harvard Medical School/Brigham and Women’s Hospital, Boston, MA, 2Ludwig-Maximilians-University, Munich, Germany
Many trauma patients suffer from multiple injuries which often include traumatic head injury. However, little is known about how traumatic head injury influences the response to extracranial trauma. We developed a combined head injury and trauma model by inducing closed-skull head injury with burn injury in mice and used this to compare the immune system effects of combined injury versus single injuries. At 1 and 7 days after injury we assessed the systemic inflammatory response to these injuries and found that the combination of burn and traumatic head injury induces a strong increase of pro-inflammatory priming of the innate immune system over single injuries, as indicated by heightened in vitro production of pro-inflammatory cytokines by splenocytes in response to inflammatory stimuli. Production of IL-1α, IL-1β, IL-6, IL-12p40 and MCP-1 by splenocytes of mice which were double-injured was found to be markedly increased over single injuries. Additionally, FACS analysis of immune cell subsets showed a significant expansion of a pro-inflammatory macrophage subset (GR-1+) in blood, lymph nodes and spleen of combined-injury mice. Additional innate immune subsets - macrophages, dendritic cells, PMNs and NK cells - were also increased significantly by combined head and burn injury. Taken together, these results suggest an increased pro-inflammatory priming of the innate immune system in double-injured mice. We next tested the effect of these injuries on anti-microbial resistance in a model of Pseudomonas aeruginosa pneumonia at 1 and 7 days after injury. Interestingly, both burn-injured and double-injured mice showed improved survival as compared to sham animals 7 days after injury. In contrast, when infected at 1 day, injured mice showed worse survival as compared to sham animals. Mice which received traumatic head injury only showed survival rates similar to sham animals at both time points. This finding provides further evidence of increased priming of the innate immune system in burn injured and double-injured mice 7 days after injury which results in enhanced anti-microbial immunity. In summary, our results provide insight into how traumatic head injury influences the immune system response to trauma. We provide new evidence suggesting that head injury exacerbates the systemic inflammatory response to extracranial traumatic injuries.
FUNCTIONAL FIBRINOGEN TEG IS A SUPERIOR ASSAY FOR DETERMINING HYPERFIBRINOLYSIS IN SEVERELY INJURED TRAUMA PATIENTS
M.P. Chapman1, 3, E.E. Moore*1, 3, C.R. Ramos1, 3, J.R. Stringham1, 3, A. Banerjee1, C.C. Silliman1, 2. 1University of Colorado, Denver, Denver, CO, 2Bonfils Blood Center, Denver, CO, 3Denver Health Medical Center, Denver, CO
Background: Our recently published data establish a Citrated Kaolin Thromboelastogram (CK-TEG) LY30 ≥3% as the critical value for clinically significant fibrinolysis in severely injured trauma patients. Unfortunately, the sub-3% range of LY30 is difficult to interpret, as it overlaps the “noise” attributable to platelet-mediated clot retraction. The FF-TEG reagent generates a fibrin-only, platelet-independent clot. We hypothesized that FF-TEG LY30 would be more sensitive and specific to fibrinolytic clot degradation than the standard CK-TEG.
Methods: First, we conducted in vitro comparisons of CK- and FF-TEG by titrating the blood of healthy volunteers with tPA. Detection threshold, sensitivity and specificity for plasmin-mediated clot lysis were determined. Next, as part of ongoing studies of fibrinolysis at our regional trauma center, we obtained admission CK- and FF-TEGs on severely injured patients, deemed likely to require a massive transfusion of ≥10 units of PRBCs in 6 hours (N=33; median ISS 25, IQR 19—34). CK-TEG measures the growth and decay of a platelet-fibrin clot using kaolin as an activator. FF-TEG measures a fibrin-only clot using tissue factor as an activator and abciximab as a platelet inhibitor.
Results: Our in vitro studies demonstrated that FF-TEG had a lower detection threshold and greater sensitivity for fibrinolysis than CK-TEG. FF-TEG also accurately discriminated platelet retraction from genuine fibrinolysis. In our clinical study, 6 of 33 patients (18%) had hyperfibrinolysis (LY30 ≥3%) as defined by CK-TEG. There was one false positive (due to platelet retraction) and one false negative which were revealed by analysis of the concurrent FF-TEG. FF-TEG had an overall PPV of 100% for massive transfusion requirement, versus 83% for CK-TEG. FF-TEG LY30 had an analytical sensitivity 86% greater in vitro and 20% greater clinically than CK-TEG. FF-TEG’s statistical sensitivity for massive transfusion prediction was 19% greater than CK-TEG.
Conclusions: FF-TEG has superior predictive value (100%) for massive transfusion related to hyperfibrinolysis than CK-TEG, using the ≥3% threshold for LY30. FF-TEG is also more sensitive for fibrinolysis than CK-TEG. FF-TEG thus represents a powerful tool for discriminating fibrinolysis from other causes of coagulopathy and should be used as the guide for anti-fibrinolytic therapy.
INFLAMMATORY LUNG INJURY FOLLOWING EXPLORATORY LAPAROTOMY RESETS AN ECRG4 SET POINT POST INJURY RESOLUTION
A. Shaterian, S. Kao, D.M. Cauvi, A. De Maio, H. Chun, T.W. Costantini*, R. Coimbra*, B. Eliceiri, A. Baird. Dept of Surgery, University of California San Diego, La Jolla, CA
Introduction: The newly recognized candidate tumor suppressor gene, Esophageal cancer related gene-4 (Ecrg4), is an epigenetically regulated, cytokine-like protein that we have recently implicated in diverse organ-specific injury responses. Ecrg4, as a protective homeostatic factor, is down-regulated in cancer and injury, but is up-regulated in lung following protective preconditioning. In the current study, we characterized the kinetics of Ecrg4 expression following exploratory laparotomy to show the time course of injury resolution.
Materials and Methods: qRT- PCR was used to characterize several well-described pro- and anti-inflammatory cytokines that function in lung injury including TNF-α, IL-1β, IL-6, and IL-10. We evaluated the referenced cytokines in healthy lung and after exploratory laparotomy as a measure of organ injury cross-talk. Ecrg4 expression levels were also compared at various time points after exploratory laparotomy and specifically evaluated for changes post-injury resolution.
Results: The expression of TNF-α, IL-1β, IL-6, and IL-10 in lung demonstrated an increase within the 6hr time point following exploratory laparotomy suggesting an induced inflammatory lung injury response. In contrast, Ecrg4 expression underwent down-regulation post surgery and was the only cytokine to rebound beyond its pre-injury expression levels at 30hrs post surgery, after the resolution of injury.
Conclusions: This study characterizes an inflammatory lung response following exploratory laparotomy suggestive of organ injury cross talk. These data also further support the proposed role of Ecrg4 as a protective injury response factor. We hypothesize that the over-correction of Ecrg4 in lung after injury resolution may serve a protective function against subsequent acute host injury. As such, pre-injury Ecrg4 expression levels may serve as a “set point” that can predict host vulnerability to subsequent inflammatory stimuli.
CHOLESTEROL TRAFFICKING PLAYS AN ESSENTIAL ROLE IN MACROPHAGE PHAGOCYTIC ACTIVITY
D.F. Niño, D.M. Cauvi, A. De Maio*. Department of Surgery and Center for the Investigation of Health and Educational Disparities, University of California, San Diego, La Jolla, CA
Background & Objective: Previous reports have demonstrated a role for cholesterol in the regulation of immune function; in particular statins have been shown to affect phagocytosis in macrophages (Møs). The aim of the current studies was to determine the role of intracellular cholesterol trafficking on the phagocytic ability of Møs.
We report that the use of the small molecule inhibitor of intracellular cholesterol trafficking (Imipramine - IPN) significantly decreases Fcγ receptors (FcγRs)-mediated phagocytosis by altering cell surface expression of FcγRs in a murine Mø cell line (J774.1 cells).
Methods & Results: Mø’s were treated with IPN (15 μM) or DMSO for 24 h. Phagocytosis of opsonized fluorescently labeled E. coli particles (at bioparticles:cell ratio of 10:1 for 15 and 30 min) and Fcγ III/II receptor (total and cell surface) expression were determined using flow cytometry. IPN decreased cell surface and total FcγRs expression to 35 and 30% of control (CTRL) levels, respectively. Phagocytic ability was reduced in IPN treated cells to 15 and 20% of CTRL by 15 and 30 min, respectively. In addition, IPN led to an increase in the mRNA expression of C/EBP homologous protein (CHOP) indicating the presence of ER stress. Furthermore, the observed effect of IPN on phagocytosis and FcγRs was rescued to CTRL levels by coadministration of low-density lipoprotein (LDL) at a dose of 100 μg/mL.
IPN led to accumulation of cholesterol in late endosomes and lysosomes, generating a phenotype reminiscent of the autosomal recessive lipid storage disorder Niemann-Pick disease type C.
Conclusion: The current report demonstrates the critical role of normal cholesterol homeostasis in the regulation of phagocytosis.
TOLL-LIKE RECEPTOR INDUCED RESPONSES BY CIRCULATING LEUKOCYTES ARE SUPPRESSED IN TRAUMA ICU PATIENTS
T.L. Holloway1, M. Rani*1, T. Craig1, Q. Zhang1, A.P. Cap*2, 1, M.G. Schwacha*1, 2. 1University of Texas Health Science Center, San Antonio, TX, 2US Army Institute of Surgical Research, San Antonio, TX
Traumatic injury often is associated with profound inflammation and activation of the innate immune system, which may involve alterations in Toll-like receptor (TLR)-mediated responses. To study this, a prospective observational study was designed. Following IRB approval, 14 severely injured (ISS = 16-41) and mechanically-ventilated trauma ICU patients were enrolled along with 6 healthy normal volunteers that served as controls. Heparinized whole blood was collected at 2-12 days after ICU admission and incubated for 3 hrs in the presence of media alone (baseline), zymosan (TLR2 agonist) or LPS (TLR4 agonist). Inflammatory cytokine levels (IL-1β, -6, -10, TNF-α) in the supernatants were measured by Luminex multiplex assay. Whole blood cultures from both healthy volunteers and subjects were responsive to TLR2 and TLR4-mediated activation displaying elevated cytokine levels over that observed at baseline. Subject cultures with media alone (baseline) had significantly elevated levels of TNFα, IL-6, and IL-10, as compared to healthy volunteers. In sharp contrast, healthy volunteer cultures stimulated with zymosan (TLR2 agonist) or LPS (TLR4 agonist) had 2-3-fold greater levels of IL-6 and TNFα than those of subjects. IL-1β and IL-10 levels did not differ significantly between healthy volunteers and subjects. In conclusion, these findings show that TLR-mediated activation of circulating leukocytes from trauma ICU patients is markedly suppressed, which may play a role in the development of subsequent infectious complications.
BETA1-ADRENERGIC ANTAGONISTS IN SEPSIS: SURVIVAL BENEFIT DUE TO IMMUNOMODULATION?
I. Ibrahim-zada, C. Gomez*, R. Means, J. Weller, P.M. Rhee*, R.S. Friese. University of Arizona, Tucson, AZ
Introduction: Prior studies demonstrate potential benefit for non-selective beta-adrenergic antagonists in sepsis. However, there is a paucity of data on β1-selective antagonists (B1A) in sepsis. The aim of the study was to describe 1) B1A effects on survival in sepsis; 2) the difference between the molecular profiles of septic animals treated with B1A versus control.
Methods: 8-12 week C57BL/6 mice received IP injection of 12.5mg/kg lipopolysaccharide (LPS). Pumps intravenously delivered B1A (esmolol; 6.7ug/kg/min) or equal volume of saline (control). Outcome was survival at 5 days post LPS. Kaplan-Meier survival curves were generated.
Six animals were sacrificed 48 hours after LPS for whole blood collection and microarray expression analysis on Affymetrix Mouse Gene 1.0 ST. Data was normalized by RMA and analyzed using ANOVA. Genes with at least 1.3-fold change in expression were included for Ingenuity pathway analysis (IPA).
Results: Mean survival difference for the 43 control and 44 experimental mice was 23.6 hours (p=0.002). B1A group had increased survival at 5 days post LPS (p=0.001). Expression analysis identified 348 genes. None of the genes were significant after FDR correction. IPA identified 36 genes involved in immunological disease, cell death and survival as significantly associated with better survival outcomes in septic mice treated with B1A (p=0.0001-0.036). Of those, CAMP (-2.9), TNFSF10 (-2.4), LY6I (-2.4), IL18BP (-1.7), EDA2R (-1.7) and BCL2L14 (-1.7) were genes down-regulated in the esmolol group.
Conclusions: B1A improved survival in septic mice. Benefits may be due to down-regulation of genes in immunologic pathways. Further studies are necessary to understand this observed immunomodulatory effect.
ABSENCE OF INDUCIBLE NITRIC OXIDE SYNTHASE ATTENUATES INSULIN RESISTANCE IN ENDOTOXEMIA
L.M. House1, 2, Y.F. Otero1, R.T. Morris3, T.M. Lundblad1, O.P. McGuinness1. 1Vanderbilt University, Nashville, TN, 2University of Tennessee Health Science Center, Memphis, TN, 3Missouri State University, Springfield, MO
Sepsis is a state marked by decreased mean arterial blood pressure (MAP), cardiac output, and insulin sensitivity. Nitric oxide (NO) regulates muscle blood flow and insulin-stimulated capillary recruitment. In endotoxemia, cytokines and lipopolysaccharide (LPS) stimulate transcription of inducible nitric oxide synthase (iNOS). We hypothesized that vasodilation from iNOS induction is the predominant cause of insulin resistance during endotoxemia. To test this we measured insulin sensitivity, tissue glucose uptake, and insulin signaling activity in conscious LPS-treated wild type (WT) and global iNOS knock out (KO) mice after a hyperinsulinemic-euglycemic clamp.
Following jugular and carotid catheterization, WT and iNOS KO C57BL6/J male mice aged 2-4 mo were fasted for 5 hr (t=-600 min). At t=-240 min mice received a bolus of 2.0 ug/g of LPS or saline. A primed infusion of [3-3H]-D-glucose was initiated (t=-120 min) prior to clamping. Blood glucose was sampled every 10 min. Tracer-determined tissue 2-deoxy [14C] glucose uptake was assessed. MAP was recorded continuously.
LPS lowered MAP (in mmHg) in WTs vs. LPS-treated iNOS KOs (97±1 vs. 115±1). Glucose requirements (GIR, in mg/kg/min) were lower in WTs after LPS treatment (36.9±0.9 vs. 54.3±1.0, LPS vs. saline), but not in iNOS KOs (42.7±1.0 vs. 50.3±0.9, LPS vs. saline). Plasma insulin did not differ among groups (for t=-10 and 120 min). Akt phosphorylation (S473) in skeletal muscle was increased by LPS in iNOS KOs only (p<0.05). LPS did not affect Akt (T308) or insulin receptor substrate 1 activity (S307, Y895) in muscle.
Thus, iNOS KOs were protected from LPS-induced insulin resistance, but without impairments in muscle insulin signaling. iNOS KOs were also protected from LPS-mediated hypotension. Our findings suggest that skeletal muscle insulin resistance in response to acute endotoxemia is driven primarily by accompanying cardiovascular and microcirculatory events.
MULTIPLE ORGAN DYSFUNCTION AND ACUTE PHASE MORTALITY IN MURINE INTRA-ABDOMINAL SEPSIS
K.N. Iskander, F. Craciun, E.L. Chiswick, D.M. Stepien, J. Henderson, D.G. Remick*. Boston University Medical Center, Boston, MA
Objective: The etiology of acute phase death in the murine sepsis model of cecal ligation and puncture (CLP) remains unclear making it difficult to develop targeted therapies to improve survival outcomes. The purpose of this study was to determine if significant liver, pancreatic, renal or cardiovascular injury develops in septic mice predicted-to-die (Die-P) after CLP compared to those predicted-to-live (Live-P).
Methods: Female outbred mice underwent CLP using a 16 gauge needle for double puncture to induce polymicrobial sepsis. Mice were stratified into Live-P and Die-P groups based on plasma IL-6 levels. Organs were harvested for histology at 24 and 48 hours, and blood samples obtained for biochemical assays at 6 and 24 hours. Non-invasive telemetry monitoring was used to assess cardiovascular function.
Results: No significant histological differences were seen in the liver, pancreas, or kidney between Live-P and Die-P mice. Plasma AST and amylase demonstrated less than a two-fold increase over normal at 24 hours. In contrast, BUN levels were almost five times higher at 24 hours indicating greater kidney injury in Die-P mice. Cystatin C, a more specific kidney function biomarker, was also measured and demonstrated a significant elevation detected in the last 24 hours prior to death (levels 2.4 times higher than normal). Cardiovascular impairment was evident as determined by hemodynamic parameters. Interestingly, pulse distension (RR:19.0, 95% CI: 2.8-128, p<0.0001) and heart rate (RR:16.2, 95% CI: 2.4-110, p<0.0001) at 24 hours was associated with greater mortality and able to predict murine death during the first five days after sepsis.
Conclusion: We conclude that limited liver and pancreatic dysfunction develops 24 hours into the evolution of murine CLP induced sepsis, while significant kidney injury is present which becomes exacerbated closer to death. Cardiovascular impairment as measured by decreased pulse distension and heart rate was an excellent predictor of death in the acute phase of sepsis.
EARLY HEMODYNAMIC PARAMETERS BETTER PREDICT MORTALITY THAN IL-6 IN A MURINE PNEUMONIA MODEL OF SEPSIS
E.G. King, D.M. Stepien, G. Bauza, J. Mella, K.N. Iskander, E. Duffy, D.G. Remick*. Boston University Medical Center, Boston, MA
Introduction: Interleukin-6 measured within 6 hours is an accepted prognostic factor of mortality in the cecal ligation and puncture (CLP) model of murine polymicrobial sepsis. However, pneumonia remains the leading cause of sepsis compared to the peritoneum. This study investigated IL-6, peripheral blood counts and hemodynamic parameters as mortality predictors in a Pseudomonas pneumonia murine model of sepsis.
Methods: Pseudomonas aeruginosa (1 x107 CFU - 2.5x107 CFU) was intratracheally instilled in adult, female ICR mice. At 6 hours and 24 hours following administration of Pseudomonas, non-invasive hemodynamic measurements were recorded using a cervical collar pulse-oximeter and 20uL of blood was obtained via facial vein to measure cytokine levels and complete blood count. Mortality was followed over 14 days.
Results: In previous studies an IL-6 level > 13ng/ml at 6 hours following CLP predicted 5-day mortality (AUC-Receiver Operator Characteristic ROC = 0.92). IL-6 levels measured at 6 hours after administration of Pseudomonas did not show a significant difference between mice that survived and those that died (3.2ng/ml ± 0.9 vs 4.2ng/ml ± 0.8). However, any detectable level of IL-6 at 24 hours predicted mortality (p<0.0001). Hemodynamic measurements accurately predicted mortality at the earlier time point. Heart rate decreased by 6 hours in mice that died compared to those who lived (p<0.0001) and a heart rate of less than 430 beats per minute resulted in a 6.5 fold increased risk of death (AUC-ROC = 0.85). Heart rate at 24 hours also accurately predicted mortality. A decrease in respiratory rate at 6 hours was observed in mice that died (147 ± 9 vs 112 ± 5) and a respiratory rate of less than 117 resulted in an 8 fold increased risk of death (AUC-ROC = 0.82). Pulse distention, though not significantly different at 6 hours, was decreased in the mice that die (p<0.0001) by 24 hours (AUC-ROC = 0.93). Peripheral blood neutrophils at both 6 hours (p = 0.01) and 24 hours (p<0.0001), as well as peripheral blood lymphocytes at 24 hours (p= 0.002), were significantly lower in the mice that died.
Conclusion: Heart rate, respiratory rate and PMN count at 6 hours after the onset of sepsis are better predictors of mortality than IL-6 in a Pseudomonas pneumonia model of sepsis.
HUMANIZED SP-B TRANSGENIC MICE AND A PNEUMONIA MODEL TO STUDY FUNCTIONAL EFFECTS OF HUMAN SP-B GENETIC VARIANTS
L. Ge1, 2, X. Liu1, R. Cooney*1, G. Wang*1. 1SUNY Upstate Medical University, Syracuse, NY, 2Tianjin Medical University, Tianjin, China
Surfactant protein B (SP-B, gene name: sftpb) is essential for normal lung function. A single nucleotide polymorphism (SNP, rs1130866) SP-B 1580 C/T is associated with multiple lung diseases and altered an N-linked glycosylation site at Asn129 of SP-B. Humanized transgenic mouse model is an ideal method to study functional effects of human SP-B genetic variants.
Objective: To generate humanized sftpb transgenic mice and establish a pneumonia model.
Methods: The cDNA of each human SP-B genetic variant was cloned into a basic 3.7-hSP-C/SV40 vector. The DNA was directly microinjected into fertilized FVB/N oocytes from wild-type (WT) FVB/N mice. Positive transgenic pups were identified by genotyping analysis and were backcrossed with SP-B KO mice to eliminate mouse SP-B gene. The expression of human SP-B (hSP-B) in the lung was examined by Western blot. To establish pneumonia model mice were infected intratracheally with 50μl of P. aeruginosa solution (1X107 CFU) or 50μl saline (Sham). The mice were sacrificed 24 hours post-infection, then histological, cellular and molecular analyses of the lungs were performed. Differences were considered significant when P<0.05 by t-test or ANOVA.
Results: 1) The humanized sftpb transgenic founder lines carrying human SP-B C allele or T allele were generated; and the transgenic mice show normal phenotypes as WT mice. The level of hSP-B protein expression in the lung of transgenic mice is comparable with that in the human lung. These indicate hSP-B is functional in transgenic mice. 2) In the bacterial pneumonia model, 24 hrs after infection with 1X107 CFU P. aeruginosa transgenic mice with either the C or T allele exhibited the characteristics of pneumonia pathology. A number of inflammatory cells (most of them are neutrophils) infiltrated into the alveoli. Significant decreases of surfactant proteins (SP-A, hSP-B, SP-C, SP-D) were observed in the infected mice compared to Sham mice.
Conclusion: Humanized transgenic mouse model is an effective method to study the functional effects of human SP-B genetic variants in the pneumonia model.
EFFECT OF P38 PATHWAY ACTIVITY ON PHAGOCYTOSIS OF CRYPTOCOCCUS NEOFORMANS BY ALVEOLAR MACROPHAGES IN VIVO
O. Abdel Razek1, X. Liu1, 2, R. Cooney*1, G. Wang*1. 1Upstate Medical University, Syracuse, NY, 2College of Biotechnology, Tianjin, China
Introduction: SP-A and SP-D are involved in the innate immunity and host defense against various pathogens. In normal lung, SP-A and SP-D bind to signal regulatory protein α (SIRPα), inhibit p38 mitogen-activated protein kinases (p38 MAPK) activation, and prevent inflammatory mediator production. SP-A and SP-D double knockout (KO) mice show higher levels of phosphorylated p38 (p-p38) in the lung and better survival after infection with C. neoformans compared to wild type (WT) mice.
Objective: The current study examines 1) the effects of p38 MAPK activity on the phagocytic activity of C. neoformans by alveolar macrophages in vivo; and 2) the effects of SP-D on the process of phagocytosis of C. neoformans.
Methods: SP-A and SP-D KO, SP-D transgenic mice and WT mice (9-12 weeks) were used. To study the effects of p-p38 activity on phagocytosis, KO mice were treated ± p38 inhibitor prior to intratracheal injection with 1x106 CFU/mouse of C. neoformans and mice were sacrificed six hours post-infection. Pulmonary p-p38 levels were measured by Western blot, the phagocytosis of C. neoformans was measured using phagocytic index of macrophages and colony-forming units (CFU) from BAL fluid. Next, the effects of SP-D on phagocytosis were studied with KO and SP-D transgenic mice using the same methods. Data are means +SE and p<0.05 by t-test was considered significant.
Results: Baseline p38 MAPK phosphorylation was significantly higher (150%) in KO mice than in WT mice; but it is decreased to similar level of WT mice in KO mice after p38 inhibitor treatment. The administration of p38 inhibitor in KO mice significantly reduced phagocytic activity of C. neoformans by macrophages in vivo (32.5 ±5.4) in comparison to the sham group (52±5.7) (p<0.05). The p38 inhibitor treated group shows significantly higher CFU count (496±53.5) than the control (Sham) group (274±54.7) (p<0.05). Furthermore, the phagocytic activity of C. neoformans by macrophages in vivo is significantly higher in SP-D transgenic mice than that in KO mice (p<0.05).
Conclusion: p38 MAPK activity plays a critical role in the phagocytic activity of C. neoformans by macrophages in vivo, and SP-D can enhance the phagocytosis of C. neoformans by macrophages.
CASPASE 1 PLAYS A CRITICAL ROLE IN SURVIVAL, MYOCARDIAL FUNCTION, AND INFLAMMATORY RESPONSE IN COMMUNITY-ACQUIRED MRSA SEPSIS
J. Hume1, D.L. Carlson*2. 1University of Minnesota Medical School, Minneapolis, MN, 2University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Community-acquired methicillin-resistant S. aureus (CA-MRSA) sepsis continues to be a significant cause of morbidity and mortality. CA-MRSA sepsis physiology has not been extensively studied in animal models. The role of the inflammasome and the key inflammasome components caspase 1 and IL-1beta in CA-MRSA infection are unclear.
Methods: Wild type (WT) and caspase 1 knockout (Cas1KO) mice were infected with TCH1516, a sequenced USA300 CA-MRSA strain. Serum and hearts were collected from sacrificed animals for Bioplex analysis of serum IL-1beta and qPCR analysis of myocardial IL-1 beta expression. Fractional shortening (FS) was measured using M-mode echocardiography.
Results: There was a trend towards reduction in survival time in Cas1KO mice as compared to WT as CFU of the inoculum increased from 5x107 to 1x109. At 1x109 CFU survival time was significantly less in Cas1KO mice than in WT mice (mean survival 24.2 hours versus 51 hours, p=0.039). There was a significant reduction in fractional shortening in caspase 1 KO mice relative to WT mice at 18 (WT 69.9% vs. 56.7%, p=0.046) and 24 hours (WT 64.9% vs. 51.4%, p=0.025) using 5x107 CFU. Over 48 hours, WT animals infected with 5×107 CFU showed low production of serum IL-1 beta relative to positive controls injected with 4 mg/kg LPS. At 24 hours, WT heart expression of IL-1beta was upregulated in CA-MRSA.
Discussion: Lack of caspase 1 results in decreased survival and worsening myocardial function in mice with CA-MRSA sepsis, associated with low levels of serum IL-1beta production. Upregulation of IL-1beta (activated by caspase 1) in the myocardium suggests an important role for caspase 1 and inflammasome activation in CA-MRSA sepsis.
TOPICAL NANOEMULSION THERAPY CLEARS GRAM POSITIVE AND NEGATIVE BACTERIA AND ATTENUATES BURN WOUND PROGRESSION
V. Dolgachev*1, S.M. Ciotti2, S. Wang1, Y. Fan1, J.R. Baker Jr2, M.R. Hemmila*1. 1University of Michigan, Ann Arbor, MI, 2NanoBio® Corporation, Ann Arbor, MI
Objective: Nanoemulsions (NE) are novel oil-in-water emulsions formulated from non-toxic materials. They have broad anti-microbial properties and can be used in burn wounds. We have shown that a topically applied NE (NB-201) decreased Pseudomonas aeruginosa growth, associated inflammation, and hair follicle apoptosis in a partial thickness burn model. Here we investigate the efficacy of additional NE formulations against Gram positive and negative bacteria.
Methods: Male Sprague-Dawley rats received a 20% TBSA partial-thickness scald burn. Animals were resuscitated with Ringer’s lactate and the wound covered with an occlusive dressing. At 8 hours after injury the burn wound was inoculated with 1x106 colony-forming units (CFUs) of Pseudomonas aeruginosa or 1x108 CFUs of Staphylococcus aureus. NB-201, NB-402, NB-402 placebo, or 0.9% saline (control) were applied 16 and 24 hours after burn. Skin was harvested 32 hours post burn for quantitative wound culture and determination of inflammatory mediators in tissue homogenates. In a separate set of experiments we examined burn wound progression over 3 days when treated with NE.
Results: Treatment of burn wounds infected with Pseudomonas aeruginosa or Staphylococcus aureus with NB-402 decreased bacterial counts by 3-4 logs compared to the saline control (P<0.0001). NB-402 and NB-201 significantly decreased neutrophil recruitment and sequestration measured visually by histology and quantitatively by myeloperoxidase assay (59.2, 59.4 vs 322.9, p<0.05). NE also reduced the dermal level of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and the neutrophil chemoattractants CXCL1 and CXCL2 compared to control. Both NB-201 and NB-402 demonstrated evidence of suppressing 72 hr burn wound progression visually in photographs.
Conclusions: Topical NB-201 and NB-402 are effective in decreasing Gram positive and negative bacteria growth in burn wounds. These NE formulations also reduced inflammation and burn wound progression. Nanoemulsion therapy is a potential new breakthrough treatment for early burn wounds.
UNCOVERING COMMON BENEFICIAL TREATMENT FEATURES FOR INJURY-INDUCED IMMUNE DYSFUNCTION USING A SYSTEMS IMMUNOLOGY APPROACH
V.M. Stoecklein1, 2, S. Ishikawa1, J. Lederer*1. 1Harvard Medical School/Brigham and Women’s Hospital, Boston, MA, 2Ludwig-Maximilians-University, Munich, Germany
Trauma disrupts immune function predisposing injured patients to infectious complications. Although pre-clinical research has uncovered beneficial immune-enhancing treatments, a view of common features associated with improved experimental outcomes in mice has not yet been studied. In this report, we used a systems immunology approach to screen for commonality among three immune response modifier treatments for injury in mice to better understand immunophenotypic changes associated with protection. To achieve this, groups of male outbred mice underwent burn injury and were treated with Rapamycin (RAPA), CpG ODN 2336 (CpG), or IL-12 after injury using treatment approaches known to protect mice from sepsis. At 7 days after injury lymph nodes (LN) and spleens (SPL) were harvested and stained with antibody panels to identify multiple immune cell subsets by high-throughput flow cytometry. Spleen cells were stimulated with LPS, αCD3, or LPS+ATP to measure treatment influences on cytokine production. Furthermore, mice were immunized with ovalbumin and were treated as described above to measure treatment effects on antigen-specific immunity. Results of immune cell phenotyping studies showed that the number of T regulatory cells (Tregs) was increased by all treatments in the LN and SPL. In the innate system, we observed that the GR-1+ macrophage subset was increased by all treatments in the SPL. All treatments caused large increases in NK cells in SPL. Production of inflammatory cytokines was blunted by CpG and IL-12 treatment while RAPA treatment had no such effect. Antigen-specific stimulation of splenocytes yielded increased production of T cell cytokines by RAPA-treated mice and importantly, we discovered that IFN-γ production was increased by all three treatments. Antibody isotype analysis showed that RAPA and CpG increased antigen-specific IgG1 and IgG2b production. In summary, we demonstrate commonality among 3 seemingly different types of immunomodulatory treatments that protect mice from sepsis mortality. These include the modulation of Tregs, GR-1+ macrophages, and NK cells and suppressing the inflammatory behavior of the innate immune system. Moreover, we demonstrate that a system immunology approach can aid in discovering new insights into how to restore normal immune system balance following trauma.
INFLUENCE OF AGE AND MORTALITY ON BURN LENGTH OF STAY: DEATH MAKES A DIFFERENCE
T. Palmieri*1, 2, S. Taylor1, S. Sen1, 2, D.G. Greenhalgh*1, 2. 1University of California Davis, Sacramento, CA, 2Shriners Hospitals for Children Northern California, Sacramento, CA
Introduction: Traditionally, estimated length of stay for a burn patient is one day per percent burn. These estimates are based on data spanning all ages from both survivors and non-survivors. The purpose of this study was to evaluate the accuracy and identify factors that may change this estimate.
Methods: We validated and analyzed the National Burn Repository, consisting of >300,000 records from burn centers across the United States and Canada. Records prior to the year 2000 or missing age, total body surface area burn, survival, and mortality were eliminated. We used ordinary least squares to estimate the slope of a linear relationship between length of stay (days) and TBSA (%) for each facility. The intercept was fixed at 0. Slopes were estimated using all patients, and only patients that lived. We further investigated slope differences by age groups and burn size among patients that lived.
Results: A total of 95,579 records were eligible for review. Median length of stay was less than 1 day per 1% TBSA using all patients (median = 0.73); however, length of stay varied substantially among facilities (range: 0.278 to 1.247). When restricted to patients who survived injury, length of stay estimated slopes were close to the 1 day per 1% TBSA rule of thumb (median = 1.09; 25th 0.95; 75th 1.28). The relationship between length of stay and TBSA was generally greater than 1:1 for older patients and patients with larger burns (> 50%).
Conclusion: Variability in hospital length of stay exists between burn centers, but overall length of stay is less than one day per percent burn. However, burn mortality serves to shorten mean hospital length of stay to the frequently quoted 1 day per percent burn. Increasing burn size and patient age lengthen hospital stay and resource utilization.
RELATIONSHIP BETWEEN IL1RA VNTR POLYMORPHISM AND THE OUTCOME OF SEVERE SEPSIS PATIENTS AS WELL AS BLOOD CYTOKINE EXPRESSION IN A JAPANESE POPULATION
T. Kimura1, E. Watanabe*1, T. Sakamoto2, T. Ikeda3, J. Kotani*4, S. Oda*1. 1Department of Emergency and Critical Care Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 2Kurume University Hospital Advanced Emergency Medical Center, Kurume, Japan, 3Specific Intensive Care Division, Tokyo Medical University Hachioji Medical Center, Tokyo, Japan, 4Emergency, Department of Disaster and Critical Care Medicine, Hyogo College of Medicine, Hyogo, Japan
Objective: To determine the genetic effects on pathophysiology of severe sepsis, we investigated whether interleukin-1 receptor antagonist variable number of tandem repeat (IL1RA VNTR) polymorphism bears influences on the susceptibility to and the outcome of severe sepsis. Also, we evaluated association between the VNTR and ex vivo expression of cytokines.
Materials and Methods: 1) Case-control study: Two hundred and sixty one patients who had admitted to an intensive care unit (ICU) between October 2001 and September 2008 were enrolled into a discovery cohort. Seven hundred and seventy ICU patients admitted to the 5 different institutes between October 2008 and September 2012 were enrolled into a validation cohort. The polymorphic region within intron 2 of IL1RA contains a VNTR polymorphism which is caused by variation of the repeated region of 86 base pairs. Genotypic distributions of IL1RA VNTR were determined with polymerase chain reaction (PCR) for the both cohorts. Susceptibility to severe sepsis and the outcome in relation to the IL1RA VNTR were examined. IL-6 blood levels for patients were daily measured with chemiluminescence enzyme immunoassay. 2) Ex vivo assay of cytokine expression: Whole blood samples of 80 healthy volunteers were stimulated by lipopolysaccharide (LPS) in vitro, then serum cytokine production was measured with enzyme-linked immunosorbent assay. Cytokine mRNA expression was analyzed using quantitative real-time reverse transcription PCR. IL1RA VNTR genotype was also determined for all the volunteers. Correlation between the genotype and cytokine expressions was examined.
Results: 1) In both cohorts, RN*2 allele was significantly associated with susceptibility to severe sepsis and the poor outcome. The RN*2 allele carriage showed trend toward up-regulated peak IL-6 blood levels during their ICU stay. 2) Although there was no significant difference in both serum IL-6 and IL-1ra after LPS stimulation between carriers and non-carriers of RN*2, IL1RA mRNA expression was significantly lower in carriers of RN*2 than in the non-carriers (P<0.01).
Conclusions: IL1RA RN*2, which was suggested to be a less IL-1ra producer, was associated with adverse outcome of severe sepsis via lower regulatory potential against some inflammatory cytokine production in a Japanese population.
IL-7 TREATMENT AMELIORATES THERMAL INJURY-INDUCED T CELL DEPLETION
C.C. Caldwell*, B.L. Johnson. Department of Research, Shriner’s Hospital for Children, Cincinnati, OH
Following burn injury, patients are at risk for infections that are normally cleared by healthy individuals. Thus, prevention of nosocomial infections is an important issue in the Shrine Hospital and other burn units to reduce mortality rates. The high rate of mortality and morbidity after severe thermal injury is believed to be related to suppression of the immune system. In particular, T lymphocyte function and numbers are commonly decreased in critically ill burn patients. IL-7 is a potent anti-apoptotic cytokine that is essential for T lymphocyte development and survival. Strikingly, our previous data show that administration of IL-7 to infected / septic mice improves overall animal survival, significantly ameliorates loss of sepsis induced T cell numbers and function, and significantly decreases the pathogen load. Based upon this, we hypothesized that IL-7 treatment after burn injury would protect against lymphocyte deletion. One day after a 25% TBSA dorsal scald burn injury, we enumerated and characterized T cells and their progenitors from bone marrow, thymus, and spleen by flow cytometry. We observed that IL-7 treatment protected against bone marrow common lymphoid progenitor, double negative thymocyte, splenic naïve CD4, and regulatory T cell depletion. Taken together, these data indicate that IL-7 treatment can reduce immune suppression observed after thermal injury. This study is sponsored, in part, by Shrine project 85000-CIN.
RELEVANCE OF MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF)-THYROXINE INTERACTIONS DURING SEPSIS
E.J. Miller*1, 2, H.M. Linge1, K. Cheng1, K. Ochani1, K. Lin1, J. Lee3, 1, Y. Al-Abed1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2Hofstra North Shore-LIJ Medical School, Hempstead, NY, 3Elmezzi College of Molecular Medicine, Manhasset, NY
Pulmonary infection with Gram-positive bacteria is a leading cause of sepsis, particularly in the older individual. Progression of the infection can lead to acute lung inflammation and injury, and hypoxemia. Although effective ventilator and fluid management strategies have been developed, mortality remains high in these patients. Our previous studies, demonstrate that macrophage migration inhibitory factor (MIF), a known myocardial depressant factor, accumulates within the lung during sepsis. The lung then acts as a major source of the MIF released into the systemic circulation leading to cardiac dysfunction. In addition, we have described a previously unrecognized interaction between MIF and the thyroid hormone thyroxine (T4). In sepsis, both in humans and our rodent models, there is an inverse relationship between plasma concentrations of MIF and T4. Furthermore, supplementation with the hormonally inactive D-form of T4 was able to significantly decrease the mortality rate in a murine sepsis model. These studies suggest a clinically relevant interaction between T4 and MIF, two key molecules in the critically ill patient. Therefore, we have hypothesized that MIF-T4 interactions may have a profound effect on the inflammatory response, particularly in the older individual. We have examined the affect of age on both plasma MIF and free T4 concentrations following intratracheal instillation of the major components of Gram-positive bacterial cell walls, lipoteichoic acid (LTA 1.5mg/kg) and peptidoglycan (PGN 5mg/kg). Six hours after the staphylococcal challenge in Fischer 344 rats (Young (6m) or Old (>18m) approximately equivalent to humans of 18 and 60 yrs respectively), there was a significant increase in plasma MIF and decrease in free-T4 (p<.05) and both of these changes were more profound in the older animals. To examine possible consequences of these changes we challenged human fibroblasts (CCL210) with MIF, T4 or a combination of the two, and examined nuclear localization of specific NFkB isomers. MIF induced significant activation of NF-KB as revealed by increased translocation of RelA/P65 to the nucleus (p=0.02). This increase was ablated in the presence of T4 (p=0.005). The data suggest that sepsis-induced changes in the normal MIF-T4 balance may have profound effects at the cellular level, and may be exaggerated in the older individual.
TANSHINONE IIA DERIVATIVE INHIBITS HMGB1-INDUCED CHEMOKINE RELEASE AND PROTECTS AGAINST LETHAL ENDOTOXEMIA
S. Zhu*, A. Jundoria, W. Li*, S. Tsai, M.F. Ward, A.E. Sama, H. Wang*. The Feinstein Institute for Medical Research, Manhasset, NY
Background: We recently discovered that tanshinone IIA sodium sulfonate (TSN-SS) selectively inhibited bacterial endotoxin-induced HMGB1 release, and conferred protection against lethal endotoxemia and sepsis when given intraperitoneally. Objectives: To determine whether TSN-SS inhibits HMGB1-induced chemokine release in vitro, and protects against lethal endotoxemia if given via a clinically feasible route.
Methods: Murine macrophage-like RAW 264.7 cells or human monocyte U937 cells were stimulated with recombinant HMGB1 (2-4 μg/ml) in the absence or presence of TSN-SS (100 μM) for 16 h, and extracellular levels of various cytokines and chemokines were determined by Cytokine Antibody Arrays. Male Balb/C mice (20-25 g, 6-7 weeks old) were subjected to lethal endotoxemia, and TSN-SS was given intravenously to evaluate its long-term therapeutic efficacy.
Results: In vitro, TSN-SS effectively inhibited HMGB1-induced release of IL-8 and MCP-1 (by > 80%) in U937 monocytes, and similarly suppressed HMGB1-mediated release of RANTES (by >75%) in RAW 264.7 macrophages. In vivo, intravenous administration of TSN-SS at 0.5 h, 24 h, and 48 h post endotoxemia significantly increased animal survival rates from 14% (in control group receiving LPS + saline, N = 28 mice/group) to 50% (in experimental group receiving LPS + 15 mg/kg TSN-SS, N= 20, P < 0.05).
Conclusions: TSN-SS effectively inhibited HMGB1-induced release of chemokines in innate immune cells, and protected animals against lethal endotoxemia even when given via a clinically feasible route.
(Supported by the National Institutes of Health Grants R01GM063075 and R01AT05076).
HEMODYNAMIC IMPACT OF NITRITE UNDER HYPOXIC CONDITIONS
P. Dungel*, C. Penzenstadler*, A. Zifko, H. Redl*, A.V. Kozlov*, S. Bahrami*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
Hemorrhage and resuscitation after trauma are associated with decreased hematocrit and hypoxia. Previously we showed that nitric oxide (NO) supplemented resuscitation (Sobhian et al., Am J Surg. 2011), temporarily decreased mean arterial pressure (MAP) but significantly improved perfusion in various organs with a strong trend to improve survival. Recently nitrite was identified as a potent oxygen-dependent NO-source. The aim of this study was to investigate the hemodynamic effects of nitrite under hypoxic conditions in a volume replacement model in rats.
Anesthetized rats were bled (0.5 min/ml) and Ringer’s solution was infused simultaneously (1.5 ml/min) to reduce hematocrit to 20 ± 2 %. Animals were randomly assigned to normoxia (NOX: pO2 >80 mmHg) or hypoxia (HOX: pO2 approx. 55 mmHg). Hypoxia was induced by inhalation of nitrogen/air containing 15% O2. After 10 min of hypoxia or equal normoxic period, animals received a bolus dose of nitrite (15μmol/kg). After 10 and 20 min blood samples were taken for blood gas analysis and to determination of NO-hemoglobin (NO-Hb) complexes. Hemodynamic parameters were determined by pulse wave analysis.
Hypoxia led to a significant decrease in pO2 (132.1 ± 7.0 vs. 58.9 ± 5.1 mmHg) and sO2 (95.4 ± 0.4 vs. 69.1 ± 4.9 %) in blood. Nitrite-derived NO assessed as NO-Hb complexes by electron paramagnetic resonance spectroscopy was 3.5 times higher in HOX compared to NOX group. Nitrite injection led to an immediate drop in mean arterial pressure (MAP) in both groups. While this was transient in the NOX group, in the HOX group MAP stayed significantly reduced (P<0.05) until end of experiment. In the HOX group vessel elasticity was significantly increased and vascular resistance decreased. This was associated with enhanced peripheral skin perfusion assessed by laser Doppler imaging (P<0.01).
Similar to NO-supplemented resuscitation nitrite under hypoxic conditions decreased MAP but increased peripheral perfusion. Additional studies will determine the impact on short- and long-term survival. FWF grant P21121.
MK-886 PREVENTS TRAUMA / SHOCK INDUCED BINDING OF FLAP AND 5-LO IN LUNG TISSUE
J.R. Stringham1, E.E. Moore*2, 1, F. Gamboni1, C.R. Ramos1, T.L. Chin1, M. Fragoso2, C.C. Silliman*1, 3, A. Banerjee*1. 1University of Colorado-Denver, Aurora, CO, 2Denver Health Medical Center, Denver, CO, 3Bonfils Blood Center, Denver, CO
Background: Leukotrienes are central in the pathogenesis of acute lung injury (ALI) following trauma and hemorrhagic shock (T/HS). Leukotriene production is catalyzed when 5-lipoxygenase (5-LO) binds its cofactor 5-lipoxygenase activating protein (FLAP). While MK-886 will bind FLAP and attenuate ALI, it is unclear how MK-886 affects 5-LO/FLAP interactions in the lung after T/HS. Hypothesis: MK-886 prevents 5-LO and FLAP binding in the lung after T/HS, thereby reducing leukotriene production and subsequent ALI.
Methods: Rats given MK-886 (6 mg/kg) or vehicle (DMSO) underwent T/HS (laparotomy, femoral artery cannulation, hemorrhage to MAP of 30 mmHg, and resuscitation). Control and trauma / sham shock (T/SS) rats were obtained (n = 3 for each group). Fluorescence resonance energy transfer (FRET) microscopy was used to visualize 5-LO/FLAP associations. Lung sections labeled for 5-LO and FLAP were imaged. FRET signal was determined and compared via ANOVA with Tukey-Kramer post hoc analysis.
Results: T/HS + vehicle showed increased FRET signal vs. Control and T/SS (p < 0.001). MK-886 abrogated the 5-LO/FLAP complexes during T/HS. Normal tissue architecture was preserved in Control and T/SS. T/HS + vehicle showed alveolar damage, while little was seen in T/HS + MK-886.
Conclusion: Pre-treatment with MK-886 caused less tissue damage than T/HS pretreated with vehicle. MK-886 also abrogated 5-LO/FLAP complexes. By decreasing the activating complexes of FLAP and 5-LO, MK-886 may limit leukotriene production and attenuate ALI following T/HS.
MMP-9 MEDIATED CHANGES IN INFLAMMATION AND CARDIAC DYSFUNCTION ASSOCIATED WITH SEVERE BURN
D.L. Carlson, S. Wolf*. University of Texas Southwestern, Dallas, TX
Introduction: MMP’s are endopeptidases that affect cardiac remodeling. We have shown that with severe burn injury (40% TBSA+ fluid resuscitation [4ml/kg/% burn]) protein levels of MMP9 are systemically increased significantly at 2,4 hrs after injury and are depressed particularly in the heart.
Purpose: To define the contribution of MMP9 in the inflammatory response and cardiac dysfunction associated with burn.
Experimental Design: FVB.Cg-Mmp9tm1Tvu/J (MMP9 deficient) and FVB/NJ (control) mice were divided into sham/ burn groups. ECHO (4, 8,12,24,48 hr) was used to assess systolic function. Cardiac and circulating cytokines were measured and RNA analyzed (IL-6, IL-10, TNF, IL-1β, MIF, caspases [1, 3, 8], and MMP [2, 3, 9]) Zymography was used to assess MMP activity alterations.
Results: Systemic analysis demonstrated that only TNF was increased by the loss of MMP9 (692 pg/ml transgenic, 356 pg/ml control). Cardiac specific RNA; levels more than doubled at 4 hr after burn compared to control. Cardiac changes were also recorded in MIF, MMP2, and caspase-1. Caspase-1 cardiac RNA was significantly increased over control animal expression at all time points. Fractional shortening decreased and was more severe in MMP-9 deficient animals (32% at 4 hr) as compared to a 12% drop at 8 hr in control animals.
Conclusions: MMP-9 deficiency led to increased TNF expression both systemically and locally, as well as increased cardiac expression of caspase-1. These changes are associated with a decrease in cardiac function as compared to control, suggesting that MMP9 may contribute through either caspase-1 and/or TNF to affect cardiac function and inflammation after severe injury.
VARIATIONS IN HEMOGLOBIN IN BURNED PATIENTS
K. Manchanda, M.J. Lee, J. Triantafyllou, S. Wolf*. University of Texas Southwestern, Dallas, TX
Burn patients are initially managed by stabilizing the Airway, Breathing, and Circulation. Because of severe blood loss from the injury, transfusions with packed RBCs have been used to prevent anemia. But there is no strict protocol as to when transfusions should be administered and little research has been done to analyze the variations in hemoglobin over time.
In this retrospective study, 63 random samples were collected from an inpatient database in 2011-12. The patients studied were at least 18 years old and had at least 20% TBSA burns. Hemoglobin measurements are recorded on routine CBCs and this data is recorded across hospitalization along with the time of blood withdrawal. Of the 63 patients, 24 were placed on a defined transfusion protocol. Group A (n=13 patients) was on the traditional protocol with hemoglobin maintained at 10g/dL and Group B (n=11 patients) on the restrictive protocol, maintaining a hemoglobin concentration of 8g/dL. Group C (n=39 patients) was not on a strict protocol.
The data was plotted for all 3 groups to demonstrate variability in hemoglobin with time for the first 50 days of hospitalization. The hemoglobin concentration was initially high (>15g/dL), most likely reflecting the loss of both fluid and RBCs and then decreases markedly. In Group A, the hemoglobin stabilizes between 10-11 g/dL, while in Group B & C it is between 7-8 g/dL within 3-4 days. After this, the hemoglobin value stays relatively constant with little variability among subjects despite blood withdrawals and transfusions. The data from Group C follow a first order curve which fits closely to a logarithmic regression with an r∧2 value of 0.8. While, the data from the transfusion protocols had very poor regressions (r∧2 value in Group A: 0.37 & Group B: 0.44).
Maintaining fluid and cellular volume are key to stabilizing burn patients when admitted. We showed that enforcing a protocol elicits a bad correlation, and that it did not work in stabilizing the hemoglobin across the population. The group that did not undergo any protocol showed a great correlation that hemoglobin concentration decreases logarithmically in the first few days of hospitalization and then stabilizes into a narrow range. This is perhaps related to monitoring and intermittent transfusion to compensate for ongoing losses and inadequate synthetic response. These findings will help define a more useful approach to transfusions in burn victims.
AUGMENTATION OF VISCERAL BLOOD FLOW FOLLOWING HEMORRHAGIC SHOCK ATTENUATES POST-SHOCK ACUTE LUNG INJURY
J.W. Smith1, P.J. Matheson1, 2, T. Rogers1, B.G. Harbrecht*1, R.N. Garrison1, 2. 1University of Louisville, Louisville, KY, 2Louisville VA Medical Center, Louisville, KY
Augmentation of visceral bloodflow (VBF) with peritoneal dialysis solution (DPR) during resuscitation from hemorrhagic shock (HS) mitigates visceral organ injury, but, the effect on the lungs has not been evaluated. We hypothesized that DPR would decrease acute lung injury (ALI) after HS. Using a rodent model of HS, rats were bled to 40% MAP for 60 min to induce HS and were randomly assigned to CR group (shed blood + NS, n=8), or DPR group (CR + 30 ml of intraperitoneal 2.5% Delflex, n=8). Proteomic, physiologic, and immuno-histological section were evaluated. Significance was assessed via one-way ANOVA and Tukey-Kramer at p < 0.01.
VBF was enhanced in the DPR group. Lung sections are shown below. Pulmonary MPO levels were reduced in the animals treated with DPR. Wet-to-dry weight ratios for the DPR group were decreased compared to CR. (Sham 4.92+0.08; CR 5.37+0.12; DPR 4.91+0.07, p<0.001) Cytosolic migration of HMGB-1, consistent with the breakdown of nuclear/cellular integrity, was present in CR animals, but not noted in DPR animals. Selected pulmonary and serum monokines are shown in the table below. Following HS, animals with enhanced VBF had less pulmonary edema and ALI compared to animals receiving CR alone. Thus, DPR decreases lung injury following HS. Selected Pulmonary and Serum Cytokine Data.
ALTERATION OF THE INFLAMMATORY RESPONSE AFTER BLUNT CHEST TRAUMA BY INTRATRACHEAL INSTILLATION OF APOPTOTIC NEUTROPHILS
A. Palmer, U. Niesler, A. Rittlinger, M.S. Huber-Lang*, F. Gebhard*, D.H. Seitz. University of Ulm, Ulm, Germany
Blunt chest trauma induces severe local and systemic inflammatory alterations. In this context, the pathophysiological consequences of apoptotic polymorphonuclear granulocytes (PMN) are unknown. Thus, in this study the effect of an intratracheal instillation of apoptotic PMN or heat inactivated E. coli 2, 24 and 48 hours after blunt chest trauma was investigated.
Male CD rats were subjected to blunt chest trauma, induced by a single blast wave. For instillation PMN were isolated from whole blood from untreated rats. Cells were labeled with CM-DiI, and apoptosis was induced by UV radiation. Fluorescent labeled apoptotic PMN or E. coli were instilled into the lungs 2, 24 and 48 hours after blunt chest trauma or sham procedure. Two hours after instillation rats were sacrificed. Phagocytosis rate of the alveolar macrophages was evaluated by FACS analysis and concentrations of IL-6, IL-1beta, TNF-alpha, RANTES, CINC-1 as well as LIX were determined in bronchoalveolar lavage fluids (BAL).
After blunt chest trauma, the capacity of alveolar macrophages (AM) to ingest apoptotic PMN remained nearly unchanged. In contrast, the ingestion rate of E. coli by AM was significantly increased from 16%±4% to 33%±4% 24 hours after trauma and remained elevated. In regard to the cytokine profile in BAL fluids, instillation of E. coli into the lungs led to an additional increase of the proinflammatory cytokines, such as IL-1beta and IL-6, in addition to the cytokine response induced by the blunt chest trauma alone. However, instillation of apoptotic PMN revealed some anti-inflammatory effects, shown by significantly decreased concentrations of the proinflammatory cytokines IL-6 and TNF-alpha as well as reduced concentrations of the chemokines RANTES, CINC-1 and LIX.
The results indicate that the capacity of AM to ingest and eliminate is unaltered for apoptotic PMN but enhanced for E. coli after blunt chest trauma. Furthermore, apoptotic PMN in the lungs attenuate the posttraumatic inflammatory response. (Supported by DFG KFO200, KN 475/5-1).
DEFINING POST-INJURY HYPERFIBRINOLYSIS WITH THROMBOELASTOGRAPHY: TECHNOLOGY IN EVOLUTION
C.R. Ramos1, E.E. Moore*1, M.P. Chapman1, C.C. Silliman*1, 3, E. Villalobos-Menuey2, M. Manco-Johnson2, A. Banerjee*1, A. Sauaia1. 1Trauma Research Center, Department of Surgery, University of Colorado Denver, Aurora, CO, 2Department of Pediatrics: Hematology/Oncology/BMT, University of Colorado Denver, Aurora, CO, 3Bonfils Blood Center, Denver, CO, Aurora, CO
Introduction: Trauma-induced coagulopathy due to hyperfibrinolysis is a major factor affecting survival in the critically injured. The definition of hyperfibrinolysis is highly debated. Thromboelastography (TEG) can be used as a point of care test to detect hyperfibrinolysis. The gold standard for evaluating fibrinolysis is the euglobulin lysis time (ELT). This lengthy procedure is impractical for evaluation of the bleeding trauma patient. We therefore sought to determine the level of fibrinolysis as reported by this viscoelastic assays that corresponds to this gold standard definition of hyperfibrinolysis, an ELT<90 minutes.
Methods: 105 trauma patients admitted to our regional trauma center were included in this prospective observational study. Citrated blood was collected in the field following injury or at admission. In vitro activation of the intrinsic pathway with kaolin (TEG) was performed within 2 hours of sampling. TEG parameters including percent lysis at 30 (LY30) and 60 (LY60) minutes were measured. Plasma was stored and the ELT was performed. The threshold for hyperfibinolysis via TEG was determined using receiver operating characteristic curve (ROC) analysis according to each sample’s corresponding ELT.
Results: ROC analysis of the 105 trauma patients (median ISS = 11, mortality = 8.6%) revealed that performance of TEG and ROTEM were below desired levels for detection of hyperfibrinolysis . A subgroup analysis of those at risk for shock, defined as an arrival base deficit ≥ 6, systolic blood pressure less than 90, or any transfusion requirement (median ISS = 22, mortality = 17.3%, n=46), showed an improved performance of both parameters.
Conclusion: These data have implications for the diagnosis of hyperfibrinolysis using TEG in trauma patients with currently accepted cutoffs being too high (LY30 = 7.5% and LY60 = 15%), but at lower levels results are confounded by the independent effects of platelets on clot stability, particularly in the less severely injured. Further investigation to determine entrance criteria for future clinical trials treating hyperfibrinolysis based on this viscoelastic assay is needed. These data corroborate with recent outcomes based studies which show that different cutoffs predict mortality.
NEUTROPENIA DELAYS ACUTE KIDNEY INJURY IN A MURINE STX2 TOXEMIA MODEL
C.S. Leibowitz, C. Mayer, S. Kurosawa*, D. Stearns-Kurosawa*. Boston University School of Medicine, Boston, MA
Introduction: Neutrophils interact with many bacterial toxins including LPS, pertussis toxin, anthrax toxins, and toxin A from Clostridium difficile. Enterohemorrhagic Escherichia coli (EHEC) which produce ribosome inactivating Shiga toxins (Stx1, Stx2) cause ∼73,000 infections annually in the U.S. and inflict a large economic burden. Stx from this intestinal infection are potent EHEC virulence factors, causing complications such as hemolytic uremic syndrome (HUS) and acute kidney injury (AKI), and inflammation accompanied by cellular stress responses. Mechanisms of toxin distribution to kidneys are not well understood, but chemokines are up-regulated after EHEC infection.
Objective: Determine if neutrophils contribute to pathogenesis in a mouse model of Stx2 toxemia by inducing neutropenia prior to challenge.
Methods: Mice were challenged i.p. with a lethal dose of either Stx2 alone (1∼1.2 ng/20 g b.w. on days 0 and 3) or following i.p. cyclophosphamide (100 mg/mouse; days 0 and 3; n=10) or anti-Ly6G (250 ug/20 g b.w.; days -2 and 1; n=10). Controls received sterile normal saline. Neutropenia was confirmed on days 0 and 3 by CBC (0.05-0.44 K/uL; normal 0.17-1.09 K/uL). Weight and kidney function (BUN) were monitored. Animals that reached euthanasia criteria were sacrificed and organs harvested for mRNA analysis by qPCR.
Results: All Stx2 challenged mice developed AKI, inflammatory responses and death by day 5. Plasma BUN increased in to 376 ± 52% of pre-treatment values by day of death. Mean mRNA levels of kidney chemokine KC and cell stress marker CHOP at time of death were 163-fold (42%-404%) and 7-fold (7.4-31.3%) of saline controls, respectively. On Day 3, animals receiving Stx2 alone had higher plasma BUN (50.67 ± 22.2 mg/dL; n=19) compared to those treated with cyclophosphamide (24.11 ± 5.4 mg/dL; p<0.005) or Anti-Ly6G (35.8 ± 9.8 mg/dL; p<0.05).
Conclusions: Delay of AKI in neutropenic animals suggests an early role for neutrophils in the pathophysiology of Stx2 toxemia in mice. Survival and increased expression of inflammatory marker KC and cell stress marker CHOP due to Stx2 were unchanged as a consequence of neutropenia, however AKI was consistently delayed. Manipulation of neutrophil trafficking pathways early in disease time course may represent a novel treatment option.
NONINVASIVE OPTICAL MEASURE OF MUSCLE OXYGENATION IN SUSPECTED SEPTIC SHOCK
K.A. Schenkman, D.J. Carlbom, E.M. Bulger*, W.A. Ciesielski, D.M. Fisk, K.M. Asplund, K.L. Sheehan, L.S. Arakaki. University of Washington, Seattle, WA
Introduction: In impaired cardiac output and decreased peripheral perfusion, muscle oxygenation may decrease before brain and central organs due to preferential shunting of blood. We hypothesize that muscle oxygenation may serve as an important early indicator of shock in sepsis.
Methods: A novel noninvasive optical monitor was developed to measure muscle oxygenation from the thenar muscles of the hand. A bifurcated optical probe was designed to illuminate the muscle and return light to a cooled CCD array detector. Optical spectra were analyzed by multiwavelength analysis to determine muscle oxygenation. The probe was placed on the hand of subjects admitted to the ED or ICU with suspected sepsis as soon as possible after identification, and optical data were collected for 15 minutes. Clinical shock severity was determined by a score based on the highest heart rate, lowest systolic pressure and highest lactate recorded during a 1 hour period in which optical data were collected.
Results: Muscle oxygenation (Mox) was determined in 32 patients, and was 79 +/- 24% in patients with mild shock (n=8), and 43 +/- 21% in patients with moderate shock (n=23). One patient had severe shock, and had a Mox of 36%. Healthy control subjects had a mean Mox of 94+/-6%.
Conclusions: Muscle oxygenation, measured noninvasively upon identification of possible sepsis correlated with shock severity determined by conventional clinical measures. We believe our novel optical monitor of muscle oxygenation may be useful in identifying patients at risk for worsening shock, and may be helpful in monitoring response to therapy.
STUDY OF THE EFFECTS OF METOPROLOL THERAPY IN EXPERIMENTAL SEPTIC SHOCK
A.L. Corrêa*1, D.T. Fantoni*2, J.O. Auler*1, D.A. Otsuki*1. 1LIM08-Anesthesiology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil, 2Department of Surgery, Faculty of Veterinary Medicine, University of São Paulo, São Paulo, Brazil
Introduction: Recent studies have demonstrated beneficial effects of β-blockers in septic patients, providing cardioprotection, attenuating systemic inflammation and reducing the mortality rate. This study was the first one to look for beneficial effects of metoprolol therapy, a selective β1-receptor blocker, in an experimental model of septic shock.
Methods: Twenty pigs in which septic shock was induced through intravenous E. coli infusion (6x109 c.f.u/kg in 2h) were randomly assigned (n=10 per group) to the Metoprolol group (214.2μg kg-1 of metoprol infused in 45 minutes) or Control group, which received a correspondent volume of normal saline. Hemodynamic parameters were then evaluated at baseline, T120 (end of bacteria infusion), T180, T240, T300 and T360 minutes. During this period, a resuscitation protocol with lactated Ringer’s solution, norepinephrine and dobutamine was used to maintain the mean arterial pressure > 65mmHg, the central venous pressure between 8-12mmHg and the mixed venous oxygen saturation > 65%. The hemodynamic data were analyzed using the two-way repeated measures ANOVA while the total volume of fluid and total amount of norepinephrine and dobutamine infused were analyzed by the Student’s t-test. Survival analysis was performed using Kaplan-Meier log-rank test.
Results: A similar behavior was observed in both groups and most of the hemodynamic parameters did not show any significant difference between groups, except for the higher values of pulmonary vascular resistance index (PVRI) at T300 and T360 (p=0.004 and p<0.001, respectively) in the animals that received metoprolol. No difference was observed between groups in relation to the total volume of fluid (p=0.914), the total amount of norepinephrine (p=0.069) and dobutamine (p=0.560) infused, and the survival rate (Control=5 and Metoprolol=4).
Conclusions: Although some studies have demonstrated several benefits with the use of β-blockers in patients with sepsis and show promising results, no benefit was observed when analyzing the hemodynamic parameters, the quantity of fluid, vasopressor and inotropic agents used, and the survival rate. Further analysis are being performed to look for possible advantages in using metoprolol in septic patients and maybe future studies are needed to establish an optimal dose of this drug in this specific situation.
Acknowledgments: Grants from FAPESP 11/21327-2 and CAPES.
AMINOGLYSIDE ACTIVITY IS MODULATED BY THE NUTRIENT CONCENTRATION IN DEVELOPING BIOFILMS
M.J. Henry-Stanley*, D.J. Hess*, C.L. Wells*. University of Minnesota, Minneapolis, MN
Critically ill patients are at high risk for developing biofilm infections associated with catheters, sutures, and other indwelling devices. Within biofilms, bacteria are antibiotic resistant, although if removed from the biofilm, these cells have antibiotic susceptibilities similar to planktonic (free-living) counterparts. The mechanism responsible for biofilm antimicrobial resistance is unclear, and may involve metabolically inactive cells resulting from nutrition depletion, restricted antibiotic penetration of bacteria within the matrix, and differences in bacterial gene expression. Because environmental factors may be important, experiments were designed to study the effect of nutrient concentration on the susceptibility of developing Staphylococcus aureus biofilms to gentamicin, vancomycin, ampicillin and streptomycin. S. aureus was incubated with silk suture in tryptic soy broth with 0.2% glucose (TSBg) in 1/3x, 1x, and 3x concentrations supplemented with antibiotic that included the minimum inhibitory concentration (MIC) for planktonic cells, and up to 10x the MIC. After overnight incubation, sonicated biofilms were quantified for viable bacteria, and crystal violet was used to quantify biofilm biomass. Gentamicin uptake was viewed using gentamicin-Texas red. Increased nutrients (3x TSBg) overcame the bactericidal effect of gentamicin, quantified by bacterial numbers and biomass (P<0.01), and this modulation was not due to differences in glucose or NaCl concentration, and 3xTSBg was associated with decreased (P<0.01) uptake of Texas red-gentamicin. Comparable results were obtained with streptomycin (another aminoglycoside) but not the cell wall active vancomycin or ampicillin. Thus, increased nutrition could overcome the bactericidal effect of aminoglycosides in developing S. aureus biofilms. These data have important implications for designing susceptibility testing of biofilms and for understanding how variations in the in vivo environment might affect antimicrobial susceptibility.
GLUTAMINE TREATMENT INCREASES CLAUDIN-3 EXPRESSION AND NUCLEAR LOCALIZATION OF OCCLUDIN IN HEAT STRESSED INTESTINAL EPITHELIAL CELLS
J.L. Van Swol, C.H. Baird*, A. Kallweit, L. Khailova*, P.E. Wischmeyer*. University of Colorado Denver, Aurora, CO
Background: Previous studies have shown that glutamine (GLN) can decrease patient mortality in clinical critical illness and can also protect intestinal epithelial cells (IEC) in vitro from a multitude of stressors. GLN has been shown to be essential to gut barrier function however specifics on tight junction proteins remain to be elucidated.
Purpose: The purpose of this study was to investigate the effects of GLN on tight junction protein expression and cellular localization in both non-stressed and HS IEC-18 cells.
Methods: IEC-18 cells were treated for 15 min +/- 10 mM GLN and exposed to heat-stress for 45 min. Cells recovered for 2.5 hours and then were evaluated for MATH1, occludin, claudin-1, claudin-3, and ZO-1 expression via western blot analysis. Cell morphology and the localization of claudin-1, claudin-3, and occludin were assessed via microscopy. Cell survival was evaluated via cleaved caspase-3 (CC3) expression.
Results: GLN treatment decreased CC3 expression by 87% in HS cells (p<0.004 vs. non-treated GLN groups). GLN increased claudin-3 expression in HS IEC-18 cells by 53% (p=0.03 vs. non-GLN treated cells). Interestingly, microscopy showed higher levels of nuclear occludin in cells treated with GLN both in the presence and absence of stress. MATH-1, claudin-1, and ZO-1 expression and cellular localization were not affected by GLN treatment.
Conclusion: This is the first reported indication that GLN affects the expression of the claudin-3 tight junction protein in HS IEC-18 cells. GLN also increased the nuclear localization of occludin in both HS and non-stressed cells. Further studies are needed to determine what implications nuclear occludin may have on GLN’s mechanism of cellular protection.
ESTROGEN ENHANCEMENT OF THE FETAL INTESTINAL MUCOSAL BARRIER: IMPLICATION FOR PREVENTING NECROTIZING ENTEROCOLITIS
L. Diebel*, D. Liberati, C.W. Manke. Wayne State University, Detroit, MI
Objective: The pathogenesis of necrotizing enterocolitis (NEC) in premature infants is incompletely understood but likely involves immaturity of the physical and innate immune barriers of the gut, inappropriate gut microbial colonization and hypoxic-ischemic events to the intestine. Estrogen (E2) has been found to modulate intestinal epithelial barrier function and intestinal mucus layer protection in vitro and in adult rodent models. The high E2 levels in third trimester placental blood may be important in gut barrier maturation. We studied the effect of E2 on physical and innate barrier properties of fetal intestinal epithelial cells in vitro.
Methods: Confluent FHs74 fetal intestinal epithelial cell (IEC) monolayers were established. Experimental conditions included E2 pretreatment for 72 hours and/or hypoxia-reoxygenation (H/R). TNFα, IL-6 and TGFβ were determined by ELISA at 240 minutes. Intestinal monolayer permeability was determined using FITC-dextran (FD4). Mucus thickness and storage modulus (G’), an index of mucus gel elasticity, were determined using an ARES rheometer. Additional experiments were conducted using Cronobacter sakazaki to assess barrier function in our model.
Conclusions: E2 pretreatment improves fetal IEC barrier function and decreases cytokine release following H/R and/or bacterial challenge. This is likely due to enhancement by E2 of the physical and mucus barriers in the fetal intestine. Additional in vitro studies are warranted.
Results: Mean ± S.D., N=4 for each group
CELL DEATH IN PORCINE MODEL OF NECROTIZING ENTEROCOLITIS IS NOT OXIDATIVE STRESS MEDIATED APOPTOSIS
A.Q. Sugihara1, Q. Meng1, S. Roy1, G. Nieman1, D. Clark2, R. Cooney*1. 1SUNY Upstate Medical University, Syracuse, NY, 2Children’s Hospital at Albany Medical Center, Albany, NY
Introduction: Necrotizing enterocolitis (NEC) is a leading cause of neonatal morbidity and mortality among premature infants. Known risk factors include prematurity, early enteral feeding and bacterial colonization. Existing NEC models commonly rely upon hypoxic or ischemic insult, however the necessity of this is unclear. In our novel porcine model, the NEC phenotype of severe intestinal necrosis is produced when weanling pigs are tube fed formula fermented by non-pathogenic bacteria, but not when they are given formula or bacteria alone. We hypothesized that short chain fatty acids produced by fermentation cause the NEC phenotype through oxidative stress induced apoptosis. To assess this and further characterize the model, we measured the expression of oxidative stress and apotosis related and genes.
Methods: Standard infant formula was fermented with non-pathogenic E Coli bacteria from human infants with NEC to a pH of 5.5. Three groups were studied, weanling pigs tube fed either bacteria plus formula (B+F), unfermented formula alone (F) or bacteria alone (B). Small intestine segments were harvested at 6 hours and frozen in liquid nitrogen. We examined the oxidative stress Relative levels of RNA and protein were measured by RT-PCR and Western blot normalized to β-actin. Data expressed as mean, ±SE and analyzed via ANOVA and t-test (p<0.05)
Results: All pigs treated with B+F developed clinical and histological signs of NEC. However there were no statistically significant differences in mRNA or protein expression of Catalase or HO-1 between treatment groups. SOD-1 protein levels were one third lower in the B+F group (p<0.05 vs B). However, this was not true of mRNA levels, which were similar. Caspase-3 mRNA levels were one third lower in the B+F group, (p<0.05 vs B, p<0.01 vs F) but protein levels were nearly equal. There was no difference in BAX, BCL-2 or the BAX/BCL-2 ratio between groups.
Conclusion: Although the porcine model recapitulates the clinical and histological phenotype of NEC, oxidative stress does not appear to play a major role in mediating apoptotic cell death in this model. Other potential mechanisms, e.g., direct effects of short chain fatty acids on cell membranes should be explored.
A PORCINE MODEL OF EXSANGUINATION CARDIAC ARREST
J.S. Berry, A. Batchinsky*, S. Belenkiy, C. Kendrick, C. Necsoiu, K.M. Ivey, D. Thomas, M.A. Dubick, J. Salinas, T.E. Rasmussen, L. Cancio*. United States Army, Fort Sam Houston, TX
Background: Hemorrhage remains the major cause of preventable death in combat. A model of exsanguination was developed to monitor cardiac function until death as a prelude to novel interventions.
Methods: Eleven anesthetized Yorkshire swine (61.0±3.1kg) had sensors (EKG, left ventricular pressure (LVP) and volume, aortic blood pressure (BP)) and a tracheostomy placed. Stroke volume (SV), heart rate (HR), LVP, LV change in pressure over time (dP/dt), respiratory rate (RR), and tidal volume (TV) were monitored. Animals were awakened, consciously sedated with midazolam while spontaneously breathing, then hemorrhaged over 20 min until 80% total blood volume hemorrhage (TBVH) or death. Measurements were taken at baseline (BL); 25%, 44%, 62% TBVH; and cardiac arrest (CA). CA was defined as systolic BP < 20 mmHg with EtCO2 < 10 mmHg. Death was defined as BP=0. During hemorrhage, if minute ventilation decreased 25% from BL for >20 seconds, volume control (VC) ventilation was initiated. One-way, repeated measures ANOVA with Dunnett’s tests were performed.
Results: Mean TBVH at death was 66.6±2.8%. There were no changes in RR or TV until 62% TBV was reached; then, all subjects were on VC (RR BL: 33.7±2.2 vs 62% TBVH: 18.6±2.8, p<0.001 and TV BL: 279.7±18.8 vs 62% TBVH: 469.1±82.8, p<0.04). Mean times to CA, death, and unorganized rhythm, ventricular fibrillation or asystole, were 23:05±1:23, 25:30±00:15 and 34:30±2:30, respectively. Between BL and 62% TBVH, SV decreased 63% (p<0.004) and HR increased 49% (p=0.01). Mean LVP fell 50% between BL and 25% TBVH (p<0.001). Contractility as measured by dP/dt was maintained even after clinical cardiac arrest.
Conclusions: A novel, reproducible model of exsanguination in consciously sedated swine was developed. The maintenance of myocardial contractility after clinical cardiac arrest and the persistence of organized cardiac electrical activity after death suggest a return of spontaneous circulation may be possible.
PATHOPHYSIOLOGIC REACTION OF AWAKE, UNRESTRAINED RATS TO HEAT STROKE
Y. Zhao1, 2, J. Gao3, B. Luo2, S. Liu1, W. Wan2, Y. Wu1, J. Guo2, X. Ding1, M. Lin4, F. Zou2, M. Fan1. 1Beijing Institute of Basic Medical Sciences, Beijing, China, 2School of Public Health and Tropical Medicine, Southern Medical University, Guang Zhou, China, 3Department of Physiology, Jilin Medical College, Jilin, China, 4Department of Medical Research, Chi Mei Medical Center, Taibei, China
Purpose: To describe the physiologic and pathophysiologic reaction of awake, unrestrained rats to heat stroke in hot environment, and to discuss awake, unrestrained rat’s heat stroke model.
Methods: Heat stroke was induced by putting the adult male Wistar rats in a hot chamber with or without pentobarbital-anaesthetization, and mean arterial pressure (MAP), heart rate (HR) and core temperature were continuously monitored. For anaesthetized rats the onset of heat stroke was taken as the time at which MAP fell to about 25mmHg from peak level, and for awake, unrestrained rats the onset of heat stroke was taken as two time points, one was the time at which MAP fell to about 25mmHg from peak level as anaesthetized rats, and the other was MAP begin to fell from peak level. After the onset of heat stroke, rats were sacrificed for organs injury inspection.
Results: MAP, HR and core temperature of awake, unrestrained rats increased much quickly than that of anesthetized rats, and MAP and HR dropped from the peak level much quickly than that of anesthetized rats. Heat treatment resulted into liver edema, pulmonary hyperemia and hemorrhage, brain edema, cerebellar subarachnoid hemorrhage, neural cell necrosis, renal dysfunction, and myocardium injury. Awake, unrestrained, rats got much severe pulmonary hemorrhage, renal dysfunction and liver injury at the time point of MAP fell to about 25mmHg from peak level than heat stroke of anesthetized rats, however, injury upon awake, unrestrained, rats at the time point of MAP begin to fall from peak level was similar to heat stroke of anesthetized rats.
Conclusions: Anesthetized rats and awake, unrestrained rats exhibited nearly same pathophysiologic reaction to heat treatment, and the latter were more sensitive to hot environment. For awake, unrestrained rats’ heat stroke model, the onset of heat stroke should be defined as the time point of the MAP begin to fall from its peak level.
PERIOPERATIVE VISCOELASTIC COAGULATION TESTING AS A PREDICTOR OF POST-DISCHARGE VTE AFTER MAJOR SURGERY
S. Cheng, C.H. Baird, A. Almagro, E. Luzier, N. Pearlman, C. Gajdos, M. McCarter, Z. Tran, K. Nordenholz, D. Matero, P.E. Wischmeyer*. University of Colorado School of Medicine, Aurora, CO
Background: Major abdominal surgery patients have increased risk of subsequent venous thromboembolism (VTE). Hypercoagulable state contributes to the pathogenesis of VTE. It is unclear if perioperative viscoelastic coagulation testing is predictive of post discharge VTE. We performed a posthoc analysis of a study on major abdominal surgery patients to assess if severity of hypercoagulable state on viscoelastic coagulation tests was associated with VTE at 6 months after surgery.
Methods: This study was conducted with IRB approval. Patients were randomized to 1 of 2 heparin regimens for postoperative VTE prophylaxis while in hospital (subcutaneous or low-dose intravenous heparin). Blood samples were drawn in the OR prior to incision, and daily for 5 days. No patients received VTE prophylaxis after hospital discharge. Patients were followed for 6 months for symptomatic VTE (any pulmonary embolism + symptomatic DVT).
Results: 110 patients were randomized. Patients became hypercoagulable for 5 days after surgery as evidenced by increased clot rate on Sonoclot analysis. Follow-up was completed in 100% of patients. No inpatients developed symptomatic VTE, while 18 patients (16%) had symptomatic VTE at 6 months. In bivariate analysis of the intention-to-treat population, age, gender, ICU length of stay, smoking status, inpatient prophylaxis regimen, and BMI were not associated with 6 month VTE. The Sonoclot clot rate on day 5 was increased in patients subsequently diagnosed with subsequent 6 month VTE (40.4±15.1 U/min in patients with VTE vs. 31.9±16.4 U/min in patients without VTE, p=0.07), although this difference did not reach statistical significance. Within the group randomized to low-dose heparin infusion, this difference became significant (48.4±11.8 U/min in patients with VTE vs. 29.9±17.4 U/min in patients without VTE, p=0.01).
Conclusions: In this small exploratory study, clinical variables showed no association with delayed postoperative VTE after major abdominal surgery. However, the Sonoclot clot rate, a measure of activated fibrin polymerization, showed a trend towards elevation after surgery in patients who eventually developed symptomatic VTE within 6 months. Further prospective study of the Sonoclot and other viscoelastic coagulation tests for the identification of surgical patients at high risk of VTE after hospital discharge is necessary.
TRAUMA-HEMORRHAGE-INDUCED ACUTE LUNG INJURY IS ASSOCIATED WITH T-CELL-MEDIATED TH2 AND TH17 IMMUNE RESPONSES
M. Rani*1, E.M. Galvan1, Q. Zhang1, M.G. Schwacha*1, 2. 1University of Texas Health Science Center, San Antonio, TX, 2US Army Institute of Surgical Research, San Antonio, TX
Trauma-hemorrhage (TH) is frequently associated with inflammation-induced acute lung injury (ALI). While studies have implicated a role for T-cells in the development of inflammatory complications after major injury, it is unknown whether T-cells play a role in the development of ALI after TH. To study this, C57BL/6 male mice (7-8/group) were subjected to TH via a mid-line incision and withdrawal of 30% of the total blood volume. This procedure was followed by fluid resuscitation 1 h later. Sham mice underwent neither blood loss nor resuscitation. Lungs and bronchoalveolar lavage fluid (BALF) were collected 2 h after resuscitation. Lung tissue was assayed for myeloperoxidase (MPO) activity and subjected to collagenase/DNase digestion to isolate single cells for analysis. At 2 h after resuscitation, ALI was evident in the injured mice with a 5-fold increase in BALF protein levels and a profound increase in lung MPO activity, as compared with shams. The isolated lung cells were phenotyped and evaluated for cytokine profiles by flow cytometry. After TH, a large influx of CD3+ T-cells into the lungs was observed. Intracellular staining of T-cells for cytokines revealed a 3-fold increase in both IL-10+ Th2 and IL-17+ Th17 cells, as compared with shams. In contrast, the numbers of Th1 (IFNγ+) T-cells were significantly lower than Th2 and Th17 T-cells. In conclusion, these data demonstrate a profound influx of activated T-cells into the lung after TH that support a mixed Th2/Th-17 immune response that likely contributes to the development of ALI.
DAMAGE CONTROL RESUSCITATION WITH FIBRINOGEN IN NEAR-LETHAL UNCONTROLLED HEMORRHAGE WITH DELAYED EVACUATION
N. White*, X. Wang, N. Bradbury, S.A. Stern*. University of Washington, Seattle, WA
Background: Models of uncontrolled hemorrhage (UH) demonstrate improved short-term mortality with initially limited resuscitation as compared to standard of care. These studies largely simulate clinical scenarios with rapid evacuation. In the setting of battlefield and rural trauma, arrival to definitive care is often delayed necessitating enhanced damage control resuscitation strategies in order to reduce mortality. Early fibrinogen (FBG) depletion is associated with increased blood loss and poor outcomes in these settings and is an important component of the coagulopathy of trauma.
Objectives: 1) To develop a clinically relevant model of near-lethal UH inflicted via aortic tear with delay to definitive care for the development and study of novel damage control resuscitation strategies; 2) To evaluate the efficacy of damage control limited resuscitation supplemented with FBG concentrate in this same model of near-lethal UH with delayed evacuation.
Methods: 14 anesthetized swine (20[0.5]kg were bled to, and maintained at, a mean arterial pressure (MAP) of 30mmHg for 15 min (T=R0) in the presence of a 4mm aortic tear. At T=R0, animals were randomized to one of 2 groups based on resuscitation fluid: Hextend® (HEX; N=7) or HEX+FBG (N=7). All animals received 10ml/kg of HEX infused over 10 min at T=R0 and T=R40. HEX+FBG additionally received 120 mg/kg FBG at T=R0 only. At 180 min (T=R180; begin in-hospital phase), the aortic tear was repaired and all animals were aggressively resuscitated with 0.9%NaCl and shed blood to normalize physiologic parameters. Animals were observed for a total of 360min (T=R360).
Results: % 6-hour survival was 29% (2/7) and 86% (6/7) (p=0.035; Kaplan Meier Log Rank), while mean survival times were 155 (55) and 319 (41) minutes in the HEX and HEX+FBG (p=0.048; Kruskal-Wallis) groups respectively. Total hemorrhage volumes were 47 (4) and 38 (4) ml/kg (p= 0.08; T-test), while hemorrhage volume corrected for survival times were 0.8 (0.24) and 0.3 (0.07) ml/kg/min (p=0.03; T-test) in the HEX and HEX+FBG groups respectively.
Conclusion: We have developed a model of near-lethal UH with an extended simulated evacuation time (180 min). In this model, supplementation of damage control limited resuscitation with fibrinogen concentrate as a hemostatic adjunct significantly reduced blood loss and extended survival time.
RAC1 REGULATES THE PUMP FUNCTION OF ISOLATED LYMPHATICS FROM HEMORRHAGIC SHOCK RATS
Y. Si, C. Niu*, L. Zhang, Z. Zhao, Y. Zhang. Hebei North University, Zhangjiakou, China
The purpose of the present study was to investigate the regulating effect of small G protein Ras-related C3 botulinum toxin substrate 1 (Rac1) on the contractility and reactivity of lymphatics obtained from hemorrhagic shock rats. The isolated lymphatics from the control, 0.5 h- and 2 h-shocked rats were prepared for the observation of the contractility and reactivity to substance P (SP) with an isolated lymphatic perfusion system at a transmural pressure of 3 cmH2O. At the same time, lymphatics isolated from 0.5 h- and 2 h-shocked rats were incubated with agonists and antagonists of Rac1 signaling pathway. Contractile frequency (CF), end-diastolic and end-systolic diameter, and passive diameter were recorded and used to calculate lymphatic tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF). After stimulation with a gradient of SP, the differences between the pre- and post- administration values of CF, CA, TI and FPF were calculated to further assess lymphatic reactivity. The contractility and reactivity of 0.5 h-shocked lymphatics were significantly reduced by the platelet derived growth factor (PDGF), an agonist of Rac1, and ML-7, an inhibitor of myosin light chain kinase (MLCK), respectively; and the decreased effect of PDGF in contractility was reversed by SP, an agonist of MLCK. Meanwhile, the contractility and reactivity of 2 h-shocked lymphatics were significantly increased by the NSC 23766, an antagonist of Rac1, and PAK-18, an inhibitor of - p21-activated kinase (PAK), respectively; moreover, SP decreased the CA of 2 h-shocked lymphatics. These results suggest that Rac1 is involved in the modulation of lymphatic pump function during hemorrhagic shock and that its effects may be mediated by PAK and MLCK. This work was supported by the National Natural Science Foundation of China (30770845) and the Natural Science Foundation of Hebei Province (C2008000503).
IMPACT OF DIFFERENT HAEMODYNAMIC RESUSCITATION STRATEGIES ON BRAIN PERFUSION AND TISSUE EDEMA MARKERS IN A MODEL OF SEVERE HAEMORRHAGIC SHOCK
L.M. Malbouisson1, K.K. Ida1, D.A. Otsuki1, L.U. Castro2, T.R. Sanches2, M.M. Shimizu2, L.C. Andrade2, M.C. Carmona1. 1LIM08 Anesthesiology Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 2LIM12 Nephrology Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
Introduction: Fluid resuscitation may worsen neuronal injury associated to hemorrhagic shock (HS). This study investigated the impact of terlipressin (TERLI) on brain perfusion, apoptosis and edema markers in a model of HS.
Methods: HS was induced in 29 piglets to target MAP of 40 mmHg and maintained for 30 min. Animals were randomized into Sham (n=2), HS (n=9), LR (3x volume bled; n=9) or TERLI groups(4mg bolus; n=9). Brain tissue oxygen pressure (PbtO2) and intracranial pressure (ICP) were assessed by an intraparenchymal oxygen sensitive probe. Hemodynamics and metabolic variables were assessed at baseline, after HS and 60 min after treatment. Tissue markers of brain edema [aquaporin-4 (AQP4) and Na-K-Cl cotransporter-1 (NKCC1)] and apoptosis [pre-apoptotic protein (Bax)] were also measured. Data were analysed with ANOVA followed by Tukey test; p<0.05 was considered significant.
Results: Sham animals had (mean±SD) CPP of 65±4 mmHg, PbtO2 of 45±7 mmHg, ICP of 5±1 mmHg, MAP of 70±3 mmHg and blood lactate levels of 1.1±0 mmoL/L at 60 minutes after treatment. At this time-point, HS had decreased CPP (37±10 mmHg), PbtO2 (25±8 mmHg) and MAP (39±10 mmHg) compared to sham (p < 0.001). LR increased ICP (7±1 mmHg, p < 0.05) and decreased CPP (43±8 mmHg) and MAP (51±8 mmHg) at 60 minutes compared to sham (p < 0.001). TERLI resulted in no significant differences in CPP (58±8 mmHg) and MAP (61±10 mmHg) at 60 minutes compared to sham. Both treatments restored PbtO2 values (TERLI 34±8 mmHg; LR 31±8 mmHg) at 60 minutes post treatment. Blood lactate levels were increased in all groups at 60 minutes (HS 8.5±4.4 mmol/L; TERLI 7.2±3.2 mmol/L; LR 5.2±2.7 mmol/L; p < 0.001) compared to sham. The HS and LR groups had an increased expression of cerebral AQP4 (HS 167±54% of sham; LR 210±56% of sham), NKCC1 (HS 237±47% of sham; LR 163±32% of sham) and Bax (HS 167±44% of sham; LR 137±24% of sham), which were restored in response to TERLI (AQP4 100±6% of sham; NKCC1 100±1% of sham; Bax 102±6% of sham).
Conclusions: In our model, LR did not recover CPP probably due to an increased ICP associated with the increased brain water and sodium content, which may have contributed to the cerebral apoptosis. TERLI recovered cerebral perfusion and oxygenation with no significant changes in ICP and cerebral markers of edema and apoptosis.
CHANGES OF THE RENAL PROTEOME INDUCED BY MESENTERIC LYMPH DRAINAGE IN HEMORRHAGIC SHOCK RATS
Z. Zhao, J. Li, C. Niu*, L. Zhang, Y. Lv, Y. Zhang, Y. Si. Hebei North University, Zhangjiakou, China
Previous studies have indicated the post-shock mesenteric lymph (PSML) was an important contributor to acute kidney injury (AKI) after hemorrhagic shock. The aim of this study was to investigate the effect of PSML drainage on the proteome in renal tissue. Twenty-seven male Wistar rats were divided into sham, shock and shock+drainage groups. In shock and shock+drainage groups, the controlled hemorrhagic shock model (40±2 mmHg) was established; after 1 h of hypotension, the fluid resuscitation was implemented within 30 min; meanwhile, the PSML was drained in shock+drainage group. After 3 h of resuscitation, the renal tissue was taken out and the high-abundant protein was removed. And the proteome of kidney was analyzed using two-dimensional fluorescence difference gel electrophoresis. Proteins that increased or decreased in relative concentration ≥ 1.5-fold were selected for trypsin digestion and analysis by liquid chromatography-tandem mass spectrometry. Compared with the sham group, the heterogeneous nuclear ribonucleoprotein C (HNRNPC) and serine-threonine kinase receptor-associated protein (STARP) were decreased in shock group; the trifunctional enzyme subunit alpha (Hadha), solute carrier family 25, member 13 (Slc25a13), ATP synthase subunit beta, mitochondrial (Atp5b), HNRNPC, STARP, Actin alpha (Acta1) and 40S ribosomal protein S3 (Rps3) were down regulated in shock+drainage group. Meanwhile, the Atp5b and Acta1 were decreased in shock+drainage group than that of shock group. The findings of this study provide a starting point for investigating the functions of these proteins in AKI induced by hemorrhagic shock, and hold great promise for the development of potential therapeutic interventions. This work was supported by the Natural Science Foundation of Hebei Province (C2010001433) and Excellent Creative Talents Foundation of Hebei Province (CPRC047).
ADIPOKINE RESPONSES TO BURN AND DISUSE IN RATS
L.A. Baer1, X. Wu*2, S. Wolf*3, C. Wade*1. 1University of Texas Health Science Center-Houston, Houston, TX, 2US Army Institute of Surgical Research, Fort Sam Houston, TX, 3University of Texas Southwestern Medical Center, Dallas, TX
Introduction: Trauma, including severe burns, results in dramatic endocrine responses associated with systemic inflammatory and metabolic changes. These changes are biphasic and include the initial inflammatory ebb phase, which encompasses up to the first 72 hours after injury, then transitions to the hypermetabolic flow phase, which can persist for over a year. Adiponectin (ADPN) and leptin (LEP) are adipose tissue-derived cytokines (adipokines), which play important roles in the regulation of inflammation and metabolism after burn. The purpose of this study was to determine the association of changes in adipokines in relation to inflammation and metabolism during the ebb and flow phases post-burn using a rat model of burn with and without disuse.
Methods: Male rats: sham control (SA; n=4/ time point), burn (40% TBSA) without hindlimb unloading (BA; n=6/ time point) or burn with hindlimb unloading (BH; n=6/ time point). Plasma inflammatory cytokines (IL-6 and IL-10) and adipokines (ADPN and LEP) were measured on days 1, 3, 7 and 14.
Results: IL-6 and IL-10 were increased (p<0.05) in BA and BH on day 1 indicative of the inflammatory ebb phase. The increases were significantly greater in BH. Beyond day 3 there were no differences between groups in cytokine concentrations. Food intake per 100 g of body mass was reduced in BA and BH on day 1 and significantly increased in BH after day 3 indicative of the hypermetabolic flow phase. On day 1, LEP was increased 3-fold in BH. Subsequently, LEP was decreased in both BA and BH compared to SA. ADPN was reduced in BA and BH at all time points (p>0.05).
Conclusions: Burn injury, accompanied by long-term disuse, causes complex inflammatory and metabolic changes. During the ebb phase, an acute onset of inflammation in BH rats appears not to be a direct result of the burn injury alone, but a combination of the injury and disuse. During the hypermetabolic flow phase there is a reduction LEP and ADPN that is counter-intuitive suggesting dysregulation of these systems, therefore adipokines and inflammatory cytokines may play independent roles in inflammatory and metabolic changes. A complete understanding the changes of adipokines during the ebb and flow phases following burn injury may assist in treating the metabolic derangements observed in burned patients. Grant information: US Army MRMC & Juvenile Diabetes Foundation.
CHRONIC STRESS IMPAIRS BONE MARROW FUNCTION
L.E. Bible, L.V. Pasupuleti, Z.C. Sifri, W.D. Alzate, D.H. Livingston, A.M. Mohr. UMDNJ-New Jersey Medical School, Newark, NJ
Introduction: Rodent models of trauma and hemorrhagic shock have been shown to result in acute bone marrow (BM) dysfunction that has nearly resolved in one week. In contrast, patients that sustain severe traumatic injury and remain in the intensive care unit have sustained physiologic stress that is associated with persistent BM dysfunction that lasts more than two weeks. The aim of this study is to evaluate a model of restraint stress to examine the effects of chronic stress on rodent bone marrow (BM) function.
Methods: Male Sprague-Dawley rats (N=3-10/group) underwent a two hour period of restraint stress (RS) and every thirty minutes the rats were provoked by repositioning and an alarm. Day 1 RS animals were sacrificed three hours after a one period of RS (Day 1). Chronic RS animals were sacrificed on day 7 after six daily morning RS periods (Day 7). Naïve animals were used as controls. BM was harvested to obtain BM cellularity and growth of BM HPC (GEMM, BFU-E, CFU-E). Peripheral blood flow cytometry, using c-kit and CD71, was used to determine mobilization of HPCs. Data presented as mean±SD. *p <0.05 by ANOVA
Results: One day of RS had no impact on BM function or mobilization of HPCs (Table). Seven days of daily RS led to significant suppression of BM cellularity and BM HPC colony growth as compared to day 1 and naïve animals. Seven days of RS also led to significant mobilization of HPCs from BM to peripheral blood. There was no difference in hemoglobin between any of the groups.
Discussion: The duration of stress plays a key role in persistent BM dysfunction. Chronic stress leads to significant BM dysfunction, manifest by decreased BM HPC growth and increased mobilization of HPCs to the peripheral blood. Therefore, regulation of the chronic stress response may be important in ameliorating BM dysfunction in critically ill trauma patients. In addition, this model may be added to current injury and shock models to mimic the stress effects of critical illness after traumatic injury.
ACUTE INSULIN RESISTANCE DUE TO ECTODOMAIN INSULIN RECEPTOR CLEAVAGE BY PANCREATIC DIGESTIVE PROTEASES
F. DeLano1, 2, G. Schmid-Schönbein*1, 2. 1University of California San Diego, La Jolla, CA, 2UCSD Microcirculation Laboratory, La Jolla, CA
The small intestine following breakdown of the mucosal barrier plays a central role in physiological shock and its associated inflammation. We recently presented evidence that autodigestion by pancreatic digestive enzymes leads to multiorgan failure after shock (Science Translational Medicine 5(169):169ra11, 2013). Shock is also accompanied by acute insulin resistance and hyperglycemia and adverse metabolic changes which affect energy supply and organ blood flow. But the mechanisms responsible for the pathogenesis of insulin resistance in hemorrhagic shock are unknown. We hypothesize that decreased responsiveness of tissues to insulin involves cleavage of the extracellular domain of the insulin receptor mediated by pancreatic digestive proteases from the intestine. The ectodomain insulin receptor cleavage prevents insulin binding and signalling of glucose transport. To determine whether blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (at 35 mmHg) for two hours at which time all shed blood volume was returned with and without a protease inhibitor. Digestive proteases in the intestinal lumen were blocked with a serine protease inhibitor one hour after start of hypotension and the density of the insulin receptor was measured using immunohistochemistry (with an antibody targeting the extracellular insulin binding domain) in circulating leukocytes, cells in intestinal villi and in the mesentery microcirculation. Hemorrhagic shock produced a significant reduction of the extracellular binding domains of the insulin receptors in all cells we studied together with a reduced glucose transport determined by glucose tolerance test. Blockade of the digestive proteases in the lumen of the small intestine served to restore insulin receptor density compared to shock controls without protease blockade. These results indicate a significant protection against proteolytic cleavage of the insulin receptor ectodomain by enteral blockade of digestive enzymes in the small intestine after severe hypotension. Insulin resistance produced by proteolytic insulin receptor cleavage in acute shock may be similar to insulin resistance in chronic metabolic syndrome (Hypertension, 52: 415, 2008).
Supported by an unrestricted gift from Leading BioSciences Inc. and by NIH grant GM 85072.
CALCIUM CHANNEL BLOCKADE AND INTESTINAL BARRIER FUNCTION: AN IN VITRO STUDY
A. Ali*, T. Kamash, D. Liberati, L. Diebel*. Wayne State University/ Detroit Medical Center, Detroit, MI
Objective: Calcium (Ca2+) is a regulator of many cellular functions. Ca2+ homeostasis is disturbed following shock conditions. Intestinal epithelial cells (IEC) express L-type calcium channels. IEC actively transport secretory IgA (SIgA); the principle Ig in mucosal defense into mucosal secretions. Transcellular transport of IgA is dependent on Ca2+. Further, Ca2+ homeostasis is known to affect IEC cytoskeleton.
Methods: Confluent HT-29 IEC monolayers grown in a two-chamber cell culture system were held under hypoxic (5% CO2) conditions for 90 minutes followed by normoxia (21% O2) (H/R). Cell subsets were exposed to lipopolysaccharide (LPS-10μg/ml). Verapamil (8μM) was added to HT-29 cell subsets prior to H/R treatment. Dimeric IgA was added to the basal compartment and apical media was sampled at intervals to quantitate IgA transcytosis by ELISA. HT-29 cells held under normoxic conditions served as controls. HT-29 permeability to FD4 was assessed at the end of each experiment. In a separate experiment, HT29 cells were stained for F actin using rhodamine-labeled phalloidin.
Conclusions: Verapamil and LPS increased IgA transcytosis in both control and H/R treatment groups. Intestinal monolayer permeability was increased following treatment with H/R and/or LPS. Verapamil treatment prevented increased permeability likely due to protection of HT29 cytoskeleton integrity.
Results: mean ± SD, N = 5.
DOSE RESPONSES OF ACETAMINOPHEN AND MELOXICAM ON PLATELET AGGREGATION AND COAGULATION IN HUMAN BLOOD IN VITRO
A.K. Martini1, C. Moreno2, J. Guerra2, W. Martini*2, M.A. Dubick*2. 1Rice University, Houston, TX, 2US Army Institute of Surgical Research, Ft Sam Houston, TX
Introduction: Acetaminophen (Ace) and Meloxicam (Mel) are two types of analgesic and antipyretic medications that are included in the pill pack in the medic first aid bag for Soldiers in the deployed environment. Ace has been known to inhibit platelet aggregation, but its dose responses on clotting are unclear. Mel is a prescription drug and its effect on hemostasis is unknown. This study investigated the dose responses of Ace and Mel on platelet aggregation and coagulation function in human blood in vitro.
Hypothesis: Clinically relevant doses of Ace and Mel inhibit platelet aggregation and coagulation function in vitro.
Methods: Blood samples were collected in citrated tubes from normal healthy humans (n=3). Upon collection, blood samples were centrifuged and processed to make platelet adjusted (100 × 1000/μl) blood samples. Ace (Tylenol®, Q-PAP, 100mg/ml) was added to the samples at the doses of 0 μg/ml (control), 200 μg/ml (the recommended oral dose, referred as 1×), 4×, 8×, 10×, 12×, 16×, and 20×. Similarly, Mel (Metacam®, 5mg/ml) was added at doses of 0 μg/ml (control), 3μg/ml (1×), 4×, 8×, 10×, 12×, 16×, and 20×. Platelet aggregation was stimulated with collagen (2μg/ml) or arachidonic acid (0.5mM) and assessed using Chrono-Log 700 aggregometer. Coagulation function was assessed by initiation time (CT), clot formation time (CFT), clotting speed (α) and clot strength (MCF) using Rotem® thrombelastogram with Extem reagents at 10 min after the addition of Ace or Mel. All samples were run at 37°C.
Results: A robust inhibition by Ace or Mel was observed in arachidonic acid-stimulated platelet aggregation. At 1× dose, arachidonic acid- stimulated platelet aggregation was inhibited by Ace or Mel to 19%±7% or 28%±5%, respectively. At Ace doses of 4×, 8×, 10×, 12×, 16×, and 20×, collagen-stimulated platelet aggregation was inhibited to 74%±9%, 76%±11%, 69%±6%, 63%±8%, 49%±10%, and 22%±5% of the control values (all p<0.05), respectively, with similar inhibitory patterns shown from Mel. However, at all tested doses of Ace and Mel, there were no significant changes observed in Rotem measurements of CT, CFT, α, or MCF.
Conclusions: Ace and Mel at clinically relevant doses compromised platelet aggregation, with no effects on clotting function in vitro. Further effort is warranted to characterize the effects of Ace and Mel on bleeding in vivo.
A PREOPERATIVE SERUM HIGH HMGB-1 LEVEL IS A RISK FACTOR FOR THE POSTOPERATIVE PULMONARY DYSFUNCTION IN PATIENTS WITH ESOPHAGEAL CANCER
R. Takahata, S. Ono*, H. Tsujimoto, S. Hiraki, A. Kimura, Y. Matsumoto, K. Yoshida, H. Horiguchi, S. Nomura, M. Kinoshita*, S. Aosasa, J. Yamamoto, K. Hase. National Defense Medical College, Tokorozawa, Japan
Background: To clarify the time course of changes in the serum high mobility group box chromosomal protein-1(HMGB-1) concentrations in esophageal cancer patients following esophagectomy and to investigate whether the serum HMGB-1 levels correlate with the postoperative clinical course and the occurrence of pulmonary complications in such patients.
Methods: Sixty patients who underwent right transthoracic esophagectomy or thoracoscopic esophagectomy for esophageal cancer were enrolled in this study. The correlation between the serum HMGB-1 levels and the patients’ perioperative clinical courses were evaluated.
Results: Preoperative serum HMGB-1 concentrations in patients with severe pulmonary complications were significantly higher than those in patients without severe pulmonary complications. There was a significant correlation between the preoperative serum HMGB-1 concentrations and the PaO2/FiO2 ratio on 3rd postoperative day (POD3) (r=-0.485, P<0.005). In the univariate and multivariate analyses, the use of neoadjuvant therapy and the preoperative serum HMGB-1 levels (>4.2 ng/ml) were found to be significantly associated with the pulmonary dysfunction, and the age (>70 years) and the chemotherapy/ chemoradiotherapy-grade (CT/CRT-grade) (0 or 1a, neoadjuvant therapy non-response) were found to be significantly associated with the preoperative serum HMGB-1 levels.
Conclusions: The preoperative serum HMGB-1 levels and the neoadjuvant therapy were risk factors for the occurrence of severe postoperative pulmonary complications in esophageal cancer patients following esophagectomy.
EFFECTS OF NITRIC OXIDE DONOR (NITROPRUSSIDE) AND INDUCIBLE NITRIC OXIDE SYNTHASE ENZYME INHIBITOR IN AN EXPERIMENTAL MODEL OF SEPTIC SHOCK
D.T. Fantoni2, 1, A. Monteiro2, 1, D.A. Otsuki1, P. Carnicelli2, 1, M. Kahvegian1, J.C. Auler1. 1Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 2Department of Surgery, Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo, Brazil
Introduction: Nitric oxide plays a key role in the pathophysiology of sepsis and its modulation could be an alternative treatment in this syndrome. This study aimed to evaluate the effect of nitroprusside and iNOs inhibitor (1400W) on hemodynamic, tissue perfusion and inflammation parameters in an experimental model of septic shock.
Methods: Eighteen anesthetized pigs were randomized into 3 groups: (1) Sham - anesthesia only; (2) Shock - E. coli infusion (4.5×109 c.f.u./ml in 60min) associated to a resuscitation protocol using lactated Ringer’s solution and norepinephrine and guided by MAP, CVP and SvO2, from T0 until T240; and (3) NO/iNOs - the same protocol used in Shock, but associated to an iNOs inhibitor and nitroprusside (from T120 to T240). We evaluated hemodynamic (pulmonary artery catheter) and tissue perfusion (intestinal tonometry) parameters, blood gas and inflammatory cytokines at baseline, T0 (end of bacteria infusion) and T240 (after 240 minutes).
Results: At T0, all animals from Shock and NO/iNOs presented tachycardia, hypotension, myocardial depression, pulmonary hypertension, increased lactate, impairment of regional tissue perfusion and increase in proinflammatory cytokines. At T240, NO/iNOs was significantly different from Shock in CI (5.4±1.7 vs. 3.6±1.7 L/min/m2, p=0.025), LVSWI (31±12 vs. 20±9 gm-m/m2/beat, p=0.01), SVI (31±12 vs. 24±11 ml/m2/beat, p=0.021), PAP (38±6 vs. 45±8 mmHg, p=0.05), SVRI (1605±597 vs. 1089±215 dyne s/cm5/m2, p=0.009) and PVRI (442±153 vs. 880±429 dyne s/cm5/m2, p<0.001). The tissue perfusion improved, with a decrease in the intestinal-arterial PCO2 gap (31±8 vs. 41±9, p=0.041) and an increase in intestinal pH (7.14±0.04 vs. 7.03±0.06, p=0.005), SvO2 (80±5 vs. 68±14%, p<0.001), arterial pH (7.37±0.02 vs. 7.29±0.07, p<0.001) and urinary output (2.5±1.2 vs. 0.5±0.7 ml/kg/h, p<0.001). IL-6 and IL-1 β were significantly lower in NO/iNOs (2044±585 vs. 2580±630 pg/ml, p=0.027; and 340±131 vs. 1134±469 pg/ml, p<0.001, respectively).
Conclusion: The conventional treatment using fluid resuscitation and vasopressor associated to a nitroprusside and iNOs inhibitor infusion improved the myocardial function, pulmonary hemodynamic and tissue perfusion, and modulated the inflammatory response in this experimental model of septic shock in pigs.
Grants: LIM08, FAPESP (2008/58875-4).
CHARACTERIZATION OF ECRG4 AS A SENTINEL IN ENDOTOXIN-INDUCED ACUTE LUNG INJURY
X. Lu, T.W. Costantini, A. Baird, B. Eliceiri*, R. Coimbra*. Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California San Diego Health Sciences, San Diego, CA
Background: Esophageal cancer related gene-4 (Ecrg4) is expressed on epithelial cells and leukocytes and functions as a sentinel protein that monitors the inflammatory state. We have identified a novel role for Ecrg4 in the injury response. We have demonstrated in animal models and clinical samples that Ecrg4 is expressed on the surface of leukocytes, shed from the cell surface after injury, and chemotactic for macrophages. Based on its function as a sentinel protein, we hypothesized that lung Ecrg4 expression would decrease in response to endotoxin-induced lung injury.
Methods: We examined the tissue distribution of Ecrg4 in C57/BL6 mice using immunoblotting. To further investigate the functional role of Ecrg4 in endotoxin-induced acute lung injury, we measured changes in Ecrg4 expression in the lung after LPS challenge (50mg/kg IP) using quantitative PCR and immunoblotting. Changes in lung Ecrg4 expression were also visualized with immunohistochemistry. LPS-induced lung injury was evaluated by changes in histology, ICAM-1, and MPO expression.
Results: Ecrg4 was expressed in the lung and spleen to a greater degree than the gut. LPS-induced lung injury was confirmed by evidence of histologic injury and by increased ICAM-1 and MPO expression. Lung Ecrg4 gene expression was decreased at early timepoints after exposure to LPS. This correlated with decreased lung Ecrg4 protein expression at 90 minutes and 6 hours after LPS challenge compared to sham (Figure). These findings were supported by changes in the immunohistochemical localization of Ecrg4 in the lung.
Conclusion: Endotoxin-induced lung injury is associated with decreased lung Ecrg4 expression. The characterization of a novel sentinel factor, such as Ecrg4, that responds to inflammatory stimuli could identify novel therapeutic targets aimed at improving outcomes in patients with severe injury and sepsis.
AMP-ACTIVATED PROTEIN KINASE AUGMENTS CYTOKINE-INDUCED NITRIC OXIDE SYNTHASE EXPRESSION IN HEPATOCYTES
B.G. Harbrecht*1, 2, I. Nweze1, 2, J. Lakshmanan1, 2, J.W. Smith1, B. Zhang1, 2. 1University of Louisville, Louisville, KY, 2Price Institute of Surgical Research, Louisville, KY
Introduction: AMP-activated protein kinase (AMPK) is a highly conserved Serine/Threonine kinase that is activated following metabolic stress. AMPK is primarily regulated by the AMP:ATP ratio but is also phosphorylated and activated by upstream kinases including Protein Kinase Cz, Akt, Calmodulin-dependent kinase, and LKB1. AMPK has insulin-like metabolic effects and AMPK activators are used clinically to treat diabetes. We have shown that insulin inhibits hepatocyte iNOS expression. Others have demonstrated that AMPK decreased iNOS expression in macrophages and myocytes. We therefore investigated the role of AMPK in hepatocyte iNOS expression.
Methods: Cultured rat hepatocytes were stimulated to produce iNOS with IL-1b (200 IU/ml)/IFN (100 IU/ml) in the presence and absence of AMPK inhibition (Compound C) or activation (DHPO, oligomycin). iNOS was measured by supernatant nitrite and protein after 24 hours.
Results: Compound C dose-dependently decreased nitrite and iNOS protein production while AMPK activators increased IL-1b/IFN-stimulated iNOS (Figure). DHPO and oligomycin alone without cytokines had no effect on iNOS (not shown).
Conclusions: AMPK increases iNOS expression in cultured hepatocytes stimulated with cytokines. Use of AMPK-activating medications for diabetes may predispose patients to increased hepatic iNOS production during inflammation.
HEMODYNAMIC EFFECTS DUE TO THERAPEUTIC HYPOTHERMIA AFTER POLYTRAUMA IN A PORCINE MODEL
M. Weuster1, A. Seekamp*1, P. Mommsen2, F. Hildebrand3, I. Witte4, J. Mohr5, M. van Griensven*6. 1Trauma Surgery, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany, 2Medical School of Hannover, Hannover, Germany, 3University Hospital of Aachen, Aachen, Germany, 4University Hospital of Duesseldorf, Duesseldorf, Germany, 5 University Hospital of Marburg, Marburg, Germany, 6Experimental Trauma Surgery, TU Munich, Munich, Germany
Introduction: Beside immediate deaths at the scene due to polytrauma, hemorrhage and blunt chest trauma depict high risks for cardiopulmonary failure. In addition, penetrating abdominal trauma aggravates patients clinical course with sepsis and multiple organ failure as late complications. Hypothermia (HPT) acts as an ambivalent player in multiple-trauma. Therapeutic effects of HPT even outcome after severe trauma are still unfamiliar.
Material and Methods: Length of protocol was 15.5hrs. 40 animals were included in our study, 20 pigs served as sham pigs without trauma (normothermia vs hypothermia). Further 20 pigs were exposed to blunt chest-, penetrating abdominal trauma and hemorrhage with a MAP of 30 ± 5mmHg for 90min. We rapidly initiated fluid resuscitation afterwards, restoring hemodynamic. Then mild HPT had been induced targeting 34°C for 4hours (n=10). Re-warming and intensive care observation followed. We used non-invasive and invasive devices, i.e. Swan Ganz catheter for obtaining hemodynamic continuously.
Results: Both sham and trauma pigs survived. Triple-trauma had maximum impact as indicated by lactate, base excess and arterial pH (p<0.05). Sham pigs differed significantly from trauma animals in hemodynamic. Comparing trauma pigs in HPT with trauma pigs in normothermia (4hrs): Heart rate was decreased (p<0.05), mean arterial pressure and central venous pressure did not differ. Cardiac output was lower. Mixed venous oxygen saturation (SvO2) showed higher values (p<0.05). Stroke volume was on equal level. Pulmonary artery pressure increased whereas WP decreased.
Conclusion: In summary our new polytrauma porcine model with the use of therapeutic HPT is feasible. The cardiovascular function showed major even protective effects. SvO2 could be detected as a leading clinical parameter. Critically speaking, time and depth of HPT should be prolonged and intensified in future studies.
ABSENCE OF ENDOGENOUS TOLL-LIKE RECEPTORS (TLRS) WORSENED SECONDARY INFLAMMATORY PROCESS AFTER SPINAL CORD COMPRESSION INJURY IN MICE
M. Campolo, D. Impellizzeri, I. Paterniti*, R. Crupi*, A. Ahmad, S. Cuzzocrea*, E. Esposito*. University of Messina, Messina, Italy
Toll-like receptors (TLRs) are transmembranal proteins, which form the major pattern-recognition receptors that transduce signals in response to diverse pathogen-associated molecular patterns (PAMPS). As such, TRLs have a central role in the initiation of innate immunity against invading microbial pathogens. Toll-like receptors are ubiquitous and present both in immune and non immune cells, and their expression is rapidly altered in response to pathogens, cytokines, and environmental stressor. A large body of evidence suggests that TRLs play a central role during the activation of the inflammatory process, they possess a dual function: they are able to modulate the inflammatory process through NF-κB activation or cytokines production and in the same time they posses anti-inflammatory effects. The aim of this study was to investigate the role of TLR-4 in C57BL/10ScN (TLR-4 KO) on the modulation of the secondary events in mice subjected to spinal cord injury (SCI).
SCI is a highly devastating pathology that can lead to dramatic dysfunction, with significantly reduction of the quality of life. In this mouse model, SCI was induced by the application of an aneurysm clip to the dura via a four-level T5-T8 laminectomy, for replicating the persistence of cord compression that is commonly observed in human SCI. SCI was induced in TLR-4 in C57BL/10ScN (TLR-4 KO) and in TRL-4 wild type (TRL-4 WT) mice. All group of mice were sacrificed at 24 hours after SCI. TRL-4 WT mice developed severe spinal cord damage as showed by immunohistochemical analysis and by western blot analysis, characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, and apoptosis. While the absence of TRL-4 KO mice resulted in a significant augmentation of all the above described parameters. We have also demonstrated that the genetic absence of TRL-4 KO worsened the recovery of limb function when compared with TRL-4 WT mice group (evaluated by motor recovery score). Taken together, our results clearly demonstrate that the presence of TRL-4 reduces the development of inflammation and tissue injury events associated with spinal cord trauma.
MURINE GUT-BARRIER FILAMIN-A EXPRESSION: DERANGEMENT WITH MAJOR THERMAL INJURY AND RECOVERY WITH MELATONIN TREATMENT
S.A. Amuda, A. Azim, A. Ali, N. Fazal, W.M. Al-Ghoul*. Chicago State University, Chicago, IL
Filamins are actin-cross linking proteins that also bind to a wide range of binding partners involved in diverse dynamic cell interactions such as cell signaling and adhesion (Annu Rev Biophys. 41:227-46, 2012) that are sure to be detrimental for gut epithelial barrier morphology and integrity. In spite of the strong evidence in support of gut barrier leakiness as a mechanism for major postburn intestinal sepsis, the role of filamins in gut leakiness has never been examined. Here, we test both the normal distribution of filamin A in the colon and terminal ileum as well as its major postburn derangements in the presence and absence of daily postburn melatonin (Mel; 1.86 mg/kg, I.P.) treatment using an IACUC-approved mouse major burn model (9-second, 90 degree C water scald; 30% TBSA). Accordingly, we compared spatiotemporal gut mucosa changes of filamin A and related cytoskeletal proteins in three groups of eight-week-old male Balb/c mice: sham, thermal injury (TI), and TI+Mel. Mice were sacrificed 48 hours postburn and colon and terminal ileum samples were processed for filamin A immunohistochemistry, flow cytometry, and Western blot analyses. Our results demonstrated consistent and significant (p<0.05, N=4) Filamin A immunolabeling decreases of 20 to 28% in TI relative to control and recovery of 93 to 96% in TI+Mel relative to control (figure: dot blot, A and flow cytometry, B). The drop of filamin A postburn correlates with postburn gut barrier pathology and its stabilization by melatonin supports its importance as a therapeutic target for warding off postburn bacterial translocation and sepsis. (This work was supported by supported by NIGMS-MBRS-SCORE grant# 1SC3GM099632-01 to WMA)