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Abstracts II

doi: 10.1097/SHK.0b013e3182597b55
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Y. Yao, Q. Zhang, X. Zhu, L. Dong. Burn Institute, Beijing, China

Introduction: Gelsolin is an actin filament-severing and capping protein, affecting cellular motility, adhesiveness and apoptosis. Whether it is expressed and cleaved in the brain of burnt mice has not been characterized.

Methods: Mice were subjected to a 15% total body surface area (TBSA) scald injury on the back. Neuropathology was examined by hematoxylin and eosin staining. Cerebral gelsolin mRNA, distribution and cleavage were demonstrated by quantitative PCR, immunohistochemistry and Western blot, respectively. Cysteinyl aspartate-specific protease (caspase)-3 positive cells and activity were also measured.

Results: Pathological changes such as the leukocyte infiltration, necrosis, microabscess and gliosis were found in the brain of burned mice. Compared with sham-injured mice, gelsolin mRNA was up-regulated at 8 hrs postburn (pb) in a transient manner in the cortex and stratium, while remained consistent higher in the hippocampus till 72 hrs pb. At 24 hrs pb, gelsolin was further cleaved into 42 and 48 kDa fragments as illustrated in the hippocampus, and was widely expressed in the brain by the monocyte/macrophages, astrocytes and neurons. Accordingly, caspase-3 positive cells were noted in the stratium of burnt mice and its activity peaked at 24 hrs pb, then gradually returned to the comparable level as the sham-injured mice at 72 hrs pb. Rat pheochromocytoma cells exposed to lipopolysaccharide exhibited increased gelsolin expression and caspase-3-dependent cleavage.

Conclusions: The results suggest that burn induced gelsolin expression would be involved in the activation of both the monocytes and astroglial cells, thereby playing an important role in the subsequent inflammation-induced neural apoptosis following burn injury.

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N. Jbeily1, I. Suckert2, F.A. Gonnert2, S. Grossmann2, R.A. Claus2. 1International Leibniz Research School, Jena, Germany, 2Center for Sepsis Control and Care, Jena, Germany

Background: Ceramide formation is an indicator of cellular stress. Secreted acid sphingomyelinase (ASM) is a key enzyme for rapid ceramide production and has been found to be increased in inflammation and sepsis. In sepsis patients, levels of ASM continue to rise throughout the development of the disease. Its inhibition with desipramine presents promising results in cystic fibrosis. Here we investigate the role of ASM in sepsis and the potential beneficial effect of its inhibition.

Methods: Sepsis was induced in mice by Peritoneal Contamination and Infection (PCI). Blood and organs were collected after sepsis. Leukocyte-endothelium interactions in liver were also investigated. Genetically deficient (ASM ko) animals were compared to wt littermates and wt animals pre-treated with desipramine.

Results: We found a significant drop in leukocyte and platelet counts in all three groups six hours following sepsis. Leukocytes in wt animals dropped from 5183 to 4079 cells/μl (p<0.05). Significant differences in bacterial burden were obtained with the highest CFU’s registered in ko animals (5 and 114 CFU/200mg of liver aerobes and anaerobes respectively; p<0.05). This reflected a significant increase in monocyte phagocytosis in ko animals six hours after sepsis. We found differences in leukocyte-endothelium interaction with an increase in rolling (from 6 to 11 rollers/100 leukocytes) and sticking leukocytes (from 39 to 65 stickers/mm2) in wt animals. These changes were not observed in ko and desipramine pretreated animals. Both genotypes, wt and ko, revealed a 20% end survival. In comparison, desipramine pre-treated animals revealed a 40% end survival.

Conclusion: Here, we elucidate a potential dual function of ASM in host response. Its inhibition presents promising results with cytokine profile, liver function, and end survival.

Survival Analysis: Sepsis was induced in ASM wt (n=16), ko (n=16) and desipramine pre-treated (i

Survival Analysis: Sepsis was induced in ASM wt (n=16), ko (n=16) and desipramine pre-treated (i

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Q. Meng*1, S. Roy*1, G. Nieman*1, D. Clark1,2, T. Ahmed1, R. Cooney*1. 1SUNY Upstate Medical University, Syracuse, NY, 2Children’s Hospital at Albany Medical Center, Albany, NY

Background: Prematurity, enteral formula feeding and bacterial colonization are predisposing factors of Necrotizing enterocolitis (NEC). Short chain fatty acids (SCFAs) are produced in the gut by bacterial fermentation of carbohydrates in infant formula. We hypothesize the production of SCFAs are involved in the pathogenesis of NEC by causing inflammation of the intestinal mucosa. To test this hypothesis, we examined the ability of isobutyric acid (IA), proprionic acid (PA) and bacterial fermentation products (BF) to activate NF-κB in cultured enterocytes.

Methods: Caco-2 cells were transfected with a NF-κB promoter vector then stimulated with IA, PA and filtered BF for 30 h. Transfected cells were stimulated with HCL as a pH control. Dominant negative IκB co-transfection was used as a positive control. Luciferase activity was expressed as fold-induction, normalized to protein. NF-κB proteins were measured by Western Blot and activity was determined by EMSA.

Results: IA, PA and filtered BF increased NF-κB promoter luciferase activity in a time- and dose- dependent fashion in Caco-2 cells. NF-κB activation was blocked by co-transfection with a dominant negative IκB vector. NF-κB was not activated by HCL-induced changes in pH alone. The combination of IA and PA did not synergistically activate NF-κB in Caco-2 cells. NF-κB DNA binding activity was elevated by IA and PA. NF-κB P65 subunit protein was increased following treatment with IA and PA.

Conclusions: Bacterially fermented enteral formula and SCFAs induce NF-κB activation in cultured intestinal epithelial cells. NF-κB activation in enterocytes does not appear to be caused by changes in pH alone. These data provide evidence bacterial fermentation of enteral formula and SCFAs plays a role in the pathophysiology of NEC by activating NF-κB in the intestinal epithelium.

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Z. Peng, K. Ban, R.A. Kozar*. University of Texas Health Science Center at Houston, Houston, TX

Objective: We have shown that glutamine confers protection to the post ischemic gut via PPARγ and by preserving cell surface expression of syndecan-1. Since the promoter of syndecan-1 is recognized by PPARγ, we hypothesized that syndecan-1 protection of the epithelium was mediated by inside-out signaling via PPARγ.

Methods: Homozygous floxed PPARγ (f/f) mice with VFB background were bred with C57BL/6J then crossed into a transgenic containing the CRE gene under control of villin1 to yield mice with a specific targeted deletion of PPARγ in intestinal villi and crypt cells. Wild type (WT) or PPARγ intestinal specific conditional knockout (KO) mice were subjected to 1 hour of intestinal ischemia and 6 hours of reperfusion and compared to WT and KO shams. Jejunum was assessed for inflammation by myeloperoxidase (MPO) and apoptosis by caspase-3 mRNA and Western. Sections were immunostained with antisyndecan-1 mAb and relative fluorescence intensity quantified using Image J software (NIH). Data expressed as mean ± SEM, n=5/ group, ANOVA with Tukey post hoc.

Results: Inflammation, caspase-3 mRNA and cleaved protein were significantly increased in WT IR mice compared to sham WT or sham PPARγ KO but further increased in PPARγ KO IR mice (Table). Syndecan-1 staining was decreased in sham PPARγ KO compared to WT, but markedly decreased in WT IR and further decreased in PPARγ KO IR mice (Figure).

Conclusions: This data for the first time provides a mechanistic link between PPARγ and syndecan-1 after intestinal IR and may explain the protective effects of enteral glutamine in the post ischemic gut. Further investigation into this novel pathway is warranted.

No caption available

No caption available

Cell surface expression of syndecan-1

Cell surface expression of syndecan-1

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N.G. Yousif, L. Ao, J.C. Cleveland, D.A. Fullerton, X. Meng*. University of Colorado Denver, Aurora, CO

Increasing number of cardiac surgery with obligatory global myocardial ischemia and reperfusion (I/R) is performed in patients 65 years or older. Aging is a well known risk factor of exaggerated post-I/R myocardial injury. Thus, understanding of the mechanism of I/R injury in aging heart is important for cardiac protection in older patients undergoing cardiac surgery. Although the myocardial inflammatory response contributes to the mechanism of I/R injury, the effect of aging on myocardial inflammatory response to I/R remains to be characterized. This study tested the hypothesis that aging augments the myocardial inflammatory response to I/R through TLR4.

Methods and Results: We examined the myocardial inflammatory response to global I/R using a mouse heart transplant model. We found that myocardial production of MCP-1 following I/R was increased in hearts of old mice (18–22 months old) compared to those of adult mice (4–6 months old). Elevated myocardial levels of MCP-1 in aging hearts were associated with exaggerated mononuclear cell infiltration and troponin-I release. MCP-1 KO reduced myocardial mononuclear cell infiltration and troponin-I release caused by global I/R. While myocardial TLR4 levels were not significantly increased in aging hearts, TLR4 deficiency attenuated myocardial MCP-1 production in aging hearts following I/R, resulting in reduced age-related differences in myocardial mononuclear cell infiltration and troponin-I release.

Conclusions: These results demonstrate that aging augments the myocardial inflammatory response to I/R in mice and that myocardial TLR4 is obligatory in the mechanism underlying the augmented inflammatory response to I/R in aging hearts.

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S. Asmussen1, D.M. Maybauer*1, J.F. Fraser*2, D.S. Herzig1, D.N. Herndon*1, M.O. Maybauer*1. 1Depts. of Anesthesiology and Surgery, The University of Texas Medical Branch and Shriners Burns Hospital for Children, Galveston, TX, 2Critical Care Research Group, The University of Queensland and The Prince Charles Hospital, Brisbane, QLD, Australia

Introduction: Acute respiratory failure (ARF) after smoke inhalation injury (SII) has significant mortality rates of up to 60% in burn patients, and continues to be the major cause of death in fire victims. Specialized centers use extracorporeal membrane oxygenation (ECMO) as a rescue therapy in ARF to support gas exchange. Aim of this study was a systematic review of the literature & meta-analysis of the available clinical data to assess the present level of evidence for ECMO in ARF resulting from burn and SII.

Methods: Our search yielded 66 total citations. Only 29 met the inclusion criteria of burn and/or SII. There are no available systematic reviews/meta-analyses published that met our inclusion criteria. Only six clinical trials with a limited number of patients were available out of these 29 results.

Results: Based on the low number of studies (evidence grades IIb and III) and patients available, a Forrest-plot but no meta-analysis was performed. Analysis of the data suggests a higher survival rate (0.542) than non-survival of burn patients with ARF undergoing ECMO. In addition an ECMO run time of less than 200 hours correlates with higher survival compared to 200 hours or more. Scald burns show a tendency of higher survival than flame burns.

Conclusion: Even though the reports of the presently available literature are promising, especially in the pediatric burn population, this review highlighted the lack of evidence for the use of ECMO in this setting. Nevertheless, ECMO technology and expertise have improved over the last two decades; therefore randomized controlled trials are warranted to provide definitive recommendations.

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M. Gao, T. Ha, X. Zhang, F. Lam, X. Wang, J.L. Kelley, K. Singh, J. Kalbfleisch, R. Kao, D.L. Williams*, C. Li*. East Tennessee State University, Johnson City, TN

Cardiac dysfunction is a major consequence of sepsis which contributes to the high morbidity and mortality of this devastating disease. The TLR9 ligand, CpG-ODN, has been reported to improve outcome in sepsis. We hypothesized that CpG-ODN may attenuate cardiac dysfunction in polymicrobial sepsis. To critically evaluate our hypothesis, male C57BL/6 mice (n=6/group) were treated with CpG-ODN, control-ODN or inhibitory CpG-ODN (iCpG-ODN) one hr prior to cecal ligation and puncture (CLP) induced sepsis. Sham operation served as sham control. Cardiac function was examined by echocardiography before and six hrs after CLP. CLP sepsis induced significant cardiac dysfunction. In contrast, CpG-ODN administration significantly attenuated sepsis induced cardiac dysfunction as evidenced by maintenance of ejection fraction (EF) and fractional shortening (FS) compared with the untreated CLP group. Control-ODN or iCpG-ODN did not alter sepsis induced cardiac dysfunction. CpG-ODN administration significantly decreased sepsis induced cardiac myocyte apoptosis and increased the levels of both Akt and ERK phosphorylation in the myocardium. In vitro experiments demonstrated that CpG-ODN induces phosphorylation of myocardial Akt and ERK via increased association between TLR9 and Ras. More significantly, pharmacologic inhibition of either PI3K or ERK abolished the beneficial effect of CpG-ODN on cardiac function in sepsis. Our data suggest that modulation of TLR9 by its ligand, CpG-ODN, attenuates sepsis induced cardiac dysfunction through activation of both PI3K/Akt and ERK signaling pathways. Pharmacologic modulation of TLR9 may be an effective approach for the management and treatment of patients with sepsis/septic shock.

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S. Jiang, J.C. Chatham, J.L. Messina*. UAB, Birmingham, AL

While impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of critical illness or injury, and Type 2 diabetes, mechanisms that modulate the function of IR remain unclear. With an animal model of critical illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 minutes following trauma and hemorrhage. Possible mechanisms for this defect were examined, including reduced amount of IR and IR post-translational modifications. The total amounts of IR α and β subunits were not altered by trauma and hemorrhage as measured by Western analysis. In addition, no change in IR tyrosine nitration was found in the liver following trauma and hemorrhage. However, there was a decrease in the level of protein O-linked beta-N-acetlyglucosamine (O-GlcNAC) modification on Ser/Thr in the liver following trauma and hemorrhage. Further studies by immunoprecipitation suggest that IR was O-GlcNAcylated and the level of IR O-GlcNAC modification was decreased by trauma and hemorrhage. There may be an involvement of c-jun N-terminal kinase (JNK) since inhibition of JNK kinase increased O-GlcNAC modification. These findings suggest that a balance between O-GlcNAC modification and JNK-induced phosphorylation may exist, and decreased Ser/Thr O-GlcNAC modification following trauma and hemorrhage may result in increased access allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits insulin receptor activity. Collectively, the present studies suggest potential mechanisms of hemorrhage-induced defects in insulin receptor activity, and a potential role for decreased O-GlcNAC and increased serine phosphorylation.

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J. Duitman1, 3, M. Schouten1,3, A.P. Groot1, J.B. Daalhuisen1, S. Florquin2, T. van der Poll*1,3, C. Spek1. 1Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center Amsterdam, Amsterdam, Netherlands, 2Department of Pathology, Academic Medical Center Amsterdam, Amsterdam, Netherlands, 3Center for Infection and Immunity Amsterdam (CINEMA), Academic Medical Center Amsterdam, Amsterdam, Netherlands

The transcription factor C/EBPδ has been suggested to be an essential player in the inflammatory response but the relevance of C/EBPδ in infectious disease is not fully understood. Here, we investigated the role of C/EBPδ during infection with Streptococcus pneumoniae (S. pneu), a common cause of pneumosepsis and shock.

To this end, bacterial outgrowth, inflammatory responses and survival were assessed in wildtype (WT) and C/EBPδ-/- mice infected with S. pneu. Moreover, C/EBPδ and PAFR expression were determined in lung epithelial cells stimulated with lipoteichoic acid (LTA) or S. pneu. We show that S. pneu induced C/EBPδ expression in lungs, that survival was improved in C/EBPδ-/- mice and that C/EBPδ-/- mice had lower bacterial loads in the lungs at 48h and decreased dissemination of the bacteria into the systemic compartment at 24 and 48h after intranasal inoculation. Moreover, lung inflammation was attenuated in C/EBPδ-/- mice at 48h as indicated by reduced cytokine and chemokine levels. In contrast to i.n. instillation, i.v. injection of S. pneu resulted in similar bacterial outgrowth and inflammatory responses in WT and C/EBPδ-/- mice, suggesting that C/EBPδ does not affect bacterial clearance in the blood itself. Interestingly, expression of PAFR, which is well known to potentiate translocation of gram-positive bacteria, was significantly reduced in C/EBPδ-/- mice compared to WT controls. Moreover, both LTA and S. pneu induced C/EBPδ expression in pulmonary epithelial cells, while C/EBPδ in turn induced PAFR expression.

In conclusion, C/EBPδ is shown to play a detrimental role in the host response to S. pneu pneumonia by facilitating bacterial dissemination, most probably in a PAFR-dependent manner.

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I.C. Hoogland1, D. Westhoff1, J. Melief2, I. Huitinga2, D.J. van Westerloo4, T. van der Poll*3, D. van de Beek1, W.A. van Gool1. 1Department of Neurology, Academic Medical Centre, Amsterdam, Netherlands, 2Nederlands Institute of Neuroscience, Amsterdam, Netherlands, 3Center for Experimental and Molecular Medicine, Academic Medical Centre, Amsterdam, Netherlands, 3Department of Intensive Care Medicine, Leids University Medical Centre, Leiden, Netherlands

Introduction: Sepsis associated delirium (SAD) is a serious complication and is associated with a poor clinical outcome. We hypothesize that microglia activation in the brain is an essential step in the development and outcome of SAD. The mechanisms triggering the neuroinflammatory response need to be elucidated.

Aims: 1. To investigate microglia activation in 2 different mouse models involving either intraperitoneally (ip) injection of lipopolysaccharide (LPS) or ip injection of live E.coli; 2. To quantify microglia activation with flow cytometry.

Methods: We inoculated 30 C57BL/6 mice ip with LPS (from E.coli strain O111:B4, 5mg/kg) and 15 C57BL/6 mice ip with E.coli (K1:018, 0.9×105 CFU/ml); 18 control mice were inoculated with saline. LPS inoculated mice were sacrificed at 3h (n=15) and 48h (n=15) after inoculation. E. coli inoculated mice were sacrificed 20h after infection. Right hemispheres were used for immunohistochemical staining with an anti-Iba-1 antibody for microglia activation; left hemispheres were used for analysis of microglia with flow cytometry using antibodies against CD11b and CD45.

Results: In LPS inoculated mice microglia were mild (t=3h) to moderately (t=48h) activated as reflected by changes in morphology on Iba-1 staining (Figures 1, B and C). Microglia of E.coli infected mice expressed only a mild activation state (t=20h). Flow cytometry of microglia showed significant changes in geometric means between sham and LPS stimulated mice, but only at 48h (forward scatter p=0.021, CD45 p=0.006, CD11b p=0.012; Figure 1, D-F).

Conclusion: Microglia are activated in a mouse model involving ip injection with LPS, which can be quantified with flow cytometry. Activation of microglia differed between mice injected ip with LPS versus mice injected ip with E.coli, which may be due to lower LPS equivalents.

Fig. 1

Fig. 1

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L.F. Gentile, D.C. Nacionales, A.G. Cuenca, M. Armbruster, R.F. Ungaro, A.S. Abouhamze, F. Moore*, D. Ang, L.L. Moldawer*, P.A. Efron*. University of Florida, Gainesville, FL

Objectives: Established murine models of trauma and hemorrhagic shock suboptimally reflect human immune alterations after injury. We developed a novel polytrauma model that better recapitulates the inflammatory response of the severely injured patient by combining long-bone fracture and cecectomy with hemorrhagic shock, resulting in an equivalent Injury Severity Score of greater than 15. We compared this new polytrauma/shock model to historically used murine trauma-hemorrhage models.

Methods: 6-10 wk old B6 mice underwent 90 minutes of shock (MAP 30 mmHg) and resuscitation via femoral artery cannulation followed by either laparotomy (T/H), laparotomy with femur fracture (HLFfx), or laparotomy with cecetomy and femur fracture with muscle tissue damage (HLFfxC). Mice were euthanized on days 1, 3, 5, and 7 and their spleens, bone marrow, blood and serum were collected. Post-injury analgesics were provided.

Results: None of the models were lethal. Mice undergoing HLFfxC exhibited a more robust inflammatory response with statistically significant elevations in serum cytokines/chemokines when compared to traditional models. HLFfxC was the only model to induce neutrophilia (Ly6G+CD11b+ cells) on POD 1 and 3 (p<0.05). HLFfxC, as compared to T/H and HLFfx, induced a panlymphopenia (CD4+, CD8+, CD19+) with simultaneously increased cell activation (CD69+ and CD25+), similar to human trauma. There was a greater loss of MHCII expression in monocytes in the HLFfxC model (p<0.05), suggestive of adaptive immunosuppression.

Conclusion: We show that this novel polytrauma model better replicates the human leukocyte, cytokine, and overall inflammatory response following injury and hemorrhagic shock.

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D. Frith, M. Guerreiro, R. Pearse, C. Thiemermann*, K. Brohi. Bart’s & The London School of Medicine, London, United Kingdom

The pathophysiology of Acute Traumatic Coagulopathy (ATC) is yet to be fully elucidated. The objective of this experimental study was to characterize thrombin generation and coagulation system component responses to trauma and hemorrhagic shock.

Fifteen euthermic Wistar rats were subjected to laparotomy, lower limb fractures and had 35% of their blood volume removed without any resuscitation. After 60 minutes blood samples were obtained for analysis.

Trauma-shock produced a decline in mean pH from 7.38 to 7.15 and reduction in hematocrit from 40% to 28%. Prothrombin and partial thromboplastin times were prolonged 1.4 and 1.6 times normal, respectively (p<0.001 compared with 10 sham controls). Endogenous coagulopathy was confirmed on RoTEM analysis with a reduction in EXTEM maximal clot firmness (MCF) from 60 mm to 54 mm (p<0.01).

This coagulopathy was associated with a significant (p<0.001) decrease in the activity of procoagulant clotting factors II (59%), V (57%), VII (47%) and X (41%). However, the anticoagulant activities of protein C (49%), antithrombin (50%) and tissue factor pathway inhibitor (71%) were also reduced (p<0.001). Overall, endogenous thrombin generation was impaired with a significant increase in lag time (2.6 vs. 2.9 s, p=0.02) and reduced total potential (434 vs. 378nM, p<0.01). Trauma-shock induced a moderate, but statistically significant, reduction in fibrinogen concentration (3.0 vs. 2.3mg/ml, p=0.03) that associated with a profound decrease in FIBTEM MCF (13.5 vs. 9.1mm, p<0.01).

The pathophysiology of experimental ATC is characterized by moderate impairment of thrombin generation and significantly reduced fibrin clot strength, despite apparently adequate fibrinogen substrate. Future studies should focus on the mechanism and treatment of this anomaly.

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R. Southard, S. Ghosh, J. Hilliard, R. Hotchkiss*. Washington University School of Medicine, St. Louis, MO

Background: Severe multisystem trauma is a major cause of death, and nosocomial infections such as Pseudomonas pneumonia are common in patients who survive the initial injury. Elderly patients are more likely to develop and succumb to such infections. However, animal models have not demonstrated an increased susceptibility to infection after injury.

Objectives: We hypothesized that aged mice subjected to a severe injury in multiple organs would demonstrate an increased susceptibility to Pseudomonas pneumonia and sought to develop a mouse model of polytrauma and infection.

Methods: Aged mice (C57BL/6 retired male breeders) were subjected to liver laceration, bilateral hind limb crush injury and limited hemorrhage. After 24 hours, the mice were exposed to Pseudomonas aeruginosa (2x106 CFU) via intratracheal injection. The primary endpoint was mortality after induction of pneumonia. Uninjured animals and injured animals exposed to heat-killed bacteria were used as controls. Quantitative cultures were performed on bronchoalveolar lavage fluid (BAL) and blood.

Results: Aged mice subjected to polytrauma had a higher rate of mortality from pneumonia (8 out of 13 mice, 62%) than uninjured controls (2 of 16 mice, 12% p<0.01.) Injured mice exposed to heat-killed bacteria had a significantly lower mortality (2 of 11 mice, 18%) than injured mice treated with live bacteria (p<0.05.) In cultures of the BAL fluid and blood, there was a trend toward more colony forming units in injured animals than uninjured controls.

Conclusions: Aged animals subjected to severe multisystem injury are more susceptible to mortality from pneumonia than uninjured controls. This increase in mortality is related to inability to clear the bacterial pathogen rather than inflammation as there were significantly fewer deaths in the heat-killed group.

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A.J. Hoogendijk1, 2, S.S. Pinhanços1, 2, T. van der Poll*1, 2, C.W. Wieland1, 3. 1Center for Infection and Immunity Amsterdam, Academic Medical Center, Amsterdam, Netherlands, 2Center for Infection and Immunity Amsterdam, Academic Medical Center, Amsterdam, Netherlands, 3Laboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, Amsterdam, Netherlands

Background: AMP activated kinase (AMPK) is a highly conserved kinase that plays a key role in energy homeostasis. Activation of AMPK was shown to reduce inflammation both in vitro and in vivo in response to lipopolysaccharide induced inflammation. 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) is intracellularly converted to the AMP analog ZMP, which activates AMPK. Lipoteichoic acid (LTA) is a major component of the cell wall of gram-positive bacteria that can trigger inflammatory responses. In contrast to lipopolysaccharide, little is known on effects of AMPK activation in LTA triggered innate immune response or gram-positive pneumonia.

Aim: Here, we study the potency of AMPK activation to reduce inflammation in vitro, in vivo in LTA induced lung injury and in a model of gram-positive bacterial pneumonia.

Results: Activation of AMPK in vitro reduced TNFα production in the alveolar macrophage cell line MH-S. In vivo, AMPK activation reduced LTA induced neutrophil influx and inflammatory mediator levels (TNFα, MIP-2, KC) at 6 hours of inflammation. In pneumococcal pneumonia ACIAR treatment did not alter inflammatory mediator levels or bacterial loads.

Conclusion: With this study we demonstrate the potency of AMPK activation to diminish inflammatory responses in LTA induced lung inflammation. However, AMPK activation did not affect the immune response during pneumococcal pneumonia.

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G. Yang, L. Liu*. Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing, China

Aim: Vascular hyporeactivity played an important role in the development and outcome of many critical illness including shock or sepsis, but its mechanisms are incompletely understood. The objective of the present study was to investigate the roles of three major mitogen-activated protein kinase (MAPK) members (ERK, p38 MAPK and JNK) on vascular reactivity.

Methods: With superior mesenteric arteries from hemorrhagic-shock rats, the role of p38 MAPK, ERK and JNK in the regulation of vascular reactivity following shock and their relationship to MLC20 phosphorylation dependent pathway was observed.

Results: ERK and p38 MAPK activities in SMAs were increased at early shock and decreased at late shock. Stimulation of MAPKs with angiotensin II (AngII) increased the vascular reactivity, calcium sensitivity and MLC20 phosphorylation. The increase effect of Ang II on vascular reactivity was antagonized by ERK, p38 MAPK and JNK inhibitor, while this effect of Ang II on calcium sensitivity was only blocked by ERK and p38 MAPK inhibitor, but not by JNK inhibitor. Ang II could increase the activity of PKC-dependent phosphatase inhibitor of 17 kDa (CPI17), integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK), effect of Ang II on CPI-17 was blocked by ERK and p38 MAPK inhibitor, while effect of Ang II on ILK and ZIPK was only blocked by ERK inhibitor.

Conclusions: MAPKs participated in the regulation of vascular reactivity during shock. ERK and p38 MAPK is mainly through ILK, ZIPK and CPI17-mediated MLC20 phosphorylation dependent pathway, while JNK may be involved in regulation of vascular reactivity by other mechanisms.

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G. Botez1, G. Piraino1, P.W. Hake1, M. O’Connor1, J. Cook*2, B. Zingarelli*1. 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 2Medical University of South Carolina, Charleston, SC

Incidence and mortality of sepsis is disproportionately increased in elderly adults when compared to young adults or children. The molecular mechanisms of this high susceptibility are not yet defined. We have recently demonstrated that the nuclear liver X receptor α (LXRα) is a crucial anti-inflammatory modulator in hemorrhage-induced lung injury. Here, we investigated whether age-dependent susceptibility to sepsis might correlate with changes in LXRα expression in the lung and whether treatment with the LXRα ligand T0901317 might modulate sepsis outcome. Sepsis was induced in male mice of different ages by cecal ligation and puncture (CLP). Myeloperoxidase activity (MPO) was measured to evaluate lung neutrophil infiltration. Intraperitoneal treatment with T0901317 (30 mg/kg) reduced MPO in the lung of infant (1-1.5 months old, 95.8±17.8 U/100 mg tissue), young (2-3 months old, 253.1±39.8 U/100 mg tissue) and mature mice (6-8 months old, 158.8±17.6 U/100 mg tissue) compared with vehicle-treated mice subjected to CLP (143.9±10.3, 357.3±46.2 and 273.4±36.4 U/100 mg tissue, respectively; P<0.05). The ligand also improved survival in young mice (2-3 months old; P=0.049). However, treatment with T0901317 did not modify survival rate (P=0.941) and did not affect neutrophil infiltration in old mice (11-13 months old, 309.7±53.5 U/100 mg tissue) compared with vehicle-treated mice (234.2±22.1 U/100 mg tissue). At molecular analysis, immunoblotting of lung nuclear extracts revealed an age-dependent downregulation of LXRα expression, which was highly impaired in old animals. Our data suggest that LXRα is a crucial factor for survival in sepsis and LXRα ligands may exert therapeutic effects. However, the protective effects of LXRα activation are age-dependent and severely compromised in old animals. (Supported by NIH R01 GM067202).

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D.N. Darlington1, T. Craig2, M.D. Gonzales1, M.G. Schwacha2, A.P. Cap1, M.A. Dubick1. 1US Army Institute of Surgical Research, San Antonio, TX, 2Department of Surgery, University of Texas Health Science Center, San Antonio, TX

Acute Coagulopathy of Trauma has been described clinically as an early coagulopathic state and has been reported in patients with severe trauma and hemorrhage. To study this phenomenon, we set out to develop a rat model of polytrauma and hemorrhage that showed a coagulopathy. Sprague-Dawley rats (300-400g) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and a right femur break. The rats were then bled to 40mmHg and held there until 40% of the blood volume was removed. Hemorrhage was usually completed between 30-60min. No fluid resuscitation was given. All rats survived the 240 min experimental period. Blood samples were taken before (time 0) and after trauma at 30, 60, 120, 180 and 240min. Prothrombin Time (PT) significantly increased over time (12.7±0.2, 13.7±0.5, 13.9±0.4, 16.0±0.4, 16.8±0.5 and 16.0±1.6sec, n=4). Plasma lactate significantly increased from 0.93±0.38 at time 0 to 4.78±0.96mM at 240min, and bicarbonate significantly decreased (34.7±1.5 to 24.7±1.7mM). ROTEM thromboelastometry (Extem) showed a significant decrease in clotting time from 0min, to 30 and 240min (44.4±3.8, 33.3±1.7, 35.5±2.6sec) and Mean Clotting Firmness (54.3±1.1, 50.6±2.3, 49.9±1.5mm). However, the rate of clot formation (angle,°) did not change. In conclusion, we have successfully created a rat model of coagulopathy of trauma as evidenced by an elevation in PT and plasma lactate, a fall in bicarbonate and a decrease in clot firmness. This project was funded by MRMC and ARO-W911NF-10-1-0403.

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M.G. Schwacha2, D.N. Darlington1, T. Craig2, M.D. Gonzales1, M.A. Dubick1, A.P. Cap1. 1US Army Institute of Surgical Research, San Antonio, TX, 2Department of Surgery, University of Texas Health Science Center, San Antonio, TX

We have developed a rat model of polytrauma and hemorrhage that displays the acute coagulopathy of trauma (ACOT) as evidenced by a significant elevation in prothrombin time during the initial 4 hrs post-injury. To investigate possible changes in inflammatory markers in this coagulopathy model, 4 Sprague-Dawley rats (300-400g) were anesthetized with isoflurane and polytrauma was induced by damaging the small intestines, the right and medial liver lobes, the right leg skeletal muscle, and breaking the right femur. The rats were then bled to 40mmHg which was maintained until 40% of the blood volume was removed. Hemorrhage was completed within 30-60min. No fluid resuscitation was given and all rats survived the 240 min experimental period. Each rat served as its own control. Blood samples were taken before (time 0) and 240 min after trauma and hemorrhage. Plasma levels (pg/ml) of the following inflammatory cytokines increased significantly (p<0.05) from time 0 to 240 min: IL-1α (5±1 to 114±24), IL-1β (64±12 to 406±113), IL-6 (51±16 to 4250±1691) and TNFα (37±13 to 446±183). In addition, the anti-inflammatory cytokine IL-10 was also significantly elevated (348±24 to 7271±1185). In addition, the anti-inflammatory cytokine IL-10 was also significantly elevated (348±24 vs. 7271±1185 pg/ml). In conclusion, we have developed a reproducible rat model of ACOT that demonstrates that the coagulopathy is associated with a robust systemic inflammation. Studies are underway to investigate potentially important causative relationships. This project was funded by MRMC and ARO-W911NF-10-1-0403.

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B.P. Scicluna1, C. van ’t Veer1, M. Nieuwdorp1, K. Felsmann2, E.S. Stroes1, T. van der Poll*1. 1Academic Medical Center, Amsterdam, Netherlands, 2SIRS-Lab GmbH, Jena, Germany

TNFα is a pleiotropic pro-inflammatory cytokine that mediates both beneficial and detrimental biological processes. Signal transduction pathways induced by TNFα are complex and still not fully understood. We aimed to uncover key genes and functional biological pathways for the systemic endotoxin-induced transcriptome mediated by TNFα release.

Sixteen young and healthy male Caucasian volunteers were enrolled in this study. They were administered placebo (normal saline) (n=8) or the TNFα inhibitor etanercept (n=8; Enbrel® 50mg) prior to receiving a bolus infusion of Escherichia coli lipopolysaccharide (LPS, 1ng/kg). Whole blood leukocyte RNA was isolated from baseline and 4-hour post-LPS challenged samples for a genome-wide gene expression screen. The systems-based weighted gene co-expression network approach (WGCNA) coupled with knowledge-based analysis was applied.LPS infusion induced a profound transcriptional response for which we detected 8168 transcripts that were differentially expressed (q<0.01). We detected 4077 transcripts that differed significantly (q<0.05) between the LPS-challenged placebo and etanercept-treated groups. These 4077 transcripts clustered into 32 modules of highly correlating transcripts. Knowledge-based analysis revealed significant enrichment for distinct biological pathways that include immune response reactions, regulation of T-cell activation and RIG-I-like receptor signaling. Key genes within these TNFα influenced modules include MARCKS, CLIC4, HIVEP1, IRAK3, RELB, and C20orf160.

These findings reveal a functional transcriptomic framework of a variety of biological and cellular pathways driven by fundamental genes that respond to TNFα signaling; these constitute veritable targets for molecular analysis relevant to complex inflammatory diseases.

Genomic analysis of the systemic endotoxin-induced transcriptional response and impact of TNFα inhibition

Genomic analysis of the systemic endotoxin-induced transcriptional response and impact of TNFα inhibition

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J. Duitman1, 3, A.J. Hoogendijk1, 3, R.R. Ruela de Sousa1, A.P. Groot1, T. van der Poll*1, 3, S. Florquin2, C. Spek1. 1Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center Amsterdam, Amsterdam, Netherlands, 2Department of Pathology, Academic Medical Center Amsterdam, Amsterdam, Netherlands, 3Center for Infection and Immunity Amsterdam (CINEMA), Academic Medical Center Amsterdam, Amsterdam, Netherlands

Mounting evidence suggests an important role for CCAAT-enhancer binding protein delta (C/EBPδ) in the acute phase response upon bacterial infections. C/EBPδ levels increase rapidly after pro-inflammatory stimuli and increased C/EBPδ levels seem to be indispensable for the amplification of the inflammatory response. Indeed, it has been shown that C/EBPδ limits E. coli.-induced peritonitis. However, whether C/EBPδ also limits pulmonary infectious disease remains elusive and is the aim of this project.

To this end, bacterial outgrowth, inflammatory responses, inflammatory cell influx and survival were assessed in wildtype (WT) and C/EBPδ-/- mice infected with Klebsiella pneumoniae. Most and for all, Klebsiella-induced mortality was significantly increased in C/EBPδ-/- mice as compared to WT controls. In line, bacterial outgrowth in lung, blood and peripheral organs was elevated at 24 and 48 hours post inoculation in C/EBPδ-/- mice, suggesting that C/EBPδ is important for bacterial clearance. Moreover, we show that macrophage numbers are significantly reduced in lungs of C/EBPδ-/- mice, suggesting that C/EBPδ dependent macrophage driven bacterial killing may (partially) explain the prolonged survival in WT mice. In vitro assays however showed that C/EBPδ does not affect macrophage function (i.e.: oxidative burst and cytokine production) per se.

Overall, these data thus show that C/EBPδ boosts the host response during Klebsiella pneumoniae-induced pulmonary infection by limiting local outgrowth and subsequent bacterial dissemination.

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B.P. Scicluna, M.H. van Lieshout, D.C. Blok, C. van ’t Veer, T. van der Poll*. Academic Medical Center, Amsterdam, Netherlands

Background & Objective: Sepsis results in a complex and dynamic systemic host response. Knowledge of the local host response during sepsis, at the primary site of infection, is limited. Streptococcus pneumoniae (Spneu) is a common cause of pneumonia and sepsis. We here aimed to unmask key biological pathways and pertaining genes in the lungs during early and late stage Spneu pneumonia in mice.

Methods: C57BL6 mice were intranasally infected with Spneu D39 (2 x 107 CFU). Total RNA was isolated from lungs of uninfected (0h) and infected (6 and 48h) mice. Genome-wide transcriptional profiling (Illumina MouseRef-8 v2) coupled with knowledge-based analysis was applied. Benjamini-Hochberg p-value<0.05 defined significance.

Results: Early stage pneumonia (0 vs 6h) resulted in a marked transcriptional response in the lungs with 5058 significantly differential transcripts. Advanced infection (0 vs 48h) significantly altered the expression of 3444 transcripts in infected lung tissue. Common pathways included Toll-like receptor signaling, apoptosis and NOD-like receptor signaling. Interestingly, we detected 1849 significantly differential transcripts between 6 and 48h post-infection. Transcripts encoded by cxcl1, cxcl2, il1b and tnf were significantly reduced at 48h. Moreover, we uncovered an up-regulation in DNA repair and mitochondrial pathways, and a down-regulation in MAPK signaling.

Conclusion: We highlight the pulmonary expression of key genes and pathways involved in the early and late stages of Spneu pneumonia. These findings may aid in revealing mechanisms at play during the progressing host response during pneumosepsis at the primary site of infection.

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D. Westhoff1, J. Witlox3, I.C. Hoogland1, L. Koenderman2, D.J. van Westerloo4, D. van de Beek1, T. van der Poll*1, W.A. van Gool1. 1Academic Medical Center, Amsterdam, Netherlands, 2University Medical Center, Utrecht, Netherlands, 3Medical Center Alkmaar, Alkmaar, Netherlands, 4University Medical Center, Leiden, Netherlands

Background: Delirium is a frequently occurring complication in hospitalized elderly and is associated with poor clinical outcome. Delirium has been hypothesized to result from a neuroinflammatory response. In patients with a hip fracture, serum levels of cytokines rise due to a systemic inflammatory response caused by the fracture itself and subsequent surgery. This may result in an imbalance between pro- and anti-inflammatory mediators in the brain. The objective of this study was to find the relation between preoperative cytokine levels in cerebrospinal fluid (CSF) and the occurrence of postsurgical delirium.

Methods: CSF was obtained preoperatively from 72 patients (≥ 75 years of age) that were admitted for surgical repair of a hip fracture. All patients were free from delirium before surgery and presence and severity of delirium were assessed daily until the 5th postoperative day. CSF levels of 42 cytokines and chemokines were determined withmultiplex technology. We examined the association between CSF cytokine levels and delirium incidence by comparing levels between patients with and without postoperative delirium.

Results: Twenty-six patients (36.1%) developed postsurgical delirium. We did not find statistically significant differences between groups for any of the 42 cytokines studied. Additional analysis to compare control patients with patients that developed delirium shortly after surgery (day 1 or 2), neither showed any differences.

Discussion: Our findings do not support the hypothesis that an exaggerated neuroinflammatory response shortly after hip fracture would be associated with postoperative development of delirium. Changes in brain cytokines may be too subtle to be reflected in the CSF compartment.

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L. Qi1, 2, X. Cui1, 2, W. Dong1, 2, R. Barrera2, G.F. Coppa2, P. Wang*1, 2, R. Wu*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY

Traumatic brain injury (TBI) and hemorrhagic shock (HS), the most common causes of trauma deaths, often occur concomitantly. Ghrelin, a gastrointestinal hormone, has been demonstrated to possess multiple functions including upregulation of uncoupling protein-2 (UCP2) and stimulation of the vagus nerve. Recent evidence has indicated that ghrelin is anti-inflammatory and neuroprotective. We therefore hypothesized that ghrelin protects rats against TBI and HS through upregulation of UCP2 involving stimulation of the vagus nerve. To study this, brain injury was induced by dropping a 450g weight from 1.5 m onto a steel helmet attached to the skull of male adult rats. Immediately after TBI, a laparotomy was performed and both lumbar veins were isolated and severed at the junction with the vena cava. At 45min after TBI+HS, ghrelin (4, 8 or 16nmol/rat) or 1 ml normal saline (Vehicle) was iv administered. Brain injury was assessed for up to 28 d after TBI+HS. In additional groups of rats, genipin, a specific UCP2 antagonist, was administered iv before the injection of ghrelin after TBI+HS. The role of the vagus nerve was assessed by performing vagotomy immediately prior to ghrelin administration. Our results showed that ghrelin attenuated brain injury and facilitated function recovery after TBI+HS. Ghrelin increased UCP2 expression in the cortex and genipin abolished ghrelin’s protection after TBI+HS. Furthermore, vagotomy prevented the beneficial effects of ghrelin and eliminated ghrelin induced UCP2 upregulation after TBI+HS. In conclusion, ghrelin treatment attenuated brain injury and facilitated function recovery in a rat model of TBI and uncontrolled hemorrhage. The protective effects of ghrelin after TBI+HS appear to be related to upregulation of UCP2 expression in the brain and involving stimulation of the vagus nerve.

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B. Zingarelli*, P.W. Hake, M. O’Connor, G. Piraino. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH.

The clinical course and outcomes of sepsis are significantly impacted by the age of the patient. However, the molecular mechanisms that link age to enhanced susceptibility to sepsis are not known. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis, which controls mitochondrial biogenesis and autophagy, i.e. the disposal of defective organelles. We hypothesized that age-dependent susceptibility to sepsis might correlate with dysfunction of AMPK activation in the liver. Sepsis was induced in male young (2-3 months) and old mice (11-12 months) by cecal ligation and puncture (CLP). At Western blotting, there was a time-dependent increase of nuclear translocation of the active catalytic subunit pAMPKα in young mice, but not in old mice. 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 mice exhibited a time-dependent increase of PGC-1α nuclear translocation after CLP. Interestingly, in old mice the majority of PGC-1α was retained in the cytosol, thus suggesting an impairment of its 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 the LC3B-I/LC3B-II ratio was similar in sham mice of both ages. After sepsis, young mice exhibited an increase of LC3B-II conversion in liver at 18h, suggesting formation of autophagic vesicles. However, LC3B-II conversion was significantly reduced in old mice when compared to the young group. Thus, our data suggest that during sepsis AMPK-dependent mechanisms are activated to promote mitochondrial repair. However, this restorative process diminishes with age. (Supported by NIH R01 GM067202).

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M. Liu1, J. Simpkins2, J. Wigginton*1, S. Wolf*1, J. Minei*1, J. Gatson*1. 1UT Southwestern Medical Center, Grand Prairie, TX, 2University of North Texas Health Science Center, Fort Worth, TX

Background: In various animal studies, 17 beta-estradiol (a strong anti-oxidant, anti-inflammatory, and anti-apoptotic agent) significantly decreases the severity of injury in the brain caused by early, devastating cell death. With respect to estrone (predominant estrogen in postmenopausal women), this estrogen has shown to be extremely promising as a neuro-protective agent following neural excitotoxicity, stroke, and TBI. The overall goal of this project was to characterize the anti-inflammatory effects of estrone after TBI in rats. Here, we hypothesized that estrone reduces cortical matrix metalloproteinase-9 (mmp-9) levels and leukocyte infiltration into the brain after TBI.

Methods: In this study, 16 male rats were treated with either placebo (corn oil; n=8) or estrone (0.5mg/kg; n=8) at 30 minutes after severe TBI. In brief, using the controlled cortical impact device in rats that underwent a craniotomy, the right parietal cortex was injured using the impactor tip. Non-injured control (n=8) and sham (craniotomy only; n=8) animals were also included in this study. At 72 hours after injury, the animals were intracardially perfused with 0.9% saline followed by 10% phosphate-buffered formalin. The whole brain was removed, sliced, and stained for mmp-9 levels and CD45+ cells using the immuno-histochemical analysis.

Results: The rats treated with estrone had reduced cortical lesion volume and cortical levels of TUNEL + staining (∼80%). In addition, estrone significantly decreased the cortical levels of both mmp-9 (p=0.01) and CD45+ cells (p=0.05).

Conclusion: Estrone given acutely after injury results in a decrease in cortical cell death, mmp-9 expression, and leukocyte infiltration. These results suggest that estrone maintains the integrity of blood brain barrier and decreases neuro-inflammation after brain injury.

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J. Slack, Y. Li, J. Dalle Lucca. United States Army Institute of Surgical Research, Fort Sam Houston, TX

Blast induced neurotrauma (BINT) is the signature life threatening injury of current military casualties. Over 73% of all US military casualties are caused by explosive weaponry. Casualties from blast injuries often experience delays of one hour to several days in receiving medical evaluation and subsequent care. During this time secondary neurodegenerative molecular responses can promote further neuronal injury. Identification of molecules and pathways associated with neuronal injury after blast is of upmost importance in providing insight into the development of pharmacotherapeutic strategies aimed at decreasing the hyperinflammatory response of blast and the progression of secondary injuries associated with this condition. This study was designed to characterize the complement system and adaptive immune-inflammatory response in a rat model of BINT and the potential of complement inhibition therapy to mitigate neuronal damage in traumatic brain injury. Our results demonstrate that moderate blast triggers systemic and local complement activation as early as 3 hours, and persisting for at least 48 hours, following blast overexposure. In addition, complement activation directly correlated with blast-induce neuronal damage as well as leukocyte infiltration and TNFα production in injured rat brain tissues. These findings indicate that complement inhibitors and/or TNFa blockers are important therapeutic targets for treatment of military and civilian casualties exposed to blast. In support of this, initial results using Decay-Accelerating Factor (DAF), a regulator of the alternative complement pathway, after blast provides a protection against neuronal damage. Administration of DAF (50 μg/kg) not only rats reduces C3 deposition but attenuates brain damage 3-hours post blast.

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F.E. van den Boogaard1, X. Brands1, M.J. Schultz2, C. van ’t Veer1, T. van der Poll*1. 1Academic Medical Center, Amsterdam, Netherlands, 2Academic Medical Center, Amsterdam, Netherlands

Background: S.pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease Activated Receptor (PAR)2 can be activated by several proteases, including tryptase and clotting factors FVIIa and Xa. We examined the role of PAR2 during pneumococcal pneumonia and -sepsis in mice.

Methods: Pneumonia was induced by intranasal inoculation of S.pneumoniae in wild type (WT) and PAR2 knockout (KO) mice; animals were sacrificed at 6, 24 and 48 hrs after infection for analyses or observed for 10 days. In separate experiments mice were treated with nafamostat (a specific tryptase inhibitor), rNAPc2 (a specific tissue factor/FVIIa/FXa inhibitor) or vehicle. In addition, primary sepsis was induced in WT and PAR2 KO mice by intravenous injection of S.pneumoniae.

Results: PAR2 KO mice showed a strongly reduced mortality during pneumonia (47% vs 100% in WT mice, p<0.007) which was associated with reduced bacterial counts in BALF at 24 and 48 hrs (p<0.05) and in lung homogenates at 48 hrs (p=0.02). In addition, PAR2 KO mice had significantly less bacteremia at 24 and 48 hrs. Treatment with nafamostat did not influence bacterial loads in WT mice. rNAPc2 inhibited activation of coagulation (thrombin-antithrombin complexes) in plasma (p=0.03) and lung (p<0.001) of both WT and PAR2 KO mice, but did not impact on bacterial loads. In pneumococcal sepsis no differences in bacterial numbers were observed between WT and PAR2 KO mice.

Conclusion: PAR2 impairs the outcome of pneumococcal pneumonia by facilitating bacterial growth and dissemination from the lungs through a mechanism that does not rely on tryptase or tissue factor pathway derived proteases.

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S.J. Weber, M. Messer, C. Hohmann, P. Kellermann, M. Perl*. University of Ulm, Ulm, Germany

RIPK1-dependent necroptosis has recently been shown to be involved in the release of danger signals during sepsis. In this context, systemic knockout or silencing of RIPK1 proofed to be detrimental due to increased apoptosis. However, the pathogenetic and therapeutic value of modulating local RIPK1 activity in the lung still remains elusive.

Thus, we hypothesized that in vivo silencing of multifunctional adaptor protein RIPK1 would ameliorate trauma induced septic ALI. Blunt chest trauma was induced by a single blast wave in male C57Bl/6 mice (n=8/group, Two-way ANOVA, SNK, p<0.05). 100 μg RIPK1-siRNA or scrambled RNA (control) was delivered 12 hrs before and after trauma. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) 24 hrs after chest trauma. TNF-α, Fas, IL-6, MCP-1 and MIP-2 in bronchoalveolar lavage fluid (BALF) and G-CSF, TNF-α, MCP-1 and MIP-2 in lung tissue as well as plasma IL-10, TNF-α and MCP-1 concentrations were markedly increased at 24 hrs following ALI but were substantially reduced in response to RIPK1-silencing. At 12 and 24 hrs after CLP BALF total protein was substantially increased during ALI and was not reduced following RIPK1-siRNA delivery. Lung histology (H&E sections) showed indices of ALI, such as lung congestion and alveolar derangement, which were not ameliorated by the administration of siRNA to the lungs. Interestingly, RIPK1 silencing in lung epithelial cells did not increase lung apoptosis as evidenced by TUNEL staining of lung sections and lung tissue Caspase-3 western blotting.

Taken together, these data indicate that RIPK1 is pathogenetically involved in the inflammatory signaling cascade in trauma induced septic ALI. Silencing of RIPK1 using siRNA in vivo might be of additional therapeutic value in antiapoptotic ALI treatment strategies.

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S. Yang1, D.M. Stepien2, T.A. Pritts*3, D. Remick*4, A.B. Lentsch*5. 1University of Cincinnati, Cincinnati, OH, 2Boston University, Boston, MA, 3University of Cincinnati, Cincinnati, OH, 4Boston University, Boston, MA, 5University of Cincinnati, Cincinnati, OH

Mild traumatic brain injury (mTBI) affects more than 1 million people in the USA annually and is increasingly recognized as a major health issue. Diagnosis of mTBI is difficult and may be associated with impaired motor coordination, cognition, and host immune responses. We developed a murine model of mTBI induced by controlled impact of a steel rod onto the cranium overlying the frontal lobes. Different degrees of injury were induced using steel rods of different weights transferring 0.085J (mTBI-1) and 0.100J (mTBI-2) of energy. Mice undergoing mTBI-1 or mTBI-2 had significant deficits in motor coordination and balance (measured using a rotorod) and cognition (determined by novel object recognition), for several days after injury compared to sham controls. Furthermore, we found both mTBI-1 and mTBI-2 caused micromolecular leakage in the blood brain barrier, whereas only mTBI-2 caused macromolecular leakage. Serum biochemical markers for brain injury, glial fibrillary acidic protein and neuron-specific enolase, were elevated acutely and corresponded to the degree of injury, but returned to baseline within 24 hours after injury. From a panel of cytokines and chemokines measured, we found serum IL-6 and KC were significantly increased (up to 18-fold) for up to 6hrs and 24hrs after mTBI-1 and mTBI-2, respectively. IL-6-knockout (KO) mice undergoing mTBI-1 or mTBI-2 had no increase in serum IL-6 or KC, and no change in cognition, but showed significant deficits in motor coordination after injury compared to wild-type mice. Our study describes a murine model of mTBI that replicates many of the functional defects of mTBI in humans. Our data suggest that IL-6 levels are greatly elevated after mTBI, drives systemic production of CXC chemokines, and plays a significant role in the maintenance of motor coordination after injury.

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M.C. Soult, N.E. Lonergan, B. Shah*, L.D. Britt*, C.J. Sullivan*. Eastern Virginia Medical School, Norfolk, VA

Gram negative bacteria remain the leading cause of sepsis worldwide. Effective treatment remains elusive and antibiotic therapy is not always effective. During growth, gram negative bacteria produce outer membrane vesicles (OMVs) which contain various components, including lipopolysaccharide (LPS). In this study we sought to determine if OMVs, independent of whole-cell bacteria, are capable of initiating the immune responses associated with the early stages of sepsis. Human umbilical endothelial cells (HUVEC) were exposed to OMVs originating from Escherichia coli. HUVEC activation in response to OMVs was evaluated by NFκB nuclear translocation, expression and cell surface presentation of vascular adhesion proteins (E-selectin, ICAM), and secretion of pro-inflammatory cytokines (TNF-α, IL-6). Significant induction of NFκB translocation was seen in HUVECs after exposure to OMVs. OMV treatment increased the expression and cell surface presentation of cellular adhesion proteins. Additionally, media of OMV treated HUVECs was found to contain increased levels of pro-inflammatory cytokines. Taken together, the data indicates that OMVs are capable of initiating the inflammatory cascade in vitro, suggesting that OMVs may have a significant role in the development of sepsis.

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K. Wilhelmsen, K. Mesa, F. Xu, J. Hellman*. University of California, San Francisco, San Francisco, CA

Objectives: Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation upregulates inflammatory mediators and modulates endothelial permeability and the expression of coagulation factors. We defined TLR2-activated EC signaling pathways, and tested the hypothesis that TLR2 signaling differs in ECs and monocytes.

Methods: We probed signaling pathways using MAPK inhibitors and siRNA knockdown of MAP kinases and NF-κB in human EC and peripheral blood mononuclear cells (PBMC) stimulated with bacterial lipopeptide TLR2 agonists.

Results: We newly identified a role for ERK5 in the TLR2-mediated expression of inflammatory mediators in primary human lung microvascular ECs (HMVEC-L) and human PBMC, and in TLR2-mediated induction of PAI-1 in HMVEC-L. ERK5 has not previously been reported to participate in TLR2 signaling in any cell type. We also found that MEK1 negatively regulates EC TLR2 signaling, whereas MEK1 promotes TLR2 signaling in PBMC. We confirmed that NF-κB, JNK and p38-MAPK also mediate EC expression of inflammatory mediators and PAI-1. Finally, we observed that p38-MAPK, JNK and ERK5 promote, while MEK1 negatively regulates, the attachment of primary human neutrophils to TLR2-activated HMVEC-L. This suggests that these signaling intermediaries participate in the recruitment and adherence of neutrophils to the lung microvasculature during infection.

Conclusions: The studies identify a novel role for ERK5 in TLR2-mediated inflammatory signaling in human ECs and monocytes, and suggest the ERK5 may play an important role in lung neutrophil recruitment and coagulopathy in infection. Additionally, the finding that MEK1 has divergent roles in TLR2 signaling pathways in EC and monocytes suggests fundamental differences in inflammatory signaling pathways in EC and monocytes.

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A.H. Smith1, N. Qa’aty1, R. Harrison1, N. Brooks1, A. Arnó1, Y. Hiyama1, M.G. Jeschke*2, 1. 1Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada, 2Ross Tilley Burn Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada

Severe burn injury induces a catecholamine surge and major alterations in glucose and lipid metabolism. Previously, we connected hepatic endoplasmic reticulum (ER) stress to insulin resistance. More recently, we found that administration of propranolol (β-adrenergic receptor antagonist) attenuates ER stress, linking catecholamines to ER stress. The underlying mechanisms of this link are undefined. We hypothesized that catecholamines directly induce hepatic ER stress via β-adrenergic receptors.

Primary hepatocytes were isolated from mice fed regular chow or Western diet and treated with epinephrine, norepinephrine, or isoproterenol. Cell lysates were collected for protein and gene expression analyses of the ER stress response. Organelle morphology after catecholamine treatment was visualized with fluorescent trackers. In HepG2’s and primary hepatocytes from mice fed regular chow, ER stress markers Bip, ATF6 and pIRE-1 were increased 12hrs after norepinephrine and 24hrs after epinephrine treatment (3 to 4 fold compared to controls, p < 0.01). In primary hepatocytes from Western diet-fed mice, epinephrine, norepinephrine, and isoproterenol also increased ATF4 and CHOP, downstream targets of the pPERK branch. XBP1 splicing did not increase after catecholamine treatment, suggesting that the pIRE1 branch may be less involved. Pre-treatment with propranolol before catecholamine treatment attenuated the ER stress response. Organelle tracker experiments suggest that epinephrine influences organelle morphology more than norepinephrine.

We conclude that catecholamines induce ER stress predominantly via β-receptors. A high fat/high sucrose environment indeed augments catecholamine alteration, indicating the importance of pre-existing metabolism before an occurring injury.

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D.C. Blok1, M.H. van Lieshout1, F.E. van den Boogaard1, A.J. Hoogendijk1, S. Florquin2, C. Garlanda3, A. Mantovani3, A.F. de Vos1, C. van ’t Veer1, T. van der Poll*1. 1Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands, 2Department of Pathology, Academic Medical Center, Amsterdam, Netherlands, 3Istituto Clinico Humanitas, Milan, Italy

Background: Streptococcus pneumoniae is a common cause of pneumonia, sepsis and death in humans. Toll-like receptors (TLR) play a pivotal role in host defense against infection. Furthermore, TLR signaling is negatively regulated by single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR a.k.a. TIR8).

Objective: To determine the role of SIGIRR in pneumonia and sepsis caused by Streptococcus pneumoniae.

Methods: Wild type (WT) and SIGIRR knockout (KO) C57Bl/6 mice were infected intranasally (to induce pneumonia) or intravenously (to induce primary sepsis) with 5x104 and 5x105 S. pneumoniae respectively. Lungs, blood, spleen and liver were harvested 6, 24 and 48 hours post infection for quantitative cultures; cytokines and chemokines were measured in lung homogenates and plasma. Lung pathology was evaluated by H&E staining.

Results: SIGIRR KO mice showed a delayed mortality in a LD100 model of pneumococcal pneumonia (median survival time 63h versus 54h in WT mice, P < 0.01). In accordance, SIGIRR KO mice displayed less dissemination of the infection at 24h after induction of pneumonia, as reflected by lower bacterial loads in blood, liver and spleen; in addition, at 48h SIGIRR KO mice had lower bacterial burdens in lungs. Although cytokine and chemokine levels were similar in both mouse strains, the early lung inflammatory response was enhanced in SIGIRR KO mice as demonstrated by higher pathology scores at 6h. SIGIRR KO mice also showed lower bacterial counts in all body sites examined after direct intravenous administration of S. pneumoniae.

Conclusion: SIGIRR impairs antibacterial host defense during pneumonia and sepsis caused by S. pneumoniae.

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G. Piraino, M. O’Connor, P.W. Hake, B. Zingarelli*. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

Extracellular signal-regulated kinase 1/2 (ERK1/2) mediates a range of activity in metabolism, inflammation and aging. The expression of phosphorylated and active ERK1/2 is usually increased in several tissues during ischemia and reperfusion injury. However, its biological role is not completely defined. In this study, we investigated the role of ERK1/2 in hemorrhage-induced end organ damage in aged animals. Hemorrhagic shock was induced in male rats of adult age (6-8 months old) and senescent age (21-24 months old) by withdrawing blood to a mean arterial blood pressure of 50 mmHg. After 3 h, animals were rapidly resuscitated by infusing the shed blood. Rats were treated with vehicle or with PD98059 (10 mg/kg intraperitoneally), an inhibitor of ERK1/2 activation, at the time of reperfusion and every hour thereafter. Myeloperoxidase activity (MPO) was measured to evaluate neutrophil infiltration in lung and liver at 3 h after reperfusion. There was a marked elevation of MPO activity in lung and liver (1355.1±62.3 and 148.5±34.9 U/100 mg tissue, respectively) of vehicle-treated adult rats when compared to sham animals (243.2±4.5 and 17.3±1.9 U/100 mg tissue, respectively). Treatment of adult rats with PD98059 significantly (p<0.05) reduced MPO activity in lung and liver (1005.9±104.9 U/100 and 19.6±3.4 U/100 mg, respectively). Vehicle-treated senescent rats also had an increase of lung MPO after resuscitation (1239.4±117.1 U/100 mg tissue) when compared to sham age-matched animals (218.5±1.5 U/100 mg tissue). However, treatment of senescent rats with PD98059 did not affect lung MPO activity after hemorrhage (1478.0±84.2 U/100 mg tissue). Thus, our data demonstrated that interventions to inhibit ERK1/2 activation might have protective effects in organ injury in an age-dependent fashion. (Supported by NIH R01 AG027990).

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N. White*1,2, N. Bradbury1,2, X. Wang1,2, S. Stern*1,2. 1University of Washington, Seattle, WA, 2Harborview Medical Center, Seattle, WA

Objectives: Traumatic brain injury (TBI) impairs fluid resuscitation of hemorrhagic shock (HS) in ill-defined ways. Our objective is to characterize the primary hemodynamic, metabolic, and organ-specific derangements associated with TBI during hemorrhage and fluid resuscitation of HS.

Methods: Domestic pigs (27±3.6kg) were subjected to a three-phase uncontrolled hemorrhage model by aortic tear with (N=7) and without (N=8) TBI by fluid percussion. HS was induced by catheter hemorrhage and aortic tear to mean arterial pressure (MAP) = 30mmHg for 15-minutes. Lactated Ringers was then given (20ml/kg) every 15 minutes as needed for MAP < 60mmHg for up to 105 minutes. The aortic tear was then repaired, and resuscitation continued in a goal-directed manner using normal saline and shed blood up to 6 hours. Two-way ANOVA with Tukey HSD was used to determine the effect of TBI on hemodynamic and metabolic measurements made serially during the protocol.

Results: There was no effect of TBI on: total LR infused, intraperitoneal blood loss, shed blood infused, survival time, or lactate at the end of hemorrhage (p values>0.46). There was a significant effect of TBI on blood withdrawal required to reach MAP=30mmHg (TBI=14.9 ml/kg vs. NO TBI=19.9 ml/kg, p=0.01) during hemorrhage, cerebral oxygen consumption (TBI=4.6 vs. 3.2 ml/kg/min, p=0.0001), MAP (TBI=58.1 vs. 65.8 mmHg, p<0.0001), arterial lactate (TBI=5.0 vs. 3.3 mmol/L, p=<.0001), and renal blood flow (TBI=1.8 vs. 2.4 ml/g/min, p=0.02) during resuscitation.

Conclusions: TBI increases cerebral metabolic activity and promotes systemic shock during fluid resuscitation of HS, supporting a systems-wide impact of TBI on shock physiology.

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C. Kawasaki1, T. Kawasaki*2, K. Okamoto2, T. Sata2. 1Sugioka Memorial Hospital, Fukuoka, Japan, 2University of Occupational and Environmental Health, Kitakyushu, Japan

Objective: Heat stroke is a syndrome consisting of life-threatening central nerve system and multiple organ dysfunctions from complications of hyperthermia. Thrombomodulin (TM) is an endothelial anticoagulant cofactor that plays an important role in the regulation of intravascular coagulation. We previously reported that TM improves the liver dysfunction after heat stress. The purpose of this study was to investigate the effect of TM on the inflammatory response of the brain 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 exposure. In some experiments, minocycline (40 mg/kg, i.p.), microglia inhibitor, was administered 1 hour before heat exposure. Heat stroke was induced by exposure to ambient temperature of 38°C for 4 hours. The heat-stressed mice were returned to room temperature (25°C) after the end of heat exposure. After heat stress, the plasma and brain inflammatory mediators (TNF-alpha, IL-6, and HMGB1) levels were determined using ELISA.

Results: In CG, plasma and brain TNF-alpha and IL-6 increased significantly at 2 hr after heat stress (p<0.05). Plasma and brain HMGB1 levels also increased in CG at 2 hr after heat stress (p<0.05). In TG, these inflammatory responses ware suppressed compared to CG (p<0.05). Minocycline treatment decreased brain inflammatory responses, but plasma inflammatory mediators levels were not altered.

Conclusion: This study demonstrated that TM suppressed plasma and brain inflammatory mediators concentrations after heat stress. TM may be a beneficial treatment for heat stroke patients for down-regulating microglial cell-mediated inflammatory responses.

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I. Paterniti1, D. Impellizzeri1, E. Mazzon2, E. Esposito1, 2, S. Cuzzocrea*1, 2. 1University of Messina, Messina, Italy, 2IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy, Messina, Italy

Rho kinase (ROK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. The aim of this experimental study was to determine the role of ROK signaling pathways in the inflammatory response, in particular in the secondary injury associated with the experimental model of spinal cord trauma.

The injury was induced by application of vascular clips to the dura via a four-level T5-T8 laminectomy in mice. Fasudil was administered in mice (10 mg/kg i.p.) 1 h and 6 h after the trauma.

The treatment with fasudil significantly decreased (1) histological damage, (2) motor recovery, 3) nuclear factor (NF)-κB expression, (4) pro-inflammatory cytokines production such as Tumor Necrosis Factor (TNF-α) and Interleukin-1β (IL-1β), (5) neutrophil infiltration, (6) nitrotyrosine and poly-ADP-ribose (PAR) formation, (7) Glial fibrillary acidic protein (GFAP) expression, (8) apoptosis (TUNEL staining, FAS ligand expression, Bax and Bcl-2 expression), (9) MAP Kinase activation (P-ERK and P-JNK expression).

Our results indicate that inhibition of ROK by fasudil may represent a useful therapeutic perspective in the treatment of inflammation associated with spinal cord trauma.

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R. Crupi1, A. Ahmad1, D. Impellizzeri1, E. Mazzon2, A. Marino3, E. Esposito1, S. Cuzzocrea*1, 2. 1Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, Messina, Italy, 2IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy, 3Department of Life Sciences “M.Malpighi”, Section of General Physiology and Pharmacology, University of Messina, Messina, Italy

Traumatic brain injury (TBI) is a major cause of preventable death and morbidity in young adults. This complex condition is characterized by significant blood brain barrier leakage that stems from cerebral ischemia, inflammation, and redox imbalances in the traumatic penumbra of the injured brain. Recovery of function after TBI is partly through neuronal plasticity. In order to test whether treatments that enhance plasticity might improve functional recovery after TBI, a new mice head injury model was developed, in which a controlled cortical impactor produced full thickness lesions of the forelimb region of the sensorimotor cortex. Once trauma has occurred, combating these exacerbations is the keystone of an effective TBI therapy. The endogenous fatty acid palmitoylethanolamide (PEA) is one of the members of N-acyl-ethanolamines family maintain not only redox balance but also inhibit the mechanisms of secondary injury. Therefore, we tested whether PEA shows efficacy in a mice model of experimental TBI. Treatment of the TBI animals with PEA reduced edema and brain infractions as evidenced by decreased 2, 3, 5-triphenyltetrazolium chloride staining across brain sections. PEA-mediated improvements in tissues histology shown by reduction of lesion size and improvement in apoptosis level (assayed by TUNEL assay, Bax and Bcl-2) further support the efficacy of PEA therapy. The PEA treatment blocked infiltration of astrocytes and restored CCI-mediated reduced expression of PAR, Nitrotyrosine, iNOS, Chymase, Tryptase, CD11b and GFAP. PEA inhibited the TBI-mediated decrease in the expression of pJAK and NF-kB. PEA-treated injured animals improved neurobehavioral functions as evaluated by behavioral tests.

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F. Hu1, Z. Tang1, L. Gatto*2, Q. Meng*1, S. Hawgood3, R. Cooney*1, G. Wang*1. 1Upstate Medical University, Syracuse, NY, 2SUNY Cortland, Cortland, NY, 3University of California, San Francisco, CA

Abdominal sepsis is a risk factor for respiratory failure and mortality. Surfactant proteins A and D (SP-A, SP-D), C-type lectins, are expressed in multiple organs. They play an important role in immunity and homeostasis through interaction with pathogens and host immune cells.

Objective: Study effects of SP-A/SP-D knockout on the regulation of immune-related gene expression and signaling pathways in the lung of mice after CLP.

Methods: SP-A and SP-D double knockout (KO) and matched wild-type (WT) C57BL/6 male mice (9-12 weeks) were studied. Mice underwent CLP or sham surgery (neither ligated nor punctured). Mortality and lung gene expression were studied. Tissues were harvested at 24 hrs and 48 hrs after CLP, then analyzed by Western blot, ELISA, CFU count, and Real-time PCR Array (>2 fold change as significant). Data are means+SE, p<0.05 significant by Kaplan-meier, or t-test.

Results: Mortality at 36 hrs after CLP was decreased in CLP KO mice (30%) compared to CLP WT mice (80%) (p<0.05). Bacterial CFU were decreased in lung BAL (5.7 fold) and blood (5.6 fold) in CLP KO mice (p<0.01, vs CLP WT). Expression of 84 immunity-related genes in the lung indicated no difference at basal line in sham KO and WT mice. After CLP the expression of nine genes (Ccl2, Clec7a, IL-10, IL-1a, ILf9, Ncf4, Nfkb1, Ptafr, Tnf) was decreased in CLP KO mice (p<0.05, vs CLP WT). Expression of two genes (lyz1 and Hc) was increased in CLP KO mice (p<0.05, vs CLP WT). Signaling pathway analysis revealed CLP was associated with the acute phase response signaling pathway, specifically with the IL-1, P38, and NF-κB pathways. Compared with CLP WT mice, P38 and NF-κB pathways were downregulated in CLP KO mice.

Conclusion: SP-A and SP-D influence mortality, regulation of immune-related gene expression and signaling pathways in lung after CLP.

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N. Packiriswamy, T. Lee, P.B. Raghavendra, H. Durairaj, N. Parameswaran*. Michigan State University, East Lansing, MI

G-protein coupled receptor kinase-5 (GRK-5) is an evolutionarily conserved regulator of NFκB pathway, recently shown to modulate NFκB in a variety of species ranging from Drosophila to Zebra fish, mouse and human cells. NFκB signaling has been shown to modulate the pathogenesis of sepsis by regulating various events in sepsis including cytokine production, apoptosis of immune and non-immune cells. Therefore, we tested the role of GRK5 in sepsis using cecal ligation and puncture model (CLP). We subjected GRK5 deficient and the corresponding wild type mice to CLP and determined various parameters involved in the progression of sepsis. We found that GRK5 deficient mice have decreased inflammatory cytokine production, attenuated thymic cell apoptosis, and improved bacterial clearance compared to the wild type mice. Interestingly, peritoneal cells collected from the septic mice demonstrated enhanced immune-responsiveness in vitro in the GRK5 deficient compared to the wild type mice. To determine the signaling mechanisms and the role of NFκB activation, we assessed the NFκB activity during sepsis in various tissues in the two genotypes. We found that GRK5 deficient mice had decreased NFκB activation in liver, but not in thymus, suggesting cell/tissue-specific regulation of NFκB by GRK5. Consistent with the overall findings on inflammation, apoptosis and bacterial load, GRK5 deficient mice had significantly improved survival following sepsis. Together, these studies suggest that inhibition of GRK5 might be a potential strategy for beneficial modulation of sepsis progression.

Figure 1

Figure 1

PI- AnnexinV+ population in thymus 20 hours post CLP

PI- AnnexinV+ population in thymus 20 hours post CLP

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N. Matijevic, V. Kostousov, Y.W. Wang, C.E. Wade*, J.B. Holcomb*, B.A. Cotton*. UTHSC Houston, Houston, TX

Background: Thrombelastography (TEG) is potential tool to detect posttraumatic coagulopathy and to monitor resuscitation therapy. We registered 2.5% incidence of excessive fibrinolysis (LY30>8%) in trauma patients on admission with 81% mortality. We have created an in vitro trauma coagulopathy model with excessive fibrinolysis, resembling hyperfibrinolytic TEG in trauma patients, and explored the antifibrinolytic effect of tranexamic acid (TXA) on TEG variables.

Methods: Citrated whole blood was diluted by 15% with crystalloids or colloids and supplemented with tissue factor and tissue plasminogen activator (tPA, 225 ng/ml), with or without TXA (3.25 μg/ml). TEG analysis was performed using rapid TEG reagent and TEG 5000 analyzer.

Results: TEG variables of the diluted donors’ blood (DCM) were similar to TEG values from 29 injured patients (PTS) with excessive fibrinolysis (Table). The addition of TXA to the model reversed fibrinolysis: decrease in LY30 from 36.6 to 1.0%, p<0.001. TXA also slightly improved MA, and G, p = 0.02 for both.

Conclusion: We have developed an in vitro model of trauma coagulopathy with excessive fibrinolysis that is potentially useful for testing the effects of different resuscitation modalities. Low dose TXA reversed hyperfibrinolysis in this model. Further clinical research is needed to explore whether the normalization of trauma induced hyperfibrinolysis by antifibrinolytics is associated with improved outcomes.

No caption available

No caption available

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D.S. Herzig1, G. Fang1, E. Sherwood*1, 2. 1UTMB, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX

Introduction: Interferons primarily induce leukocyte activation by stimulation of the STAT1 signaling pathway, which induces anti-viral immunity and inflammation. However, the importance of STAT1 signaling during septic shock caused by bacterial infections has not been rigorously studied.

Methods: In this study, survival, bacterial clearance, core body temperature, and systemic cytokine production were evaluated in STAT1 knockout mice and in matching wild type controls. Sepsis was induced by cecal ligation and puncture (CLP). In some experiments mice received Primaxin (12.5 mg/kg) as antibiotic treatment. All mice received lactated Ringer’s (1 ml, IP) as fluid resuscitation at the time of CLP.

Results: Our study shows that survival (80 vs 10%) and core body temperature (33.4 vs 23.2°C) were significantly improved in STAT1-deficient mice compared to wild type controls. In addition, MIP-2, IL-6 and CXCL10 concentrations in plasma and peritoneal lavage fluid were significantly lower in STAT1 knockout mice. Without antibiotics, bacterial counts were significantly lower in STAT1 knockout mice compared to control mice but were not significantly different when Primaxin was given.

Conclusion: These results indicate that STAT1-deficent mice are resistant to CLP-induced immunopathology, particularly when antibiotics are administered.

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K.G. Shah, N. Kohn, C.P. Marini, R. Barrera. Hofstra North Shore - LIJ School of Medicine, Manhasset, NY

Background: Body mass index (BMI) is a commonly used proxy to measure body fat in individuals. Extremes in BMI, either high or low, have not been well studied and may contribute to prolonged ventilator days in the surgical intensive care unit (SICU). We sought to determine if these extremes in BMI could be an independent predictor of mechanical ventilation duration.

Methods: Data from patients admitted to our SICU were collected for analysis. Days from start of ventilation to discontinuation of ventilation in the SICU, or discharge from the SICU on a ventilator (whichever came first), was estimated using the product-limit method. BMI, classified as Underweight (<18 kg/m2), Normal (18 to <25 kg/m2), Overweight (25 to <30 kg/m2), and Obese (≥30 kg/m2), were compared using the log-rank test. Upon finding a significant difference among BMI classifications, pairwise comparisons were carried out using Tukey’s HSD test. Patients who died on the same day that mechanical ventilation was removed were considered censored for this analysis.

Results: A total of 1,741 patients were included for analysis. Patients under the age of 18 were not included. The requirement for mechanical ventilation was more common post surgery, either elective or emergency, in all groups. The majority of patients (>50%) were removed from the ventilator after day 1 in all four groups. However, there were no statistically significant differences in total days on mechanical ventilation among the four BMI classifications.

Conclusions: Extremes in BMI are not independent risk factors for a prolonged stay on mechanical ventilation. Furthermore, there were no differences in the length of time spent on mechanical ventilation. Our data suggests that extremes in BMI do not affect total ventilator days, and weaning protocols need not be specific for BMI.

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I.V. Ostrova, M.S. Avrushchenko. V.A.Negovsky Research Institute of General Reanimatology, Russian Academy of Medical Sciences, Moscow, Russian Federation

Transient ischemia causes neuronal injury in the hippocampus. Endoplasmic reticulum (ER) stress has been implicated to the pathogenesis of cerebral ischemia. 78-kDa glucose-regulated protein (GRP78) is an indicator of ER dysfunction.

Objective: To reveal an association of the changes in GRP78 protein expression with the state of hippocampal neuronal populations in the post-resuscitation period.

Methods: Adult white rats both sexes underwent 10-min circulatory arrest evoked by intrathoracic clamping of the cardiac vascular bundle, followed by resuscitation. On post-resuscitative days 1, 7, and 14, the density and composition of pyramidal neuronal populations in the CA1 and CA4 hippocampal fields were determined by morphometric analysis and the level of GRP78 protein expression was investigated.

Results: It was revealed sex-depended differences in neuronal damage caused ischemia-reperfusion. Thus, in resuscitated males loss of neurons was observed in both fields of the hippocampus: in CA1 - on the 7th day, in CA4 - on the 1st day after cardiac arrest. In females neuronal loss was revealed only in field CA1 (14th day). There were gender differences in the post-resuscitative changes in the immune responsiveness of hippocampal neuronal populations to GRP78 protein. Complex analysis indicated that neuronal loss occurred in neuronal populations with lower immunoreactivity to GRP78. Elevated expression of GRP78 protein on the 1st day after cardiac arrest promoted the prevention of neuronal dystrophic changes and/or death.

Conclusion: The process of neuronal death in the postresuscitation period are closely correlated with the change of neuronal populations immunoreactivity to the protein GRP78. The presence of immunoreactivity to GRP78 is an important factor in ensuring resistans of nerve cells to ischemia-reperfusion.

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A. Ayub, H. Kim, G. Zhai, W.J. Hubbard, C.L. Floyd, N.L. Day, K. Zinn, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL

We examined the efficacy of E2-SO4 treatment for TBI in a lateral fluid percussion (LFP) model. This involved 6 groups of rats (n=5/group). All rats received a craniectomy. Groups 1, 2, 4 and 5 received LFP injury. Groups 1 and 4 were treated with E2 (1 mg/kg) 1 hr post-injury, groups 2 and 5 were vehicle (Veh)-treated, groups 3 and 6 were sham. Groups 1-3 underwent intracranial pressure (ICP), cerebral perfusion pressure (CPP) and partial oxygen pressure (pbtO2) monitoring. Groups 4-6 underwent fludoexoyglucose (FDG)-PET/CT and diffusion tensor imaging (DTI) analysis on days 1 and 7 post-induction. Relative standard uptake values (SUV) of upper half of brain to central value, fractional anisotropy (FA) and apparent diffusion coefficient (ADC) maps were calculated. Results indicate that ICP, CPP and pbtO2 in sham, Veh and E2 groups (mmHg) were 4.7±0.4, 17.4±0.5, 7±0.4, 82.1±1.3, 65.2±1.4, 75.7±1.4, 39.6±0.9, 15.8±1.3, and 34.1±1.6, respectively (differences significant at p<0.05). Relative % SUV of Veh, treated and sham groups on day 1 were 89.9±0.02, 94.0±0.01, and 96.9±0.02, respectively, and on day 7 were 93.6±0.01, 92.4±0.01, and 96.1±0.01, respectively. Day 1 SUV in Veh group was significantly lower than treated and sham groups (p=0.045 and 0.013, respectively); no statistical significance was found on day 7. Edema sizes on days 1 and 7 in Veh and treated groups were (mm3) 23.9±5.1 and 10.3±2.1 and 10.4±2.6 and 1.2±0.7, respectively. No edema was found in sham group. Edema size in Veh group was significantly larger compared to treated group on days 1 (p=0.038) and 7 (p=0.010). No significant FA change was found between Veh and treated groups. Thus, E2 promotes recovery from TBI by increasing brain metabolism, resolving edema and improving physiological conditions if injected 1 hr post-TBI. (W81XWH-08-2-0153).

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F.A. Rivera-Chavez*, A. Burris, M. Liu, S. Wolf*, J. Minei*. UT Southwestern Medical Center, Dallas, TX

Clinical data have demonstrated that the ability of the elderly to respond to infection is diminished in comparison with young adults. Impairment of multiple neutrophil functions has been observed in the elderly. A novel anti-bacterial mechanism in the PMN has been recently described; the formation of neutrophil extracellular traps (NETs) for the containment of infection and inflammation. NET formation is reduced in neonates compared with adults, indicating that this aspect of neutrophil function is modified through development. Thus, we investigated the possibility that NET formation is altered, or deficient, in neutrophils from elderly individuals.

Methods: a) Human peripheral blood neutrophils were isolated from healthy young (n =5) and older (n =5) (> 65y) volunteer donors b) PMN stimulation with Lipopolysaccharide (LPS, 100 ng/mL) for 1 hour at 37°C. c) NET detection by immunolabeling d) fluorescent microscopy, e) NET quantification; Experiments were analyzed using a paired, one-tailed Student’s t test.

Results: Mean age was 35 young Vs 76 older. We found that neutrophils from elderly donors form significant fewer NETs when activated by LPS, in contrast to PMN from young adults. (Graph 1).

Conclusions: This is the first report of neutrophil extracellular trap formation in older adults. NET formation may play a role in the diminished ability of the elderly to respond to infection. The clinical implications of these findings are unknown.

The proportion of NETs per total amount of neutrophils was calculated for comparison of NET formation by each group

The proportion of NETs per total amount of neutrophils was calculated for comparison of NET formation by each group

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E. Esposito, R. Crupi, A. Ahmad, M. Campolo, I. Paterniti, S. Cuzzocrea*. Clinical and Experimental Medicine and Pharmacology, University of Messina, Messina, Italy

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 complicated by the cross talk and coordinated regulation between autophagy and apoptosis. The role of autophagy in the pathogenesis of renal diseases has not been identified. In this study, we determined the contribution of autophagy in renal tubular cell injury using in vivo models of renal ischemia/reperfusion (I/R). C57BL/6 mice were subjected to sham operation or 30 minutes of bilateral renal ischemia, followed by 6 or 24 hours of reperfusion. A basal level of LC3-II was shown by immunoblotting in the sham kidney homogenates. 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. Autophagy is induced in a time-dependent manner during renal I/R. Moreover, I/R increased mitochondrial cytochrome C release, renal O2(-*) level and renal 3-nitrotirosine accumulation, Beclin-1 expression, Bax/Bcl-xL ratio, caspase 3 expression and poly-(ADP-ribose)-polymerase fragments. Treatment with SB216763, an inhibitor of GSK-3β, decreased renal tubular dilatation, vacuolization and sloughing, blood urea nitrogen, creatinine and renal cell apoptosis. The inhibition of GSK-3β significantly inhibited the markers of autophagy. We show that autophagy is induced in this model and is critically regulated by GSK-3β.

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R. Elbaz, C. Culberson, M. Clemens*. UNC Charlotte, Charlotte, NC

The gaseous mediators nitric oxide (NO) and carbon monoxide (CO) are recognized as important mediators in shock and sepsis. More recently hydrogen sulfide (H2S) has been shown to be protective in ischemia, but its role in sepsis in controversial. Recent reports suggest an interaction between H2S and CO in modulating macrophage inflammatory response. Therefore, we tested whether induction of heme oxygenase-1 (HO-1) might modulate the expression of cystathionine γ lyase (CSE) the enzyme responsible for H2S production in the liver. RAW 264.7 cells were treated with cobalt protoporphyrin (COPP, inducer of HO-1), LPS or COPP + LPS and expression of HO-1, iNOS and CSE were determined at 4 and 24 hours. COPP induced HO-1 mRNA and protein. This was associated with a decrease in the LPS-induced iNOS and an increase in CSE expression at 24 hours (10.9 fold). LPS also induced CSE (3.97 fold, p< 0.05) and this was greatly potentiated by COPP pretreatment (20.1 fold, p<0.01) suggesting the possibility that CSE induction is dependent upon HO-1 activity. To test this, following induction with CoPP, HO-1 activity was inhibited using tin protoporphyrin (SnPP). SnPP cotreatment completely prevented the induction of CSE message and protein by CoPP. This suggests that products of HO-1 activity are responsible for the induction of CSE in murine macrophages. In separate experiments, we tested whether H2S might reciprocally regulate HO-1 expression. Neither Na2S (fast release H2S donor) nor GYY4137 (slow release H2S donor) had any effect on HO-1 expression. These results demonstrate a relationship between CO and H2S generating enzymes and suggest that induction of HO-1 in endotoxemia is necessary for upregulation of CSE and increased H2S production in sepsis while downregulating iNOS.

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M. Starr*, B. Evers, H. Saito*. University of Kentucky, Lexington, KY

Background: The underlying mechanisms for age-associated increases in sepsis mortality remain largely unknown. Our previous studies using murine endotoxemia (LPS) and peritonitis (CLP) models of sepsis showed that augmented thrombosis and elevated inflammatory cytokine production are closely associated with increased mortality in the aged. We recently reported that adipose tissue is a major source of multiple inflammatory and coagulant factors during sepsis/endotoxemia and that these factors are more abundantly produced in aged compared to young mice.

Objective: The objective of this study was to determine the cell origin of adipose tissue-derived cytokines and coagulation factors during systemic inflammation.

Methods: Young (4 months) and aged (24 months) C57BL/6 mice were sacrificed 0, 6, or 12h after injection with LPS (2.5mg/kg) and epididymal adipose tissues were harvested for histological analysis and collagenase digestion to separate cell types into adipocytes and stromal cells. RNA was subsequently isolated from cell fractions for gene expression analysis by qRT-PCR.

Results: Histological examination and analysis of inflammatory cell markers (F480, CD11b, CD11c, CD206) did not reveal any evidence of inflammatory cell infiltration into the adipose tissue. RNA analysis of separated cell fractions revealed that LPS- and age-dependent changes in gene expression of cytokines and coagulation factors (IL-6, TNFα, IL-1β, PAI-1, PAI-2, Thbs-1, tissue factor) occur predominantly in stromal cells, rather than in adipocytes.

Conclusions: (1) During systemic inflammation, adipose tissue strongly expresses many cytokines and coagulation factors. (2) Induced levels of these factors are augmented by aging. (3) The major cell sources of these factors are resident stromal cells, not adipocytes or infiltrating inflammatory cells.

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D. Okamura, M. Starr*, H. Takahashi, B. Evers, H. Saito*. University of Kentucky, Lexington, KY

Introduction: Activation of coagulation and inhibition of fibrinolysis are important mechanisms promoting thrombosis during sepsis. We previously reported that an age-associated increase in mortality of mice during endotoxemia is partly due to enhanced coagulation in the aged, caused by insufficient activation of the protein C (PC) anti-coagulant pathway.

Objective: The purpose of this study was to investigate the mechanisms for age-dependent coagulation during cecal ligation and puncture (CLP)-induced sepsis.

Methods: Intra-abdominal peritonitis was induced by CLP using either 21 or 16 gauge (G) needles on young (4-6 months) and aged (23-25 months) male C57BL/6 mice. Coagulation was assessed by measuring fibrin formation through Western blot analysis and immunohistochemistry. Plasma d-dimer and plasminogen activator inhibitor type-1 (PAI-1) were measured to evaluate fibrinolysis. Activated protein C (aPC) was measured to evaluate activation of the PC anti-coagulant pathway. Degree of inflammation was assessed by plasma IL-6 levels.

Results: Compared to young mice, aged mice showed significantly increased mortality, coagulation and inflammation after 21G CLP. Young mice with 16G CLP showed a mortality rate and inflammation levels equivalent to aged mice with 21G CLP; however, fibrin formation in the lung and kidney was prominent only in aged mice. Levels of pulmonary tissue factor, plasma d-dimer and plasma PAI-1 after CLP were equivalent in young (16G) and aged (21G) mice. Plasma aPC levels were elevated after CLP in young (16G and 21G) mice but remained low in aged (21G) mice.

Conclusions: Disseminated intravascular coagulation during CLP-induced sepsis is enhanced in an age-dependent fashion and likely due to reduced anti-coagulant PC pathway function in the aged; fibrinolysis seems largely unaffected by aging.

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K.R. Zettel, R. Eid, M. Hoffman, Q. Wang, A. Tsung, T. Billiar*. University of Pittsburgh, Pittsburgh, PA

REDD1 (Regulated in Development and DNA damage response1) is a hypoxia-inducible gene that suppresses mTOR expression through dissociation of the TSC2 from 14-3-3. Increased REDD1 expression is associated with skeletal muscle atrophy from hypoxia in in-vitro studies, and disruption of this gene causes improved hepatic recovery after isolated hepatic ischemic reperfusion injury. Hemorrhagic shock is associated with hypoxia that leads to organ injury, though the mechanisms are not well understood. It is postulated that a hypoxia-induced gene would account for some of the cell death. Our extensive gene array analysis (Edmonds et al. Physiol Genomics, 2011) showed upregulation of cell death pathways in the liver. We surveyed our gene array for specific genes induced by hypoxia and found that REDD1 was upregulated. We then further evaluated the involvement of this gene in hemorrhagic shock.

Both C57Bl/6 and REDD1 knockout mice were subjected to hemorrhagic shock of a mean arterial pressure equal to or less than 25mm Hg for 1.5 hours followed by resuscitation with lactated ringers. At 4.5 hours after resuscitation, surviving mice were analyzed. There was a significant increase in mortality in wild type mice (55%) compared to REDD1 knockout (0%). ALT levels were significantly decreased in the REDD1 KO hemorrhagic shock group compared to wild type (266 U/I vs. 928 U/I, p<0.05) while AST levels were trending toward a protective effect in hemorrhagic shock with REDD1 knockout (788 U/I vs. 1268 U/I).

In summary, REDD1 is a regulatory gene upregulated in hypoxia, as well as in hemorrhagic shock. The diminished release of hepatic enzymes and decreased mortality after disruption of this gene in hemorrhagic shock demonstrates that REDD1 plays a negative role in the cellular response in global hemorrhagic shock.

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E.D. Fox1, S.F. Monaghan1, C.J. Chamberlain2, J.T. Machan3, W.G. Cioffi1, S. Gravenstein2, A. Ayala*1, D.S. Heffernan*1. 1Department of Surgery, Brown University, Rhode Island Hospital, Providence, RI, 2Division of Geriatrics, Department of Medicine, Providence, RI, 3Department of Orthopedics, Providence, RI

Background: Trauma typically induces a neutrophilia. Geriatric trauma patients have higher mortality than younger patients. Geriatric patients have altered neutrophil responses and function. They are extremely susceptible to the inflammation associated with shock and trauma. How neutrophil response in the elderly relates to outcomes, especially mortality, remains unknown.

Methods: Post-hoc analysis of 5 years of prospective data on moderate and severely injured (Injury Severity Score >/=15) geriatric (>/=65yrs) and young (18-35yrs) patients who survived at least 3 days. Neutrophil counts for the first 4 hospital days were grouped by elevation/lack of elevation and return/failure to return to normal. We used proportional hazards regression with time-varying covariates and Kaplan-Meier curves on changes of the neutrophil count to predict risk of death, and time to healthy discharge.

Results: Of 2,448 moderately and severely injured patients, geriatric patients had higher mortality than young patients. The neutrophil profile did not associate with mortality for young patients. Geriatric patients lacking neutrophilia had improved survival compared to those with neutrophilia which resolved (8% versus 21.5%; p<0.001) or with sustained neutrophilia (8% vs 21.7%; p=0.026). Mortality in geriatric patients without neutrophilia over the first four days was comparable to that of young trauma patients. Among geriatric survivors within all groups, lack of elevated neutrophil response was associated with faster time to healthy discharge.

Conclusions: Geriatric patients are more susceptible to the effects of inflammation. Young patients tolerate sustained inflammatory responses to trauma, whereas a neutrophilic inflammatory response is more ominous for geriatric trauma patients.

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I.C. Nweze, J.W. Smith*, J. Lakshmanan, C.M. Klinge, B. Zhang, B.G. Harbrecht*. University of Louisville Hospital, Louisville, KY

Endothelial NO synthase (eNOS) maintains microvascular perfusion and prevents shock-induced organ injury. Excessive nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) during sepsis plays a significant role in the pathophysiologic sequelae of sepsis and hemorrhagic shock. Limiting excessive NO from iNOS improves hepatic function and reduces mortality after sepsis. Estrogen administration in rats improves shock induced hepatocellular injury and also modulates NO production by regulating iNOS expression. However, the pathway leading to this regulation of iNOS is unknown. We hypothesized that regulation of NO by 17β-estradiol will be mediated by the activation of one of the described estrogen receptors.

Methods: Isolated rat hepatocytes were stimulated in-vitro with pro-inflammatory cytokines to induce NO synthesis with or without estrogen and in the presence of selective estrogen receptor agonists and antagonists. Nitrite was detected after 24hours. INOS protein was determined by western blot and iNOS mRNA was analyzed using rt-PCR.

Results: Estrogen dose dependently decreased cytokine stimulated NO production in hepatocytes by decreasing iNOS mRNA and protein levels. However, none of the estrogen receptor agonist hadas potent an effect as estrogen by itself and none of the receptorswere demonstrated to be exclusively responsible for estrogen effect.

Conclusion: 17β-Estradiol decreases cytokine-stimulated iNOS expression and NO production. Understanding the mechanisms involved in the pathway initiation and signaling of this regulation will help to elucidate the mode of Estrogen modulation of NO in sepsis and shock.

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P. Greiffenstein*1, J. Sulzer2, P. Molina*2. 1LSUHSC-Dept of Surgery, New Orleans, LA, 2;LSUHSC-Dept of Physiology, New Orleans, LA

Previous studies from our laboratory have demonstrated that binge alcohol intoxication (Alc) accentuates peripheral blood mononuclear cell (PBMC) pro-inflammatory cytokine response to LPS challenge in the late (5days) post-injury (trauma-hemorrhage; Tx-Hem) phase. This study examined the impact of Alc-Tx-Hem on tissue (spleen) and PBMC inflammatory cytokine levels and myeloperoxidase (MPO) activity in the spleen as well as the PBMC phagocytic activity at 5 days post Tx-Hem. Rats were administered Alc (5g/kg, 30% w/v intragastrically) daily for 3 consecutive days followed by 2.5g/kg x 30 min prior to Tx+Hem. Time-matched controls received isocaloric/isovolumic dextrose (Dex). Animals were subjected to TxHem (muscle crush followed by hemorrhage; MABP 40mmHg for 60 min) and resuscitated with Lactated Ringer’s (2.4 x total volume removed). Animals were allowed to recover for 5 days prior to excision of spleens and PBMC harvesting for determination of cytokine levels, MPO activity, and phagocytic activity. In the spleen, Alc+TxHem accentuated IL-6 content compared to Dex-TxHem (148±42 vs 44±17 pg/mg protein; p<0.05) but decreased MPO activity (82±3 vs 108±3 U/min/mg protein in Dex-TxHem; p<0.05). Likewise, in PBMCs Alc+TxHem accentuated IL-1 (377.1±50.03 vs 241.1±55, p<0.05) and TNF (5290±780 vs. 3600±755, p<0.05) levels, but attenuated PBMC phagocytic activity (p>0.05). These results indicate that upregulation of pro-inflammatory cytokines secondary to Alc+TxHem is not reflected in the inflammatory cell activity. This may reflect a significant impact on the competence of the immune system during the late recovery period following alcohol binge and trauma-hemorrhage. (DOD PR-054196, NIAAA-AA7577, NIAAA-AA19587).

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S. Narasimhan, C. Culberson, M. Clemens*. UNC Charlotte, Charlotte, NC

Hydrogen sulfide is an important mediator in septic shock but its exact role is controversial. The activity of Cystathionine gamma lyase (CSE), the enzyme responsible for H2S production in the liver, has been shown to be critical during the systemic inflammatory response in sepsis but the mechanism regulating its expression in inflammation have not been elucidated. Thus we tested whether CSE message levels might be negatively regulated by H2S levels in RAW264.7 macrophages. DL- Propargylglycine (PAG), a commonly used inhibitor acting on the pyridoxyl phosphate site was used to inhibit CSE activity. Treatment with PAG for 6 hours under normoxic conditions, significantly up-regulated of CSE mRNA (approx 5 fold). This suggested that H2S might downregulate CSE expression. To test this, we treated the cells with both PAG and GYY4137 (slow release H2S donor). Addition of H2S had no effect on CSE up-regulation. For testing if PAG exerts its effect through cysteine accumulation, the cells were treated with L-Cysteine. L-cysteine had no effect on CSE expression. Since sepsis is associated with tissue hypoxia we tested the effects of hypoxia on up-regulation of CSE expression caused by PAG. Interestingly, the up-regulation was completely abrogated by hypoxia and the same effect was observed when the cells were treated with the HIF1α activator CoCl2. This suggests that regulation of CSE expression might be due to the activity of HIF 1 alpha. Finally, we tested whether the pyridoxyl phosphate independent CSE inhibitor β cyano L-alanine (BCA), a competitive inhibitor of CSE, also exerted the same effect as PAG. In contrast to PAG, BCA had no effect on CSE expression. These results suggest that the effect of PAG on CSE expression is independent of CSE activity but that it is related to hypoxia/HIF1α activation.

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D.S. Heffernan*1, S.F. Monaghan1, E.D. Fox1, M. Kettenmann1, J.T. Machan3, S. Gravenstein2, W.G. Cioffi1, A. Ayala*1. 1Department of Surgery, Brown University, Rhode Island Hospital, Providence, RI, 2Division of Geriatrics, Department of Medicine, Providence, RI, 3Department of Orthopedics, Providence, RI

Background: Lymphocytes modulate responses to trauma, critical illness and shock. Trauma typically induces lymphopenia. We have shown that among all trauma patients, failure to normalize a lymphopenic response is associated with increased mortality. Geriatric patients display baseline lymphocyte dysfunction. We assessed how lymphocyte patterns affect mortality in geriatric trauma patients.

Methods: Post-hoc analysis of 5 years of prospective data on moderate and severely injured (Injury Severity Score >/=15) geriatric (>/=65yrs) and young (18-35yrs) patients who survived at least 3 days. Lymphocyte counts for the first 4 hospital days were grouped by depression/lack of depression and return/failure to return to normal. We used proportional hazards regression with time-varying covariates and Kaplan-Meier curves on changes of the lymphocyte count to predict risk of death, and time to healthy discharge.

Results: Of 2,448 patients, overall mortality was higher in geriatric patients. Mortality was not affected by lymphocyte response in young patients. Within Geriatric patients, any Lymphopenia was associated with significantly elevated mortality (9.1%vs22.6%; p=0.008). However, failure to normalize lymphocyte loss had the most profound association with mortality (24%vs19.7%; p=0.002). Geriatric patients without lymphopenia have mortality akin to young patients who display the typical pattern of lymphocyte loss (9.1% vs 5.8%;p=0.9). Furthermore, lack of lymphopenia was associated with a faster time to healthy discharge.

Conclusions: Lymphopenia affects mortality in geriatric trauma patients, especially persistent lymphocyte loss. Geriatric patients who retain normal circulating lymphocyte counts have mortality similar to young patients. Preventing lymphocyte loss in geriatric trauma patients may be a future therapeutic target.

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J. Cuschieri*1, S.G. Rhind2, S.B. Rizoli3, W. Junger*4, A.J. Baker5, M.Y. Shiu3, P.N. Shek2, E.M. Bulger*1. 1Department of Surgery, University of Washington, Harborview Medical Center, Seattle, WA, 2Defence Research & Development Canada (DRDC) Toronto, Toronto, ON, Canada, 3Department of Surgery & Critical Care Medicine, Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 4Department of Surgery, Beth Israel Deaconess Medical Center & Harvard Medical School, Boston, MA, 5Brain Injury Laboratory, Cara Phelan Centre for Trauma Research Keenan Research Centre, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada

Background: Cytokines play a crucial role in the pathogenesis of traumatic brain injury (TBI), contributing to delayed tissue damage. Hypertonic saline exerts anti-inflammatory actions that may attenuate secondary injury cascades and convey neuroprotection.

Objectives: To study how hypertonic resuscitation influences neuroinflammatory mechanisms in head injury, we measured multiple pro- and anti-inflammatory cytokines.

Design: Prospective randomized controlled trial conducted within the Resuscitation Outcomes Consortium enrolled 82 severe TBI patients (GCS≤ 8) and 20 healthy controls.

Intervention: A single infusion (250mL) of 7.5% hypertonic saline (HS;n=21), 7.5% HS plus 6% dextran-70 (HSD;n=22) or 0.9% saline (NS;n=39).

Measurements & Results: Serial blood samples were drawn at admission and after 12 h, 24 h and 7 d. Serum concentrations (pg/mL) of interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-1RA (receptor antagonist), and soluble TNF-receptors (sTNF-RI/II) were determined by ELISA. Patient levels of pro-inflammatory IL-6, IL-8, and TNF-α were significantly (P<.05) elevated (4-5-fold) above control values at admission. TNF-α peaked at 24 h and was highest in patients with poor outcome (GOSE≤4 or death). Patients resuscitated with HS or HSD had lower levels of pro-inflammatory cytokines at all times compared to NS. Admission levels of anti-inflammatory IL-10 and IL-1RA were elevated up to 75-fold and 15-fold, respectively. Maximal increases of IL-10 and IL-1RA were observed in NS-treated patients. Sustained increases in sTNF-RI/II were evident at all times.

Conclusions: These data provide insight into the distinct temporal profile of cytokine release after TBI and demonstrate that hypertonic fluids can attenuate pro- and anti-inflammatory mediators. Funded by Defence R&D Canada & NIH R01GM076101.

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Q. Zhang, L. Dong, X. Zhu, Y. Yao. Burn Institute, Beijing, China

Introduction: Recent studies have suggested that glucagon-like peptide 1(GLP-1) analogue, exendin-4 (Ex-4), could manage hyperglycaemia in critically illness. Whether it could reduce burn-induced hyperglycaemia has not been explored yet.

Methods: Male Balb/c mice were randomly selected to receive either bovine serum albumin as placebo (2 mg/kg) or recombinant Ex-4 at low (0.24 nmol/kg), medium (2.4 nmol/kg) or high doses (24 nmol/kg) intraperitoneally immediately after burn injury. Mice were sacrificed 8, 24, 48 and 72 h postburn (pb) after tail glucose levels were recorded. T cell functions were evaluated by culturing the splenocytes with concanavalin A for 48h followed by detection of the proliferation and cytokines. Hepatic myeloperoxidase was examined by colorimetry. In addition, naïve splenocytes were treated with Ex-4 in vitro and nuclear factor kappa B (NF-κB) was determined by ELISAs, while the supernatants for cytokines examination.

Results: Unexpectedly, exogenously administered Ex-4 exacerbated burn-induced mortality with attenuated hyperglycemia but increased expressions of both pro-and anti-inflammatory cytokines. Augmented hepatic leukocyte infiltration and enhanced pancreatic CD11b expression were also found. An ex vivo study revealed that the early exacerbated inflammatory response provoked by Ex-4 is likely mediated, at least in part, by a shift in cytokine expressions toward Th1 via activating splenic NF-κB followed by compensated immunosuppressive Th2 response.

Conclusions: These data support a pathogenic role for GLP-1 signaling during burn-induced immune response, warranting its clinical application in crucial illness.

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Y. Sumi1, L. Li2, Y. Kuroda1, M. Morikawa1, Y. Inoue1, S. Matsuda1, K. Okamoto1, H. Tanaka1, W.G. Junger*2. 1Juntendo University Urayasu Hospital, Urayasu, Chiba, Japan, 2Beth Israel Deaconess Medical Center, Boston, MA

ATP has been recognized as a danger signal in various clinical conditions. We have previously shown that ATP release and feedback via P2Y2 receptors are involved in neutrophil activation in vitro and in a mouse sepsis model. However, little is known about the role of plasma ATP in clinical sepsis and its role in neutrophil activation.

Here we investigated in 15 sepsis patients (age: 67 ± 14 years, APACHE: 23 ± 5), whether plasma ATP levels correlate with neutrophil activation. Sepsis was predominantly due to pneumonia, urinary tract infection, cellulitis, or cholecystitis. Arterial blood was drawn via a radial line and plasma ATP concentrations and neutrophil activation were determined by HPLC and assessing CD11b expression, respectively.

On days 0-1 after sepsis, ATP levels were significantly higher in patients than in normal controls (NC). These levels decreased over time (Fig; mean ± SEM, Student’s t test, ** p<0.01). Patients with positive blood cultures (n=10, 115 ± 14 nM) had higher ATP levels than patients with negative cultures (n=5, 84 ± 11 nM). Neutrophil CD11b expression in sepsis patients on days 0-1 was significantly higher than in NC (Fig; mean ± SEM, Student’s t test, * p<0.05) and diminished over time corresponding to plasma ATP levels.

We conclude that ATP release into plasma may be involved in neutrophil activation. Therapeutic interventions to modulate ATP levels could reduce neutrophil-induced host tissue damage after sepsis.

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P. Mommsen1, M. Weuster7, I. Witte2, J. Mohr3, M. Fröhlich4, C. Keibl5, S. Flohé2, H. Pape4, A. Seekamp7, S. Ruchholtz3, M. van Griensven*6, F. Hildebrand*1. 1Trauma Department, Hannover Medical School, Hannover, Germany, 2Department of Trauma and Hand Surgery, University Hospital Duesseldorf, Duesseldorf, Germany, 3Department of Trauma, Hand and Reconstructive Surgery, University Hospital Marburg, Marburg, Germany, 4Department of Orthopaedic and Trauma Surgery, University Hospital Aachen, Aachen, Germany, 5Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria, 6Department for Experimental Trauma Surgery, Technical University Munich, Munich, Germany, 7Department of Trauma Surgery, University Hospital Kiel, Kiel, Germany

Objectives: Induced hypothermia is known to reduce pro-inflammatory reactions after elective surgery and single insults (e.g. hemorrhage). The effects of induced hypothermia on inflammatory response in a porcine multiple trauma model are not fully elucidated.

Methods: 40 male pigs (aged 12-16 weeks, weighing 30 ± 3 kg) were assigned to 4 different study groups (each consisting of 10 animals). Beside sham animals, experimental animals underwent multiple trauma with blunt chest injury, liver laceration and pressure-controlled hemorrhage (MAP 30 ± 5 mmHg) followed by fluid resuscitation. Afterwards, either normothermia was maintained or hypothermia of 34°C was induced via endovascular cooling for 3 hours with subsequent rewarming in sham and experimental groups. Animals were sacrificed 15.5 hours after trauma induction. White blood cell count, leukocyte activation (CD11r3 expression) and respiratory burst were determined by flow cytometry. Systemic IL-6 levels were assessed by ELISA.

Results: Multiple trauma resulted in a transitory increase of leukocyte count in normothermic animals, whereas persistent leukocytosis was observed following hypothermia. Reduced CD11r3 expression and respiratory burst was found in the presence of hypothermia, but without reaching statistical significance. Hypothermic trauma animals showed significantly attenuated IL-6 plasma levels.

Conclusions: In the present porcine multiple trauma model, hypothermia led to a reduction of pro-inflammatory immune response with significantly decreased systemic IL-6 levels and apparently attenuated leukocyte activation. The latter and the potentially adverse effects of immunosuppression with increased susceptibility to infection have to be validated in further studies with extended and deeper hypothermia.

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K. Iskander, F. Craciun, D. Stepien, E. Duffy, R. Moitra, J. Kim, D. Remick*. Boston University Medical Center, Boston, MA

Objective: Sepsis is the leading cause of acute lung injury in patients. In the murine cecal ligation and puncture (CLP) model, we investigated whether significant lung injury develops in septic mice predicted-to-die (Die-P) compared to those predicted-to-live (Live-P).

Methods: Polymicrobial sepsis was induced by CLP in female outbred mice. Mice were stratified into Live-P and Die-P groups based on plasma IL-6 levels at 24 hours post-CLP. Measures of pulmonary inflammation and injury were quantified at 24 and 48 hours after sepsis induction. Mice receiving intratracheal normal saline (no surgical intervention) were negative controls. Positive controls were mice given Pseudomonas aeruginosa to elicit significant lung injury.

Results: Plasma and bronchoalveolar lavage (BAL) levels of pro and anti-inflammatory cytokines were significantly elevated in Die-P mice compared to Live-P, indicating pulmonary inflammation. Die-P mice maintained normal oxygen saturation on arterial blood gas compared to Live-P mice at 24 and 48 hours, while mice with Pseudomonas were clearly hypoxic (p<0.0001). BAL total protein, albumin, and IgM in Pseudomonas mice were significantly increased indicating vascular permeability from endothelial damage (p=0.0001), but Die-P and Live-P values were both low and not different than saline. No significant neutrophil infiltration on BAL or change in myeloperoxidase activity between Die-P and Live-P mice was seen. Histology confirmed the absence of lung injury in Die-P and Live-P mice, yet histological scores were markedly elevated for Pseudomonas mice (p<0.0001).

Conclusion: Mice predicted-to-die from sepsis have greater lung inflammation but no significant lung injury at 24 and 48 hours post-CLP. The etiology of death in the acute phase of the CLP model of sepsis is not due to pulmonary injury.

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E. Boelke*1, C. Matuschek1, M. Pelzer1, M. Peiper1, D. Hermsen1, G. Schieren1, M. van Griensven*2, G. Steinbach3, G. Lammering1, W. Budach1. 1Heinrich Heine University, Duesseldorf, Germany, 2Klinikum rechts der Isar, Muenchen, Germany, 3Universitaetsklinikum Ulm, Ulm, Germany

Background: Evaluation of renal function is important in cancer patients receiving chemotherapy to avoid renal insufficiency and in sepsis. Limitations of creatinine as marker for the glomerular filtration rate (GFR) led to the proposal of cystatin C as a better biomarker especially in patients with low muscle mass. Furthermore it is a good marker for renal dysfunction in sepsis. We compared the accuracy of cystatin C- and creatinine-based equations to estimate GFR in head-and-neck cancer (HNC) patients receiving platinum based radiochemotherapy (model).

Methods: 50 HNC patients (GFR range 37-105 ml/min/1.73 m2), complemented by 19 patients with renal insufficiency (GFR range 10-60 ml/min/1.73 m2) were enrolled. Intra-Class-Correlation-coefficients were calculated between the reference method (51) Cr-EDTA clearance and estimated GFR by creatinine clearance and equations based on creatinine (Cockroft-Gault, Modified-Diet-in-Renal-Disease, Wright) or cystatin C (Larsson, Dade-Behring, Hoek). Sensitivity and specificity to discriminate GFR < 60 ml/min/1.73 m2 were evaluated by receiver-operating-characteristic-curve (ROC).

Results: The highest correlation coefficients were found for the cystatin C-based estimates in comparison with creatinine-based estimates or creatinine clearance. Bland-Altman plots revealed GFR overestimation for all equations tested. The cystatin C-based Hoek formula exhibited the highest overall precision and accuracy.

Conclusion: Cystatin C-based GFR estimates showed the overall strongest correlation to the reference method and seems to be a perfect and inexpensive biomarker in clinical practice for determining kidney function.

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H. Liang, Y. Gu, C. Zhang, X. Fan, X. Yang. Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, China

Objective: To investigate the protective effects of agmatine (AGM) on zymosan (ZYM)-induced systemic inflammation and acute lung injury (ALI) in mice.

Methods: C57BL/6 mice were randomly divided into four groups: normal control (SHAM+PBS), AGM control (SHAM+AGM), inflammation (ZYM+PBS), AGM treatment (ZYM+AGM). Mice received either intraperitoneally ZYM (500 mg/kg) or vehicle, with which AGM (200 mg/kg, i.p.) was administered simultaneously. Blood samples and peritoneal exudates were collected 12h after drug administration for measuring tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and nitric oxide (NO). The myeloperoxidase (MPO) activity, TNF-α, IL-6 levels and nuclear factor-kappaB p65 (NF-kappaB p65) activity in the lung tissues were determined, and the pulmonary histological changes and apoptosis were also observed.

Results: Treatment of mice with AGM for 12h not only attenuated the increase of TNF-α, IL-6 and NO levels in peritoneal exudates and serum, but also alleviated the elevation of MPO, TNF-α, IL-6 levels and NF-kappaB p65 DNA-binding activity in lung tissues caused by ZYM. Histological examination demonstrated that ZYM treated mice had severe inflammatory reaction in lung tissues, manifested as vasodilatation, neutrophils infiltration and apoptosis, AGM treatment significantly reduced such injury.

Conclusions: Agmatine can attenuate systemic inflammation and ALI induced by zymosan in mice.

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J. Wiersinga1, 2, T. Weehuizen1, T. van der Poll*1, 2. 1Center of Experimental and Molecular Medicine (CEMM), Academic Medical Center, Amsterdam, Netherlands, 2Division of Infectious Diseases and Department of Medicine, Academic Medical Center, Amsterdam, Netherlands

Introduction: TLRs recognize bacterial virulence factors and initiate immune responses. Melioidosis, caused by B. pseudomallei is an important cause of sepsis in SE-Asia and a recognized biothreat agent. Previously we have shown that TLR5 - which recognizes bacterial flagella - is highly upregulated in melioidosis patients. Since flagella is regarded as a key virulence factor of B. pseudomallei we aimed to dissect the role of TLR5 in melioidosis.

Methods: We performed stimulation experiments with HEK-TLR5, murine alveolar macrophage (MH-S) and murine lung epithelial (MLE-15) cells. In addition, we inoculated wild-type and TLR5 knock-out mice intranasally with B. pseudomallei 1026b wild type strain or its flagellin-lacking mutant MM36 (courtesy Dr. D Woods). Mice were sacrificed at 24, 48 and 72 hr to assess bacterial loads and inflammation.

Results: B. pseudomallei flagellin was confirmed to signal through TLR5. In vitro we found flagellin-lacking B. pseudomallei MM36 to be less potent in eliciting inflammatory responses compared to WT strains. However, in vivo no difference in bacterial counts or inflammatory response was found between WT mice receiving B. pseudomallei 1026b or its flagellin-lacking MM36 mutant. Furthermore, TLR5 did not seem to play a role during pulmonary B. pseudomallei infection: equal lung bacterial counts were seen in WT and TLR5 KO mice after infection. Interestingly, TLR5 KO mice infected with B. pseudomallei 1026b or MM36 demonstrated higher systemic bacterial loads compared to their WT counterparts.

Discussion: This data suggest a surprisingly limited role for both TLR5 and flagella during B. pseudomallei induced pneumonia. This could be in correspondence with known low pulmonary TLR5 expression. However, TLR5 does seem to play a protective role in preventing systemic bacterial spread.

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R.A. Namas*1, 2, M. Mikheev1, 2, J. Yin1, 2, P. Over2, M. Young2, G. Constantine3, R. Zamora*1, 2, J. Gerlach2, Y. Vodovotz*1, 2. 1University of Pittsburgh, Pittsburgh, PA, 2McGowan Institute for Regenerative Medicine, Pittsburgh, PA, 3Departments of Mathematics and Biostatistics, Pittsburgh, PA

Objective: Properly-regulated inflammation facilitates recognition and reaction to injury or infection, but inadequate or overly-robust inflammation can lead to disease. Endotoxin causes inflammation driven and regulated by TNF-α, which is in turn negatively regulated via its endogenous inhibitor, soluble TNF-α receptor (sTNFR). We hypothesized that endogenous sTNFR is produced too late or at a low level to attenuate the positive feedback cycle of inflammation → damage → inflammation, and that continuous provision of sTNFR via a biohybrid device will attenuate this positive feedback and reduce inflammation.

Methods: We generated stably-modified variants of human HepG2 hepatocytes, using lentiviral constructs coding for mouse sTNFR driven by the constitutive cytomegalovirus promoter, and seeded them in a 0.8 mL liver bioreactor. The bioreactor was then connected to anesthetized, cannulated rats subjected to endotoxin infusion and maintained solely by the animals’ circulation for a 6 h period. Serial blood samples were collected for cytokine, NO2-/NO3-, AST/ALT and sTNFR analysis. In addition, the mean arterial pressure was recorded continuously.

Results: The biohybrid device elevated circulating sTNFR, reduced the levels of TNF-α and other key inflammatory mediators, alleviated hypotension, and reduced circulating markers of organ damage.

Conclusion: This novel class of biohybrid devices may be modified for patient- and disease-specific application, and thus may represent a novel strategy for rational inflammation reprogramming.

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M. Rani*, Q. Zhang, M.G. Schwacha*. University of Texas Health Science Center at San Antonio, San Antonio, TX

Introduction: Trauma-hemorrhage (TH) can induce acute lung injury (ALI) and other pulmonary complications of an inflammatory nature. Previously, we have shown that there is an influx of activated γδ T-cells into the lung after TH. It remains unknown whether these γδ T-cells are causative in the development of ALI.

Methods: C57BL/6 wild type (WT) and δ TCR-/- (γδ KO) mice were subjected to TH or sham treatment. A 2-cm incision was made along the mid-line to induce soft-tissue trauma and 30% of total blood was collected via the vena cava to induce hemorrhagic shock. The incision was closed and mice were resuscitated 1 h later. Sham mice underwent neither blood loss nor resuscitation. Two hours after resuscitation lung cells were isolated and analyzed using flow cytometry.

Results: TH induced a significant influx of αβ T-cells into the lungs that was markedly attenuated in γδ KO mice. The suppressed T-cell infiltration in γδ KO mice after TH was associated with a significantly increased influx of granulocytes (Gr1+) and monocytes (CD11b+), as compared with WT mice. Further analysis of the myeloid cells revealed that they were myeloid suppressor cells (MDSC; CD11b+Gr1+). MDSC influx was present in both WT and γδ KO mice after TH, but the increases were more profound in the γδ KO group.

Conclusions: Gamma-delta T-cells are protective against pulmonary inflammation after TH through the regulation of T-cell influx which in turn appears to be critical in the suppression of granulocyte, monocyte and MDSC influx.

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S.M. Coldewey1, 2, A.I. Khan1, M. Rogazzo3, M. Collino3, A. Kapoor1, N.S. Patel1, C. Thiemermann1. 1Queen Mary University of London, The William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, London, United Kingdom, 2Hannover Medical School, Clinic for Anaesthesiology and Intensive Care Medicine, Hannover, Germany, 3University of Turin, Department of Drug Science and Technology, Turin, Italy

EPO reduces acute kidney injury (AKI) and improves survival in mice with endotoxemia or sepsis caused by cecal ligation and puncture (CLP). The erythropoietic effects of EPO are mediated by the ‘classical’ EPO-receptor homodimer, while the tissue-protective effects may be mediated by a heterocomplex between EPO-receptor monomer and the β-common receptor (βcR). Here we investigate the mechanisms by which EPO reduces the AKI caused by lipopolysaccharide (LPS) or CLP. In male C57/BL6 mice (2 months), LPS (9mg/kg i.p. for 18h) caused a rise in urea and creatinine and, hence, AKI. EPO (1000IU/kg s.c. 1h after LPS) significantly attenuated the AKI caused by LPS. In kidneys from mice subjected to LPS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase 3-β (GSK-3β; inhibition), and endothelial nitric oxide synthase (eNOS; activation). In the kidney, LPS also caused an increase in phosphorylation (activation) of ERK1/2 and nuclear translocation of p65 (activation of NF-κB), both of which were attenuated by EPO. To elucidate whether the beneficial effects of EPO observed in endotoxemia were mediated by activation of the βcR, βcR-knock-out mice were subjected to LPS and treated with EPO. In βcR-knock-out mice, LPS caused the development of AKI, but this was not significantly affected by EPO. In male C57/BL6 mice (8 months), CLP caused the development of AKI, which was also attenuated by EPO (1000IU/kg s.c. 1h after CLP). In conclusion, therapeutic administration of EPO attenuates the AKI caused by LPS (young mice) and CLP (old mice). The mechanism of action of EPO involves the activation of the Akt/eNOS survival pathway and inhibition of activation of ERK1/2, GSK-3β and NF-κB. We propose that these effects of EPO are secondary to the activation of the βcR.

SMC and AIK equally contributed to this study.

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P. Wacharasint, T. Nakada, J.H. Boyd, J.A. Russell, K.R. Walley. Critical Care Research Laboratories, Institute for Heart + Lung Health, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada

Objective: It is known that interleukin (IL)-10 plays a role in immunosuppression in sepsis. We hypothesize that single nucleotide polymorphisms (SNPs) of IL-10 family members (IL-10, IL-19, IL-20, and IL-24) genes, clustered within 200 kb on chromosome 1, may be associated with increased mortality in septic shock.

Methods: We investigated 13 tag SNPs of IL-10 family members in 1,205 septic shock patients, combining two septic shock cohorts: a single-center (St. Paul’s Hospital; SPH, n=589) and a multicenter cohort (Vasopressin in Septic Shock Trial (VASST, n=616), and checked for deleterious effects by genotype in a separate independent cohort of acute inflammation (postoperative cardiac surgery; CSICU, n=985). DNA was extracted from blood samples and 39 cytokines were measured, including IL-13, an anti-inflammatory cytokine normally up-regulated by IL-19 and IL-20. The primary outcome was 28-day survival.

Results: Polymorphisms in IL-19 (rs2243191) and IL-20 (rs2981573) genes were associated with survival. Patients with genotypes rs2243191[TT] or rs2981573[GG] had increased mortality compared to patients with either the CT or CC genotypes (p=0.001) or the AG or AA genotypes (p=0.001), respectively. In addition, these patients had significantly fewer days-alive and free of organ failure (p values<0.05) as well as decreased serum IL-13 levels (p=0.002 and 0.009, respectively). Patients in the CSICU cohort with genotypes rs2243191[TT] or rs2981573[GG] had a significantly increased risk of prolonged ICU stay (p=0.011 and 0.017, respectively).

Conclusions: Septic shock patients with genotypes rs2243191[TT] and rs2981573[GG] of the IL-19 and IL-20 genes respectively have significantly more mortality and organ dysfunction.

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O. Takasu*1, J. Gaut2, E. Watanabe*3, E. Fagley1, I. Efimov4, P. Swanson5, R. Hotchkiss*1. 1Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO, 2Department of Pathology, Washington University School of Medicine, St. Louis, MO, 3Department of Emergency and Critical Care Medicine, Chiba University, Chiba, Japan, 4Department of Biomedical Engineering, Washington University School of Medicine, St. Louis, MO, 5Department of Pathology, University of Washington School of Medicine, Seattle, WA

Purpose: This study examined the role and type of cell death and organelle injury in sepsis-induced cardiac and renal failure.

Methods: Rapid postmortem tissue harvest was performed in 35 septic patients. Twenty six non-septic cardiac tissues and 20 non-septic kidney tissues served as controls. Tissues were examined for necrosis by conventional light microscopy (LM) and for apoptosis by immunohistochemical (IHC) staining. Kidney injury molecule-1(Kim-1) and p-mammalian target of rapamycin (p-mTOR) were quantitated in kidney. Autophagy and ultrastructural effects were evaluated with electron microscopy (EM).

Cardiac Results: LM from septic patients appeared normal without cell injury. IHC demonstrated low levels of apoptotic cardiomyocytes (<1-2 cells/thousand) that were not different in sepsis versus controls. EM showed rare focal mitochondrial injury in 41% of sepsis and 29% of controls. Autophagolysosomes were uncommon in both groups.

Renal Results: Septic kidneys showed focal changes including tubular dilatation, tubular cells vacuolization and injury under LM. Tubular injury occurred in cortex of 77% of patients, and affected 6% of cortico-medullary junction (CMJ) tubules and 1.2% of cortical tubules. Kim-1 and p-mTOR staining were positive in 31%, 38% CMJ tubules in septic kidneys, consistent with a high degree of tubular injury. Apoptosis occurred in <1-2% of tubules in both septic and control kidneys. EM showed focal tubular and organelle injury in septic kidneys.

Conclusion: Cardiac dysfunction in sepsis is not secondary to cardiomyocyte injury, cell death or gross contractile apparatus abnormalities. Although there was a high incidence of renal tubular cell injury, it was present focally and cell death was rarely present. Thus, renal failure in sepsis is not explained solely by cell injury or cell death.

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M. Kinoshita, H. Miyazaki, S. Ono*, A. Kimura, K. Nishikawa, D. Saitoh, S. Seki. National Defense Medical College, Saitama, Japan

We investigated the efficacy of IL-18 treatment in post-burn Methicillin-resistant Staphylococcus aureus (MRSA) infection. Alternate-day injections of IL-18 into burn-injured C57BL/6 mice significantly increased their survival after MRSA infection and after methicillin-sensitive S. aureus infection. Although IL-18 treatment of burn-injured mice augmented natural IgM production before MRSA infection and IFN-γ production after MRSA infection, neither IgM nor IFN-γ significantly contributed to the improvement in mouse survival. IL-18 treatment increased/restored the serum TNF, IL-17, IL-23, G-CSF, and MIP-2 levels, as well as the neutrophil count, after MRSA infection of burn-injured mice; it also improved impaired neutrophil functions, phagocytic activity, production of reactive oxygen species, and MRSA-killing activity. However, IL-18 treatment was ineffective against MRSA infection in both burn- and sham-injured neutropenic mice. Enhancement of neutrophil functions by IL-18 was also observed in vitro. Furthermore, when neutrophils from IL-18-treated burn-injured mice were adoptively transferred into non-treated burn-injured mice 2 days after MRSA challenge, survival of the recipient mice increased. NOD-SCID mice that have functionally intact neutrophils and macrophages (but not T, B, or NK cells) were substantially resistant to MRSA infection. IL-18 treatment increased the survival of NOD-SCID mice after burn injury and MRSA infection. An adoptive transfer of neutrophils using NOD-SCID mice also showed a beneficial effect of IL-18-activated neutrophils, similar to that seen in C57BL/6 mice. Thus, although neutrophil functions were impaired in burn-injured mice, IL-18 therapy markedly activated neutrophil functions, thereby increasing survival from post-burn MRSA infection.

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T. Hommes1, A. Achouiti1, M. Dessing1, M. Colonna2, C. van ’t Veer1, A. de Vos1, T. van der Poll1. 1AMC, Amsterdam, Netherlands, 2Washington University School of Medicine, St. Louis, MO

Background: Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Triggering receptor expressed on myeloid cells 1 (TREM-1) is an activating receptor belonging to the Ig-superfamily that is primarily expressed on neutrophils, monocytes and macrophages. Through an unknown ligand TREM-1 enhances Toll- and NOD-like receptor signaling.

Aim: To determine the role of TREM-1 in host defense during pneumococcal pneumonia.

Methods: Pneumonia was induced in TREM-1 knockout (KO) and wild-type (WT) mice by intranasal inoculation with viable S. pneumoniae. TREM-1 expression was investigated using qRT-PCR and flow cytometry. Lungs, spleen, liver and blood were harvested at 6, 24 and 48 hour post-infection for quantitative cultures. Cytokines, chemokines and MPO were measured in lung homogenates. Responsiveness towards S. pneumoniae of TREM-1 positive and TREM-1 negative murine alveolar macrophages and human leukocytes was tested in vitro.

Results: WT mice demonstrated enhanced TREM-1 expression during pneumococcal pneumonia. Alveolar macrophages lacking TREM-1 produced less TNF-α than WT cells upon stimulation with S. pneumoniae in vitro. Purified human TREM-1 negative granulocytes showed reduced responsiveness to S. pneumoniae relative to TREM-1 positive granulocytes. TREM-1 KO mice were more susceptible to pneumococcal pneumonia, as reflected by a reduced survival and increased bacterial loads in lungs, blood and distant organs at 24 and 48 hours post infection. At 6 hours post-infection, pulmonary TNF-α, IL-1β and MPO levels were lower in TREM-1 KO mice despite equal bacterial loads, pointing to an impaired initiation of the innate immune response.

Conclusion: TREM-1 contributes to protective immunity during pneumococcal pneumonia.

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T. Brin1, D. Mierzwiak, A. Ambrosch2. 1Dept. of Surgery, St. Joseph-Hospital, Bremerhaven, Germany, 2Inst. of Laboratory Med. and Microbiology, St. Joseph-Hospital, Bremerhaven, Germany

Clinicians are in need of better diagnostic markers for rapid diagnosis of severe infections. Therefore, we studied the clinical significance of mean cell volume in monocytes (MMV), Interleukin 6 (IL-6), C-reactive protein (CRP), procalcitonin (PCT) and white blood cell (WBC) count to identify patients with a severe systemic inflammatory response from infection.

Methods and patients: A total of 74 hospitalized patients with systemic and non-systemic clinical infections were categorized according to the SIRS score and fatal outcome (mortality). At the day of clinic diagnosis, blood was sampled from each patient and MMV and WBC count were obtained by a laser scanning technique on a Coulter DXH hematology analyzer. In addition, IL-6, CRP and PCT were analysed by EIA and immunotubidimetry (CRP), respectively.

Results: According to the SIRS score, 37 patients with severe infections had more than 2 points. A total of 8 patients (11 %) died from infections. However, patients with a high score (SIRS score > 2 points) had significantly higher levels of PCT (p = 0.032), CRP (p=0.028), a higher WBC count (p=0.01) and an increased MMV (p=0.041). From these parameters, only MMV was significantly associated with mortality (p=0.018).

Conclusion: Among different classical markers of inflammation, MMV seems to be a potential parameter to identify patients with a severe systemic inflammatory response. In addition, an increased MMV is associated with mortality in the present study. Since our data are preliminary, more patients should be investigated to underline our findings.

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L. Lee1, E.E. Moore*1, 3, M. Kelher2, N.J. McLaughlin1, 2, D.J. Elzi1, 2, A. Banerjee*1, C.C. Silliman*1, 2. 1University of Colorado, Aurora, CO, 2Bonfils Blood Center, Denver, CO, 3Denver Health Medical Center, Denver, CO

Background: Packed red blood cell (pRBC) transfusion is an independent risk factor for the development of post-injury multiple organ failure (MOF). Arachidonic acid (AA) accumulates during routine pRBC storage; however, its effects on endothelium remain unknown.

Hypothesis: pRBC AA activates human microvascular endothelial (HMVECs) and liver sinusoidal endothelial cells (LSECs) via the G-protein coupled (GPCR) leukotriene receptor B2 (BLT2).

Methods: HMVECs and LSECs (80-100% confluence) were stimulated with heat treated plasma (10-40%) from day 1 (d1) [AA 1μM] or 42 (d42) [AA 5μM] leukoreduced-pRBC plasma for 6hrs. LSECs were also stimulated with lipopolysaccharide (LPS) (20ng-2μg/ml), tumor necrosis factor α (TNFα) (1-100ng/ml), or interferon δ (IFNδ) (1-100ng/ml), for 2-6hrs. ICAM-1 surface expression (SE) was measured using flow cytometry, and neutrophil (PMN) adherence was quantified by myeloperoxidase assay. β-arrestin-1, a GPCR signaling protein, was immunoprecipitated from HMVECs stimulated with AA 5μM 1-5mins and co-precipitated with BLT2 and PKC δ and β1.

Results: TNFα and IFNδ increased LSEC ICAM-1 SE (1.8-2.1 fold, p<0.05) with only a transient increase with LPS. HMVECs and LSECs stimulated with d42 pRBC plasma had a significantly increased ICAM-1 SE (table 1) and PMN adherence vs. d1 (>1.5-6 fold, p<0.05). Immunoprecipitation of β-arrestin-1 co-precipitated with BLT2 (fig.1), PKCδ, and PKCβ1.

Conclusion: PRBCs induce pro-inflammatory activation of LSECs and HMVECs. AA, the major non-polar lipid in stored pRBCs, signals through BLT2 and a downstream PKC-dependent kinase cascade. These data provide mechanistic insight into the role of stored pRBCs in propagating post-injury MOF, and inhibition of their generation or signaling may lead to safer resuscitation strategies.

Increased ICAM-1 Surface Expression (MFI) in HMVECs and LSECs stimulated with d42 pRBC plasma vs

Increased ICAM-1 Surface Expression (MFI) in HMVECs and LSECs stimulated with d42 pRBC plasma vs

Co-precipitation of BLT2 and β-arrestin-1 in HMVECs that were stimulated with AA

Co-precipitation of BLT2 and β-arrestin-1 in HMVECs that were stimulated with AA

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A. Kimura, S. Ono, H. Tsujimoto, S. Hiraki, R. Takahata, H. Miyazaki, M. Kinoshita, K. Hatsuse, D. Saitoh, K. Hase, J. Yamamoto. National Defense Medical College, Tokorozawa, Japan

Objective: To clarify the time course of changes in the serum interleukin-15 (IL-15) concentrations in septic patients undergoing emergency surgery for abdominal infection, and to investigate whether the serum IL-15 levels correlate with the postoperative clinical course of septic patients.

Methods: Twenty-four septic patients who had intra-abdominal infection and underwent an emergency operation were enrolled in this study. The serum IL-15 levels were measured before surgery, and on postoperative day 1(POD1), POD3, and POD5, and the relationship between the serum IL-15 levels and the postoperative clinical course estimated by the Systemic Inflammatory Response Syndrome (SIRS) criteria, APACHE-II (Acute Physiology and Chronic Health Evaluation) score and the parameters of organ function were evaluated.

Results: The time course of changes of the serum IL-15 levels were significantly different between survivors and non-survivors (p<0.05, by repeated measures ANOVA). There was a statistically significant relationship between the serum IL-15 levels on POD1 or POD3 and the duration of SIRS (R=0.50, p<0.05, R=0.65, p<0.01, respectively). Furthermore, a significant positive correlation was observed between the serum IL-15 levels on POD1 and the creatinine levels on POD1 or POD3 (R=0.48, p<0.05, R=0.50, p<0.05, respectively), and a significant negative correlation between the serum IL-15 levels on POD1 or POD 3 and the PaO2/FiO2 on POD3 (R=-0.51, p<0.05, R=-0.69, p<0.01, respectively).

Conclusions: The measurement of post-operative serum level of IL-15 might be useful for predicting the severity of SIRS and organ dysfunction, especially renal and pulmonary dysfunction.

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H.K. de Jong1, 2, A. Achouiti1, A.M. Vollaard3, G.C. Koh4, 5, J.J. Roelofs6, J.T. van Dissel3, J. Roth7, T. Vogl7, T. van der Poll*1, J. Wiersinga1. 1Center for Infection and Immunity Amsterdam (CINIMA), Center of Experimental and Molecular Medicine (CEMM) and Division of Infectious Diseases, Academic Medical Center, Amsterdam, Netherlands, 2Chittagong Medical College Hospital, Chittagong, Bangladesh, 3Department of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands, 4Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom, 5Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 6Department of Pathology, Academic Medical Center, Amsterdam, Netherlands, 7Institute of Immunology, University of Muenster, Muenster, Germany

Background: Typhoid fever, which is caused by the Gram-negative bacterium Salmonella (S.) enterica serovar Typhi and can be complicated by shock, is a major cause of illness and death worldwide. Mrp8 (S100A8) and Mrp14 (S100A9) are endogenous activators of Toll-like receptor 4 promoting lethal, endotoxin-induced shock and multi-organ failure. We aimed to characterize the expression and function of Mrp8/14 in typhoid fever.

Method: Mrp8/14 expression was determined in leukocytes and plasma from 60 typhoid patients and 60 controls, and in mice infected with S. Typhimurium. In addition to in vitro experiments, Mrp8/14 function was investigated in experimental wild type (WT) and Mrp14 deficient mice (which also lacks Mrp8) orally infected with low and high doses of S. Typhimurium. Mice were sacrificed 2- and 5- days post infection to determine bacterial loads and inflammation.

Results: Patients demonstrated markedly increased Mrp8 gene expression and Mrp8/14 concentrations in plasma. Mice inoculated with S. Typhimurium displayed a robust increase in liver and systemic Mrp8/14 production. In vitro Mrp8/14 inhibited the growth of S. Typhimurium in a dose dependent matter. Surprisingly Mrp14 knock out (KO) mice were indistinguishable from WT mice with respect to bacterial loads and inflammatory responses, as determinedbycytokine levels, histopathology and liver function tests. Mrp8recovery in Mrp14 KO mice may potentially contribute to this phenotype.

Conclusion: Mrp8/14 complexes are markedly elevated during typhoid fever. In experimental Salmonella infection Mrp8/14 only modestly influences antibacterial defense mechanisms. We did not find any evidence that Mrp14 contributes to bacterial dissemination or liver injury during Salmonella infection.

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J. Wu*, Y. Lai*, L. Zeng*, J. Lu*, S. Moochhala*. DSO National Laboratories, Singapore, Singapore

The objective of this study is to investigate the effects of hypertonicsaline (HTS) on combat-related multiple trauma in swine model.Swine multiple trauma model described by Cho et al (2009) was slightly modified: pigs received femur facture, 60% of blood loss,and hypothermia at 33 degree of core body temperature. After 30min of shock, HTS (7.5% NaCl, 4ml/kg) or normal saline (NS) of 3times volume of shed blood was given as fluid resuscitation. At 15min after resuscitation, a grade V liver laceration was induced. Sham operation animals underwent surgical procedure without anytrauma. Untreated animals suffered all trauma without fluid resuscitation.

All untreated animals died within 5 hours (3.4±1.3 hr), while most of pigs in HTS and NS groups survived more than 16 hours (16.5±6.7 and 17.4±4.7 hr, respectively). Multiple trauma significantly increased blood lactate level (untreated group, 2hr after shock: 6.36±2.26 mmol/L, 4hr after shock: 9.85±0.00 mmol/L), which was reduced more effectively by HTS than NS resuscitation (HTS vs. NS group, 4 hr after shock: 4.67±1.41 vs. 5.22±3.17 mmol/L, 8 hr after shock: 6.62±7.01 vs 7.89±5.38 mmol/L, 16 hr after shock: 2.83±0.00 vs 5.78±2.21 mmol/L). Similarly, base excess (BE) reduced by multiple trauma was also reversed by HTS, but not NS treatment (HTS vs. NS group, 4 hr after shock: 2.0±2.8 vs. -5.0±4.7 mmol/L, 8 hr after shock: 8.0±0.0 vs. -8.0±6.7 mmol/L, 16 hr after shock: 4.0±0.0 vs. -7.5±2.1 mmol/L).

In conclusion, HTS more effectively corrected acidosis caused by multiple trauma, although HTS and NS had similar effects on animal survivability. Considering the benefits of HTS in small resuscitation volume and shorter infusion duration, it can be used in far forward battlefield and pre-hospital first response settings.

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D. de Boer1, C. van ’t Veer1, M. Schouten1, J. Roelofs2, J. van der Zee3, B. Isermann4, C. Esmon5, T. van der Poll*1. 1Center for Experimental and Molecular Medicine, Academic Medical Center University of Amsterdam, Amsterdam, Netherlands, 2Department of Pathology University of Amsterdam, Amsterdam, Netherlands, 3Department of Pulmonology, Onze Lieve Vrouwe Gasthuis, Amsterdam, Netherlands, 4Department of Medicine and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany, 5Cardiovascular Biology Research Program, Oklahoma Medical Research Program, Oklahoma Medical Research Foundation, Oklahoma, OK

Background: Dysregulation of the Protein C system is a common feature of diseases associated with lung inflammation, such as pneumonia or acute lung injury. Activated Protein C (APC) has both anticoagulant and anti-inflammatory effects. The endothelial protein C receptor (EPCR) strongly augments PC activation.

Objective: To study the effects of modulation of the Protein C system in the pulmonary compartment during allergic lung inflammation.

Methods: Wildtype (Wt), transgenic mice with overexpression of APC (APC+) or EPCR (EPCR+) and EPCR knockout (KO) mice were sensitized intranasally on day 0, 1 and 2 with 25 μg house dust mite (HDM) extract and challenged with 6.25 μg HDM extract on day 14, 15, 18 and 19. Mice were sacrificed on day 21 for analysis.

Results: Compared to Wt mice, APC+ and EPCR+ mice displayed attenuated allergic lung inflammation as reflected by a decreased influx of cells in bronchoalveolar lavage fluid (BALF) (Wt 1.4 vs EPCR+ 0.4 x 10e6 cells/ml; p=0.003, Wt 4.9 x 10e5 vs APC+ 2.9 x 10e5 cells/ml; p=0.05), lower influx of eosinophils in BALF (Wt 87% vs EPCR+ 55%; p=0.005, Wt 92% vs APC+ 42%), lower levels of IgE in plasma (Wt 891 vs EPCR+ 284 pg/ml; p=0.01, Wt 1280 vs APC+ 14 pg/ml; p=0.006), and lower levels of cytokines in BALF (IL-4; 11 pg/ml vs not detectable, IL-5; 30 vs 26 pg/ml; IL-6; 10 vs 5 pg/ml in Wt vs EPCR+ respectively, IL-4 Wt 11 vs APC+ 4 pg/ml; p=0.04, IL-5 Wt 33 vs APC+ 17 pg/ml; p=0.01, IL-6 Wt 9 vs APC+ 4 pg/ml; p=0.03). EPCR KO mice did not have an altered phenotype. The protection against allergic lung inflammation in EPCR+ mice was associated with a 74% reduction of D-dimer levels in BALF compared to Wt mice (p=0.001).

Conclusion: Transgenic overexpression of human APC or EPCR attenuates allergic lung inflammation in a murine asthma model with house dust mite allergen.

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A.I. Khan1, S.M. Coldewey2, 1, A. Kapoor1, N.S. Patel1, C. Thiemermann1, M. Rogazzo2, M. Collino3. 1Queen Mary University of London, The William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, London, United Kingdom, 2Hannover Medical School, Clinic for Anaesthesiology and Intensive Care Medicine, Hannover, Germany, 3University of Turin, Department of Drug Science and Technology, Turin, Italy

Erythropoietin (EPO) ameliorates organ dysfunction/injury in endotoxemia. The erythropoietic effects of EPO are mediated by the ‘classical’ EPO-receptor homodimer, while the tissue-protective effects may be mediated by a heterocomplex of the EPO-receptor and the β-common receptor (βcR). We investigated the mechanisms by which EPO attenuates the cardiac dysfunction caused by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). Wild-type (WT) male C57BL/6 mice (2 months) subjected to LPS (9 mg/kg i.p. for 18 h) had a significant reduction in ejection fraction, fractional shortening and fractional area of change and, hence, cardiac dysfunction. Hearts obtained from mice subjected to LPS exhibited a reduction in left ventricular developed pressure (Langendorff) ex vivo. EPO (1000 IU/kg s.c.) 1 h post LPS significantly attenuated this cardiac dysfunction. The cardiac dysfunction caused by LPS in βcR knock-out mice was not affected by EPO. In hearts from WT mice subjected to endotoxemia, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase 3-β (GSK-3β; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In addition, EPO attenuated the endotoxemia induced nuclear translocation of p65 (NF-κB; activation). All of these effects of EPO were lost in βcR knock-out mice subjected to endotoxemia. Furthermore, WT male C57BL/6 mice (8 months) subjected to CLP also developed cardiac dysfunction at 24 h, which was attenuated by EPO (1 h post CLP). Thus, EPO attenuates the cardiac dysfunction induced by LPS or polymicrobial sepsis. This protective effect is (at least in part) attributable to the activation of the Akt/eNOS survival pathway, and inhibition of activation of GSK-3β and NF-κB, secondary to the activation of the βcR receptor.

AIK and SMC contributed equally to this study.

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A. Achouiti1, T. Vogl2, C.F. Urban3, T.J. Hommes1, M.A. van Zoelen1, 4, S. Florquin5, J. Roth2, C. van ’t Veer1, A.F. de Vos1, T. van der Poll*1. 1Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands, 2Institute of Immunology, University of Münster, Münster, Germany, 3Laboratory for Molecular Infection Medicine Sweden, Umea University, Umea, Sweden, 4Department of Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands, 5Department of Pathology, Academic Medical Center, Amsterdam, Netherlands

Background: K. pneumoniae is a common causative pathogen in sepsis and pneumonia. Invasive infection is associated with enhanced release of MRP8 and MRP14 that together form MRP8/14 heterodimers. During sepsis, MRP8/14 enhances pro-inflammatory responses via Toll-like receptor 4 and in addition acts as a direct growth-inhibitor of pathogens through chelation of trace metals in infectious environments.

Aim: To determine the role of MRP8/14 in murine Klebsiella pneumonia.

Methods: Wildtype (WT) and MRP14 knockout (KO) mice were intranasally infected with K. pneumoniae. Mice were sacrificed 6, 24 and 48 hours after infection for the determination of MRP8/14, cytokines and bacterial loads in blood and organs or monitored in a survival study. Growth-inhibitory effects of MRP8/14 were assessed in vitro by incubation of Klebsiella with recombinant MRP8/14 in the presence or absence of zinc. In addition, antimicrobial effects were determined in neutrophil extracellular traps (NETs) induced in WT and MRP14 KO neutrophils and in human NETs treated with anti-MRP14 or control antibody.

Results: K. pneumoniae infection resulted in elevated MRP8/14 levels in lungs and plasma. MRP14 KO mice showed enhanced growth of bacteria in blood and organs and a decreased survival when compared to WT mice. Contribution of MRP8/14 to the Klebsiella cytokine response was only minimal. In vitro, recombinant MRP8/14 dramatically inhibited Klebsiella growth which was reversed by the addition of zinc. NETs from MRP14 KO neutrophils showed impaired Klebsiella growth inhibition relative to WT NETs which was analogous to the effect of anti-MRP14 on human NETs.

Conclusion: MRP8/14 contributes to a protective innate defense in Klebsiella pneumonia by virtue of its capacity to inhibit Klebsiella growth in NETs through the chelation of zinc.

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H. Ito1, S. Asumusen1, P. Enkhbaatar*1, L.D. Traber*1, A.L. Salzman3, D.N. Herndon*2, D.L. Traber*1. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX, 3Radikal Therapeutics, Inc., West Tisbury, MA

Introduction: Septic shock represents a leading cause of death among intensive care patients resulting in multiple organ failure, specifically pulmonary failure. We hypothesized that treatment with R-100 (catalyzes superoxide degradation and releases nitric oxide) in sheep that suffer from Pseudomonas aeruginosa (PSA) induced septic pneumonia, would suppress superoxide, augment NO resulting in improved pulmonary function.

Methods: Eleven female sheep were operatively instrumented for chronic study and randomly allocated to vehicle control (n=6) or treatment group (n=5). Smoke inhalation injury (48 breaths of cotton smoke) was induced and 2.4×1011 CFU of PSA were instilled into the lung via bronchoscope. The treatment animals received a total of 80mg/kg of R-100 i.v., diluted in 500ml of 5% dextrose. A bolus (30min) of 10mg/kg of R-100 was injected 1 h post-injury and followed by the continuous infusion of 70mg/kg for 22.5hrs. The control group received same amount of Dextrose. Data are expressed as mean ± SEM. Statistical analysis: two-way ANOVA and Bonferroni post hoc comparison.

Results: All sheep treated with R-100 showed significant improvement in PaO2/FiO2 (24h: 245.8±28.8 vs 137.0±40.1mmHg) and peak ventilatory pressures (24h: 25±0.9 vs 36±2.2cmH2O) and pause ventilatory pressures (24h: 21±0.6 vs 35±3.3cmH2O). Increase in pulmonary artery pressure was markedly reduced by R-100 (24h: 25±0.9 vs 30±1.9mmHg). Increased activated clotting time in the control animals was markedly reduced by R-100(24h: 176±8.8 vs 208±13.9sec). Additionally, the R-100 treated sheep had a significantly lower total fluid balance at 24h after injury compared to the control group (190±44.5 vs 595±233ml/3hr).

Conclusion: Infusion of R-100 stabilizes the pulmonary function and reduces massive fluid imbalance in ovine PSA septic shock.

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B.P. Yoseph1, E.R. Breed1, Z. Liang1, J. Dominguez2, K. McConnell*1, C.M. Coopersmith*1. 1Emory University School of Medicine, Atlanta, GA, 2University of Colorado Denver, Denver, CO

Background: Barrier dysfunction of the intestinal mucosa has been hypothesized to be a major problem in sepsis, and contribute to the development of multiple organ failure. The objective of this study was to evaluate intestinal permeability and localization of tight junction protein occludin in the intestine following sepsis.

Methods: FVB/N mice underwent 2X23 CLP or sham laparotomy and were gavaged with 22mg/ml of fluororescein isothiocynate-dextran (FD-4), 5 hr prior to harvest. Intestinal permeability was assayed in vivo by measuring FD-4 concentration in the plasma at 0, 6, 12, 24, and 48 hr (n= 6/sham and 9/CLP/timepoint) by using fluorospectrometry. Gene expression of the tight junction protein Occludin was analyzed by real-time PCR. Data were compared using Mann-Whitney tests.

Results: CLP resulted in significantly elevated serum FD-4 concentration at 6 hr (655 vs sham 274; p=0.007), 12 hr (668 vs sham 239; p=0.002), 24 hr (492 vs sham 192; p= 0.02) and 48 hr (404 vs sham 196; p=0.01) (fig 1). Intestinal gene expression of occludin was significantly decreased at 6 hr (p< 0.001), 12 hr (p< 0.05), and 24 hr (p< 0.001) after CLP compared to sham (fig 2).

Conclusions: Increase of FD-4 concentration and disappearance of transmembrane protein occludin in the gut lumen after CLP strongly suggest sepsis induces barrier dysfunction in mice by altering the tight junction, resulting in intestinal hyperpermeability.

FITC Dextran (FD-4)

FITC Dextran (FD-4)



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F.E. van den Boogaard1, K.P. van Gisbergen3, 1, J.J. Roelofs1, J.H. Vernooy2, H. Endeman4, M.A. van Zoelen4, 5, D.H. Biesma4, L. Boon6, C. van ’t Veer1, A.D. Vos1, T. van der Poll*1. 1Academic Medical Center, Amsterdam, Netherlands, 2University Hospital, Maastricht, Netherlands, 3Sanquin, Amsterdam, Netherlands, 4Antonius Hospital, Nieuwegein, Netherlands, 5University Medical Center, Utrecht, Netherlands, 6Bioceros, Utrecht, Netherlands

Background: S.pneumoniae is the most common causative pathogen in community-acquired pneumonia (CAP). Granzyme A (GrzmA) is able to modulate the host immune response during infection via different mechanisms; CD8+ T cells and natural killer (NK) cells are considered major producers of GrzmA. We here soughtto determine the role of GrzmA during pneumococcal pneumonia.

Methods: GrzmA was measured in bronchoalveolar lavage fluid (BALF) harvested from CAP patients (N=7) from the infected and contralateral uninfected site and in serum from patients and controls (N=8). Lung tissue slides of CAP patients and controls were stained for GrzmA expression. Pneumonia was induced by intranasal inoculation of S.pneumoniae in wild type (WT) and GrzmA deficient (GrzmA-/-) mice; animals were sacrificed at 6, 24 and 48h after infection for analyses, or observed in a survival study. In separate experiments WT and GrzmA-/- mice were treated with CD8+ or NK1.1 cell depleting antibodies.

Results: In CAP patients, GrzmA levels were increased in serum and in BALF obtained from the infected lung. GrzmA was expressed by leukocytes, pneumocytes, bronchial epithelial cells and alveolar macrophages in human lung tissue. Upon infection with S.pneumoniae, GrzmA-/- mice showed a better survival (p=0.007), lower bacterial counts in BALF (6h) and distant body sites (24 and 48h), and lower lung histopathology scores (48h) compared to WT mice. Although CD8+ T-cells and NK1.1 cells produced GrzmA during pneumonia, depletion of these cells did not influence bacterial loads.

Conclusion: GrzmA is produced by several parenchymal and nonparenchymal cells in the lungs. GrzmA plays an unfavorable role in host defense during murine pneumococcal pneumonia by a mechanism that does not depend on CD8+ or NK1.1 cells.

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D.C. Blok, A.F. de Vos, S. Terpstra, C. van ’t Veer, T. van der Poll*. Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands

Background: Interleukin (IL)-33, a newly identified member of the IL-1 family and ligand of the IL-1 like receptor ST2 (a.k.a. IL-1RL1), has been implicated in sepsis pathophysiology as a potential endogenous danger signal. In addition, another study shows that administration of recombinant IL-33 can evoke protection against lethality caused by polymicrobial abdominal sepsis.

Objective: To determine the effect of recombinant IL-33 treatment during pneumosepsis caused by Klebsiella pneumoniae.

Methods: Wild type (WT) and ST2 knockout (KO) BALB/c mice were intravenously treated with recombinant IL-33 or vehicle 24 and 1 hour prior to intranasal inoculation with 1.5×104 K. pneumoniae. Lungs, blood, spleen and liver were harvested 6, 24 and 48 hours post infection for quantitative cultures; cytokines and chemokines were measured in lung homogenates.

Results: IL-33 treatment improved antibacterial defense in WT mice as reflected by markedly lower bacterial loads 24 and 48 hours after infection in all body sites examined. This effect was mediated by ST2, since IL-33 did not influence bacterial growth or dissemination in ST2 KO mice. At 6 hours after infection, lung levels of IL-6 and IL-1β were higher in IL-33 treated WT mice. At later time points, IL-33 administered mice displayed lower cytokine and chemokine concentrations in lungs, most likely due to the lower bacterial counts and thus diminished pro-inflammatory trigger.

Conclusion: IL-33 treatment strongly attenuates bacterial growth and dissemination during pneumosepsis caused by K. pneumoniae in a ST2 dependent manner.

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H. Hardway1, D.J. Stearns-Kurosawa*2, S. Kurosawa*2. 1Boston University, Boston, MA, 2Boston University School of Medicine, Boston, MA

Introduction: Monitoring the course of a response to septic challenge either in the clinic or an experimental setting typically involves accumulating vast amounts of data that report host physiology, immune and inflammation status. We asked if it was possible to mathematically transform grouped physiology data and cyto/chemokine data obtained over time in a way that would provide a map reporting status (improvement vs deterioration) and consequently predict survival. As a test system, we used data from Papio baboons challenged with ribosome-inactivating Shiga toxins-1 and -2 from enterohemorrhagic E.coli. Intestinal infection with these bacteria and subsequent toxemia can induce hemolytic uremic syndrome, and are the primary cause of acute renal failure in otherwise healthy US children.

Methods & Results: Physiology (n=24) and inflammation (n=17) parameters were grouped, normalized and analyzed without knowledge of survival outcome as a function of time using principal component analysis (PCA). PCA space analysis revealed distinct regions corresponding to survival or non-survival of the subject as well as toxin type.

Major results: 1) Stx1 and Stx2 exert differing impacts on physiology and while physiology parameters separated survivors and non-survivors, the separation was late with little value for prognosis; 2) Cytokine time-map paths (PC1 vs PC2) separated survivors and non-survivors by 10 hours post-challenge giving confident prognosis by day 2-3 post-challenge. Associated with non-survival were G-CSF, IL-1Ra and IL-6 (inflammation); high ALT, AST, LDH, BUN, creatinine and thrombocytopenia (physiology).

Conclusions: Mathematical transformation of large data groups over time provides maps that follow changes in host status toward healing or increased disease severity and non-survival.

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N. Hutchins1, 2, C. Chung*2, A. Ayala*2, 1. 1Brown University, Providence, RI, 2Rhode Island Hospital, Providence, RI

Endothelial cell (EC) dysfunction contributes to multiple organ injury, the primary cause of fatality seen in critically ill patients. Although the development of EC dysfunction in the heart and lungs has been well studied, it remains understudied in the liver. It is proposed that liver sinusoidal endothelial cells (LSECs) maintain immune tolerance by suppressing local inflammation-induced injury/cell death. LSECs ligate the co-inhibitory receptor, programmed-death receptor-1 (PD-1), on leukocytes via the cell surface expression of PD-ligand-1 (PD-L1). Our laboratory has recently reported that PD-1 expression contributes to increased septic (CLP) morbidity. However, whether PD-1:PD-L1 signaling effects septic mouse LSEC function and/or survival is unknown. Thus, we hypothesized that PD-L1 protects LSECs from injury and the breakdown of an immune tolerant state. To address this, we initially phenotyped LSECs as CD146+CD45- expressing cells and found an increased expression of PD-L1 24h post-CLP in BL6 mice. To determine if PD-L1 alters LSEC injury, we enriched for CD146+ cells from PD-L1-/- or BL6 mouse livers and stained LSECs for Annexin V, or apoptosis. Our results indicate that LSECs from CLP PD-L1-/- mice exhibit significantly less Annexin V staining compared to CLP BL6 mice. However, PD-L1’s role in LSEC apoptosis during sepsis appears independent of Fas:FasL signaling, since Fas receptor expression remains unchanged between CLP BL6 & PD-L1-/- mice. We also noted that LSECs from CLP BL6 mice had significantly lower expression of angiogenic VEGF-R2 vs. Sham controls, and this is rescued in CLP PD-L1-/- mice. Our data, surprisingly, suggests that in sepsis, PD-L1 ligation contributes to LSEC injury/death and represses angiogenic signaling—exacerbating (not maintaining) the immune tolerant state.

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J.G. Younger*1, M.M. Thornton1, C. Babcock1, H.M. Chung-Esaki1, D.M. Bortz2, M.J. Solomon1. 1University of Michigan, Ann Arbor, MI, 2University of Colorado-Boulder, Boulder, CO

Background: While the importance of fluid dynamical conditions is well-recognized in the growth of biofilms, their role during bacteremia is unknown. We examined the impact of physiological fluid shear forces on the development of multicellular aggregates of Klebsiella pneumoniae.

Methods: Wildtype and O-antigen or capsular mutants of K. pneumoniae were grown as broth culture in a Taylor Couette flow cell configured to provide continuous shear forces comparable to those encountered in the human arterial circulation (i.e., on the order of 1.0 Pa). The size distribution and antibiotic resistance of aggregates formed in this apparatus were determined, as was their ability to persist in the bloodstream of mice following intravenous injection.

Results: Unlike growth in shaking flasks, bacteria grown in the test apparatus readily formed aggregates, a phenotype largely absent in capsular mutants and to a lesser degree in O-antigen mutants. Aggregates were found to persist in the bloodstream of mice. Importantly, organisms grown under physiological shear were found to have an antibiotic resistance phenotype (for ciprofloxacin and ceftriaxone) that was intermediate between fully planktonic and biofilm states.

Conclusions: When grown under intravascular-magnitude fluid dynamic conditions, K. pneumoniae spontaneously develops into multicellular aggregates that are capable of persisting in the circulation and exhibit increased antibiotic resistance.

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P. Raeven*1, K. Weixelbaumer*1, S. Drechsler*1, H. Redl*1, M. van Griensven*1, 2, S. Bahrami*1, P.J. Declerck3, M.F. Osuchowski*1. 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Trauma Research Center, Vienna, Austria, 2Clinic for Trauma Surgery, Technical University Munich, Munich, Germany, 3Laboratory for Pharmaceutical Biology, KU Leuven, Leuven, Belgium

Plasminogen activator inhibitor-1 (PAI-1) peaks in dying septic patients, but its exact contribution to outcome in abdominal sepsis is unclear. We aimed to test the effects of treatment with anti-PAI-1 monoclonal antibody MA-MP6H6 upon polymicrobial sepsis. Immediately, or 18h after cecal ligation and puncture (CLP), 3 month old female mice were intravenously treated (T) with either 10 mg/kg MA-MP6H6 or control antibody (MA-31C9), sampled daily (at 6, 24, 48, 72, 96h) and followed for 14 days. For data analysis, mice were additionally divided into the dying (DIE) and survivors (SUR).

In a separate CLP group, circulating PAI-1 in acutely DIE mice (within 24h of death) was 3-fold higher vs. SUR. After injection, MA-MP6H6 blocked 100% of active plasma PAI-1 6h later (co-and post-T) and 60% 24h later (co-T). MA-MP6H6 co-T restored fibrinolytic activity of plasma at 24h (by 5-fold, p<0.05 vs. MA-31C9). Yet, 14-day survival was 33% in MA-MP6H6 vs. 50% in MA-31C9 (co-T; p>0.05), and was 60% in both groups after post-T. MA-MP6H6 co-T (but not post-T) enhanced the release of circulating IL-6, KC, and MCP-1 at 24h post-CLP (3.5, 3 and 1.5-fold, p<0.05), but not of IL-1β, TNFα and IL-10. Notably, the first effect occurred only in DIE (but not SUR) groups: at 24h, IL-6 and KC in MA-MP6H6 DIE mice were 3 and 2-fold higher (p<0.05) vs. MA-31C9 DIE. Although urea rose (2-fold) at 24h, MA-MP6H6 did not further alter post-CLP organ dysfunction (ALT, AST, LDH), blood glucose, platelet/cell counts between 0-96h.

Restoration of fibrinolytic activity by inhibition of active PAI-1 in acute sepsis was concurrent with both amplification of the pre-lethal pro-inflammatory cytokines and lack of survival benefit. This implies that early systemic hypofibrinolysis is not a key trigger of acute mortality in abdominal sepsis. WWTF Grant LS07-065.

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K.R. AbdelFattah, B. Johnson, J. Gatson*, D. Maass*, J. Minei*, A. Idris*, S. Wolf*, J. Wigginton*. UT-Southwestern Medical Center, Dallas, TX

Background: Recent evidence has suggested the importance of hepatic inflammation and damage leading to significant morbidity and mortality following burns. The mechanism by which this inflammation arises have not been fully elucidated, however one suggested mechanism is that the unfolded protein response (AKA ER stress) may play a role. Estrogens have been implicated in reducing markers of the ER stress response, evidenced by anti-estrogen therapies that may be dependent on the process for apoptosis. We hypothesized that estrogens can mitigate the hepatic ER stress response following severe burns.

Methods: Adult male Sprague-Dawley rats were divided into three groups. Following either a sham burn or 40% TBSA scald burn, animals were resuscitated using intraperitoneal LR, and the treatment groups received either placebo or 17β-estradiol. Twenty-four hours following injury, animals were sacrificed and liver tissue was harvested. Protein levels were determined using Western Blot. ImageJ software was used to analyze blot data, and GraphPad Prism was used to compare groups.

Results: We identified a significant reduction in ER stress response signaling protein pPERK (p<0.01), however did not observe a statistically significant difference in the parallel pathway mediated by pIRE1 (p=0.2). We also demonstrated a significant reduction after treatment with 17β-estradiol group in the apoptotic marker Bcl2 (p=0.02).

Conclusion: Our findings suggest a possible mechanism by which 17β-estradiol may exert anti-inflammatory and anti-apoptotic effects in hepatic tissue following a severe thermal injury. Ongoing studies will determine if parallel pathways of ER-stress mediated apoptosis can be attenuated via estrogens, with the ultimate focus of tissue salvage and restoration of normal function.

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A. Bihorac*, T. Ozrazgat Baslanti, A. Cuenca, Y. Agosto Collazo, R.F. Ungaro, P.A. Efron*, L.L. Moldawer*. University of Florida, Gainesville, FL

Background: Tissue hypoxia in severe trauma (ST), through upregulation of angiogenic factors (AF), may provide the stimulus for mobilization of angiogenic endothelial progenitor cells (EPCs) and monocytes carrying Tie-2 receptor for angiopoietin 2 (TEMs) from the bone marrow, leading to restoration of microcirculatory perfusion.

Objective: Blood EPCs and TEMs, soluble AFs and urinary markers of tubular injury were measured in the first 12 hours of ST as markers of response to hypoxic tissue injury.

Methods: Prospective study of 17 patients with ST (early hypotension with hemorrhage in the field or at initial hospital evaluation requiring blood transfusions), 7 healthy controls (HC) and 2 patients with severe sepsis (SS). EPCs and TEMs were quantified using flow cytometry. Urinary markers and AFs were determined using ELISA.

Results: Plasma levels of erythropoietin (Epo), SDF1, angiopoietin 2 (Ang 2) and sFLT1 (soluble VEGF receptor 1) were significantly elevated in patients with ST and SS compared to HC. Furthermore, ST non-survivors were characterized by persistently higher levels of Ang 2, sFLT1 and Epo (first 12 hours of injury and day 2). This increase was associated with a paradoxical decrease in mobilized angiogenic cells (TEMs and EPCs) among non-survivors (Table 1 and Figure 1). Urinary tubular markers reflecting kidney hypoxia, neutrophil gelatinase-associated lipocalin (NGAL) and interleukin 18 (IL18), were also elevated among non-survivors early after ST.

Conclusion: The upregulation of AF by tissue hypoxia and mobilization of angiogenic progenitor cells from the bone marrow into the circulation may be inadequate in non-survivors of ST. Measurement of angiogenic cells, AF and urinary hypoxic markers may provide a new tool for the assessment of the extent of hypoxic injury in early ST.

Angiogenic factors and progenitor cells among severe trauma survivors and non-survivors

Angiogenic factors and progenitor cells among severe trauma survivors and non-survivors

Change in soluble angiogenic factors in the first 48 hours after severe trauma stratified by survival status

Change in soluble angiogenic factors in the first 48 hours after severe trauma stratified by survival status

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F. Venet1, 2, A. Foray1, A. Villars-Méchin1, A. Lepape2, 3, G. Monneret1, 2. 1Hospices Civils de Lyon - Cellular Immunology Lab, Lyon, France, 2Hospices Civils de Lyon - Université Claude Bernard Lyon I, EAM 4174, Lyon, France, 3Hospices Civils de Lyon -Intensive Care Units - Lyon-Sud University Hospital, Pierre-Bénite, France

Rationale: Septic syndromes are the leading cause of mortality in intensive care units (ICUs) and are characterized by the development of a stage of profound immunosuppression associated with delayed mortality. Sepsis-induced lymphocyte dysfunctions include increased apoptosis leading to lymphopenia, anergy and increased percentage of naturally regulatory T cells. Interleukin-7 (IL-7) is an homeostatic cytokine for resting lymphocytes and is involved in peripheral expansion in lymphopenic hosts. Recombinant human IL-7 (rhIL-7) is currently tested in phase I and II clinical trials in other diseases with no major side effect observed so far.

Objective: In septic shock patients, we investigated ex vivo the capacity of recombinant human IL-7 (rhIL-7) to restore normal lymphocytic functions.

Methods: After mononuclear cell purification and T Cell Receptor (TCR) stimulation, the effect of rhIL-7 on cell proliferation, IFN-γ production, Bcl-2 expression and STAT-5 phosphorylation were measured by flow cytometry in patients in comparison with healthy volunteers.

Measurements and Main Results: After T cell stimulation, septic shock patients presented with marked lymphocyte dysfunctions: decreased CD4+ and CD8+ T cell proliferation, decreased IFN-γ production by CD8+ T cells, decreased Bcl-2 and pSTAT5 expressions in T cells in comparison with healthy volunteers. Importantly, these sepsis-induced lymphocyte alterations were totally reversed after incubation with rhIL-7.

Conclusion: Our results show for the first time that rhIL-7 is able to restore ex vivo sepsis-induced lymphocyte alterations in patients. These observations suggest that rhIL-7 therapy could promote functional T cell responses after sepsis. Upon patients’ stratification, rhIL-7 may improve survival of patients with septic shock.

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F. Venet1, 2, O. Filipe-Santos3, A. Lepape2, 4, N. Plantier3, N. Pasqual3, G. Monneret1, 2. 1Hospices Civils de Lyon - Cellular Immunology Laboratory, Lyon, France, 2Hospices Civils de Lyon - Université Claude Bernard Lyon I, EAM 4174, Lyon, France, 3ImmunID Technologies, CEA/iRTSV, Grenoble, France, 4Hospices civils de Lyon - Intensive Care Units - Lyon-Sud University Hospital, Pierre-Bénite, France

Rationale: Septic syndromes are the leading cause of mortality in intensive care units (ICUs). In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality although the exact role of lymphocyte dysfunctions is not well established.

Objective: In septic shock patients, we investigated T Cell Receptor (TCR) diversity, a novel aspect of sepsis-induced lymphocyte alteration and assessed its association with deleterious outcomes (death, nosocomial infection).

Methods: Using a novel molecular biology technique, the combinatorial diversity of human TCRβ chain (TRB locus) was measured in peripheral blood from 41 patients prospectively included from two adult ICUs in a university hospital and sampled twice after the onset of shock (early after inclusion [D1] and at the end of the first week [D7]).

Measurements and Main Results: Septic shock patients presented with a decreased TCR diversity after the onset of shock associated with mortality (Log Rank test, p = 0.0058) and the development of nosocomial infections (p<0.05, Mann Whitney U-test). In paired samples, we observed a very steep recovery slope of TCR diversity never described in other clinical situations (p<0.0001, Wilcoxon paired test).

Conclusion: Our results show for the first time that septic patients present with a marked decreased TCR diversity associated with increased risk of death and nosocomial infection. TCR diversity measurements may become a crucial tool to monitor immune functions in ICU patients. Moreover, our results open novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis.

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M. Sigman, J.L. Rendon*, S. Akhtar, J. Eberhardt, R.L. Gamelli, M.A. Choudhry*. Loyola University Chicago Health Sciences Campus, Maywood, IL

Background: Intestinal inflammation in burn injury is a well-recognized mediator of multi-organ failure. We hypothesized that markers of intestinal inflammation are detectible non-invasively and that variation in the profiles of these measurements may correlate with degree of burn injury.

Methods: In 7 severely burned patients and 15 control patients we quantified and compared the concentration of several inflammatory cytokines in fecal samples at various time points in the course of hospitalization using a multiplex assay kit (Bio-Rad). Fecal levels of myeloperoxidase (MPO) and elastase were measured using standard procedures.

Results: Compared to a control group, levels of inflammatory cytokines were significantly increased in the burn group. IL-6 was increased 2.16 ± 0.61 pg/mg, (mean ± SEM) to 3.81 ± 0.49; p<0.05, as were IL-8 (3.32 ± 0.76 pg/mg to 20.51 ± 6.65 p<0.05), IL-12 (4.457 ± 0.98pg/mg to 7.371 ± 0.95 p<0.05), IL-13 (3.86 ± 0.32 pg/mg to 11.83 ± 1.47 p<0.05), MPO (13.41 ± 1.40 units/mg protein to 24.52 ± 4.31 p<0.05) and elastase (2.46 ± 0.38 pg/ml to 5.08 ± 0.72 p<0.05).

Conclusions: Our results suggest that markers of intestinal inflammation are measurable by non-invasive means and are increased following burn injury compared to controls. Of note, increased IL-8 correlated with increased MPO and elastase activity, suggesting a role for neutrophil activation in burn-mediated intestinal inflammation. Thus, these inflammatory cytokine profiles may be valuable biomarkers of intestinal inflammation following burn injury. (Support: NIH R01AA015731 and Dr. Ralph and Marian C. Falk Medical Research Trust).

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E.L. Chiswick, D. 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. But by 24 hours post-CLP, Die-P mice have significantly more peritoneal phagocytes and bacterial CFUs than Live-P mice. This suggests a cellular defect of phagocytes in Die-P mice. We investigated whether there are differences in phagocytosis and reactive oxygen species (ROS) between Live-P and Die-P mice at 6 and 24 hours post-CLP.

Methods: Sepsis was induced by CLP in female outbred mice. Peritoneal cells were harvested by lavage at 6 (T-6) or 24 (T-24) hours post-CLP. Mice were stratified into Live-P and Die-P groups by plasma IL-6 levels. ROS was measured with diHydroRhodamine-123 (DHR-123) in response to stimuli. Rhodamine-123 (R-123) was used as a control to simulate maximum DHR-123 to R-123 conversion. Phagocytosis was measured with pHrodo® (pH sensitive probe) or Alexafluor-488® labeled E.coli. Cells were analyzed by flow cytometry and probe fluorescence was normalized to unstimulated controls. Cell type was determined with cell specific markers.

Results: By T-6, Die-P mice show less functional (phagosome acidification) and total phagocytosis than Live-P mice in both PMNs and monocytes (Fig.1).The ROS burst is not substantially different at T-6, but by T-24 PMNs had significant impairment. Control R-123 ratios are similar at T-6 but diverge by T-24 (Fig. 2). This suggests there are fewer functional mitochondria in cells at T-6, or basal ROS generation is already near maximum capacity, hence the reduced R-123 ratio and reduced response of cells to stimuli.

Conclusion: Peritoneal phagocytes from Die-P mice display decreased effector functions as early as 6 hours post-CLP and this contrast increases with time.

FIG. 1

FIG. 1

FIG. 2

FIG. 2

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Z. Liang1, Y. Xie2, J.A. Dominguez*3, E.R. Breed1, B.P. Yoseph1, N.O. Davidson*2, C.M. Coopersmith*1. 1Emory University, Atlanta, GA, 2Washington University, St. Louis, MO, 3University of Colorado Denver School of Medicine, Aurora, CO

Background: Mttpflox/flox villin-Cre-ERT2 (Mttp-IKO) mice exhibit a complete block in chylomicron assembly together with lipid malabsorption. Our previous studies with young Mttp-IKO mice show that blocking intestinal chylomicron secretion improves outcome in sepsis. However, 80% of mortality in sepsis occurs in patients over age 65.

Objective: To determine whether blocking chylomicron secretion has the same effects in aged septic mice as in young septic mice.

Methods: Aged Mttp-IKO mice (22 months, n=38) and WT mice underwent intratracheal injection with P. aeruginosa and were followed for survival for 7 days. Another cohort of mice (n=27) were sacrificed after 24 hours to explore potential mechanisms.

Results: Aged septic Mttp-IKO mice exhibited increased seven-day mortality, with 66% dying compared to 38% septic WT aged mice (p<0.05). This contrasts with young septic Mttp-IKO mice which have significantly decreased mortality compared to WT mice (p<0.05) Aged septic Mttp-IKO mice have increased villus length, crypt depth, increased serum cholesterol and triglycerides and jejunal triglyceride. Cytokine levels (IL-1β, IL-6, IL-10, IL-13, MCP-1, TNF-α) in BAL and serum from aged septic Mttp-IKO mice were similar to young septic Mttp-IKO mice.

Conclusions: Gut-derived lipids play an important role in the progression of sepsis. Decreasing intestinal lipid transport by preventing chylomicron assembly and secretion is beneficial in young mice but detrimental in aged mice following P. aeruginosa pneumonia. The host response appears to involve different mechanisms in young and aged mice and highlights the importance of age following a septic insult.

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M.J. Henry-Stanley*, D.J. Hess*, M.B. Sivertson, C.L. Wells*. University of Minnesota, Minneapolis, MN

Introduction: Surgical site infections and systemic infections associated with central venous catheters are common causes of morbidity and mortality in ICU patients, and often arise from skin contamination with Staphylococcus aureus. Deposition of S. aureus onto abiotic surfaces often triggers biofilm formation, and biofilm-associated bacteria are typically more antibiotic resistant than their planktonic (free-living) counterparts.

Methods: Using an in vitro model of suture-associated biofilms, experiments were designed to determine if exposure of S. aureus to antibiotics might facilitate subsequent biofilm formation. S. aureus RN6390 was incubated overnight in subinhibitory (0.1 μg/ml) and bactericidal (0.5 μg/ml) concentrations of ampicillin (AMP). To cultivate in vitro biofilms, 1-cm segments of 3-0 silk suture were inoculated with 104 washed, AMP-treated S. aureus. The S. aureus inocula were stained with two fluorophores to label DNA blue (Hoechst 33342) and to label the cell surface red (wheat germ agglutinin conjugated to AlexaFluor 594 that binds N-acetyl-glucosamine).

Results: After 6-hr, there were greater numbers (log10 avg ± SE) of biofilm-associated S. aureus on sutures (n=6-7) inoculated with S. aureus that survived overnight incubation with 0.1 μg/ml AMP (5.1 ± 0.5), compared to sutures inoculated with bacteria that survived 0 μg/ml AMP (2.0 ± 0.1) or 0.5 μg/ml AMP (2.1 ± 0.1), P<0.01, ANOVA with Fisher’s post hoc. Confocal microscopy revealed the S. aureus inoculum incubated in subinhibitory AMP (0.1 μg/ml) consisted primarily of large cells or “pseudomulticellular forms”, known to result from inhibition of cell separation during cell division.

Conclusion: Prior incubation with subinhibitory AMP and/or altered cellular morphology facilitated development of S. aureus biofilms.

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H.L. Van Sweringen, N. Sakai, R. Quillin, C. Caldwell*, A.B. Lentsch*. University of Cincinnati, Department of Surgery, Cincinnati, OH

Hepatic ischemia/reperfusion (I/R) is a cause of significant morbidity and mortality after hemorrhagic shock and complex vascular surgery. We have recently shown that CXC chemokines, signaling through the CXCR2 receptor, are critical to the process of recovery and regeneration of functional liver mass after I/R. CXCR2 is expressed on both neutrophils and hepatocytes, however, the cell-specific roles of this receptor is unknown. Using chimeric mice, we sought to determine the contributions of myeloid and hepatocyte CXCR2 in the recovery of the liver after I/R injury. Using wild-type and CXCR2-/- mice, bone marrow transplantation was used to generate chimeric mice that expressed CXCR2 selectively on hepatocytes (Hep) and/or myeloid cells (My) in the following combinations: Hep+/My+; Hep-/My+; Hep+/My-; Hep-/My-. Four weeks later, mice were subjected to 90 minutes of 70% hepatic ischemia and reperfusion. Chimeric phenotypes were confirmed by flow cytometry. Liver recovery and regeneration were determined by histology and PCNA staining. Serum CXC chemokine levels were markedly and significantly increased in chimeric mice that lacked myeloid CXCR2 (Hep+/My- and Hep-/My-), indicating a regulatory role for myeloid CXCR2 in the systemic expression of its ligands. CXCR2 Hep+/My+ mice showed the least amount of recovery of hepatic parenchyma and lowest degree of hepatocyte proliferation. CXCR2 Hep+/My- and Hep-/My+ mice had modestly increased liver recovery and hepatocyte proliferation, whereas CXCR2 Hep-/My- mice had the greatest degree of liver recovery and moderate hepatocyte proliferation. The data suggest that CXCR2 on myeloid cells regulates the production of its ligands, and that expression of CXCR2 on both myeloid cells and hepatocytes has detrimental effects on the recovery of the liver parenchyma after I/R injury.

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E.R. Breed, Z. Liang, M.E. Wagener, B.P. Yoseph, I.R. Ferrer, M.L. Ford, T.G. Buchman*, C.M. Coopersmith*. Emory University, Atlanta, GA

Background: Recovery from critical illness has been hypothesized to depend, in part, on recoupling of biological clocks, which can be dose-dependently disrupted by replacement of H2O with D2O. Our previous studies suggest that survival after cecal ligation and puncture (CLP) is significantly reduced in mice exposed to 10% D2O. D2O disrupts cell proliferation and can induce apoptosis in high-turnover cells.

Objective: To examine the physiological effects of D2O that decrease survival in mice recovering from sepsis.

Methods: Male C57bl/6J mice were provided with either normal drinking water or water containing 10% D2O 10 days prior to 2×25 CLP. Splenocyte apoptosis was quantified at 24 hours, measuring PI and Annexin V expression via flow cytometry (n=13/group). Gut epithelial apoptosis was quantified using H&E and active caspase-3 staining (n=8-13/group). Proinflammatory cytokine response in serum was assayed via 6-plex analysis (n= 5-6/group). 7-day survival studies comparing mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice exposed to D2O were also performed (n=5-8/group).

Results: Lymphocyte apoptosis was markedly increased in animals receiving D2O (p<0.0001 for CD4+s and p=0.0004 for CD8+s). D2O did not induce gut apoptosis. The cytokine response (IL-1, IL-6, IL-10, IL-13, MCP-1, and TNF-α) was similar in both cohorts. There was no difference in survival between Bcl-2-Ig and WT mice.

Conclusion: Mice exposed to D2O experienced an increase in lymphocyte apoptosis. Preliminary survival studies suggest that increased lymphocyte apoptosis does not account for increased mortality from CLP. We therefore speculate recovery from CLP in mice provided with D2O is mitigated due to disruption of biological clocks that must recouple during recovery from sepsis.

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J.S. Brady, K.T. Seale, J.H. Weitkamp, J.P. Wikswo, A.K. May, P.R. Norris. Vanderbilt University, Nashville, TN

Introduction: Diverse biomolecules, pathways, and environmental factors mediate the human response to critical illness or injury; the cell is the fundamental unit for integrating these factors into organ- and system-level responses. We implemented novel microfluidic and image processing technologies to study the dynamic physiology of human immune cells. These measurements may provide new insights into the pathophysiology of critical illness and injury, and illuminate the link between molecular phenomena and clinical symptoms. In this comparative pilot study, we tested the hypothesis that leukocyte calcium (Ca++) responses would differ between healthy volunteers and critically injured patients.

Methods: We obtained whole blood samples from 3 healthy volunteers and 3 trauma ICU patients and isolated leukocytes using similar technique. Leukocytes were incubated with Flou-4, a Ca++ sensitive fluorescent dye, for 30 min. Cells were introduced into the multi-trap nanophysiometer (MTNP), a flow-through microfluidic device designed to hold cells stationary for extended experimentation. After a baseline period, cells were stimulated with ionomycin and Ca++ flux over time was measured fluorescently.

Results: Ca++ fluxes were measured in each of 809 cells across all 6 subjects. Individual cellular Ca++ responses were higher in trauma cells (mean 99.8 v. 44.4, p<.001, N=809). Inter-patient mean Ca++ response was also higher in trauma samples but the difference was not statistically significant (mean 66.1 v. 23.7, p=0.15, N=6).

Conclusions: 1) The MTNP can isolate and measure physiological dynamics of human leukocytes; 2) Leukocyte Ca++ responses were altered in trauma ICU patients; 3) The MTNP permits a wide range of future studies to understand the dynamic cellular physiology of critical illness or injury.

LEFT: DIC Image of Trauma Patient Cells in MTNP

LEFT: DIC Image of Trauma Patient Cells in MTNP

Trauma Patient Cell Calcium Flux Profile in Response to Ionomycin

Trauma Patient Cell Calcium Flux Profile in Response to Ionomycin

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D.J. Stearns-Kurosawa*, C. Mayer, C. Leibowitz, S. Kurosawa*. Boston University School of Medicine, Boston, MA

Introduction: Ingesting enterohemorrhagic E.coli (EHEC) from contaminated food or water gives rise to hemorrhagic colitis, inflammation, neurologic complications and 5∼15% of patients develop potentially lethal hemolytic uremic syndrome (HUS) with renal failure. New more virulent strains are emerging and the 150 million patients per year infected globally have no therapeutic recourse other than supportive critical care. The bacterial Shiga-like toxins (Stx1, Stx2) cause most of the organ damage. Our nonhuman primates are the only animal models that develop HUS from Stx challenge.

Objectives: Characterize: 1) mixed Stx1+Stx2 toxin because most EHEC secrete both toxins; 2) EHEC oral challenge models to mimic natural infection; and 3) identify biomarkers that report HUS development.

Methods & Results: Anesthetized baboons were challenged with Stx1+2 (25+25 ng/kg, i.v.) or with EHEC Stx1+Stx2+ bacteria and development of disease monitored. Challenge with mixed toxins elicited a significantly more severe response (60hrs median survival) than the same toxin load given separately (Stx1, 72hrs; Stx2, 116hrs). Plasma cytokines shifted toward Stx1 profile (high G-CSF, IL-12/23) or were repressed (IL-6, IL-1Ra) after Stx1+2. After clearing commensal flora, EHEC challenge induced HUS with thrombocytopenia, thrombotic microangiopathy and renal injury. DAMP biomarkers in plasma (HMGB1, mtDNA) and urine (HMGB1) increased after Stx1 challenge earlier than traditional BUN or creatinine levels.

Conclusions: Challenge with both toxins elicited a synergistic response, consistent with clinical observations that EHEC strains secreting both toxins elicit severe disease. Urinary DAMPs released during cell damage may be early indicators of HUS with wide clinical utility to identify high risk patients for earlier intervention.

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J. Hellman*, F. Xu, K. Mesa. University of California, San Francisco, San Francisco, CA

Background & Objectives: Activation of endothelial cell (EC) Toll-like receptor 2 (TLR2) by bacterial lipoproteins upregulates inflammatory mediators, facilitates neutrophil-endothelial interactions, and modulates coagulation pathways. We tested the hypothesis that bacterial lipopeptides and heat-killed Staphylococcus aureus (HKSA) induce lung vascular permeability in vitro and in vivo via TLR2.

Methods: Electric Cell-substrate Impedance Sensing (ECIS) was used to assess the permeability of monolayers of mouse and human lung microvascular EC (LMVEC) incubated with HKSA, or with bacterial lipopeptides, including Pam3Cys and FSL. In vivo experiments assessed the effects of intratracheal (IT) challenge with Pam3Cys or HKSA on lung permeability to albumin.

Results: Treatment with bacterial lipopeptides, including Pam3Cys (TLR1/2) and FSL (TLR2/6), and HKSA increased permeability of human and mouse LMVEC monolayers (p<0.001). Treatment with anti-TLR2 antibody fully blocked the effects of bacterial lipopeptides (p<0.001) and partially blocked the effects of HKSA on permeability of human LMVEC (p<0.001). Similarly, LMVEC from TLR2 KO were unresponsive to bacterial lipopeptides, and had reduced responsiveness to HKSA, indicating that TLR2 participates in HKSA-induced EC permeability (p<0.001). IT challenge with bacterial lipopeptides and HKSA TLR2-dependently induced lung vascular leak in mice (p<0.01 and p<0.05 respectively).

Conclusions: Bacterial lipopeptides and HKSA modulate lung microvascular endothelial permeability in vitro and induce lung vascular leak in vivo via TLR2. These studies suggest that endothelial TLR2 contributes to increased lung vascular permeability in bacterial sepsis.

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J. Sondeen, D. Prince, A. Polykratis, R. DeGuzman, A.P. Cap, M.A. Dubick*. US Army Inst Surg Research, San Antonio, TX

This study tested if the addition of platelets (PLT) or cryoprecipitate (CRYO) to FFP reduced post-resuscitation blood loss (PRBL) better than FFP alone in a swine model of non-compressible hemorrhage. Anesthetized, instrumented female pigs (12/grp) had a controlled hemorrhage of 24 ml/kg, 20-min shock period, splenic injury with 15-min initial uncontrolled hemorrhage, and administration of test fluids. The hypotensive (MAP ≤ 65 mmHg) resuscitation treatments were 15 ml/kg for Hextend, FFP, PLT&FFP, and CRYO&FFP. Two control groups were sham time controls (TC) and definitive care (splenectomy (SPLENEX)) prior to FFP. PRBL, hemodynamics, coagulation status, hematocrit, and oxygen metabolism were measured post injury for 5h. PLT were collected via plateletpheresis and CRYO was made from FFP according to standard methods. Doses were equivalent to human standards: six-pack PLT or 50 mg/kg CRYO. While PRBL was greater in the Hextend group compared to all other groups (22 ± 2 ml/kg), there was no significant difference among PLT+FFP (11 ± 3), CRYO+FFP (12 ± 2), and FFP alone (15 ± 2 ml/kg). There was no significant difference in survival time (HEX:135±24; FFP:176±28; PLT:181±32; CRYO:184±30) while the control groups (TC, SPLENEX) survived 300±0 minutes. All fluids produced similar changes in hemodynamics, oxygen metabolism. Compared with all other fluids, HEX significantly lowered hematocrit (i.e., a greater hemodilution) and reduced coagulation measures, possibly due to an indirect dilutional effect or a direct hypocoagulable effect. CRYO+FFP raised fibrinogen concentrations. PLT+FFP raised PLT count at 30 min. A standard dose of either PLT or CRYO did not enhance the hemostatic effect beyond that of FFP alone. Overall, no clear benefit of adding PLT or CRYO to FFP was observed in this model, but FFP was superior to HEX.

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N. Shubin1, 2, S.F. Monaghan2, 1, L. Irwin2, C. Chung*2, 1, A. Ayala*2, 1, D.S. Heffernan*2, 1. 1Brown University, Providence, RI, 2Rhode Island Hospital, Providence, RI

Epidemiological studies in humans have indicated that we become more susceptible to sepsis with age. Recently we have reported that increased expression of the immune-suppressive receptor, B and T Lymphocyte Attenuator (BTLA) on monocytes/macrophages not only associates with increased incidence of sepsis in humans, but also contributes to increased myeloid cell dysfunction and septic morbidity/mortality in mice. It is unclear, however, if the expression of BTLA isaltered further on leukocytes of systemic immune response syndrome (SIRS) or septic ICU patients with age. To address this, blinded/de-indentified peripheral blood samples from SIRS or septic ICU patients were collected with IRB approval, and tested for leukocyte BTLA expression via flow cytometry. While SIRS and septic patients all showed high mean fluorescent intensity (MFI) and % of BTLA+ lymphocytes, the differences were not marked between different age groups. However, patients >65 of age displayed a significantly higher % of BTLA+ monocytes and granulocytes, compared to SIRS patients <40 (Fig). Furthermore, we found that the MFI of BTLA was also increased nearly 4-fold on monocytes and 6-fold on granulocytes of SIRS patients >65 compared to SIRS patients <40. Surprisingly, during sepsis the inverse was seen, in which patients <40 had a higher level of BTLA on both monocytes and granulocytes compared to septic patients >65. While BTLA function on lymphocytes seems to be classically anti-inflammatory, much less is known of its impact on myeloid cell function. Thus, the changes in myeloid and granulocyte BTLA expression (but not lymphoid) in SIRS vs septic ICU patients suggest BTLA expression may not only be a unique index of immunological/morbid changes related to age, but may be a reflection of yet to be defined myeloid cell functional changes.

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S.G. Rhind1, S.B. Rizoli2, W. Junger*3, J. Cuschieri*4, A.J. Baker5, M.Y. Shiu2, P.N. Shek1, E.M. Bulger*4. 1Defence Research & Development Canada (DRDC) Toronto, Toronto, ON, Canada, 2Department of Surgery & Critical Care Medicine, Sunnybrook Health Sciences Centre, Toronto, Ontario, ON, Canada, 3Department of Surgery, Beth Israel Deaconess Medical Center & Harvard Medical School, Boston, MA, 4Department of Surgery, University of Washington, Harborview Medical Center, Seattle, WA, 5Brain Injury Laboratory, Cara Phelan Centre for Trauma Research Keenan Research Centre, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada

Background: Neuroinflammation after traumatic brain injury (TBI) plays a critical role in the pathogenesis of secondary injury. This is mediated in part by upregulation of cell adhesion molecules (CAM) on cerebral vascular endothelium, which are involved in postinjury leukocyte adhesion, microcirculatory disturbances and edema formation.

Objectives: To evaluate changes in endothelial CAMs in relation to injury severity (ISS), 6-month neurologic outcome (GOSE; dichotomized as >4 or ≤4) and resuscitation strategy.

Design: Prospective randomized controlled trial conducted within the Resuscitation Outcomes Consortium, assessed soluble CAMs obtained on a subset of 81 patients with severe TBI (GCS≤8) and 20 healthy controls. Intervention: A single, prehospital bolus (250mL) of 7.5% hypertonic saline (HS;n=21), 7.5% HS plus 6% dextran-70 (HSD;n=22) or 0.9% saline (NS;n=38).

Measurements & Results: Serum concentrations (ng/L) of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), sE-selectin and sP-selectin were measured by ELISA in blood samples taken at hospital admission, 12 h, 24 h, and 7 d. Mean (±SEM) levels of sICAM-1 (198±17) were normal on admission, but rose significantly (P<.05) by 24 h (295±31) and 7 d (408±44). In contrast, levels of sVCAM-1 (746±68), sE-selectin (42±1) and sP-selectin (83±9) were elevated on admission and rose progressively over time. Peak sICAM-1, sVCAM-1 and sE-selectin values were related to ISS and poor neurologic outcome. Although the highest values for all sCAMs were observed in NS-treated patients, no differences were found between fluid groups.

Conclusions: Soluble adhesion molecules are promising inflammatory markers of injury severity and outcome in patients with severe TBI. Funded by Defence R&D Canada and NIH R01GM076101.

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O.M. Palmer*, S. Yende, C. Song, F. Mayr, L. Weissfeld, D. Angus*, G. Tseng. University of Pittsburgh, Pittsburgh, PA

Introduction: Severe sepsis is not only a public health concern, but the incidence is disproportionally higher in Black compared to white Americans. We recently showed that this disparity persists despite adjustment for crude healthcare delivery and social variables. Thus it is plausible that underlying biological responses may partly explain the disparity. This area is a poorly understood controversial concept. We hypothesis that host transcriptomic responses to infection differ across racial/ethnic populations.

Methods: We conducted a nested case-control study within the IRB approved, prospective observational cohort study, Genetic and Inflammatory Markers of Sepsis (GenIMS) study (N=2,320). Self-identified Black (case) and white (control) subjects diagnosed with community-acquired pneumonia were paired using the propensity score method. mRNA was extracted from whole blood samples (n=22 pairs) and analyzed using the Human RefSeq8 Expression BeadChips. RMAExpress and BRB array tools were used to analyze the raw expression data. RT-PCR was performed to validate microarray findings.

Results: Interestingly, out of 24,500 genes, twenty-one genes (involved in transcription, zinc and iron homeostasis) were differentially regulated in black compared to white subjects (FDR<0.05) (R and Significance Analysis of Microarray, paired analysis). Furthermore, the transcriptome profile within each population was distinctly different. Only two genes were differentially expressed within the black population compared to sixty-four genes in the white subjects (FDR <0.2, unpaired SAM).

Conclusion: In the setting of CAP and severe sepsis, racially diverse populations exhibit differential transcriptomic signatures. Further Investigation is necessary to ascertain their potential as biomarkers of susceptibility for infection and sepsis.

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X. Huang, Y. Chen, C. Chung*, A. Ayala*. Rhode Island Hospital/Brown University Alpert Medical School, Providence, RI

Recent studies indicate PD-1 plays a significant role in the development of immune suppression associated with sepsis, in that PD-1 deficient mice were markedly protected from the lethality of sepsis, showed improved bactericidal capacity, reduced inflammatory cytokine levels, and less tissue damage than wild-type (WT) mice. B7-H1(PD-L1) is one of the major ligands of PD-1 and while deficiency of B7-H1 gene also accords significant survival benefit to septic mice, it is less marked than that seen in PD-1 gene deficient mice (Huang X [2010] Shock 35:82 [Suppl. 1]). In terms of histological development of multiple organ damage and inflammatory cytokine levels in circulation or infectious site, PD-1 deficient and B7-H1 deficient mice showed a similar reduction in these indices when compared with WT mice. However, when the bacterial burden in the body was assessed, B7-H1 gene deficient mice, unlike PD-1 knockout mice, had little effect on the rise in bacterial colony counts when compared to WT mice. To get at the mechanistic basis underlying this difference, alterations in septic mouse peritoneal macrophage associated characteristics were examined in macrophages from WT, PD-1 or B7-H1 knockout septic or control mice. We report 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, protection of ex vivo macrophage phagocytic function was only seen in septic PD-1 knockout mice cells, but not in septic WT or B7-H1 knockout mice. Thus, while we need to determine what other difference exist in leukocyte function in these animals, we speculate that such differences in macrophage function may contribute to the differences seen in their respective survival patterns. (Supported by NIH-GM046354).

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M. Sakai. Shintakeo Hospital, Takeo, Japan

Purpose: CHDF using with a polymethymethacrylate membrane is currently widely applied for non-renal indications in Japan, this technique is used in the treatment not only of patients with sepsis, but also of those with cytokine-induced critical illness such as ARDS and pancreatitis. This study aimed to investigate the clinical efficacy of PMMA-CHDF in the treatment of patients with sepsis and ARDS.

Methods: Thirty-five patients diagnosed with sepsis (ARDS [n=10], Pyelonephritis [n=5], Cholangitisn [n=5], Tsutugamusi in Scrub typhus disease [n=1], Snake Mamushi bitten [n=1], haemophagocytic syndrome [n=1], anti neutrophil cytoplasmic antibody (ANCA) lung disease [n=1], beriberi heart disease [n=1] and unknown causes [n=8]) were enrolled in this study between August 2010 and November 2011. The common cause for ARDS in elderly patients is aspiration pneumonia. Our study group composed 15 men and 20 women, aged 35–85 years (median age 68 years).

Results: Before initiating treatment with the PMMA-CHDF, the average Acute Physiology and Chronic Evaluation (APACHE) score of these patients was 17.5+/-3.6, whereas the average Sepsis-related Organ Failure Assessment (SOFA) score was 6.5+/-1.3. The duration of PMMA-CHDF treatment was 5.2+/-2.3 days. Following initiation of PMMA-CHDF treatment, early improvement of hemodynamics was observed, along with an increase in the urine output. The average survival rates of patients were 75.6%. The low survival rate among diseases 35% belonged to the Unknown group. The highest survival rate for patients with ARDS was 95%.

Conclusion: The present study suggests that cytokine-oriented critical care using PMMA-CHDF might be effective in the treatment of sepsis and ARDS, particularly, in the treatment of ARDS associated with aspiration pneumonia in elderly patients.

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A.J. Hoogendijk1, 2, M.T. Kuipers2, 3, T. van der Poll*1, 2, M.J. Schultz3, 4, C.W. Wieland3, 4. 1Center for Infection and Immunity Amsterdam, Academic Medical Center, Amsterdam, Netherlands, 2Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands, 3Laboratory of Experimental Intensive Care and Anesthesiology, Academic Medical Center, Amsterdam, Netherlands, 4Department of Intensive Care, Academic Medical Center, Amsterdam, Netherlands

Background: Mechanical ventilation can induce lung damage in healthy lungs or aggravate existing lung injury. Polymorphonuclear cell (PMN) recruitment plays an important role in driving the inflammatory response in VILI. The CDK inhibitor r-roscovitine has been shown to induce apoptosis in PMNs in vitro and in vivo.

Aim: In this study, we investigated the potential of r-roscovitine treatment in reducing lung damage in a mouse model of VILI using low (∼7.5 mL/kg) or high (∼15 mL/kg) tidal volume (VT) mechanical ventilation (MV).

Results: R-roscovitine treatment enhanced apoptosis in PMNs in vitro. VILI was associated with pulmonary PMN influx in low and high VT MV. During high VT (but not low VT) MV R-roscovitine treatment reduced the number of PMNs and lowered levels of the lung damage markers sRAGE and total IgM in bronchoalveolar lavage fluid. R-roscovitine did not influence cytokine or chemokine levels in the bronchoalveolar space.

Conclusion: R-roscovitine treatment reduced lung damage in VILI. Our data suggest that interventions reducing PMN presence, like r-roscovine, could prove a beneficial therapy in PMN driven lung injury.

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C. Zhang, H. Liang, X. Fan, X. Yang. Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, China

Objective: To investigate the in vivo effect of evodiamine (EVO) on splenic dendritic cells (DC) in mice following trauma.

Methods: Closed femur fracture combined with hemorrhage in Balb/c mice was performed. Simultaneously EVO (10mg/kg) was injected into peritoneal cavity of traumatized mice for 4 times (once/day). Splenic DC were isolated 4 day after trauma, the number of DC in splenocytes, DC apoptosis and DC functions were determined. Aryl hydrocarbon receptor (AhR) and CYP1A1 mRNA levels were also detected.

Results: The number of DC in splenocytes from trauma group was reduced and the occurrence of apoptosis in DC was significantly increased, the capacity of DC from trauma group in inducing allogeneic T cells responses was inhibited, the expressions of MHC II, CD40, CD80 and LPS-stimulated secretion of TNF-alpha, IL-6, IL-12 p40 and IL-12 p70 were obviously decreased, and the levels of AhR mRNA and CYP1A1 mRNA were reduced. EVO administration could significantly decrease DC apoptosis induced by trauma, and promote the renewal of DC number, elevate its capacity in inducing allogeneic T cells responses. EVO showed no reversal effect on suppressed expressions of MHC II, CD40, CD80, and reduced secretion of cytokines following trauma, while strengthening the depression of AhR and CYP1A1 transcription.

Conclusions: EVO could maintain the invariableness of DC number through exerting its anti-apoptotic action and correct, at least partially, suppressed antigen presentation function of DC from traumatized mice by inducing more depressed AhR and CYP1A1 mRNA transcription.

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M.K. Brunialti1, A.B. Alves1, M.L. Fernandes1, F.R. Machado1, 2, O. Rigato1, 3, E. Silva4, R. Salomao*1, 2. 1Universidade Federal de Sao Paulo, Sao Paulo, Brazil, 2Hospital Sao Paulo, Sao Paulo, Brazil, 3Hospital Sirio Libanes, Sao Paulo, Brazil, 4Hospital Israelita Albert Einstein, Sao Paulo, Brazil

A modulation of monocytes’ function, with low production of proinflammatory cytokines upon LPS stimulus, was found in septic patients (SP) that resembles the phenotype of alternatively activated macrophages. In this study we evaluated the percentages of monocytes expressing markers of alternatively activation (AAM), in PBMC from SP by flow cytometry. Blood samples were obtained from SP at admission (D0, n=62) and after seven days of therapy (D7, n=33). Thirty-two healthy volunteers (HV) were included as controls. CD163 and CD206 were measured on CD14 positive stained monocytes and expressed as percentage of positive cells. A striking difference in the percentage of CD14 positive monocytes expressing CD163 (0.0 and 23.7%) or CD206 (0.5 and 2.4%) was found between HV and SP, with the highest values observed in SP (P<0.001). CD163 and CD206 were detected as separate subsets as well as co-expressed on monocytes from SP. The percentage of monocytes expressing only CD163 was 24.4%, the percentage of cells expressing only CD206 was 0.8%, and the percentage of cells expressing both receptors was 2.0%. Co-expression was not observed in HV (0.0%). The percentage of monocytes expressing CD163 or CD206 remained high in patients’ follow-up samples, with similar results obtained in D7 samples compared with D0 samples; the percentages at both time points (D0 and D7) were higher than in HV (P<0.001). Similar percentages of CD163 and CD206-expressing monocytes were found at admission in survivors and non-survivors. Also, no differences were found between the D0 and D7 samples in surviving and non-surviving patients for either of the receptors analyzed. SP showed a markedly increased proportion of monocytes expressing CD163 and CD206 indicating that monocytes may acquire the AAM phenotype during sepsis.

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S. Drechsler*1, K. Weixelbaumer*1, M. Jafarmadar1, M. van Griensven*1, 2, S. Bahrami*1, M.F. Osuchowski*1. 1Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Trauma Research Center, Vienna, Austria, 2Clinic for Trauma Surgery, Technical University Munich, Munich, Germany

Age/gender may affect progress of secondary septic complications post-trauma. In a 2-hit model of trauma/hemorrhage (TH, 1st hit) and cecal ligation and puncture (CLP, 2nd hit), we studied the influence of age/gender on the response of circulating cytokines (CC), cells and organ function (OF) parameters. We also tested if pre-CLP events can predict post-CLP deaths.

3, 15 and 20 month (m) old female (♀) and male (♂) mice underwent sublethal TH (at -48h) and CLP (17G needle) 48h later (0h). Blood was sampled at -48h, -24h and 0h prior to, and 6, 24, 48 and 72h post-CLP. Survival was followed for 14 days. For comparisons, we calculated two global response scores: CC (combining: KC, MIP-1α, TNFα, MCP-1, IFNγ, IL-1β, 5, 6, 10) and OF score (Urea, ALT, AST and LDH). Mice were also divided into survivors (SUR) and dying (DIE) based on CLP outcome.

Pre-CLP: TH caused no obvious changes in OF score among age/gender groups (except 40% rise in 20m♂ vs. younger ♂ at -24h). After TH, CC score was lowest (by 50-100%) in 3m♂ at 0h vs. others. In 15m♂ SUR, IFNγ was 1.7-fold higher vs. DIE at -24h: its predictive accuracy for outcome was 0.99 (ROC test). Post-CLP: ♀ mice survival was always better (3m-53%, 15m-46%, 20m-25%) than males (28%, 18%, 7%) and independent of estrus cycle phases. OF score increase across age/genders was identical. The height of OF raise (∼3-fold) in DIE (vs. SUR) was alike among all groups. In contrast to ♂, CC score in ♀ decreased with age but only at 48h (20m 3-fold lower vs. 3m). Differences in CC score between SUR and DIE were high (∼3.5-fold) but similar across age/genders.

Pre-CLP: IFNγ predicted post-CLP deaths but only in 15m♂ mice. Strong survival disparity among age/genders was not clearly reflected by either changes in OF/CC scores, or extent of differences in SUR vs. DIE responses. WWTF Grant LS07-065.

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E. Boelke*1, C. Marquardt2, C. Matuschek1, M. Peiper1, P. Gerber1, D. Höfer3, J. Seydlitz-Kurzbach1, M. van Griensven*4, M. Schauer1, W. Fleischmann1. 1University of Duesseldorf, Duesseldorf, Germany, 2Klinikum Ludwigsburg, Ludwigsburg, Germany, 3Institut fuer Hygiene und Biotechnologie, Boennigheim, Germany, 4Klinikum rechts der Isar, Muenchen, Germany

Introduction: There are new effective strategies to manage the healing of complicated wounds. One option is to treat them with Lucilia sericata larvae (maggot debridement therapy, MDT). Observations have indicated that maggots have the ability to debride wound beds, provide anti-microbial activity and also stimulate the wound healing. There are no studies investigating the question how long should chronic wounds be treated with maggots.

Materials and Method: To examine this question, we investigated the enzymatic activity of the excretory - secretory (ES) products from 1000 unsterile Lucilia sericata larvae over 4 ½ days in vitro by enzymatic catalization of N-benzoyl-DL-arginyl-p-nitroanilid (BAPNA). Trypsin from pork pancreas served as positive control medium.

Results: The enzymatic activity decreased continuously on every day, when the maggots were kept without food source. In contrast to this the tryptic activity increased during the first days in the life of the maggots and reached its maximum on the 3rd day. On day 4 it decreased to a level of 46% of the maximum and after 4 ½ days the activity decreased below the initial activity of day 1.

Conclusion: Maggot debridement therapy is an effective treatment of chronic wounds. According to our results, maggots for biosurgical debridement should be kept in the wound for 2 - 3 days. Larvae are applied in the wound on the 2nd day of their life and should then stay in the wound for a minimum duration of 2 days, to unfold their full enzymatic activity on the 3rd day of their lives.

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S. Ono*, A. Kimura, S. Hiraki, R. Takahata, H. Tsujimoto, M. Kinoshita, H. Miyazaki, J. Yamamoto, K. Hase, D. Saitoh. 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 Tregs-mediated immunosuppression and the effect of PMX-F on sepsis-induced immunosuppression.

Objective: To investigate the role of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in septic patients, and to evaluate the effect of hemoperfusion with polymyxin B-immobilized fiber (PMX-F) on the recovery from immunosuppression due to septic shock.

Methods: Thirty-two septic patients who had an identified focus of infection in the abdominal cavity were enrolled in this study. Peripheral blood mononuclear cells (MNCs) in the septic patients were examined to evaluate the roles of Tregs and the serum cytokine levels. We also examined the effects of PMX-F therapy on the CD4+T cells, especially Tregs, and serum cytokine levels in patients with septic shock.

Results: The percentage of Tregs in the CD4+T cell population, and the serum IL-6 and IL-10 levels were significantly higher in patients with septic shock compared to those without septic shock, and PMX-F therapy significantly decreased the number of Tregs, as well as the serum IL-6 and IL-10 levels. Furthermore, a significant increase in the number of CD4+T cells, a significant decrease in the percentage of Tregs in the CD4+T cell population, and a significant decrease in the serum IL-6 and IL-10 levels 24 hours after PMX-F therapy were observed in septic shock survivors compared to non-survivors.

Conclusions: We found a major increase in the percentage of Tregs in peripheral blood circulating CD4+T cells from patients with septic shock, and observed that the removal of Tregs by hemoperfusion with PMX-F might represent a novel strategy for inducing recovery from the immunosuppression associated with sepsis.

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T. Nakao1, Y. Murao*1, N. Tsuda1, I. Ota1, M. Hamaguchi1, K. Maruyama1, T. Uejima1, I. Sakata1, T. Sato2. 1Kinki University Faculty of Medicine, Osakasayama, Japan, 2Kinki University Faculty of Medicine, Pathology, Osakasayama, Japan

We examined the effects of hypertonic saline and IL-10 deficiency on histological damage of kidney tissue after hemorrhagic shock and resuscitation in mice.

Methods: Male C57BL6/J mice and IL-10 knockout mice (IL-10 KO) weighing 20 to 30 g were anesthetized and the left femoral artery was cannulated. Heparin of 100 units was injected through the catheter. Blood was withdrawn until a mean arterial pressure of 40±5 mmHg was attained. The level of hypotension was maintained for 60 min, and the resuscitation procedure was as follows: HS+SB; hemorrhage shock for 60 min and followed by resuscitation with hypertonic saline (4 ml/Kg of 7.5% NaCl :HS) and shed blood (SB) at the same time, 2LR+SB; hemorrhage shock for 60 min and followed by resuscitation with lactated Ringer’s solution (2 times the volume of the shed blood) and SB. Kidney samples were harvested at 48 h after hemorrhage and resuscitation. Injury score was defined as the extent of injury on the kidney histology as assessed by neutrophil infiltration, lymphocyte infiltration, renal tubular vacuolation, hemorrhage, renal tubular epithelial cell necrosis and glomerulus congestion, which were graded as follows: 0.5=minimal injury, 1.0=mild injury, 2.0=moderate Injury, 3.0=severe injury.

Results: Histological damage shows the highest score in wild type of HS+SB group at 48h and IL-10 KO of HS+SB at 48h group shows significant decrease compared to wild type of HS+SB group at 48h (P<0.05), and also IL-10 KO of 2LR+SB group at 48h shows a trend to decrease compared to wild type of 2LR+SB group at 48h (P=0.0654). On the contrary hypertonic saline resuscitation has no effect on histological damage of kidney.

Conclusion: It was suggested that IL-10 deficiency might reduce acute kidney injury.

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T. Palmieri*1, 2, S. Sen1, 2, D.G. Greenhalgh*1, 2. 1Shriners Hospitals for Children Northern California, Sacramento, CA, 2University of California Davis, Sacramento, CA

Introduction: Hyperglycemia has been associated with increased morbidity and mortality in burn patients. Although continuous insulin infusions are frequently used to control blood glucose levels, there are conflicting reports on outcomes, and the optimal duration of glycemic control is undefined. The hypothesis of the study was that glucose variability in the first two weeks as well as admission glucose levels are associated with outcomes after severe burn injury.

Methods: All children admitted within a 2 year period with a burn ≥30% total body surface area (TBSA) treated with an insulin infusion were included in the study. Parameters measured included demographics (age, gender, ethnicity), treatment (operations, insulin rate, hourly glucose measurements), and outcomes (length of stay, ventilator days, survival).

Results: A total of 34 children, mean age of 9.2±0.9 years, TBSA 52.6±2.8%, PRISM score 7.4±9.4, were included in the study. Mean ICU length of stay was 58.3±9.3 days, ventilator days 30.6±7.4, and mortality 11.7%. A total of 9384 hourly accucheck glucose measurements, mean value of 126.9±36.4, were recorded. A total of 40 glucose readings were <60 g/dL, and 319 glucose readings >200 g/dL. On multivariate analysis, glucose variability in the first two weeks was associated with increased hospital and ICU length of stay (p<0.05). There was no association between glucose variability at 4 weeks and length of stay, mortality, or ventilator days (p>0.05).

Conclusion: Variability in hourly glucose levels in the first two weeks post-injury are associated with longer ICU stay and increased hospitalization. Early glucose variability may be a marker for prolonged illness, and efforts should be made to reduce glycemic variability in the first two weeks post injury.

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L.V. Gerasimov1, V.V. Moroz1, Y.V. Marchenkov2. 1V.A.Negovsky Research Institute of General Reanimatology, Russian Academy of Medical Sciences, Moscow, Russian Federation, 2Botkin’s Hospital, Moscow, Russian Federation

Introduction: Anemia is common in trauma patients and is associated with the use of blood transfusion, which are correlated with poor clinical outcomes. Two prospective, randomized studies from Corwin et al. (EPO-2, 2002; EPO-3, 2007) demonstrated that the use of epoetin alfa in critically ill patients does not reduce the incidence of red-cell transfusion but may reduce mortality among patients with trauma. In that studies epoetin was given subcutaneously.

Objectives: The aim of our study was to evaluate the influence of intravenous using of epoetin alfa in trauma patients on hemoglobin level, incidence of red- cell transfusions and the rate of thromboembolic events.

Methods: 48 patients with trauma and blood loss aged 36,9±9,1 years were examined. Hemoglobin level for 2nd day less than 11 g/dl was criterion of inclusion. Patient were treated randomly with either only 100 mg intravenous iron (control group, n=23), or intravenous iron plus intravenous epoetin alfa (study group, n=25). 40,000 units epoetin alfa administered at 2nd day after admission. Study period was 21 days.

Results: Level of hemoglobin at day 1 after admission was 105,8 ± 2,9 in the study group and 100,8 ± 3,7 - in control group. During the study period level of hemoglobin did not differ between the groups, except for the period from 12th to 14th days. So, for 13th day hemoglobin level in the study group and control group was 101±6,49 and 77,2±7,44 (p < 0,05) respectively. The total amount of RBC did not differ in groups, but significantly reduction was found in group with EPO in comparison with control on 2nd week. (Table 1). Thrombotic vascular complications didn’t differ in groups (Table 2).

Conclusions: Single use of 40000 ED epoetin alfa intravenously provide the significant but short-term gain of hemoglobin in trauma patients.

Table 1

Table 1

Table 2

Table 2

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A. Achouiti1, T. Vogl2, M.A. van Zoelen1, 3, H. Endeman3, 5, S. Florquin4, J. Roth2, C. van ’t Veer1, A.F. de Vos1, T. van der Poll*1. 1Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, Netherlands, 2Institute for Immunology, University of Münster, Münster, Germany, 3Department for Internal Medicine, University Medical Center Utrecht, Utrecht, Netherlands, 4Department of Pathology, Academic Medical Center, Amsterdam, Netherlands, 5Intensive Care Department, Onze Lieve Vrouwe Gasthuis, Amsterdam, Netherlands

Background: Streptococcus (S.) pneumoniae is the most common cause of community-acquired pneumonia (CAP). Severe infection is associated with enhanced release of MRP8 and MRP14, that together form MRP8/14 heterodimers. MRP8/14 enhances pro-inflammatory responses via Toll-like receptor 4 and in addition acts as a direct growth-inhibitor of pathogens through the chelation of trace metals in infectious environments.

Aim: To determine the role of MRP8/14 in CAP and murine pneumococcal pneumonia.

Methods: MRP8/14 levels were determined in serum and bronchoalveolar lavage fluid obtained from CAP patients in both infected and contralateral healthy lung. Pneumonia was induced in wild type (WT) and MRP14 knockout (KO) mice by intranasal inoculation with S. pneumoniae. Mice were sacrificed 6, 24 and 48 hours after infection for the determination of MRP8/14, cytokines, MPO and bacterial loads or monitored in a survival study. The lungs were analyzed by (immuno)histochemistry for pathology and granulocyte influx.

Results: CAP patients showed significantly higher levels of MRP8/14 in the infected lung compared to the contralateral healthy lung. Serum levels were also increased and remained elevated until 30 days after time of diagnosis. Murine pneumococcal infection resulted in increased MRP8/14 levels in lungs and plasma. Strikingly, MRP14 KO mice showed diminished growth of bacteria in blood and organs and an improved survival when compared to WT mice. Lung cytokines and pathology were unaffected. Lung MPO levels and granulocyte numbers were increased in MRP14 KO mice.

Conclusion: Human CAP leads to increased MRP8/14 levels at the primary site of infection. In mice, lack of MRP8/14 leads to enhanced neutrophil influx into lung tissue and an improved outcome in pneumococcal pneumonia.

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T. Shimizu*, T. Obata, H. Akabori, T. Miyake, H. Sonoda, Y. Eguchi, Y. Endo, T. Tani. Shiga University of Medical Science, Otsu, Japan

A novel Limulus amebocyte lysate assay for detecting endotoxins has recently been developed, named endotoxin scattering photometry (ESP) method. We report a case of septic shock in which plasma endotoxins were detected using ESP method. A 56-year-old man who had a traffic accident was diagnosed with peritonitis caused by perforation. Emergency surgery revealed perforation of the descending colon by contusion, and the patient underwent Hartmann’s procedure. Since the patient presented with septic shock after surgery, 13 h of direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX) was employed. Plasma endotoxin level was measured using the ESP method following surgery and PMX treatment. Reduction in the endotoxin level before and after direct hemoperfusion was also observed when ESP method was used. In conclusion, ESP method may more sensitively detect endotoxins than the widely used ordinary turbidimetric method. Moreover, the ability of endotoxin adsorption remained, despite the longer duration of PMX. To the best of our knowledge, this is the first report demonstrating the ability of endotoxin adsorption in a longer duration use of PMX.

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L.A. van Vught, B.P. Scicluna, A.J. van der Meer, F.M. de Beer, H. Belkasim, M.A. Huson, A.J. Hoogendijk, C. van ’t Veer, J. Horn, M.J. Schultz, T. van der Poll*. Academic Medical Center, Amsterdam, Netherlands

Background & Objective: Sepsis represents a complex clinical condition that arises from multiple interacting tissue and cell-specific host response mechanisms. We aimed to define central genes in the systemic transcriptional response induced by intravenous LPS in healthy humans that may aid in discriminating non-infectious critical illness and sepsis at ICU admission.

Methods: Eight healthy men were enrolled for an Escherichia coli LPS (4 ng/kg) intravenous challenge. Whole blood leukocyte RNA was isolated from baseline and 4-hour post-LPS for a genome-wide gene expression screen. Weighted gene coexpression network analysis (WGCNA) was applied. Polymorphonuclear cells (PMNs) were purified from blood of patients on admission to ICU for either non-infectious illness (N=6) or sepsis (N=11). Total RNA was isolated from PMNs presenting CD15+ purity>90% (FACS) for quantitative PCR (qPCR) analysis.

Results: WGCNA of the genome-wide transcriptional profiles in response to LPS-challenge identified a number of putative phenotype drivers (q<0.01) that included MARCKS, IL1RN, GPR84, GPR109A, and TNFAIP3. In ICU patients these candidate transcripts showed marked inter-individual variability in CD15+ PMNs; nonetheless, TNFAIP3 transcript abundance was significantly higher (p=0.039) in PMNs from septic patients.

Conclusion: We predict TNFAIP3 (A20), a negative regulator of NF-kB activity, as a LPS-response transcriptional network driver gene. Peripheral blood PMN-specific TNFAIP3 gene expression was higher in sepsis than in non-infectious critical illness. Our data lend further weight to the key concept of immunosuppression underlying the onset of sepsis among critically-ill.

This research was supported by the Center for Translational Molecular Medicine (MARS).

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M. Nakamura, S. Oda*, T. Sadahiro, E. Watanabe*, R. Abe, T. Nakada, Y. Morita, H. Hirasawa*. Graduate School of Chiba University, Chiba, Japan

Introduction: To investigate the relationship between blood interleukin (IL)-6 level, blood glucose level and glucose control in septic patients.

Methods: This retrospective observational study in a general ICU of a University Hospital included a total of 153 patients with sepsis, severe sepsis, or septic shock who were admitted to the ICU between 2005 and 2010, stayed in it for seven days or longer, and did not receive steroid therapy prior to or after ICU admission. The severity of stress hyperglycemia, status of glucose control and correlation between those two factors in these patients were investigated using blood IL-6 level as an index of hypercytokinemia.

Results: A significant positive correlation between blood IL-6 level and blood glucose level on ICU admission was observed in the overall study population (n=153) (r=0.24, p=0.01), and was stronger in the non-diabetic subgroup (n=112) (r=0.42, p<0.01). The rate of successful glucose control (blood glucose level of <150 mg/dL maintained for six days or longer) decreased with increase in blood IL-6 level on ICU admission (p<0.01). The blood IL-6 level after ICU admission remained significantly higher and the 60-day survival rate was significantly lower in the failed glucose control group than in the successful glucose control group (p<0.01, p<0.01, respectively.).

Conclusions: High blood IL-6 level was correlated with hyperglycemia and with difficulties in glucose control in septic patients. These results suggest that there is possibility that hypercytokinemia might be involved in the development of hyperglycemia in sepsis and that thereby it might affect the success of glucose control.

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R.M. Huebinger*1, S.E. Dowd3, A. Burris1, B.D. Arnoldo1, J.L. Hunt1, J. Sterling1, G.F. Purdue1, S.E. Wolf*1, R.C. Barber2. 1UT Southwestern Medical Center, Dallas, TX, 2University of North Texas Health Science Center, Fort Worth, TX, 3Molecular Research, LP, Shallowater, TX

Objective: To examine the microbiome of blood samples in burned patients using next generation sequencing (NGS) technology to determine the presence and diversity of bacteria in the blood across time after injury.

Methods: Blood samples were collected in the BICU as a part of an IRB approved protocol from patients with >20% TBSA. Blood samples were collected on post burn injury days 1, 3, 7, and 14 were sequenced for the 16s region utilizing the Roche 454 titanium FLX sequencer to identify the presence of unique bacterial sequences. These were analyzed through a data analysis pipeline to identify each 16s sequence to its appropriate taxonomic designation (∼species).

Results: Blood samples from 20 individuals in the Burn ICU with a >20% TBSA were examined. Bacteria were detected in all samples. Number of phylotypes (∼species) in the samples ranged from 25 to 262. Additionally, the number of phyla ranged from 4 to 14 in the blood samples. When examined across time, these diminished in number but persisted throughout the study period.

Conclusions: Following severe burn, a large and diverse bacterial population circulates in the blood stream which declines with time, but persists for at least 2 weeks following burn injury.

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S. Jacob, Y. Zhu, J. Carmical, D. Herndon*, H. Hawkins*, R. Cox*. Shriners Hospital for Children and the University of Texas Medical Branch, Galveston, TX

Airway epithelial cells are essential in the innate airway defense against inhaled pathogens and activation of the adaptive immune response. This study tests the hypothesis that cutaneous burn injury modifies airway epithelial cell expression of genes associated with innate airway defense.

Methods: Anesthetized Sprague Dawley rats were subjected to a 60% total body surface area scald injury (n=3) or anesthetized without burn injury (n=3). Twenty four hours after injury, the trachea were removed and RNA lysis buffer was flushed through the open lumen for collection of epithelial cell specific RNA. Total cellular RNA was extracted and converted to cRNA for GeneChipTM microarray analysis. Gene expression changes were identified using iReportsTM (Ingenuity® Systems,) following the default settings; fold change (FC) > 1.5, false discovery rate (FDR) applied, p-value<0.05.

Results: Fifty-nine genes were differentially regulated between sham and burn injured animals. Six genes were up-regulated and 53 were down regulated. Ten genes were identified as having significant roles in innate defense, one of which was up-regulated and nine exhibited a significant decrease in expression. Of the 49 genes not associated with innate defense, their translational products function in DNA repair, exogenous agent metabolism, bacterial killing, lysosomal function, cell cycle regulation, solute and ion transport, oxidative stress, glycoprotein synthesis, extracellular nucleotide processing and RNA degradation.

Conclusions: Burn injury induced acute changes in epithelial cell gene expression that may significantly impact epithelial homeostasis and innate defense properties of the airways. Further studies are needed to define the temporal changes in gene expression and how these changes may increase susceptibility to airway infection.

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A.C. Sherburne, K. Iskander, Z. Wang, S. Agarwal, D. Remick*, P. Burke*. Boston University Medical Center, Boston, MA

Introduction: The hepatic acute phase response (APR) is an organ-specific response to a number of insults, including sepsis. It is largely under transcriptional control via hepatic transcription factors (HNFs) and leads to a shift of hepatic phenotype, resulting in alterations in hepatic gene production and eventually plasma acute phase protein (APP) levels. Previous studies from this lab showed peritoneal sepsis induced by cecal ligation & puncture (CLP) in mice decreased DNA-binding activity of HNF-4α & HNF-1α. There were also significant differences in the early kinetics of DNA-HNF binding when mice were stratified by predicted mortality, based on plasma IL-6 levels.

Objectives: To determine if similar kinetic patterns after CLP are present in mRNA and plasma levels of positive APPs, fibrinogen-γ (FGG) & serum amyloid A (SAA).

Methods: 54 mice underwent CLP with plasma IL-6 levels obtained at 6h to allow stratification into groups predicted to die (P-DIE) or live (P-LIVE). Mice were sacrificed at 6h, 24h & 48h (n=28,16,10, respectively) to obtain liver tissue for mRNA extraction and RT-PCR for FGG & SAA mRNA levels. Blood was also obtained at 24h & 48h to measure plasma FGG & SAA protein levels via ELISA.

Results: Liver FGG & SAA mRNA levels increased significantly after CLP with peaks at 24h. At 6h, FGG mRNA was significantly higher in P-LIVE (RQ 11.1±2.1 vs 7.1±0.9, p=0.04). A similar trend was noted in the SAA group at 6h (RQ 1226±235 vs 818±91, p=0.06). Plasma FGG & SAA levels markedly increased after CLP, but no differences were found with regards to predicted mortality.

Conclusion: Predicted mortality is associated with delayed intrahepatic APR. This relationship was not observed in plasma, underscoring the importance of the hepatic phenotype in this clinically-relevant model of intraabdominal sepsis.





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C. Baird*, A. Kallweit*, S. Niederlechner*, P.E. Wischmeyer*. University of Colorado Denver, Aurora, CO

Background: Threonine (THR) is an essential amino acid that is important in intestinal mucin synthesis and normal gut function. It has been utilized clinically in inflammatory disease but its mechanism of protection is currently unknown. P38 mitogen activated protein kinase (MAPK) and cleaved caspase-3 (CC3) are both important mediators of programmed cell death, or apoptosis. The purpose of this study was to evaluate THR’s potential to protect intestinal epithelial cells in a model of heat stress (HS) injury and to elucidate its effects on CC3 expression and P38 MAPK pathway activation. Since THR is an osmotically acting amino acid, its effect on protective heat shock protein (HSP) expression was also evaluated.

Methods: Cells were treated for 15 min +/- THR and subsequent lethal or non-lethal HS injury. Survival was evaluated via MTS assay and CC3 expression. Western blot analysis was used to evaluate phosphorylation of P38 MAPK, total HSP25, and total HSP70 levels.

Results: MTS assays showed THR increased cell survival in a dose dependent manner (p<0.02 vs. HS alone). THR decreased CC3 more than 54%, and decreased phosphorylated P38 MAPK more than 65% in HS cells (p<0.05 vs. HS alone). HSP25 and HSP70 levels increased more than 5 fold in HS cells treated with THR (p<0.02 vs. HS alone).

Conclusion: This is the first demonstration that THR can protect by decreasing apoptosis in heat stressed intestinal cells. THR also increases protective HSP25 and HSP70 expression during heat stress, which may be an integral component of THR’s mechanism of cellular protection.

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V. Dolgachev*, Y. Bi, K. Raghavendran*, L. Sun, M. Hemmila*. University of Michigan, Ann Arbor, MI

Objective: Chest injury/lung contusion predisposes the patient to pneumonia. IL-10 is known to down regulate the pro-inflammatory response in the setting of acute lung inflammation. Blockade of IL-10 improves survival from pneumonia. We hypothesize that increased levels of IL-10 in the setting of lung contusion (LC) and pneumonia impacts survival by altering macrophage function.

Methods: A bi-transgenic, doxycycline inducible, human IL-10 overexpression (IL-10OE) mouse model and single transgenic (TG-) control were used. Mice underwent LC injury at time -6 hrs. At time 0 animals were inoculated intratracheally with 500 CFU of Klebsiella pneumoniae (LC+Pneu) or vehicle (LC). Bronchoalveolar lavage fluid (BAL), lung tissue specimens or purified macrophages were collected. Lung tissue and blood bacteria levels were quantified. Cytokine levels were assayed by ELISA. Cell type identification and quantification was done using RT PCR and flow cytometry.

Results: IL-10OE mice showed decreased 5 day survival compared to TG- mice after LC+Pneu (0 vs. 25%, p=0.0003). IL-10OE mice had significantly higher lung bacteria counts (p=0.02) and levels of bacteremia (p=0.001) at 24hrs. IL-10OE mice recruited fewer neutrophils into the alveoli as measured in BAL fluid compared to TG- mice. Lung parenchyma macrophages from IL-10OE mice displayed increased alternative activation (M2 macrophages, p=0.046). Macrophages from TG- mice exhibited classical activation (M1 macrophages) and much higher bacterial killing potential (p=0.03). IL-6 and MIP2 levels were significantly elevated in IL-10OE LC+Pneu animals (p<0.05).

Conclusions: hIL-10 over expression in the setting of LC and pneumonia caused a strong and persistent alternative activation of lung macrophages with suppressed ability to kill bacteria, resulting in accelerated mortality.

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H. Akabori, T. Shimizu*, T. Miyake, H. Yamamoto, T. Obata, Y. Endo, T. Tani. Shiga University of Medical Science, Otsu, Japan

Background: EAA, a novel assay for endotoxin that is approved by the Food and Drug Administration in Europe and the USA, is proven clinical utility in the detection and risk stratification of clinically ill patients with suspicion of sepsis. In this study, we assessed the accuracy of EAA as an early marker of perioperative infection for general surgery.

Methods: 31 patients were submitted to gastrointestinal surgery that comprehend emergency operation. The patients were divided into the following three types of groups to analyze: “Elective (n=24)” or “Emergency (n=7)” operation; “Complication (n=7)” or “Complication-free (n=24)” after surgery; “Laparotomy (n=18)” or “Laparoscopic (n=13)” surgery. Serum EAA levels were measured at 2 times (T1=preoperative, T2=postoperative day 1). EAA-values were compared to inflammatory mediators (leukocyte, neutrophil count, C-reactive protein, and procalcitonin).

Results: Emergency surgery significantly increase in EAA (T1) levels compared to elective surgery (0.62±0.08 vs 0.30 ±0.04; p<0.01). In addition, EAA (T2) levels in complication group were markedly higher than those of complication-free group (0.60±0.05 vs 0.40±0.04; p<0.05). 7 patients had postoperative complications that were: 3 surgical site infection, 2 deep abdominal infections, 2 cholangitis, 1 pneumonia, 2 ileus. There were strong correlation between EAA (T1) and other laboratory variables (neutrophil, CRP, PCT) in “laparotomy” group as well as postoperative “complications-free” group.

Conclusion: In the preoperative period, EAA itself appears to be an important aid not only for a marker of inflammation but also for early diagnosis of postoperative complications when it is used with the other indexes.

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N. Oka1, T. Ueda2, M. Aoyama1, 2, T. Inoue1, M. Usami1, J. Kotani*2. 1Kobe Graduate University School of Health Sciences, Kobe, Japan, 2Hyogo College of Medicine, Nishinomiya, Japan

Introduction: Antithrombin III (AT III) plays a central role in anti-coagulation, and also has an anti-inflammatory property. It has been reported that AT III attenuated lung injury by inhibiting neutrophil recruitment into the lung in sepsis, but the mechanism is still not understood.

Purpose: To elucidate the effect of AT III on lipopolysaccharide (LPS)-induced inhibition of neutrophil apoptosis in vitro.

Methods: Neutrophils were isolated from healthy volunteers (5 men and 5 women). Neutrophils were incubated with RPMI medium for 20 hr (37°C, 5% CO2) with or without AT III (10 IU/mL) in the presence or absence of LPS (1 μg/mL). The AT III were added after 0, 1, 3, or 6 hr after LPS stimulation. In the inhibitor groups, caspase inhibitors z-VAD-fmk (caspase-3), z-IETD-fmk (caspase-8) or z-LEHD-fmk (caspase-9) were pre-incubated for 1 hr before LPS stimulation, followed by administration of AT III at 6 hr after LPS stimulation. Apoptosis was assessed by annexin-V using flow cytometry.

Result: LPS inhibited neutrophil apoptosis. Administration of AT III at 1, 3, or 6 hr, but not 0 hr, after LPS stimulation significantly recovered LPS-induced inhibition of neutrophil apoptosis (p<0.05). This effect of AT III was more significant in women than in men. The caspase-3, 8, or 9 inhibitors did not influence on this effect of AT III.

Conclusion: Posttreatment with AT III may recover LPS-induced inhibition of neutrophil apoptosis by the mechanisms other than caspase-dependent pathway. There might be gender difference in this effect of AT III, i.e., female neutrophils may have higher sensitivity for this effect of AT III than male neutrophils.

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T. Kawasaki*1, C. Kawasaki2, K. Okamoto1, T. Sata1. 1University of Occupational and Environmental Health, Kitakyushu, Japan, 2Sugioka Memorial Hospital, Fukuoka, Japan

Objective: Previous studies demonstrated that surgical stress induces immunosuppression. We also showed that neutrophil (PMN) functions decreased from the early period of surgery. Since antimicrobial peptides have been described as important effector molecules on host antimicrobial innate immunities, in the present study, the influence of PMN from patients with upper abdominal surgery on the beta-defensins (BD) production by human keratinocytes has been studied.

Methods: PMN were isolated from patients who had elective upper abdominal surgery (pre.OPE; pre-operative, OPE2H; 2h after incision, post.OPE; the end of surgery, POD1; postoperative day 1, POD4; postoperative day 4) or normal volunteers. Normal human epidermal keratinocytes (NHEK: 5×105 cells, Lonza) were cultured with PMN (1×105 cells, upper chamber) in dual-chamber transwells. Culture fluids harvested 24 hrs after cultivation were assayed for BD by ELISA. Also, these culture fluids were analyzed for their antimicrobial activities by a standard colony counting method using Pseudomonas aeruginosa.

Results: The production of BD2 and BD3 by NHEK was suppressed by PMN from surgical-stressed patients (OPE2H and post.OPE). The culture fluids of NHEK transwell-cultured with patient PMN (OPE2H and post.OPE) showed decreased antimicrobial activities, as compared with controls (culture fluids of NHEK transwell-cultured with healthy PMN). PMN isolated from patients (OPE2H and post.OPE) produced IL-10 and CCL2, but not IL-12 and CCL3.

Conclusion: Our results indicate that PMN appeared in association with surgical stress contribute to the decreased production of BD. These results in part explain the impairment of host-defense mechanisms seen in the perioperative period.

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T. Ueda1, M. Aoyama2, 1, N. Oka2, M. Usami2, J. Kotani*1. 1Hyogo College of Medicine, Nishinomiya, Japan, 2Kobe University Graduate School of Health Sciences, Kobe, Japan

Introduction: We defined the neutrophil score (n-score) as a useful marker of ICU mortality at the 34th annual SHOCK meeting. In this study, we analyzed n-score in the ICU septic patients as the predictor of the mortality.

Methods: Approval for this study was granted by the ethics committees at both universities. Blood samples, the APACHE II scores, and the mortality in ICU were collected from 116 ICU patients. The neutrophil score was defined for 5 consecutive days as the neutrophil number increase/decrease as follows; 1 point score means 1,000-cell/μL-increase or 300-cell/μL-decrease compared to 3,120 cells/μL, the median of normal range in our laboratory (WBC; 4,000-9,000 cells/μL and segment neutrophils 38–58%). When the number of neutrophils was decreased, 10-point was added as the basal score.

Results: Among 116 patients, 50 were non-septic patients with 34.8% mortality (23/66, age; 57 ± 19, male; 34 and female; 16, APACHE II; 19 ± 10) and 66 were septic patients with 16.0% mortality (8/50, age; 70 ± 14, male; 33 and female; 33, APACHE II; 24 ± 9). N-score in the septic patients was 14.9 ± 9 whereas that in the non-septic patients was 10 ± 6. The n-score≧16 indicated higher mortality (50%; 12/24) compared with n-score<16 (26%; 11/42) in sepsis patient (p=0.05). The n-score of surviving patients was 8 ± 6 and of non-surviving patients was 16 ± 8 (p<0.05) in the septic patients who requiring emergency surgery and high APACHE II score (≧21) on admission. N-score was not correlated with APACHE II and SOFA on admission. However, n-scores were significantly higher in septic non-surviving patients than surviving patients for 5 days (p<0.01).

Conclusion: The neutrophil score could be a useful and convenient prognostic marker in septic patients.

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T. Oshiro, Y. Nagura, T. Harada, M. Takeda, A. Yaguchi*. Tokyo Women’s Medical University, Tokyo, Japan

The effectiveness of pulmonary artery catheter (PAC) still has a controversial issue. In our previous study, we showed the initial data of PAC had no differences except for mean arterial pressure (MAP) between survivors and non-survivors. The purpose of the present study is to more assess outcomes with PAC in critically ill patients. The study design is a retrospective database review. From July 1996 to June 2010, 220 patients received PAC in our ICU. The initial, the second and the last data of PAC, including mean arterial pressure (MAP) (mmHg), pulmonary capillary wedge pressure (PCWP) (mmHg), cardiac index (CI) (l/min/m2), mixed venous oxygen saturation (SvO2) (%), oxygen delivery and consumption (DO2 and VO2) (mL/min) were compared between survivors and non-survivors. Values are expressed as median. Data was analyzed by Kruskal-Wallis test, Mann-Whitney U test, and Wilcoxon signed-rank test. P values less than 0.05 were considered significant. There were 159 survivors and 61 non-survivors (148 men, 72 women; age 66[19-94]). The initial, the second and the last MAP were significantly different between survivors and non-survivors (81 vs. 63, 89 vs. 65, 91 vs. 73 mmHg, p < 0.0001, respectively), although the last MAP significantly increased than the initial’s in non-survivors (73 vs. 63 mmHg, p=0.042). There were no significant differences in SvO2, PCWP, CI, and DO2 at the initial, the second, and the last measurements of PAC between survivors and non-survivors. However, the last VO2 was significantly lower in non-survivors than in survivors (183.5 vs. 219 mL/min, p=0.013). There were no significant differences in SvO2 and DO2 between survivors and non-survivors, but the last VO2 evidently decreased in non-survivors. Not only MAP but VO2 in PAC data could suggest poor outcome.

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T. Lustenberger, B. Relja, B. Puttkammer, I. Marzi. Goethe University Hospital, Frankfurt am Main, Germany

Introduction: The thrombin-activatable fibrinolysis inhibitor (TAFI) has been recognized as a potent inhibitor of fibrinolysis. However, the time course of TAFI and its activated form (TAFIa) following trauma, in particular in patients suffering trauma-induced coagulopathy, has been barely described.

Methods: A total of 26 severely injured trauma patients were prospectively enrolled. TAFI and TAFIa levels were measured upon arrival and throughout hospital day 1-10. Trauma-induced coagulopathy was defined as elevated international normalized ratio (INR) and/or prolonged activated partial thromboplastin time (aPTT) and/or thrombocytopenia within 1 day of admission.

Results: TAFI and TAFIa levels demonstrated the most prominent decrease on hospital day 2 and 1, respectively, with a progressive increase thereafter. Overall, 11 patients developed coagulopathy. No statistically significant differences were found for TAFI levels comparing the two groups. However, for TAFIa, coagulopathic patients demonstrated significantly lower levels on admission and hospital day 6 to 8 (all p<0.05). Statistically significant Spearman correlations were noted between TAFIa level on admission and the amount of packed red blood cells (p=0.011; coefficient=-0.5) and fresh frozen plasma (p=0.044; coefficient=-0.405) transfused within the initial 24 hours of admission.

Conclusion: The role of TAFI in the development of trauma-induced coagulopathy and its clinical relevance warrants further investigation.

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D.S. Herzig1, B.R. Driver1, G. Fang1, E. Sherwood*1, 2. 1University of Texas Medical Branch, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX

Introduction: NK, NKT and T lymphocytes express the chemokine receptor CXCR3 and are known to facilitate systemic inflammation and physiologic dysfunction in experimental models of severe sepsis. In our previous studies, we demonstrated that CXCR3 participates in the regulation of lymphocyte trafficking during cecal ligation and puncture (CLP)-induced sepsis. In this study, we evaluated the effects of treatment with anti-CXCR3 and antibiotics on outcome during septic shock caused by CLP.

Methods: C57BL/6J mice were treated with neutralizing antibodies against CXCR3 (50-100 μg) plus antibiotics (Primaxin 12.5 mg/kg) at either 24 hours prior to or at 2 or 6 hours after CLP. Control mice received non-specific IgG plus Primaxin in the same regimen. Survival, core body temperature, bacterial clearance and systemic cytokine production were evaluated.

Results: Treatment with anti-CXCR3 plus Primaxin at 24 hours prior to (45 vs 5%) or 2 hours after (60 vs 10%) CLP significantly improved survival and attenuated hypothermia compared to mice receiving non-specific IgG plus Primaxin whereas treatment at 6 hours after CLP did not. Treatment with anti-CXCR3 prior to CLP also attenuated IL-6 and MIP-2 production but did not alter bacterial clearance. Treatment with anti-CXCR3 and Primaxin at both 2 hours and 6 hours after CLP did not improve bacterial clearance and systemic cytokine production compared to mice treated with IgG and Primaxin.

Conclusion: The results from this study indicate that neutralization of CXCR3 prior to or 2 hours after the initiation of CLP-induced septic shock improves survival and attenuates CLP-induced inflammation and physiologic dysfunction.

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M.E. Diebel, L. Diebel*, D.M. Liberati. Wayne State University, Detroit, MI

Objective: The intestinal mucus layer and other host defenses including secretory IgA (SIgA) interact in protecting the intestinal barrier against potential luminal pathogens. The H2 blocker cimetidine decreases mucus release by goblet like cells and may impair barrier function. We studied the interaction of mucus and SIgA under normal and hypoxia/reoxygenation (H/R) culture conditions in vitro.

Methods: HT29-MTX (a mucus producing IEC) monolayers were established in a two-chamber cell culture system. Dimeric IgA was added to the basal media of IEC monolayers to allow binding of IgA to the polyimmunoglobulin receptor on the basal side of IEC followed by uptake and delivery of SIgA to the IEC apical surface (transcytosis). Cimetidine was added to the media of a subset of the HT29-MTX intestinal cells and E. coli (EC) to the apical media of HT29-MTX cell monolayers. Either fluorescein-labelled EC (FITC-EC) or unlabeled EC were added to subsets to quantify bacterial adherence (60 minute co-culture) and bacterial passage (240 minutes) thru IEC monolayers respectively. In additional experiments cell co-cultures were subjected to a 90 minute hypoxic insult (95%N 2, 5%CO2) followed by reoxygenation. Mucus content was indexed by O-linked oligosaccharide chain (OSC) content and Western blot analysis.

Results: Mean ± S.D., N = 5 for all groups.

Conclusions: Bacterial trapping in the intestinal mucus layer enhances SIgA interaction with luminal bacteria and contributes to intestinal barrier integrity. Loss of the intestinal mucus layer associated with cimetidine administration impairs barrier function in this model. Use of cimetidine may contribute to systemic inflammation and remote organ dysfunction in at risk patients especially with episodes of gut H/R.

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Y. Hiyama1, R. Kraft2, A. Arno1, A.H. Smith1, D.N. Herndon*2, M.G. Jeschke*1. 1Sunnybrook Health Sciences Centre, Toronto, ON, Canada, 2Shriners Hospital for Children, Galveston, TX

Severe burn injury causes hepatic dysfunction that result in major metabolic derangements such as hypercatabolism and insulin resistance, and is associated with hepatic endoplasmic reticulum (ER) stress. We have previously shown than insulin improves burn induced ER stress and insulin resistance. To determine whether this was due to insulin per se or glucose modulation, we utilized metformin, an anti-diabetic drug that has been recently shown by others to improve patient outcome by attenuating post-burn stress responses. The aim of the present study is to determine the effects of metformin on post-burn hepatic ER stress and metabolism. We randomized rats to sham, burn injury, and burn injury plus metformin. The animals received 60% total body surface area burn and were sacrificed at various time points. There was a significant increase in pAMPK levels in the metformin treated livers (1.8±0.2 vs 1.0±0.1 shams, relative expression levels, p<0.05), verifying proper drug administration. Burn-induced decrease in albumin and increase in alanine transaminase levels were not normalized by metformin treatment. In addition, ER stress markers ATF6, BIP, XBP-1 spliced, and CHOP were increased similarly in burn injury with or without metformin compared to sham. Burn injury alone increased the expression of genes involved in fatty acid and glucose metabolism. Although metformin did not have an effect on this increase after 24 hours, there was a further upregulation of these genes after 48 hours, suggesting a time-dependent response with metformin in burn injury.

Our results indicate that, while thermal injury results in hepatic ER stress and dysfunction, metformin does not ameliorate post-burn stress responses by correcting them. Further studies are necessary to understand the precise molecular mechanisms.

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S. Yang, S. Hu, R. Raju*, K.I. Bland, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL

T-H induced a decrease in cardiac nuclear estrogen receptors (ERs), ERRs and PGC-1α in rats, which are associated with cardiac depression; estrogen (E2) administration after T-H restored cardiac function as well as cardiac ERs, ERRs and PGC-1α. It has been reported that ERRβ-mediated growth inhibition may be potentiated by an ERRβ/γ agonist DY131 in in vitro study; however, the role of DY131 in T-H model is not known. To study this, DY131 was administered after T-H was induced in male adult Sprague-Dawley rats. Seven groups were established: sham, sham+DY131, T-H+vehicle, T-H+E2 (1 mg/kg body weight [BW]), T-H+ICI 182,780 (ICI, 3 mg/kg BW)+E2, T-H+DY131 (0-80 μg/kg), and T-H+ICI+DY131. Either E2 or DY131 was administered at the onset of resuscitation, IV, and the ER antagonist ICI was given 30 min before E2 or DY131, IP. LV performance was determined; blood and heart tissue were harvested 2 hrs after T-H and resuscitation. Cardiac nuclear ERs, ERRs and PGC-1α were determined by Western blot. Our results showed that DY131 restored LV performance as well as all of the above mechanisms except for cardiac ERs. Unlike E2, ICI did not alter the effect of DY131 on the heart. Moreover, DY131 acted in a dose-dependent manner on cardiac function as well as on signaling mechanisms. Thus, it may be concluded that the mechanism of DY131-derived salutary effect on the heart after T-H is not the same as that of E2; DY131-produced cardioprotection is through restoration/enhancement of cardiac nuclear ERRs and PGC-1α (NIH R01 GM39519).

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D.J. Hess, M.J. Henry-Stanley, C.L. Wells. University of Minnesota, Minneapolis, MN

Introduction: Bacterial biofilms cause serious infections in critically ill patients including those with burn wounds, endocarditis, ventilator-associated lung infections, infected devices such as joint prostheses, heart valves, stents, catheters, and infected surgical sites. Because biofilm microbes are more antibiotic resistant than their planktonic (free-living) counterparts, experiments were designed to test the ability of gentamicin to prevent biofilms initiated with Staphylococcus aureus RN6390.

Methods: Biofilms were cultivated by incubating high (107) and low (104) inocula of S. aureus with 1-cm segments of 3-0 silk suture, and biofilm growth was assessed by quantifying viable suture-associated bacteria after 16 hr. Incubation medium was 0.66% tryptic soy broth with 0.2% glucose plus concentrations of gentamicin (0 to 50 μg/ml) that ranged from subinhibitory to >10 times the minimum bactericidal concentration (MBC, 1-2 μg/ml) for planktonic cells.

Results: After inoculation with 104 bacteria, suture-associated S. aureus biofilms were prevented only if the growth medium contained at least 0.25 μg/ml gentamicin. However, after inoculation with 107 S. aureus (a number compatible with typical skin contamination), S. aureus biofilms were prevented only in samples incubated with at least 5 μg/ml gentamicin, a concentration above the MBC and difficult to achieve clinically. (In companion experiments, similar results were obtained with S. aureus ATCC 25923.)

Conclusions: Taken together, these data indicate that biofilm development depended both on the antibiotic concentration and the size of the bacterial inoculum. Prophylactic gentamicin could prevent biofilm infections if the bacterial inoculum was low, but would not be effective in many clinical situations where the inoculum is relatively high.

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A.K. Jaehne1, S. Brown1, A. Suarez1, 3, P. Koti1, D. Figueora1, L. Eichhorn-Wharry2, V. Coba1, 2, A. Garcia1, 4, E.P. Rivers1, 2. 1Henry Ford, Detroit, MI, 2Henry Ford Hospital, Surgery, Detroit, MI, 3Henry Ford Hospital, Anesthesia, Detroit, MI, 4Henry Ford Hospital, Internal Medicine, Detroit, MI

Septic shock (SS) can be accompanied by hypothalamic pituitary axis (HPA) dysfunction. The cosyntropin stimulation test (CST) consists of baseline cortisol levels, the admin of 250 μg cosynthropin which is followed by cortisol levels at 30 & 60 min. Failure to have a normal response to the CST is an indication for steroid replacement which has been associated with a decrease in vasopressor dependency and mortality.We prospectively analyzed clinical and laboratory data of 122 vasopressor dependent SS patients (pt) admitted to the ICU who received the CST test (Oct 2010 to Jan 2011). Steroid use was determined by the treating clinician’s discretion. The results of the CST were not available at the time of the decision to treat. The data was analyzed using univariate test by means of chi2 or Fisher’s exact as appropriate (p-value of <0.05 considered statistically significant).Of the 122 pt, 69 received steroid treatment during their SS episode and 43 pt did not receive steroids. The groups were similar in regards to age, gender, ethnicity, APACHE II, SAPS II and SOFA score. In hospital, 28 day and 60 day mortality for pts treated with steroids was 33% vs 30%, 38% vs 37% and 45% vs 41%, respectively. CST results were similar in pts who received steroid treatment and those who did not receive steroid treatment. Baseline, peak levels and Δ cortisol levels did not distinguish mortality.

At the moment, the CST remains the only test to assess adrenal function in SS. The CST does not help to stratify pts, who will benefit from steroid replacement. This is consistent with the SSC recommendations for steroid use in vasopressor dependent shock with CST, an optional measurement. Further research is needed to elucidate the mechanisms of adrenal dysfunction in septic shock which may improve upon the diagnostic and therapeutic precision.

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B. Miyazawa, C. Ho, M. Cohen*. University of California San Francisco, San Francisco, CA

Background: Hemorrhagic shock induced ischemia/reperfusion (I/R) can result in endothelial and epithelial injury, which can lead to organ failure and infection. Our group has shown that activated protein C mediates acute traumatic coagulopathy after injury and that later maintenance of plasma protein C leads to organ protection and reduced infection. This cytoprotective function of activated protein C (aPC) has been described but its relationship to injury and early activation is not well understood. The purpose of this study was to examine the mechanisms for activation of PC and its protection of endothelial barrier function.

Methods: HUVEC monolayers were subjected to anoxia followed by reoxygenation. Thrombomodulin (TM), Endothelial Protein C Receptor (EPCR), and the functional capacity to activate PC were assayed by on cell western and enzyme capture assay. In related experiments we assessed the effect of aPC on endothelial permeability using collagen coated transwell chambers.

Results: Cell surface thrombomodulin was increased 43% after 60 minute anoxia (Fig 1). This increase in TM expression results in increased functional ability to activate protein C. 60 min of anoxia resulted in a 78% increase in protein C activation (Fig 1). Anoxia results in a 3.7 fold increase in endothelial permeability. aPC treatment significantly protected against this increased permeability (Fig 2).

Conclusion: Our I/R model, which mimics that seen in patients after injury, results in increased cell surface expression of TM and EPCR, which can functionally activate protein C. This aPC protects against hypoxia induced barrier dysfunction. Further experiments are warranted and underway to examine the relationship between activation of the protein C system, acute traumatic coagulopathy and cytoprotection after injury.

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T. Brooks1, S. West*2, C. Mold3. 1University of New Mexico School of Medicine, Albuquerque, NM, 2University 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

After surviving the initial trauma, severely injured trauma patients become susceptible to infectious complications. This has been attributed in part to dysregulation of the immune system. Soluble CD163 (sCD163) is an anti-inflammatory marker that has been shown to be a predictor of susceptibility to sepsis in burn patients. We hypothesized that soluble CD-163 could be used to predict susceptibility to infections in trauma patients. The Human Research Review Committee approved all protocols prior to sample collection. Severely injured trauma patients defined by an ISS of 16 or greater or requiring ICU admission were consented and blood samples were collected at enrollment, 48 and 72 hrs. Patients were followed through their hospital stay and any infectious complications were recorded. Soluble CD163 levels were determined in the plasma from all three blood draws. The mean values for trauma patients on day 1 were similar to healthy controls. We found significantly decreased soluble CD163 on day 2 and day 3 when compared to day 1 (p<0.05) or to controls. There were significantly higher levels of sCD163 in plasma of patients who became infected during their hospital stay compared to those who did not (p<0.05). For a 50 ng/ml increase in the amount of sCD163 from the mean, there was a 1.6 greater chance of developing an infection (p = 0.04). This study confirms the utility of sCD163 as a potential biomarker for infectious complications in severely injured trauma patients. Identification of at risk patients could lead to potential preventative and therapeutic interventions.

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P.G. Perakis, I. Rubinfeld, A. Narayana, M. Cumba, L. Takis, V. Coba*, D. Blyden, M. Horst. Henry Ford Health System, Detroit, MI

Introduction: Following the first sepsis insult, the recurrent incidences of sepsis events and hospital readmissions are problematic. We sought to identify the incidence of hospital readmission associated with septic patients from the surgical intensive care unit (SICU).

Methods: Retrospective cohort study of all septic patients discharged from the SICU between 2007 and 2010 at an urban academic tertiary care center. Septic patients were identified according to Angus criteria, ICD-9 sepsis infection codes and acute organ dysfunction. Hospital readmission was defined as after 24 hours and within 30 days from hospital discharge.

Results: During the study period, 9,259 patients were screened and we identified 1,497 septic patients discharged from the SICU. The mean age was 61.5 yrs (±15.8), 58% were male, the average hospital LOS was 10 days, and Charlson Comorbidity Index was 3.6 (±2.4). Disposition of the septic patients were 50% home ±home health care, 28% skilled nursing facility, 12% rehab facility, 5% long term care facility and 5% other. 418 were readmitted within 30 days and 116 of 418 (28%) were recurrent septic related readmissions. The hospital mortality for the recurrent septic and non-septic related readmissions was 14.7% and 6.3%, respectively (p<0.01). ICU readmissions for recurrent septic and non-septic related conditions were 44% and 22%, respectively (p<0.001).

Conclusion: Septic patients from the SICU are at high-risk for 30-day hospital readmission. Recurrent septic related readmissions had a significantly higher ICU readmission rate and hospital mortality compared to non-septic related readmissions. Further research is warranted to identify associated factors.

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S. Komak1, 2, L. Porro1, 2, A. Miller2, S. Komak2, J.O. Lee*1, 2, D.N. Herndon*1, 2. 1Shriners Hospital for Burned Children, Galveston, TX, 2University of Texas Medical Branch, Galveston, TX

Introduction: Sepsis and burn injury are one of the most prevalent causes of acute kidney injury (AKI) in the pediatric population– with mortality rates of 50% and higher. We examined AKI in the pediatric burn population focusing on overall mortality rates and specific factors important in survival vs. non-survival of patients with AKI.

Patients and Methods: Patients with AKI admitted from Jan 1997 to Jan 2010 were included in the study. All patients had a > 30% TBSA burn. AKI was defined as serum Cr>1.5, UOP <0.5cc/kg/hr, or initiation of renal replacement therapy (RRT).

Results: The average age of patients with AKI was 7.7 +/- 5.5 yrs, with a 65.4 +/-19.9 % TBSA burn. The overall mortality in the group was 34% (n=57). Non-survivors were older than survivors (9.9 +/-5.6 yrs vs. 6.7+/- 5.1 yrs P <.001), and had a larger TBSA burn (73.3% vs. 60.4% P<.001). Forty-eight percent of non-survivors presented with AKI on admission compared to 20% of survivors (p <.001). Renal replacement therapy was required more often in non-survivors vs. survivors (79.5% vs 19%, P<.001). The base deficit on arrival was significantly worse in non-survivors compared to survivors (BD: 5.9 Meq/l vs. BD: 3.2 Meq/l P<.01). The duration of renal replacement therapy was also longer in non-survivors vs survivors (9.5 days vs. 5.1 days P<.01). Non-survivors had an increased incidence of sepsis and multi-drug resistant infections compared to survivors (P<.05).

Discussion: Burn injury is a leading cause of AKI in children. Here we report an overall mortality rate of thirty-four percent. We furthermore demonstrate that children with AKI who eventually succumb have a significantly higher rate of AKI on admission, worse base deficit, higher use of RTT, longer duration of RRT, larger TBSA burn, and a higher rate of sepsis.

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M. Bohan Brown, L. Li, J.L. Messina*. University of Alabama at Birmingham, Birmingham, AL

Hyperglycemia and insulin resistance develop rapidly following acute injury or infection and contributes to increased mortality and morbidity. However, the mechanisms of this acute insulin resistance are not understood. Blocking the renin-angiotensin system (RAS) has been shown to improve insulin sensitivity in chronic insulin resistance and decrease the risk for type 2 diabetes.

However, it is not known whether RAS contributes to the development of acute insulin resistance. In this study, we have demonstrated that losartan, an antagonist of angiotensin II type 1 receptors (AT1R), improved insulin signaling in adipose tissue of rats following trauma and hemorrhage (mean arterial pressure of 35–40 mmHg for up to 90 minutes). Rat adipose tissue developed acute insulin resistance 60 and 90 minutes following hemorrhage, as evidenced by decreased insulin-induced phosphorylation of the insulin receptor (IR), insulin receptor substrate 1 (IRS-1), and Akt. When losartan was administered 60 minutes prior to the start of surgery, rats developed less insulin resistance following hemorrhage, resulting in increased insulin-dependent phosphorylation of IR, IRS-1, and Akt, and almost complete recovery of insulin-induced glucose transport. Aldosterone concentrations increased precipitously following hemorrhage, but to a much lesser extent in losartan pretreated rats. Thus, there is a significant role of the renin-angiotensin system, which may include aldosterone, in the hemorrhage-induced insulin resistance and decreased insulin-dependent glucose transport. Inhibiting the action of angiotensin II is a potential approach to reverse the insulin resistance that develops following trauma and hemorrhage.

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L.J. Porro, S. Komak, J.O. Lee, D. Herndon*, O.E. Suman, W.J. Meyer. University of Texas Medical Branch and The Shriners Hospitals for Children, Galveston, TX

Introduction: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are uncommon mucocutaneous adverse reactions to medication which carry a mortality rate of up to 30%. Both of these life-threatening disease processes have effects on multiple end-organs. Here we examine the incidence of acute kidney injury (AKI) in SJS/TENS and focus on the associated morbidity and mortality.

Patients and Methods: Thirty three patients admitted to the Shriners Hospitals for Children with a diagnosis of SJS and TEN between the years 1990–2010 were included. Patient’s demographics, hospital course, development of AKI and mortality were evaluated.

Results: The average age of the patients was 8.5 +/- 5.0 years. Total body surface area affected was 62.2 ± 33.6 %. The time from beginning of disease process to admission was 7.0 ± 5.9 days and the length of acute hospital stay was 19.1 ± 18.0 days. Of the 33 patients, six (18%) presented AKI as defined by a serum Cr >1.5, urinary output <0.5cc/kg/hr, or need for renal replacement therapy. One patient presented with AKI on arrival. The overall mortality in patients with SJS/TENS who subsequently developed AKI was 33%.

Discussion: SJS/TEN is an allergic mucocutaneous process with significant morbidity and mortality. In this study we report that 18% of patients developed AKI. The mortality rate after development of AKI was 33% indicating that the end-organ effects on the kidney are significant. Preventing or modulating the nephrotoxic effects of these disease processes have important clinical consequences.

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E. Diaz, N.A. Rodriguez*, J.O. Lee, C.C. Finnerty*, D. Herndon*. University of Texas Medical Branch/Shriners Hospitals for Children, Galveston, TX

Introduction: Acute kidney injury (AKI) complicates the outcomes of children admitted to the ICU. Previous studies in non-burned populations have demonstrated AKI as an independent risk factor of mortality. However, it is not known if AKI, defined by the pRIFLE criteria can be used to predict mortality in burned children. The purpose of this study was to evaluate the development of AKI (defined as a pRIFLE classification of risk, injury, or failure) during acute hospitalization and its effect on mortality.

Methods: We evaluated 2018 electronic medical records of patients with acute admissions to our burn unit from 1996 to 2011. Renal function was assessed by applying the pRIFLE criteria during acute hospitalization. Patients were < 19 years of age, %TBSA burned ranged from 0 to 99%, 66% males, 56% flame, and 20% inhalation injury. Dichotomous and categorical variables were compared using chi-square test. Risk factors of mortality were assessed with univariate and multivariate analysis.

Results: There were 453 (22.4%) children who developed AKI by pRIFLE criteria. Patients with AKI exhibited a higher mortality rate when compared to non-AKI group (P <0.01). 68% of the nonsurvivors presented AKI. Multiple logistic regression analyses demonstrated AKI as an independent variable of mortality (P < 0.01) after controlling for Age, Gender, TBSA, Third, Inhalation, and Ethnicity.

Conclusion: The pRIFLE criteria, although currently not recommended for medical decision making, have proven to be a significant factor for prediction of survival in pediatric burned patients. Further research is necessary to characterize risk factors both demographics and biomarkers that determine AKI in this patient population.

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R.H. Tullis, R. Lefkowitz, P. Duffin, R. Lucas. Aethlon Medical, San Diego, CA

We are developing, under a DARPA contract, an extracorporeal treatment for sepsis in wounded Warfighters to reduce mortality. Toward this aim, we are developing an affinity plasmapheresis system to capture substances implicated in the progression of sepsis, such as immunosuppressive exosomes (e.g. INOS-containing, platelet-derived, macrophage-derived), bacterial endotoxins and exotoxins, sepsis-associated viruses (e.g. CMV), complement components, digestive enzymes, bacterial DNA, and viral RNA. In our prior studies, pathogenic substances were removed from blood using lectins selective for highly mannosylated targets. The primary lectin we used was Galanthus nivalis agglutinin (GNA) covalently coupled to diatomaceous earth. To date, GNA was used for successful capture of many enveloped viruses including: HIV, HCV, Dengue, West Nile, orthopox viruses such as vaccinia (related to smallpox), ebola, and other threats of interest in the interdiction of bioweapons. We have demonstrated that this same lectin can capture immunosuppressive exosomes expressed in large amounts in many types of solid tumors. Since several studies have now implicated exosomes or microvesicles in the progression of sepsis, we are now testing the ability of affinity plasmapheresis to clear these materials from circulation as a potential treatment for sepsis. In addition, we are using specific antibodies to capture INOS-containing exosomes (obs KD ≈ 10-11 M), complement component C5 (obs KD = 1.0 × 10-10 M), and E. coli DNA (obs KD = 1.1 × 10-7). We have used the irreversible protease inhibitor α1- antitrypsin to capture pancreatic digestive enzymes trypsin, chymotrypsin, and elastase. We have also demonstrated maximal complement depletion using Cobra Venom Factor and successfully captured bacteria-infected macrophage-derived exosomes. For each of these we have determined affinity and kinetic constants using biolayer interferometry. In conclusion, we have demonstrated successful in vitro capture of numerous sepsis factors. We are continuing this work for endotoxins (LPS and LTA), free fatty acids, cytokines such as TNFα- and IL-6, paving the way toward an animal trial as a test of the utility of this novel treatment approach for sepsis and septic shock.

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A. Kholmukhamedov*1, C. Czerny2, J.J. Lemasters1. 1Medical University of South Carolina, Charleston, SC, 2J.W. Goethe University, Frankfurt am Main, Germany

Background: The underlying mechanism of liver injury following hemorrhagic shock/resuscitation (HS/R) remains unclear. Here, our Aim was to determine whether the mitochondrial permeability transition (MPT) is involved in HS/R-induced liver injury and whether minocycline, an MPT inhibitor, can decrease that injury.

Methods: Under anesthesia, C57BL6 mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer’s solution with and without minocycline. In some experiments, mice were pretreated (1h) with minocycline. Serum ALT, liver necrosis and apoptosis were assessed at 6h after HS/R. To investigate if onset of MPT takes place after HS/R, intravital multiphoton imaging of tetramethylrhodamine methylester (TMRM) and calcein was performed at 4h after HS/R.

Results: After resuscitation with vehicle, ALT increased to 1988 U/L. Minocycline decreased ALT to 857 U/L. Minocycline also decreased hepatic necrosis and apoptosis and improved survival after HS/R. In sham-operated mice, TMRM fluorescence was punctate in all hepatocytes, indicating normal mitochondrial polarization. Calcein fluorescence outlined mitochondria as dark voids. In contrast at 4h after HS/R, mitochondria did not take up TMRM in many hepatocytes, indicating mitochondrial depolarization, and dark voids of calcein fluorescence disappeared, indicating mitochondrial inner membrane permeabilization and onset of the MPT. Minocycline prevented these changes in most cells.

Conclusion: The MPT plays a major role in liver injury after HS/R. Minocycline blocks the onset of MPT after HS/R, protects against hepatic injury even when administered at the end of resuscitation. Minocycline, an MPT blocker, is a safe, clinically used, FDA-approved drug that might therefore be used to mitigate liver injury after HS/R.

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K. Yamakawa1, N. Matsumoto1, Y. Imamura2, 1, T. Muroya1, J. Nakagawa1, J. Shimazaki1, H. Hosotsubo1, H. Ogura1, Y. Kuwagata*1, T. Shimazu1. 1Osaka University Graduate School of Medicine, Osaka, Japan, 2Kyoto University Graduate School of Medicine, Kyoto, Japan

This study was performed to gain insights into novel therapeutic approaches for the treatment of heat-stroke. The central nervous system regulates peripheral immune responses via the vagus nerve, the primary neural component of the cholinergic anti-inflammatory pathway. Electrical vagus nerve stimulation (VNS) reportedly suppresses pro-inflammatory cytokine release in several models of inflammatory diseases. Here, we evaluated whether electrical VNS attenuates severe heat-stroke in rats. To determine the effect of VNS in severe heatstroke, anesthetized rats were subjected to heat stress (41.5°C for 30 min) with/without electrical VNS. Exposed left cervical vagus nerves in the experimental group were stimulated for 20 minutes after the heat-stroke model induction, with constant voltage (10 V, 2 ms, 5 Hz); sham-operated animals received no stimulation. Serum concentrations of cytokines were measured at 6 hours after heat stress. In another experiment, survival of each group was monitored for 7 days after the heat stress. We observed that electrical VNS significantly decreased serum tumor necrosis factor levels (VNS group, 4810 ± 1070 pg/mL vs. control group, 1740 ± 800 pg/mL; P < 0.05) and serum interleukin-6 levels (VNS group, 7.7 ± 1.6 pg/mL vs. control group, 16.1 ± 3.3 pg/mL; P < 0.05). Mortality was also significantly improved in the VNS group compared with those in the control group (P = 0.016). These data indicate that electrical VNS attenuates the cholinergic anti-inflammatory pathway in a rat model of heat-stroke and that this protective effect is associated with improved mortality. These findings could be of particular interest in the clinical setting.

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B. Relja, A. Zachgo, K. Wilhelm, K. Eldesoqi, D. Henrich, M. Lehnert, I. Marzi*. University Hospital of the Goethe-University Frankfurt am Main, Frankfurt am Main, Germany

Objective: Hypoxia causes an excessive inflammatory response resulting in organ injury. Ethanol (EtOH) intoxication is assumed to worsen H/R-induced pathophysiologic changes by exacerbated oxidative stress. Previously, we examined the role of acute EtOH gavage upon oxidative stress, inflammatory changes and hepatic injury after H/R in vivo. Here, we investigated the potential of ethyl pyruvate (EtP) as H2O2 scavenger to mimic effects of EtOH in vitro.

Methods: 14h before H/R, rats received EtOH or saline gavage. 2, 24 and 72h after H/R, hepatic injury, neutrophil infiltration, systemic inflammation (IL-6), oxidative/nitrosative stress were evaluated. Neutrophil adhesion to hepatocellular cell lines or to lung epithelial cells after incubation for 1, 24 or 72h with various doses of EtP (2.5-10mM), sodium pyruvate (NaP, 10mM) or EtOH (85-170mM) and subsequent stimulation with IL-1beta, TNF-alpha, IL-6 or PMA were evaluated in vitro.

Results: EtOH reduced significantly oxidative and nitrosative stress after H/R compared to controls (p<0.05). H/R-induced systemic IL-6 increase, neutrophil infiltration and hepatic injury were markedly reduced by EtOH in vivo. In vitro-treatment of hepatic and lung epithelial cells with EtOH resulted in substantial reduction in cytokine-induced adhesion of neutrophils to monolayers. Likewise, treatment with EtP reduced neutrophil adhesion to monolayers in a dose- and time-dependent fashion, whereas NaP treatment conferred no reduction.

Conclusions: EtOH attenuates H/R-induced oxidative stress thereby reducing cellular injury in vivo. Exposure of hepatocellular and lung epithelial cells to EtP inhibits the adhesiveness of neutrophils to monolayers similar to ethanol`s effects in vitro - indicating anti-inflammatory potential of EtP. (Supported by DFG LE 1346/2-1).

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S.R. Carter1, 2, C.S. Davis1, 2, S.E. Janus2, M.J. Mosier1, 2, J. Gibbs1, L. Ramirez2, R.L. Gamelli1, 2, E.J. Kovacs*2. 1Department of Surgery, Loyola University Medical Center, Maywood, IL, 2Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, IL

We previously reported an enhanced pulmonary inflammation with worse grades of inhalation injury and that a blunted pulmonary immune profile was associated with mortality. The present study aimed to investigate: a) the effects of inhalation injury on systemic inflammation, b) the balance of pro- and anti-inflammatory mediators in the blood early after burn and smoke inhalation.

Bronchoscopy was performed on 80 patients admitted to the burn intensive care unit for suspected smoke inhalation from 2007 to 2011; of these, 72 met inclusion criteria. Inhalation injury was graded based on Abbreviated Injury Score criteria, grade 0 being no visible injury and grade 4 corresponding to massive injury. Plasma was collected and analyzed for 28 immunomodulatory factors.

After adjusting for age and % TBSA, plasma levels of interleukin (IL)-1RA, IL-6, IL-8, G-CSF, and monocyte chemotactic protein (MCP) -1 were higher with worse inhalation injury severity (p <0.05). Plasma IL-1RA, IL-6, IL-8, IL-15, eotaxin, and MCP-1 were elevated in deceased patients compared to survivors (p<0.05), while IL-4 and IL-7 were lower (p<0.05). Plasma IL-1RA was associated with mortality after adjusting for age, % TBSA, and inhalation injury grade (OR 3.12, 95% CI 1.03-9.44), and correlated with % TBSA, injury grade, fluid resuscitation, and multiple organ failure.

The severity of inhalation injury affects systemic inflammation and thus should be treated in a graded manner. Several plasma mediators measured early after injury correlated with mortality. Of these, IL-1RA had the strongest correlation with injury severity and outcome. This may explain the blunted pulmonary immune response previously observed in non-survivors.

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J. Lee1, 2, E.J. Miller*1, 3. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2The Elmezzi Graduate School of Molecular Medicine, Manhasset, NY, 3Hofstra University School of Medicine, Manhasset, NY

Background: Systemic vascular dysfunction in sepsis is characterized by increasingly permeable vessels affecting circulation and leading to generalized tissue hypoxia. Ang-2, produced by endothelial cells and stored in Weibel-Palade bodies (WPB), can increase vascular permeability. Plasma concentration of Ang-2 increases in sepsis and correlates with disease severity and mortality. However, the mechanisms involved in Ang-2 related vascular dysfunction remain unclear. Since Gram-positive bacteria are a common cause of sepsis, we examined the responses of human pulmonary microvascular endothelial cells (HMPECs) to Gram-positive cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN).

Methods: HPMECs were treated with LTA (50-100 μg/ml) and PGN (167-333 μg/ml) for 30min-6hr in the presence or absence of TAT-NSF700 fusion peptide (10 μΜ) to inhibit WPB exocytosis. Ang-2 in culture medium was measured by ELISA, and Ang-2-WPB exocytosis assessed using immunofluorescent (IF) staining. HPMEC monolayer permeability was measured using FITC-dextran, with or without siRNA to deplete Tie-2 (an Ang-2 receptor).

Results: LTA-PGN stimulation increased Ang-2 levels dose-dependently (up to 9 fold, p= 0.003) in the culture medium within 30 min and was blocked by TAT-NSF700 pre-treatment. If staining showed aggregation and localization of Ang-2 in the cytoplasm suggesting ongoing WPB exocytosis in 30 min. LTA-PGN induced permeability increased in two phases, at 30min and 6h, the latter being more prominent. The permeability change was augmented by Tie-2 siRNA transfection.

Conclusion: The results suggest that LTA-PGN induced rapid Ang-2 secretion via WPB exocytosis and is associated with significant changes in permeability. This suggests a possible new target to reduce vascular permeability in Gram-positive sepsis.

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L. Khailova, P. Wischmeyer*, J. Dominguez*. University of Colorado Anschutz Medical Campus, Aurora, CO

Background: Sepsis is common in infants with a mortality rate of >30%. Overexpression of inflammatory cytokines in sepsis leads to organ dysfunction, including severe respiratory disorders. Probiotics ameliorate symptoms of various intestinal diseases but their role in septic peritonitis is unknown.

Objective: To evaluate the effects of Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum (BL) treatment on systemic and local inflammatory response in a weanling mouse model of septic peritonitis.

Methods: FVB/N weanling mice were subjected to cecal ligation and puncture or sham laparotomy and treated with or without LGG (1x109 CFU/ml, o.g.) or BL (1x107 CFU/ml, o.g.). At 24h, blood, colonic and lung tissue were collected and TNF-α, IL-1β, IL-6, TLR-2, TLR-4 and MyD88 analyzed by RT-PCR and ELISA.

Results: Of the cytokines studied, only IL-6 was increased systematically in septic mice (362.8 pg/ml ± 35.2 (septic) vs 19.8 pg/ml ± 8.7 (sham); P<0.05) and LGG/BL treatment resulted in significantly reduced IL-6 serum levels (206.1 pg/ml ± 24.7 (LGG); 189.4 pg/ml ± 38.9 (BL); P<0.05). In the colon, septic mice had increased mRNA levels of TNF-α, IL-1β, TLR-2, TLR-4 and MyD88 (P<0.05) and LGG/BL treatment significantly decreased these levels (P<0.05). In the lungs, septic mice had increased mRNA levels of TNF-α, IL-1β, IL-6, TLR-2 and MyD88 (P<0.05) which were normalized to sham levels in the LGG/BL treated mice. Protein levels of TNF-α and IL-6 were elevated in the lungs of septic mice (P<0.05) but were similar to shams in septic mice treated with LGG/BL.

Conclusions: Probiotics confer a survival advantage in septic peritonitis which is associated with attenuated systemic and local cytokine expression with possible involvement of TLR pathway.

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J. Kaplan*, M. Nowell, P. Lahni, B. Zingarelli*. Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

Obesity is associated with an enhanced inflammatory response. We have previously demonstrated that the thiazolidinedione, pioglitazone (PIO), exerts anti-inflammatory effects in polymicrobial sepsis in normal chow-fed mice. We hypothesize that PIO will also reduce inflammation in high fat-fed mice subjected to polymicrobial sepsis. Six-wk old C57BL/6 mice were randomized to a high fat diet (HFD) (60% kcal fat) or a control diet (CD) (16% kcal fat). Following 6 wks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP) with 22g double puncture technique. Mice received oral gavage of vehicle (0.5% methylcellulose) or PIO (20mg/kg) at 1 and 6h after CLP and were sacrificed at various timepoints. Plasma and tissue were obtained for analysis. A p value of ≤0.05 was considered significant. Following 6wks of feeding, HFD mice had greater body weights and higher levels of the adipokines leptin and resistin compared to CD mice. Myeloperoxidase (MPO) activity, an indicator of neutrophil infiltration, increased in the lung in vehicle-treated CD and HFD mice 18h after CLP. Compared to vehicle, PIO reduced MPO in CD (366±45 vs.238±40 U/100 mg tissue, p<0.05 by t-test) but not in HFD mice. Plasma levels of the adipokine leptin were increased at 6h after CLP in CD mice compared with prior to CLP (1,290 vs. 528 pg/ml, p<0.05 by rank sum test). However, HFD mice had significantly higher plasma leptin levels at 6 and 18h after CLP when compared with CD mice. PIO reduced plasma leptin levels in HFD mice (8,347 vs. 4,0953 pg/ml, p<0.05 by t-test) but not in CD mice at 18h after CLP. PIO also reduced plasma resistin levels in HFD mice but not in CD mice. The efficacy of treatment with pioglitazone is affected by high fat feeding. Supported by NIH K08GM093135 and R01GM067202.

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E.R. Lusczek, D.R. Lexcen, N.E. Witowski, K.E. Mulier, G.J. Beilman*. University of Minnesota, Minneapolis, MN

Introduction: The disruption in oxygen delivery induced by ischemia and reperfusion as a result of trauma and hemorrhagic shock has profound consequences for cellular metabolism and the maintenance of homeostasis. We studied the pathophysiologic state associated with traumatic injury and hemorrhagic shock using a scale-invariant metabolic network and a partial least squares discriminant analysis (PLS-DA) model. These methods were used to assess functional relationships between metabolites (network) and observable relationships to experimental timecourse and phase of shock (PLS-DA).

Methods: Urinary metabolic profiles were constructed from NMR spectra of urine samples collected at set timepoints in a porcine model of hemorrhagic shock that included a pulmonary contusion, a liver crush injury, and a 35% controlled bleed. The network was constructed from these metabolic profiles using the WCGNA (Weighted Correlation Gene Network Analysis) package for R software. A PLS-DA model that discriminates by experimental timepoint was constructed with SIMCA-P (v. 12.0).

Results: Comparisons of the network and PLS-DA model revealed complementary information. First, metabolic markers of ischemia/reperfusion injury and evidence of cell death due to hemorrhage were associated with early resuscitation. Second, metabolites suggestive of protein catabolism, traumatic injury, and osmotic stress were associated with late resuscitation.

Conclusions: By combining analysis methods that identify functional relationships between metabolites and observable relationships of metabolites with phase of shock, we identify specific metabolic consequences of trauma, shock and recovery. We also confirm the potential usefulness of these tools for future analysis of metabolic networks in other tissues and compartments.

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J.L. Messina*, S. Jiang. University of Alabama at Birmingham, Birmingham, AL

Skeletal muscle becomes insulin resistant following injuries or critical illnesses. However, molecular mechanisms underlying the injury-induced insulin resistance in skeletal muscle remain unclear. In the present studies, roles of inflammatory pathways were investigated, including macrophages and tumor necrosis factor α (TNFα), in the development of acute insulin resistance following trauma and hemorrhage in skeletal muscle. Inhibition of inhibitory κB kinases (IKKs) or c-Jun N-terminal kinase (JNK) by systemic administration of adenovirus vectors expressing dominant negative IKKα, β, or JNK1 resulted in significant improvement in insulin signaling in skeletal muscle after trauma and hemorrhage for 60 minutes. Since the main target cells for adenovirus-mediated gene delivery are hepatocytes and macrophages, the observed improvement in insulin signaling in muscle are likely due to inhibition of IKKs or JNK1 either in hepatocytes or in macrophages. Depletion of macrophages by Gadolinium Chloride largely improved insulin signaling in skeletal muscle, while expression of dominant negative IKKs or JNK1 in hepatocytes on macrophage-depleted rats didn’t result in additional improvement in insulin signaling in skeletal muscle, suggesting an important role for macrophages, while a significant role for hepatocytes is unlikely. In addition, inhibition of TNFα by adenovirus mediated-expression of TNF inhibitor significantly increased skeletal muscle insulin signaling after trauma and hemorrhage. In summary, these findings suggest an important role for TNFα, likely derived from macrophages, in the acute development of hemorrhage-induced insulin resistance in skeletal muscle.

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J.A. Weinberg1, P.A. MacLennan2, M.J. Vandromme-Cusick2, L.J. Magnotti1, J.D. Kerby*2, J.M. Angotti1, C.A. Garrett1, L.W. Rue*2, M.A. Croce*1, T.C. Fabian*1, S.R. Barnum2, R.P. Patel2. 1University of Tennessee Health Science Center, Memphis, TN, 2University of Alabama at Birmingham, Birmingham, AL

Background: Previous studies identified that transfusion of older allogeneic red blood cells (RBCs) is associated with morbidity and mortality in trauma patients. The mechanisms for this phenomenon, however, remain poorly defined. It is our hypothesis that transfusion of older RBCs impedes microvascular perfusion. We evaluated sublingual microcirculation in trauma patients to determine if, in fact, transfusion of older RBCs was associated with a decrease in capillary density.

Methods: Sublingual microcirculation was imaged at bedside with sidestream dark field illumination microscope before and after transfusion of one RBC unit in stable trauma patients in the ICU. Perfused capillary vascular density (PCD) pre- and post-transfusion was determined from imaging sequences, and change in PCD (ΔPCD) following transfusion was evaluated with respect to RBC storage age.

Results: Sublingual microcirculation was observed in 62 patients. 71% were male and mean age was 48 years. Pre-transfusion mean hemoglobin was 7.5 g/dL, base excess was 3.2 mmol/L, and mean arterial pressure was 88 mmHg. Age of RBC unit ranged from 7 to 42 days (mean 29). Following transfusion, ΔPCD ranged from -6.2 to 4.5 n/mm and was found to correlate negatively with RBC storage age (Spearman correlation = - 0.26602, p = 0.0366). Mean RBC age was significantly higher among patients whose PCD decreased post-transfusion compared to those whose PCD increased (31.5 vs. 27.2 days, p = 0.048).

Conclusions: Sublingual capillary perfusion was found to be negatively affected by transfusion of relatively older RBCs. Trauma patients are often transfused in an effort to augment tissue oxygen delivery, but this therapy may in fact be inhibitory to tissue perfusion if RBC units are older. Reservation of relatively fresh RBC units for injured patients may be advisable.

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A.E. Altshuler, A.H. Penn, J.A. Yang, G. Kim, G.W. Schmid-Schonbein*. University of California, San Diego, La Jolla, CA

Objective: During hemorrhagic shock (HS), the reduced perfusion to the intestine and pancreas results in increased intestinal mucosal permeability, pancreatitis, and potential leakage of serine proteases (i.e. trypsin- a potent matrix metalloproteinase [MMP] activator) into the circulation. These proteases may degrade extracellular domains of vascular endothelial (VE)-cadherin, vascular endothelial growth receptor 2 and 3 (VEGFR-2 and VEGFR-3) in the lung. We hypothesize that after HS, both trypsin and MMP-9 enter the circulation and appear in the lung (an early damaged organ in shock) and degrade extracellular receptor components.

Methods: Male Wistar rats were grouped into No-HS or HS (N=5). Mean arterial blood pressure of HS animals was reduced to 35 mmHg (2 hr) followed by resuscitation of shed blood (2 hr). Post-HS plasma and lungs were collected. Protease activity in plasma was measured by plate zymography (trypsin-like and MMP-9 specific substrates for plasma) or by gelatin gel zymography (also for lung). Pancreatic trypsin, MMP-9, VE-Cadherin, VEGFR-2, and VEGFR-3 levels were identified by immunoblot.

Results: Trypsin-like and MMP-9 activities were elevated in plasma after HS (p<0.05). Post-HS plasma and lungs had elevated levels of pancreatic trypsin and MMP-9. There was significant reduction of VE-cadherin, VEGFR-2, and VEGFR-3 in the lung after HS (p<0.05).

Conclusion: In HS, trypsin is transported into the system circulation and may contribute to MMP-9 activation. Extracellular domains of important membrane receptors are decreased after shock, possibly due to protease activity, contributing to cell dysfunction and organ failure. Supported by GM85072.

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W. Martini*, L.H. Blackbourne, M.A. Dubick*. US Army Institute of Surgical Research, Ft Sam Houston, TX

Introduction: Military first responders seem to favor normal saline (NS) resuscitation over Lactated Ringer’s (LR) for combat casualties, while recent civilian studies favor LR. As this controversy remains to be resolved, this study compared the effects of LR and NS on hemodynamics and coagulation after severe hemorrhagic shock in pigs.

Methods: 20 anesthetized pigs were randomized into control (C, n=6), LR (n=7), and NS (n=7) groups. Hemorrhage of 60% total blood volume was induced in LR and NS groups by removing blood from the left femoral artery using a computer-controlled pump. Afterwards, the pigs were resuscitated with either LR of 3 times the bled volume, or NS to reach the same MAP as in LR group. Pigs in C were not bled or resuscitated. Hemodynamics were measured hourly and blood samples were taken at baseline, 15min and 6h after hemorrhage and resuscitation (H-R) to measure thrombin generation and coagulation function using thrombelastography®.

Results: Baseline (BL) values were similar among all 3 groups and no changes were observed in C. MAP was decreased by hemorrhage but returned to BL within 1h after resuscitation with LR (4374 ±363 ml) or NS (6090±131 ml, p<0.05). Bass excess (BE) was decreased by H-R at 15min and recovered to BL by LR at 6h but not by NS. Stroke volume returned to BL by NS but was decreased by LR from BL of 41±3 to 25±2 ml/beat (p<0.05) for 6h. Clot strength was decreased by H-R but returned to BL in 6h; clotting speed was increased for 6h by LR or NS. Similar changes were observed between LR and NS in platelets, fibrinogen, thrombin generation, and coagulation function.

Conclusions: After severe hemorrhage, NS resuscitation requires larger volume to return MAP and compromised BE. However, there were no differences in coagulation profiles between LR or NS resuscitation.

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S.F. de Stoppelaar, M.C. Schaap, R. Nieuwland, C. van ’t Veer, T. van der Poll*. Academic Medical Center, Amsterdam, Netherlands

Background: S. pneumoniae strains vary considerably in the ability to cause invasive disease (sepsis) in humans, and this is associated with the capsular serotype. The S. pneumoniae capsule inhibits complement- and phagocyte-mediated immunity. Patients suffering from sepsis often develop thrombocytopenia, which is associated with higher mortality. Besides the obvious role for platelets in hemostasis, platelets exert a role in the immune continuum through release of regulatory proteins, neutrophil complex formation and secretion of antimicrobial peptides.

Objective: To determine the direct stimulatory role of S. pneumoniae on platelets.

Methods: Human platelet aggregation was measured in citrate anticoagulated platelet rich plasma using light transmission aggregometry. Platelet P-selectin expression and platelet-neutrophil complexes were measured after whole blood incubations using flow cytometry.

Results: S. pneumoniae induced platelet aggregation in a strain dependent manner. Mutant S. pneumoniae strains missing the capsule were most potent. Aggregation was not induced by strains known for a thick capsule, LTA or LPS. Aggregation was inhibited by the GPIIb/IIIa antagonist abciximab, but not by hirudin or anti-TLR2. Whole blood incubation with all S. pneumoniae serotypes resulted in P-selectin expression on platelets and platelet-granulocyte and platelet-monocyte complex formation. This was not inhibited by abciximab or anti-TLR2. Different serotypes of S. pneumoniae induced these effects to a different extent.

Conclusions: S. pneumoniae can cause direct, TLR2 independent, platelet activation through a mechanism that is inhibited by a thick capsule. This may contribute to thrombocytopenia observed in S. pneumoniae infected sepsis patients and to the differences in virulence observed between different strains.

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V. Brod, H. Bitterman*. Carmel Hospital, Faculty of Medicine, Technion, Haifa, Israel

We studied the isolated effect of hyperoxic ventilation on long-term survival in hemorrhagic shock.

Methods: Hemorrhagic shock was induced in male Sprague-Dawley rats (250-330g) by withdrawing 35% of the total blood volume within 90 minutes. A single exposure to hyperoxic ventilation for 6 or 24 hours was started 20 minutes after the end of bleeding. Survival was followed for 7 days. Seven groups were studied: no treatment, treatment with oxygen 40%, 60%, or 100% for 6 hours, or treatment with oxygen 40%, 60%, or 100% for 24 hours.

Results: The experimental protocol resulted in hypotension, 10-12% decrease in the hematocrit, marked increase in arterial blood lactate (P<0.01), and a markedly decreased 7 days survival (38%). Inhalation of oxygen resulted in a proportional increase in arterial blood PO2, decrease in blood lactate (P<0.05 for the 60% and 100% Oxygen groups), and a significant, dose dependent, increase in long-term (7 days) survival reaching 92% in the 100%/24h oxygen group (P<0.01).

Conclusion: Early use of isolated short-term resuscitation with hyperoxia caused a significant, dose related, increase in long-term survival in a rat model of controlled hemorrhagic shock.

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T. Chin1, E.E. Moore*2, J.N. Harr1, J.L. Johnson*2, A. Banerjee1, A. Sauaia1. 1University of Colorado, Denver, CO, 2Denver Health Medical Center, Denver, CO

Background: Recent studies suggest that platelets play a critical role in postinjury hyperinflammation. Our previous work in a single institution dataset identified an association between thrombocytopenia and multiple organ failure (MOF), a major cause of morbidity and mortality after severe trauma. Thus, we queried the multi-institutional Inflammation and Host Response to Injury (Glue Grant) database to further explore the association between platelet count (PC) and adverse outcomes.

Methods: Prospectively studied 1420 high-risk, blunt trauma patients (age>16, base deficit>6mEq/L or systolic blood pressure<90mmHg, and ≥1 blood transfusion/12hrs) admitted from 2001-2011 with ICU days >4, were included. Logistic regression was used to evaluate the association of early changes in PC (days 2 to 4) with mortality, MOF, and major infections, adjusted for age, Injury Severity Score (ISS) and red blood cell transfusion/12 hours (RBC).

Results: Mean age was 43±19, median ISS was 34 (IQR:25-43) and 67% were male. Median RBC was 5.8 (IQR:3-10.5). Mortality, MOF, and infection rates were 9.8%, 18.3%, and 56.8%, respectively. PC change from day 2 to 4 was predictive of mortality (p<0.0001), MOF (p<0.0001), and infection (p=0.0017), adjusted for other risk factors. A 30% decrease in PC from day 2 to 4 carried adjusted odds ratios (OR) of 1.14 (95%CI:1.05-1.2) for infection, 1.5 (95%CI:1.3-1.7) for MOF, and 1.7 (95%CI:1.4-2.0) for death. Greater decreases in platelets result in a higher OR for adverse outcomes.

Conclusions: Failure to increase platelet count early postinjury predicts mortality, MOF, and infection in a multi-institutional cohort and is a biomarker for high risk patients. Further mechanistic research is necessary to define the role of platelets in the development of adverse postinjury outcomes.

Adjusted odds ratios for death by percent decrease in platelet count from day 2 to day 4

Adjusted odds ratios for death by percent decrease in platelet count from day 2 to day 4

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A. Matsuda*1, 2, W. Yang1, 2, A. Jacob1, 2, M. Aziz1, 2, P. Wang*1, 2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY

Rationale: Acute lung injury (ALI) is a leading cause of high mortality after intestinal ischemia/reperfusion (I/R) caused by surgery, abdominal injuries, or septic shock. The I/R intestines release proinflammatory mediators that trigger inflammatory disorders and cell death in the lungs and cause ALI. FK866, an inhibitor of visfatin (an adipokine), possesses an anti-inflammatory activity. We hypothesized that FK866 has therapeutic effects on ALI induced by intestinal I/R.

Methods: Male C57BL/6 mice were subjected to intestinal I/R (by placing a microvascular clip across superior mesenteric artery for 90 min, followed by reperfusion) or sham operation. Right after ischemia, the mice were treated with DMSO (vehicle) or FK866 at 10 mg/kg BW i.p. At 4 h after reperfusion, blood and tissues were collected for various measurements. The survival rate was assessed at 24 h after reperfusion.

Results: Visfatin levels in the plasma and lungs of intestinal I/R mice increased by 1.9- and 1.8-fold, respectively. FK866 significantly inhibited the elevation of plasma proinflammatory cytokines (TNF-α and IL-6), organ damage markers (ALT and creatinine), and number of lung apoptotic cells induced by intestinal I/R as listed in the Table. FK866 also improved the injury scores in the intestines and lungs, judged by histology. The number of cells with positive nuclear NF-κB staining in the lungs was reduced in FK886-treated mice, accompanied by reduction of IκB degradation. Finally, FK866 increased a survival rate from 14.3% to 41.7%, compared to the vehicle group (p < 0.05, n=13/group).

Conclusions: FK866 can potentially serve as a therapeutic agent to attenuate the ALI after intestinal I/R by modulating NF-κB pathway and inhibiting proinflammatory cytokine release.

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H.K. de Jong1, 2, G.C. Koh3, 4, A. Achouiti1, F. Stephan5, S. Zeerleder5, N.P. Day4, 6, S.J. Peacock3, 4, T. van der Poll*1, J. Wiersinga1. 1Center for Infection and Immunity Amsterdam (CINIMA), Center of Experimental and Molecular Medicine (CEMM) and Division of Infectious Diseases, Academic Medical Center, Amsterdam, Netherlands, 2Chittagong Medical College Hospital, Chittagong, Bangladesh, 3Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom, 4Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 5Department of Immunopathology, Sanquin and Department of Haematology, Academic Medical Center, Amsterdam, Netherlands, 6Nuffield Department of Clinical Medicine, Churchill Hospital, University of Oxford, Oxford, United Kingdom

Introduction: NETs have recently emerged as a central part of antimicrobial innate immune defense. Neutrophils release extracellular fibers that ensnare bacteria, degrade their virulence factors and, ultimately, kill their target. Major components of NETs include extracellular DNA-histone complexes (nucleosomes) and neutrophil elastase. We studied the role of NETs in B. pseudomallei infection (melioidosis), which is an important cause of sepsis in Southeast-Asia.

Methods: We assessed circulating nucleosomes, neutrophil-elastase complexes (HNE-A1AT) and Factor VII-activating protease activity (FSAP α2AP, marker for cell death) levels in 43 septic melioidosis patients and 83 controls. We also stimulated isolated human neutrophils with B. pseudomallei, visualizing NET release by neutrophils with electron- and fluorescence microscopy and in order to quantify NETosis by measuring levels of nucleosomes, HNE-A1AT and DNAse-cleaved NET components.

Results: Nucleosome, HNE-A1AT and FSAP α2AP levels were elevated in melioidosis patients, and these levels gradually normalized in those patients who did survive. Nucleosome and HNE-A1AT concentrations were strongly correlated (r=0.84, p<0.001), which suggests that neutrophils are an important source of circulating cell-free DNA. In line with the patient data, human neutrophils stimulated with B. pseudomallei were able to induce NET formation and showed increased levels of elastase and DNAse-cleaved NET components in the supernatant.

Conclusion: B. pseudomallei induces NET formation by human neutrophils which is reflected by strongly increased levels of NET related-components in patients with melioidosis. Our data point towards an important role for NETosis in the host defense against B. pseudomallei induced sepsis.

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L. Zhang, D. Lu, X. Zhu, D. Maass*, D. Carlson*, A. Idris*, J. Wigginton*, H. Yin*. UT Southwestern, Dallas, TX

Severe thermal burn is frequently associated with clinically significant oxidative stress and multiorgan dysfunction. A single dose of 17β-estradiol (E2) decreases burn-induced inflammatory cytokine release and promotes Akt activation in rats. E2 reverses oxidant damage in vitro and increases production of PIP2, an essential actin regulator and the obligate precursor to PIP3 in the E2 activated Akt signaling cascade. Phosphatidylinositol 4 phosphate 5 kinase β (PIP5Kβ) synthesizes PIP2; in vitro studies have shown that it stabilizes the cortical actin cytoskeleton and it is inhibited by oxidants.

Objective: To determine if PIP5Kβ regulates burn-induced inflammatory cytokine responses and if it contributes to E2 protection against systemic burn inflammation.

Methods: PIP5Kβ knockout (KO) and age-matched wildtype (WT) littermates were subjected to 40% total body surface burn, subsequently injected with placebo or E2 (0.5 mg/kg) at 15 min., and sacrificed at 24 h.

Results: Burned PIP5Kβ KO mice had a 1.8-fold increase in serum IL6 (see Fig.), and similarly large increases in lung and heart IL6 levels (not shown) compared with WT burned mice. TNFα level also increased but to a much smaller extent. Although E2 substantially decreased IL6 and TNFα in burned WT animals, and suppressed the abnormal IL6 surge in burned KO mice, it was less effective in rescuing the TNFα response in burned KO mice.

Conclusions: PIP5Kβ selectively protects against IL6 release in burn animals most likely by stabilizing the cortical actin barrier. The unexpected differences in IL6 and TNFα responses in PIP5Kβ KO mice suggest that these inflammatory cytokines have differential dependence on the actin cytoskeleton, and that E2 protects by mobilizing PIP5Kβ-dependent and -independent pathways. Supported by NIH P50 GM02168 Burn Center Grant.

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B. Sadowitz1, K. Cooper3, S. Roy1, B. Emr1, M. Kollisch-Singule1, L. Gatto3, B. Kubiak1, S. Albert1, J. Satalin1, K. Snyder1, Y. Vodovotz*2, G.F. Nieman*1. 1Upstate Medical University, Syracuse, NY, 2University of Pittsburgh, Pittsburgh, PA, 3SUNY Cortland, Cortland, NY

Introduction: Acute kidney injury (AKI) and acute renal failure (ARF) affect up to 20% of all ICU patients. Sepsis and shock remain the main causes of AKI/ARF in ICU patients. We hypothesized a porcine model of intestinal ischemia/reperfusion (I/R) and peritoneal sepsis (PS) would more accurately recreate the renal pathophysiology and histopathology seen in human sepsis compared to an endotoxemia model.

Methods: Two groups (n=4 each) of female Yorkshire pigs were anesthetized, mechanically ventilated, and instrumented for pulmonary and hemodynamic monitoring. The LPS group was given 100μg/kg of IV endotoxin and studied for 6 h. The PS+I/R group had the superior mesenteric artery clamped for 30 min, then a fecal clot was placed in the peritoneal cavity and studied over 48 h. Clinical markers of renal function, including BUN, creatinine, and urine output, were recorded and renal histopathology was assessed quantitatively.

Results: The LPS and PS+I/R groups both demonstrated pathophysiologic and histopathologic renal injury compared to naïve controls. However, the PS+I/R group more closely mimicked human histopathology as it displayed significantly more peritubular edema and lymphatic vessel dilation compared to the LPS group (see Table and Figure).

Conclusions: The PS+I/R model achieves comparable renal pathophysiology and histopathology to that seen in human sepsis. Thus, compared to endotoxin treatment, the PS+I/R model represents a superior method for elucidating the pathogenesis and treatment of sepsis-induced AKI/ARF in humans.

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Representative photomicrographs of kidney cortex from naive, endotoxin, and septic shock pigs

Representative photomicrographs of kidney cortex from naive, endotoxin, and septic shock pigs

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C. Wu1, S. Chen2, Y. Lin3, C. Shih4, C. Tsao5, W. Liaw6. 1National Defense Medical Center - Pharmacology, Taipei, Taiwan, 2Kang-Ning Junior College of Medical Care and Management - Nursing, Taipei, Taiwan, 3National Defense Medical Center - Physiology, Taipei, Taiwan, 4National Defense Medical Center - Medical Sciences, Taipei, Taiwan, 5Taipei Veterans General Hospital - Anesthesiology, Taipei, Taiwan, 6National Defense Medical Center - Anesthesiology, Taipei, Taiwan

Background: Sepsis and its sequelae, multiple organ dysfunction syndrome (MODS), are major contributors of mortality in critical ill. Global tissue hypoxia results in an imbalance between systemic oxygen delivery and demand, and is a key development preceding MODS anddeath. Hydralazine preferentially dilates the arterioles and increases renal blood flow. In addition, hydralazine per se has powerful free radical scavenger properties. Therefore, we evaluated the effect of low dose hydralazine on peritonitis-induced sepsis with MODS in Wistar rats.

Methods: Intraperitoneal sepsis was induced by a cecal ligation and puncture (CLP) surgery. Animals were divided into four groups: (1) sham operation (SOP), (2) SOP + hydralazine (3 mg/kg i.v. at 3 hr after surgery), (3) CLP, and (4) CLP + hydralazine (3 mg/kg i.v. at 3 hr after CLP). The changes of hemodynamics, blood glucose, blood gas, rectal temperature, lactate dehydrogenase, hepatic and renal functions were monitored over 18 hours. In addition, plasma nitric oxide (NO), interleukin-6 (IL-6), organs superoxide (O2−) production were also measured. In addition, lung, liver, kidney and colon were harvested at 18 hr after surgery to perform histo-pathological studies. The survival rate in each group was also calculated.

Results & Conclusion: Our results demonstrated that hydralazine attenuated hypotension, vascular hyporeactivity, hypoglycemia, and hepatic and renal dysfunction in peritonitis-induced sepsis rats. We suggest that beneficial effects of hydralazine on the circulatory failure and MODS caused by peritonitis-induced sepsis is attributed to amelioration of organ blood flow and reduction of plasma NO and IL-6 as well as organ O2− levels, and decreasing liver, kidney, colon PMN infiltration, thereby reducing the mortality rate in CLP-treated animals.

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L.E. Sousse*1, Y. Yamamoto1, P. Enkhbaatar*1, E.R. Kraft1, D.J. Deyo1, C.L. Wright2, A. Taylor2, M.G. Traber2, R. Cox*1, H.K. Hawkins*1, S.W. Rehberg1, L.D. Traber*1, D.N. Herndon*3, D.L. Traber*1. 1University of Texas Medical Branch, Galveston, TX, 2Oregon State University, Corvallis, OR, 3Shriners Hospitals for Children, Galveston, TX

Many burn victims suffer from smoke inhalation injury (n = 23,000) in the US annually. We have reported that airway nebulization of gamma-tocopherol (g-T) had a beneficial effect in the ovine model of acute lung injury.

Objective: We hypothesize that burn and smoke inhalation injury is induced by reactive oxygen species (ROS) that activates the arginase pathway, leading to increased collagen deposition and decreased pulmonary function. The nebulization of g-T will scavenge ROS and improve long-term pulmonary function following injury after 3 wks.

Methods: Ewes were randomly divided into the following groups (n=8): uninjured, injured, and injured with g-T treatment for 48 hrs (950 mg/g g-T). The injured animals were subjected to a 20% total body surface area, 3° burn and 36 breaths of cotton smoke under deep anesthesia. The eschar was removed at 24h and replaced with autographs, and the sheep were sacrificed after 3 wks.

Results: The expression of dimethylarginine dimethylaminohydrolase-2, which degrades asymmetrical dimethylarginine (ADMA), a nitric oxide synthase inhibitor, was significantly higher with g-T then with injured, untreated sheep (p<0.05) while ADMA decreases. Arginase activity (p<0.05), ornithine aminotransferase, which is a collagen precursor, and collagen deposition (p<0.05) significantly decrease with g-T compared to injured animals without g-T. The decreases in arginase and collagen with g-T are associated with significantly increased DLCO (p<0.05), decreased lung wet-to-dry ratio (p<0.05), and increased wound healing (p<0.05).

Conclusions: Smoke-induced chronic pulmonary dysfunction is mediated through the ROS/ADMA/arginase pathway. ROS scavengers such as g-T may be a potential alternative for management of burn patients with inhalation injury. Support: SHC No. 8450, NIH GM066312, T32-GM8256.

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F.B. Sotelo1, M.S. Della Casa2, A.M. Liberatore1, P.B. Clissa2, I.H. Koh1. 1Federal University of São Paulo, Paulista School of Medicine. UNIFESP-EPM, São Paulo, Brazil, 2Butantan Institute, São Paulo, Brazil

Microcirculatory dysfunction occurs in severe sepsis from its early phase, leading to tissue malperfusion, cellular hypoxia, organ failure and death. Platelets derived inflammatory role contributes to this process. In this study we examined antiplatelet-GPIIbIIIa, a recombinant RGD (Arg-Gly-Asp) disintegrin gluthatione conjugated from the Bothrops Insularis viper venom (Insularin) effect on organ perfusion and mortality in rats with severe sepsis.

Methods: Adult male Wistar rats were distributed in the following groups: Sepsis (1ml of 109CFU/ml E.coli R6/100g body weight, i.v.); Sham (saline 0,9%, i.v.); Insularin (365ug Insularin/kg body weight, IC50 by aggregometry, 30 minutes prior experiments); Insularin+Sepsis (as described). Organ (liver, kidney, jejunum and ileum) perfusion was monitored by laser-Doppler (0, 30 and 90 min.) and mortality was followed per 30 days.

Results: Insularin reduced 75% sepsis’ mortality to 30%. No death or hemorrhage was seen at Insularin or saline groups. Besides, Insularin minimized organ’s perfusion fall due to the sepsis and this effect was organ-specific. The small bowel compartments and liver were the mostly protected sites (perfusion levels were similar to the controls) and the kidneys were the less protected. These results suggested that by preventing splanchnic microcirculation dysfunction in the early phase of sepsis, Insularin could have minimized the gut associated lymphoid tissue activation and bacterial translocation, both related to the systemic inflammatory response amplification in sepsis. In addition, the Insularin antiplatelet aggregation factor might have minimized the platelet dependent inflammation cascade in sepsis.

Conclusion: Insularin dependent antiplatelet activation has therapeutic potentials in sepsis.

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R. Moitra, D. Remick*. Boston University School of Medicine, Boston, MA

Aim: This study tested the mechanism of bacterial killing by plasma factors present before the onset of CLP-induced sepsis in mice. A Plasma Enhanced Killing (PEK) assay was performed to evaluate the bacterial killing capacity of naïve plasma.

Methods: The PEK assay is an in vitro bacterial killing assay involving the opsonization of cecal bacteria with naïve plasma. Elicited macrophages were added to the opsonized bacteria, incubated to facilitate killing, samples plated and incubated overnight. Another assay did not include the macrophages in the PEK assay. The degree of bacterial killing was calculated as PEK = [1/Log (N)] X 100, where N = number of surviving bacteria. A higher PEK means improved bacterial killing and a high PEK (HPEK, >16) predicts better survival after the onset of sepsis. The experimental groups for both HPEK and Low PEK (LPEK) plasma involved removing the plasma after opsonization, and addition of IgG purified from LPEK to the opsonized bacteria.

Results: Addition of macrophages in the PEK assay caused no difference in bacterial killing by HPEK plasma. However, presence of macrophages caused a significant inhibition in bacterial killing by the LPEK plasma. Removal of plasma post opsonization improved killing for both HPEK and LPEK plasma, irrespective of the presence of macrophages (Table 1). When purified IgG from LPEK were added to naïve HPEK plasma, the killing capacity was inhibited. However, in the absence of macrophages, purified IgG from LPEK when added to bacteria opsonized with HPEK did not alter the PEK (Figure 1). The inhibitory capacity of LPEK IgG was only present when macrophages were included in the assay.

Conclusions: The inhibition in bacterial killing by LPEK plasma is due to an inhibitory IgG which functions by blocking macrophage killing of bacteria.

Table 1

Table 1

Figure 1

Figure 1

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S. Das1, S.V. Cherian1, E. UlHaq1, S. Roy*1, B. Sadowitz*1, J. Satalin1, K.P. Snyder1, L.A. Gatto*2, 1, G.F. Nieman*1. 1SUNY Upstate Medical University, Syracuse, NY, 2SUNY, Cortland, NY

Introduction: Our group is currently developing a mechanically ventilated rat model of hemorrhage-induced acute respiratory distress syndrome (ARDS). A critical component of this protocol was whether ketamine-xylazine or pentobarbital sodium anesthesia would be superior for use in our model.

Hypothesis: Each of these anesthesia types has potential drawbacks: Ketamine is a known anti-inflammatory drug, which might mask hemorrhagic shock pathogenesis and pentobarbital can potentially cause excessive cardiac depression and hence early mortality.

Methods: Rats were either anesthetized with intraperitoneal ketamine (90mg/kg) and xylazine (10mg/kg; n=4) or with intra peritoneal pentobarbital (50 mg/kg; n=3) and surgically prepared for hemodynamic monitoring. All rats were ventilated using ARDS-net low tidal volume protocol for 6 hours or until ventilation became impossible due to airway flooding with edema.

Results: All rats in the ketamine-xylazine group developed fulminant pulmonary edema during the 6 hr ventilation period (Fig 1 B) whereas the pentobarbital group had normal lungs (Fig 1 A) and survived the entire 6 hour experiment.

Conclusion: Unexpectedly ketamine-xylazine anesthesia did not reduce pulmonary inflammation but actually caused pulmonary edema. Pentobarbital caused no measurable lung injury and early mortality secondary to cardiac depression did not occur, thus pentobarbital is an excellent anesthesia choice for our animal model. We speculate that ketamine-xylazine-induced pulmonary edema maybe caused by: increasing permeability of pulmonary vasculature (Amouzadeh et al, Toxicol Appl Pharmacol. 1991), a significant increase in pulmonary vascular pressure, or blockage of amiloride sensitive epithelial Na+ channels (ENaC) impairing alveolar fluid clearance (Cui et al J Biomed Biotechnol. 2011).

Fig. 1

Fig. 1

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T. Christopher*, H. Su, W.B. Lau, X.L. Ma. Thomas Jefferson University, Philadelphia, PA

The mechanisms by which diabetes exacerbates myocardial ischemic (MI) injury remain incompletely understood. C1q/TNF-related proteins (CTRPs) are newly identified adipocytokines sharing structural similarity to adiponectin (APN), a potent cardioprotective molecule deficient in Type 2 Diabetes (T2D). Of the CTRP family, CTRP9 shares the greatest degree of amino acid identity (51%) with APN. Recent in vitro studies have demonstrated that CTRPs possess similar metabolic regulatory actions as APN. However, whether CTRP9 expression is decreased (similar to APN) in T2D, or has any pathological significance, remains unknown. CTRP9 mRNA and protein levels in both adipose and cardiac tissue were detected via RT-PCR and Western blot. Male adult mice fed a high-fat diet (HFD) for 8 weeks and normal diet (ND) littermates were subject to MI via LAD ligation and treated with one gCTRP9 bolus (15 μg/g, intraperitoneal) 10 minutes before reperfusion (R). Both adipose/cardiac tissue CTRP9 mRNA and protein levels were decreased in HFD mice compared to ND mice (P<0.05). HFD mice manifested significantly exacerbated MI/R injury compared to ND (P<0.01). CTRP9 administration improved cardiac function (augmenting LVEF, and maximal and minimal dP/dt; and decreasing LVEDP) post MI/R. Furthermore, CTRP9 treatment reduced infarct size and the apoptotic index in HFD mice (P<0.01). Mechanistic investigation revealed increased pAkt and peNOS in the CTRP9-treated group compared to control (P<0.05). In summary, our results demonstrate significantly reduced expression of CTRP9 in T2D. Exogenous CTRP9 exerts cardioprotective effects in the D ischemic heart via Akt and eNOS signaling. These results suggest CTRP9 deficiency may contribute to exacerbated D MI/R injury, and may provide a novel potential therapeutic target against D cardiovascular injury.

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S.F. de Stoppelaar, F.E. van den Boogaard, M. Schouten, C. van ’t Veer, T. van der Poll*. AMC, Amsterdam, Netherlands

Background: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. In patients, thrombocytopenia is correlated with an adverse outcome of pneumonia. Thrombocytes can modulate the host response to infection in several ways, i.e. by facilitating clot formation, production of antimicrobial proteins and interaction with neutrophils. We studied the effect of thrombocytopenia during murine pneumococcal pneumonia.

Methods: Pneumonia was induced in mice by intranasal inoculation of S. pneumoniae. Thrombocytes were depleted by anti-mouse thrombocyte (α-thrombo) serum; controls received non-immunogenic serum. Animals were sacrificed at 24 and 48h after infection for analyses, or observed in a survival study. In vitro, S. pneumoniae was incubated with platelet rich plasma (PRP) or platelet poor plasma (PPP) in the presence or absence of neutrophils and bacterial counts were quantified after 45 and 90 minutes.

Results: Platelet counts were reduced to <1% of controls by treatment with α-thrombo serum (p<0.0001). Thrombocytopenic mice showed a reduced survival (27% versus 75% amongst controls; p=0.003), which was associated with higher bacterial loads in lungs and spleen at 24h (p=0.065 and p=0.0017 respectively) and blood at 24 and 48h (p=0.049 and p=0.006). Thrombocytopenic mice showed enhanced coagulation activation (thrombin-antithrombin complexes) in lung homogenates and plasma at both time points (p<0.05). At 48h cytokine levels in plasma, but not in lung, were higher in thrombocytopenic mice (IL-6, TNFα, IFNγ, MCP-1, all p≤0.01). Platelets did not influence (in the presence of neutrophils) killing of S. pneumoniae in vitro.

Conclusion: Thrombocytes play a protective role during pneumococcal pneumonia which is not related to direct bactericidal effects on S. pneumoniae in vitro.

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K. Ban, Z. Peng, W. Lin, R.A. Kozar*. University of Texas Health Science Center at Houston, Houston, TX

Objective: The mammalian target of rapamycin (mTOR) plays an important role in cell growth, migration, apoptosis and immune function. We therefore hypothesized that mTOR would protect against intestinal ischemia-reperfusion (IR) injury.

Methods: p70S6K, the major effector of mTOR, was investigated along with rapamycin, a specific inhibitor of mTOR, and an immunosuppressive agent used clinically. Mice were pretreated +/- rapamycin, then underwent 60 min intestinal ischemia and 6 hour reperfusion and compared to shams. p70S6K activity was measured by Western blot, mucosal injury by histopathology, permeability using everted sac method, and inflammation by myeloperoxidase. Animal survival was assessed up to 7 days.

Results: p70S6K was stimulated by reperfusion but was abolished by rapamicin (Fig 1A). IR significantly increased intestinal inflammation, permeability, and injury, which were all further increased by rapamycin (Table). Importantly, survival was significantly increased in rapamycin treated mice (Fig 1B). This marked difference in mortality clearly demonstrates the detrimental effect of inhibition of p70S6K by rapamycin to the hypoperfused gut.

Conclusion: We demonstrate for the first time the critical role of p70S6K in protection against intestinal IR injury. Additionally, despite the well-documented immunosuppressive profile of rapamycin, intestinal inflammation was not inhibited but rather enhanced. This study provides the rationale for using p70S6K as novel intervention against intestinal I/R injury and raises concern for the use of rapamycin in patients at risk for intestinal hypoperfusion.

Table 1

Table 1

Fig. 1

Fig. 1

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E. Lee1, 2, I. Yazji1, 2, S. Chhinder2, A. Afrazi1, 2, M. Good2, C. Egan2, M.D. Neal1, 2, H. Jia2, M. Branca2, Z. Grant2, C. Ma2, T. Prindle2, D. Hackam*1, 2. 1University of Pittsburgh School of Medicine, Pittsburgh, PA, 2Children’s Hospital of Pittsburgh, Pittsburgh, PA

Introduction: Necrotizing enterocolitis (NEC) is distinct from other forms of intestinal inflammatory disease by an early and high propensity to develop intestinal necrosis. Our lab has shown that TLR4 signaling is fundamental in NEC. However, the relationship of intestinal ischemia and inflammation has not been explored.

Hypothesis: We hypothesize that the newborn intestine is predisposed to the development of NEC via an exaggerated intestinal ischemic response to TLR4 activation.

Methods: TLR4 signaling was induced in adult and newborn wild-type (WT) mice via IP LPS 2.5mg/kg. NEC was induced in newborn WT mice via combination of hypoxia and formula gavaging with or without 10uM of sodium nitrite. 3D-blood flow modeling was achieved via Tomato Lectin intra-cardiac injections.

Results: TLR4 activation induced significantly worse impairment of intestinal perfusion in the newborn gut, 78%, in contrast to the adult 50% decrease, corresponding to a 6-fold increase in iNOS expression in newborns vs. 2-fold only in adults. NEC animals had 82% decrease in intestinal perfusion. However, attenuating ischemia with NO-donor sodium nitrite (NaNO2) doubled the intestinal perfusion to the stressed gut, which led to significant protection from NEC with return to baseline expression of iNOS, apoptosis marker CC3, and proliferation marker PCNA. In support of a critical role for TLR4 signaling in the endothelium in the pathogenesis of impaired perfusion in NEC, mice lacking TLR4 in the mesenteric endothelium (Tie2-TLR4 CKO) were protected from NEC development and had normal intestinal perfusion.

Conclusion: Our results demonstrate that TLR4-mediated intestinal ischemia and inflammation is exaggerated in mice newborn gut vs. adults, and preservation of adequate blood flow is sufficient to protect the newborn gut from NEC related injury.

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J.N. Harr1, E.E. Moore*2, 1, A. Ghasabyan2, T. Chin1, A. Sauaia1, A. Banerjee1, C.C. Silliman1, 3. 1University of Colorado Denver, Aurora, CO, 2Denver Health Medical Center, Denver, CO, 3Bonfils Blood Center, Denver, CO

Background: Thrombelastography (TEG) is emerging as the standard in the management of acute coagulopathies in injured patients. While TEG is sensitive in detecting abnormalities in clot strength, one short-coming is differentiating between fibrinogen and platelet contributions to clot integrity. Current US algorithms suggest platelet transfusion, while European guidelines suggest fibrinogen concentrates for correcting low clot strength. Therefore, we hypothesized that a TEG-based functional fibrinogen assay would assess the contribution of fibrinogen and platelets in clot strength, and provide insight to transfusion regimens.

Methods: Blood samples were obtained from trauma patients admitted to the SICU (n=68). Citrated kaolin TEG, FF, and von Clauss fibrinogen levels (plasma-based clinical standard) were measured. Correlations were assessed using linear regression models.

Results: FF strongly correlates with von Clauss fibrinogen levels (R2=0.87) and clot strength (R2=0.80). The mean fibrinogen contribution to clot strength was 30%; however, there was a direct linear relationship with fibrinogen level and % fibrinogen contribution to clot strength (R2=0.83). Traditional TEG parameters associated with fibrinogen (angle and k-time) have significantly lower correlations with FF (R2=0.70 and 0.35), and platelet count had a lower correlation with clot strength (R2=0.51).

Conclusion: FF can be rapidly performed with TEG, and correlates well with von Clauss fibrinogen. Both fibrinogen and platelet contribution of clot strength can be derived from FF. Moreover, FF had the strongest correlation to clot strength and increased levels were directly associated with increased % contribution to clot strength. These data suggest fibrinogen should be addressed early in trauma patients manifesting the acute coagulopathy of trauma.

Clot Strength increases with Functional Fibrinogen levels, but plateaus at levels greater than 500 mg/dL

Clot Strength increases with Functional Fibrinogen levels, but plateaus at levels greater than 500 mg/dL

The percent fibrinogen contribution to clot strength increases proportionally with fibrinogen levels

The percent fibrinogen contribution to clot strength increases proportionally with fibrinogen levels

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S. Hu, S. Yang, R. Raju*, K.I. Bland, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL

We have previously shown that T-H induces an increase in cardiac proinflammatory cytokine IL-6, which is closely correlated with T-H-induced cardiodepression. Although E2 administration following T-H downregulates cardiac IL-6, the mechanism by which this occurs remains unknown. To study this, male adult Sprague-Dawley rats were divided into four groups: sham, T-H + vehicle, T-H + E2 (1 mg/kg), T-H + ICI 182,780 (ICI; 3 mg/kg) + E2. E2 was given at the onset of resuscitation, IV, and the estrogen receptor antagonist ICI was given 30 min before E2, IP. Two hours after resuscitation, left ventricular (LV) performance was determined; blood and heart tissue were harvested. Cardiac SOCS-1, -2 and -3 were determined by Western blot. Plasma and heart tissue cytokines were measured by ELISA. Our results indicated that T-H induced cardiac depression along with a decrease in cardiac SOCS-1, -2 and -3. In contrast, T-H induced an increase in plasma and heart TNF-α, IL-6, and IL-10. Treatment with E2 after T-H restored LV performance and restored/enhanced all of the above SOCS signaling. Moreover, E2 prevented the increase in plasma and heart TNF-α and IL-6, but induced a further increase in plasma and heart IL-10. ICI abolished the salutary effects of E2 on those parameters. Therefore, it may be concluded that the cardiac depression after T-H is induced by the upregulation of cardiac proinflammatory cytokines, which is through T-H-induced downregulation of cardiac SOCS. E2-mediated cardiac protection is via E2-derived upregulation of cardiac SOCS, which downregulated proinflammatory, but enhanced anti-inflammatory, cytokines after T-H (NIH R01 GM39519).

(n=5-6/group; mean ± SE; one-way ANOVA + Tukey’s test

(n=5-6/group; mean ± SE; one-way ANOVA + Tukey’s test

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F. Hu1, L. Gatto*2, R. Cooney*1, G. Wang*1. 1Upstate Medical University, Syracuse, NY, 2Dept of Biological Science, SUNY Cortland, Cortland, NY

Bacterial urinary tract infection (UTI) is a common septic focus, especially in woman. Surfactant proteins A and D (SP-A, SP-D) expressed in the kidney, play an important role in innate immunity and regulation of inflammation.

Objective: Investigate the effects of SP-A and SP-D on bacterial growth and renal inflammation using an E. coli (human pathogen strain CFT073) model of UTI.

Methods: SP-A and SP-D double knockout (SP-A/D KO) and matched wild-type (WT) C57BL/6 female mice (10 - 12 weeks) were used. Fifty μl of E. coli (5x106 CFU/mouse) or buffer were delivered into the bladder of female mice. After 24 and 48 hrs, the CFU of E. coli in the kidney and urine were measured. Histological, cellular and molecular analyses were performed by H/E staining, IHC, Western blot and ELISA. The effects of SP-A and SP-D on bacterial growth were studied in vitro. Data are means+SE, with analysis by t-test or ANOVA, p<0.05 is significant.

Results: Infected SP-A/D KO mice demonstrate increased susceptibility to UTI as evidenced by a) higher CFU in kidney and urine (p<0.05, vs WT) at 24 and 48 hrs, b) increased inflammatory cell infiltration in the connective tissue of the calyx and lumen adjacent to the renal papilla (p<0.05, vs WT) at 24 hrs. SP-A/D KO and WT sham mice showed normally histologic renal structure. Infected WT mice demonstrate increased keratinocyte-derived chemokine (IL-8) (p<0.01, vs sham WT) in the kidney at 24 hrs; but not for SP-A/D KO mice, suggesting SP-A and SP-D signaling are necessary for efficient upregulation of renal IL-8 expression in infected mice. In vitro E. coli growth was inhibited 3-fold by 40 μg/ml SP-A (p<0.01) and 2-fold by 20 μg/ml SP-D (p<0.01).

Conclusion: SP-A and SP-D inhibit E. coli growth and attenuate renal inflammation in this murine UTI and in vitro bacterial growth model.

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A. Zifko*, C. Penzenstadler*, A. Khadem, M. Jafamadar, W. Öhlinger, H. Redl*, S. Bahrami*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Trauma Research Center, Vienna, Austria

Aim: To determine the effects of normoxemic, restricted, and hyperoxemic reoxygenation (NR, RR, HR, retrospectively) protocols on organ dysfunction after hemorrhagic traumatic shock (HTS).

Methods: Ventilated male rats were subjected to HTS, during which hypoxia was induced by hypoventilation to achieve an oxygen partial pressure in arterial blood (pO2) of 55 mmHg. At the onset of reperfusion, animals were randomized to receive three different (n=10 each) reoxygenation strategies. Return to baseline values of pO2 was achieved in the NR group, immediately, in the RR group, gradually over the first 50 minutes of resuscitation, and in the HR group, animals received 100% oxygen at begining of resuscitation, which was reduced gradually over 50 minutes until baseline oxygenation settings were reached. The experiment was terminated 100 minutes post-shock.

Results: At the end of observation plasma creatinine (48+/-13; 38+/-5; 37+/-8 μmol/L), ALT (305+/-463; 48+/-14; 53+/-25 IU/L), LDH (125+/-44; 1164+/-1816; 112+/-31 IU/L) and CK (384+/-137; 231+/-41; 267+/-75 IU/L) levels were significantly higher in the RR versus NR and HR groups, retrospectively. The ceruloplasmin/transferrin ratio in plasma was significantly lower in the RR group compared to the HR group (0.06+/-0.02; 0.08+/-0.03, retrospectively). No differences could be seen between groups for blood cell counts, wet/dry ratio and myeloperoxidase concentration in lung, histological evaluation of organs, thiobarbituric acid-reactive substances in liver, kidney or ileum, or oxidative burst.

Conclusions: Restricted reoxygenation during resuscitation after HTS may increase cell and organ damage compared to normoxemic and hyperoxemic reoxygenation.

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K. Yin*, J. Walker, D. Temmermand, K. Mian, B. Daiutolo, B. Spur, A. Rodriguez. UMDNJ, Stratford, NJ

Lipoxin A4 (LXA4) has been shown to resolve inflammation by decreasing cytokine production as well as reducing neutrophil activity in various forms of sterile inflammation. In sepsis however, neutrophils play a vital role in bacterial clearance. Furthermore, most in vivo studies have used long-lasting LXA4 analogs rather than natural LXA4. Our previous studies showed that natural LXA4 increased survival, decreased systemic inflammation and reduced blood bacterial load. The objective of this study was to examine the different effects of natural LXA4 versus LXA4 analog on systemic inflammation, bacterial load and neutrophil recruitment in sepsis. Cecal ligation and puncture (CLP) was performed on male Sprague-Dawley rats (250-310g). 1h after surgery, either saline, natural LXA4 (1.8 μg) or 15-epi-16-parafluorophenoxy-LXA4 (1.8 μg) was injected i.v. After 24h, natural LXA4 (21±6 X 106) increased neutrophil recruitment into the peritoneal cavity but LXA4 analog (5.9±1.5 X 106) did not affect neutrophil number compared to CLP + saline (9.71±.9 X 106). Natural LXA4 but not LXA4 analog reduced blood bacterial load compared to CLP + saline. In contrast, both natural LXA4 as well as LXA4 analog reduced plasma IL-6 compared to CLP + saline. To investigate if effects of natural LXA4 on neutrophil recruitment were specific to the sepsis model, we examined effects of natural LXA4 (1.8 μg, i.v.) in sham controls, 24h after surgery. Natural LXA4 given 1h after sham surgery reduced neutrophil recruitment compared to shams given saline suggesting that LXA4 mediated neutrophil recruitment in CLP is specific to sepsis These results suggest that natural LXA4 has novel beneficial effects on neutrophil recruitment, and blood bacterial load in a model of septic peritonitis that cannot be reproduced using stable LXA4 analog.

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N. Seiser, S. Kasravi, B. Leung, F. Primus, H. Harris. UCSF, Department of Surgery, San Francisco, CA

Triglyceride-rich lipoproteins, found in VLDL and chylomicrons, play an integral part in the host defense against bacterial infection. Apoprotein E (ApoE), regulates particle hydrolysis and clearance via the LDL receptor, and has been linked to lipid antigen presentation and NKT cell activation. We previously showed that in septic mice supplied with exogenous ApoE, mortality and NKT cell activation increased, making ApoE modification an interesting target for a possible lipid-based therapy for sepsis. We hypothesized that changes in ApoE expression can alter the septic mortality through an NKT-cell-dependent mechanism in a rodent model of polymicrobial sepsis. ApoE hypomorphic mice (5% of physiologic levels), wild type mice and NKT cell deficient CD1D-knockout mice underwent cecal ligation and puncture. Mice lacking NKT cells showed the highest 28-day survival (88% versus 80% for hypomorphic mice and 63% for WT mice; p=0.0161, p=0.184 respectively). NKT cell frequencies among total lymphocytes in the liver were upregulated in septic animals and highest in septic wild type mice. Thus, ApoE deficiency appears to lead to decreased hepatic NKT cell activation, and fewer activated NKT cells translate to improved survival in sepsis. When we examined serum TH1 and TH2 cytokine production in all three groups we found a relative increase in IL10 expression in hypomorphic mice compared with wild type mice. Lymphocytes isolated from the livers of hypomorphic mice also produced higher amounts of IL10 than wild type controls, possibly linking this change in cytokine profile to the survival advantage and ApoE expression. We conclude that decreased ApoE levels and NKT cell frequency are linked to better survival in our polymicrobial sepsis model, which makes this mechanism an attractive target for lipid-based therapy for sepsis.

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K. Kojima, H. Akabori, C. Kojima, J.A. Mobley, I.H. Chaudry*. University of Alabama at Birmingham, Birmingham, AL

Traumatic injury causes 125,000 deaths yearly and is the fifth leading cause of death in the US. T-H is characterized by reduced perfusion that requires resuscitation (Res). Although beneficial, current Res procedures often fail to restore essential cellular functions, which trigger immune responses, inflammation and irreversible damage to tissues. We have shown that administration of E2 at the time of Res following T-H reduces inflammation in part by decreasing proinflammatory cytokines in the hypothalamus. Recent reports indicate that treatment of shock with histone deacetylase inhibitors (HDACI) reduces immune response and increases survival. HDACI normalizes the acetylation status of proteins, which prevents cell death and inflammation. In this study, we investigated effects of E2 on the global proteomic and acetylation status in the hypothalamus in a T-H model. Male rats were subjected to either sham or T-H (blood pressure 40 mmHg for 90 min followed by fluid Res) with and without E2 (n=5/group). Tissues were harvested at 0 and 2 hrs following Res, and global shotgun proteomics and systems biology analysis was applied. Results indicated that T-H led to activation of pro-apoptotic pathways involving c-myc (Fig. 1). E2 administration after T-H was associated with activated anti-inflammatory and pro-survival pathways including Sp1. We then compared the abundance of acetylated proteins and found that T-H decreases acetyl-lysine, which is further decreased with E2 treatment. However, acetylation of specific proteins including tubulins and ATPases were increased with E2 (Fig. 2), which are associated with increased survival following T-H. Thus E2 administration after T-H may have similar pro-survival effects to HDACI and be useful in restoring essential cellular functions following T-H (NIHT32 GM063490, RO1 GM39519).

Western analysis for c-myc and phosphorylated c-myc at 2hr (n=3/group)

Western analysis for c-myc and phosphorylated c-myc at 2hr (n=3/group)

T-H: trauma-hemorrhage; E2: 17β-estradiol

T-H: trauma-hemorrhage; E2: 17β-estradiol

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D. McDonald, S. Ashe, T. Ozment, J. Kalbfleisch, D.L. Williams*. East Tennessee State University, Johnson City, TN

We have discovered that the class A scavenger receptor (SR-A) plays a central role in mediating the morbidity and mortality of sepsis/septic shock. Specifically, we have found that SR-A deficient (SR-A-/-) mice show an attenuated pro-inflammatory phenotype and improved survival outcome in response to sepsis. However, the mechanisms by which SR-A contributes to sepsis have not been defined. Seimon et al. have speculated that in response to cellular stress SR-A inhibits the pro-survival IRF3/interferon (IFN)β pathway (PNAS 103:19794, 2006). We hypothesized that SR-A amplifies the pro-inflammatory, pro-death phenotype associated with septic shock while simultaneously inhibiting the pro-survival IRF3/IFNβ signaling pathway. To test this hypothesis, we examined the effect of sepsis on tissue levels of interferon sensitive genes (ISGs) in the presence and absence of CLP sepsis. Wild type (WT) and SR-A-/- mice were subjected to CLP sepsis. No surgery and sham surgery served as controls. Tissues were harvested at 8 hrs after CLP. ISG (Ifit1, Isg15) mRNA levels were assessed by qPCR. GAPDH was the housekeeping gene. Pulmonary ifit1 mRNA was increased by 400% in SR-A CLP vs WT control, 227% vs WT CLP and 225% vs Sham SR-A. Liver and spleen ifit1 mRNA were not increased. Lung ISG15 mRNA was increased by 95% vs WT CLP and 129% vs SR-A sham. Liver ISG15 mRNA was increased by 130% in SR-A CLP vs WT CLP. We conclude that in the absence of SR-A, ISG mRNA is up regulated in response to sepsis indicating that the Type I IFN pathway is activated. Thus, SR-A appears to be an endogenous negative regulator of the IRF3 Type I IFN pathway during sepsis. To the best of our knowledge, this is a new and novel role for SR-A in the pathogenesis of septic disease.

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S. Matsuo1, 2, W. Yang1, 2, M. Aziz1, 2, P. Wang*1, 2. 1Hofstra North Shore-LIJ School of Medicine, Manhasset, NY, 2The Feinstein Institute for Medical Research, Manhasset, NY

Introduction: Gut ischemia/reperfusion (I/R) injury is an important pathophysiologic problem after surgery of digestive organs and transplantation, and can cause severe systemic inflammatory responses. Excessive neutrophil infiltration to the inflamed sites plays a critical role in organ damage. The migration of neutrophils is mediated by integrin signaling, which can be blocked by RGD peptide. We hypothesized that administration of cyclic RGD peptide (cRGD) provided protection against tissue injury induced by gut I/R.

Methods: Gut I/R was performed by tying superior mesenteric artery with 4-0 suture for 45 min in adult male C57BL/6 mice, followed by reperfusion. cRGD (5 mg/kg BW) or normal saline (vehicle) was administered by intraperitoneal injection 1 h prior to ischemia. Blood, small intestine and lung tissues were collected at 4 h after reperfusion for various measurements.

Results: cRGD significantly inhibited the myeloperoxidase activity in the small intestine and lungs by 74.3% and 54.2%, respectively, after gut I/R in comparison to the vehicle group. The cRGD treatment reduced the levels of proinflammatory cytokine and chemokine in the small intestine and lungs after I/R. cRGD improved the microscopic structure of the small intestine and lungs, judged by histological examination. In addition, the levels of cleaved caspase-3 in the lungs of the cRGD-treated group were significantly lower than those in the vehicle group. Finally, cRGD lowered the levels of serum proinflammatory cytokines and organ injury index after I/R (Table below).

Conclusions: cRGD lowered the neutrophil infiltration to the small intestine and lungs, leading to reduction of inflammatory responses and organ injury after gut I/R. Thus, it explores the possibility of using cRGD in treating patients with gut ischemia.

No caption available

No caption available

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X. Cui1, 2, W. Dong1, 2, R. Barrera2, G.F. Coppa2, P. Wang*1, 2, R. Wu*1, 2. 1The Feinstein Institute for Medical Research, Manhasset, NY, 2North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY

Natural killer T (NKT) cells are an important early source of interleukin (IL)-4 and, therefore, crucial for the generation of Th2 responses in vivo. Reduced IL-4 levels in the gut are association with loss of effective mucosal immunity. Given that NKT cells respond to the α-galactosylceramide (α-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines including IL-4, we hypothesized that activation of NKT cells by α-GalCer attenuates bacterial translocation in the post ischemic gut. To study this, gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery for 30 min in male adult mice. After removing the clip, α-GalCer (2 μg/mouse) or normal saline containing 0.5% Tween 20 (Vehicle) was administered intraperitoneally. The mesenteric lymph node (MLN) and small intestine samples were collected 4h after reperfusion. The MLN complex was homogenized and plated on chocolate agar plates for bacterial culture. Gut injury was assessed by examining the lactate dehydrogenase (LDH) activity in the gut. Intestinal levels of IL-4 were measured by ELISA. Our results showed that bacterial translocation to the MLN was minimal in sham mice, but was extensive in gut I/R vehicle treated mice (P<0.05). α-GalCer treatment, however, reduced bacterial translocation to the MLN by 83% after gut I/R (P<0.05). Similarly, I/R induced a 49% loss of LDH from intestinal tissues in vehicle-treated mice as compared with sham mice (P<0.05). α-GalCer treatment restored intestinal LDH activity to almost sham levels (P<0.05). Moreover, α-GalCer treatment is associated with a 2.6 fold increase in intestinal IL-4 levels after gut I/R (P<0.05). In conclusion, activation of NKT cells by α-GalCer attenuates bacterial translocation in the post ischemic gut involving secretion of IL-4.

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I.E. Brown, A. Shawo, O. Bellevue, B. Raiyakanti, H.W. Harris*. University of California, San Francisco, San Francisco, CA

Sepsis is a condition characterized by a whole-body inflammatory state and the presence of infection. With progression, the inflammatory response becomes an increasingly morbid insult. While antibiotics, fluid resuscitation, and supportive therapy are the mainstay of treatment, attempts to directly address the inflammatory component have been largely unsuccessful.

We hypothesized that antibiotics plus immunosuppressants to attenuate inflammation would protect mice against severe sepsis better than antibiotics alone. We used a model of polymicrobial infection and sepsis, cecal ligation and puncture, to show that treatment with cyclophosphamide and the broad-spectrum antibiotic imipenem confers a significant survival advantage over treatment with imipenem alone (62.5% vs 32.1% p=0.0273). Analogous to a clinical setting, treatment followed the insult, and outbred mice were used to maximize subject variability. At the effective dose of cyclophosphamide, peripheral leukocyte counts did not differ between the two groups. In fact, doses resulting in leukocyte depletion increased mortality. Flow cytometry showed a higher percentage of peripheral activated neutrophils in mice treated with cyclophosphamide. Interestingly, when peripheral blood cytokines were examined, G-CSF levels were elevated in mice receiving cyclophosphamide over the first 96 hours of infection when mice were most susceptible to death. Expectedly, elevated IL-6 levels predicted mortality.

Low dose cyclophosphamide significantly improved survival in severe sepsis through a mechanism independent of peripheral leukocyte depletion. With careful patient selection and judicious use to effectively balance risks and benefits, clinical applications targeting the inflammatory component of sepsis may potentially prevent significant morbidity and mortality.

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M. Fu, M. Yamada, M. Kaneki. Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, Charlestown, MA

Metabolic derangements and muscle wasting are major complications of burn injury, affecting the recovery and clinical outcome of burn patients. We have previously shown that burn induces skeletal muscle insulin resistance, a major player in metabolic aberrations and muscle wasting. Insulin resistance causes mitochondrial dysfunction and vice versa. Little is known, however, about the effects of burn on mitochondria in skeletal muscle. Full-thickness third degree burn injury comprising 30% of total body surface area was produced under anesthesia in male CD-1 mice by immersing the trunk in 80°C water. At 3 days after burn or sham-burn, rectus abdominis was excised under anesthesia for biochemical analyses. Muscle insulin resistance, as judged by attenuated insulin signaling and insulin-stimulated glucose uptake, was accompanied by over 2-fold decrease in mitochondria DNA to nuclear DNA ratio in burned mice compared with sham-burn. Consistently, protein expression of mitochondrial respiratory chain components, complex I, III, IV and V, were markedly decreased by burn injury, while complex II expression was not significantly altered. These changes paralleled with the suppressed expression of nuclear respiratory factor 1 (Nrf1), a key transcription factor for mitochondrial biogenesis and function. These findings demonstrate that burn injury decreased mitochondrial content along with reduced Nrf1 expression in muscle. Together, our data indicate that burn-induced insulin resistance is associated with suppressed mitochondrial biogenesis. These results raise the possibility that dysfunction and disintegrity of mitochondria may be a novel potential target to reverse or ameliorate burn-induced metabolic derangements, including insulin resistance and muscle wasting, to improve the prognosis and long-term outcome of burn patients.

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S.G. Miller, S.R. Bailey, A.B. Lentsch*, T.A. Pritts*. University of Cincinnati, Cincinnati, OH

CD14 is an LPS receptor expressed in two forms; a membrane-bound form expressed on the cell surface and required for LPS binding and TLR4 activation (mCD14), and a soluble form (sCD14) representing a post-translational mCD14 cleavage product which is secreted into the serum. sCD14 is known to be a marker of inflammation and thought to sequester circulating LPS. We examined if sCD14 is secreted into other compartments, specifically the gut lumen, and if intestinal epithelial cells may contribute to luminal sCD14 secretion. In vitro, the Caco-2 cell line was used to represent intestinal epithelial cells (IECs). Caco-2 cells were grown to confluence on transwell filters and sCD14 secretion was measured in both apical (luminal) and basolateral (blood) compartments. The effect of cell differentiation on sCD14 secretion was evaluated over a 27-day time course. For in vivo studies, C57BL/6 mice were treated with intraperitoneal LPS or saline. Serum, tissue, and stool were evaluated for sCD14 expression. IECs express sCD14 in a variable pattern that is differentiation dependent. Fully differentiated IECs secrete sCD14 preferentially into the apical (luminal) compartment. Treatment of cells with basolateral LPS, to mimic endotoxemia, caused a significant increase in apical sCD14 secretion (Figure) and a significant decrease in basolateral secretion. Endotoxemic mice demonstrated increased levels of sCD14 in intestinal segments and secreted significantly increased sCD14 into the lumen of the colon (data not shown). IECs express sCD14 and secrete sCD14 preferentially into the lumen in response to endotoxin. Mice secrete sCD14 into the colonic lumen in response to endotoxemia. Increased production of sCD14 by the intestine could lead to sequestration of LPS in the gut lumen under these conditions.

Apical sCD14 secretion by fully differentiated Caco-2 monolayers in response to basolateral treatment with LPS versus serum free media

Apical sCD14 secretion by fully differentiated Caco-2 monolayers in response to basolateral treatment with LPS versus serum free media

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D.K. Stutzman, C. Baird*, A. Kallweit*, L. Khailova, P.E. Wischmeyer*. University of Colorado Denver, Aurora, CO

Background: Glutamine (GLN) has been shown to protect cells in multiple forms of injury, including heat stress (HS), by inducing heat shock proteins (HSPs). Literature shows GLN is an osmotically acting amino acid and causes cell swelling by a sodium-associated transport system. Alpha-aminoisobutyric acid (AIB) is a non-metabolizable amino acid that utilizes the same transport system, and is also known to cause cellular swelling. Since osmotic changes have been shown to induce HSPs our objective was to do a comparative study to determine what role cell swelling plays in GLN’s protective abilities using AIB as a control.

Methods: Cells were treated with either 10mM GLN or AIB for 15min and subjected to lethal HS (44°C/50min) for survival or non-lethal HS (43°C/45min) for HSP analysis. Survival was evaluated via MTS assay. HSP25, HSP32, and HSP70 were analyzed via Western Blot. Cells were also grown on slides, treated (as above), and subjected to non-lethal HS for 15 min or 30 min. Slides were stained for f-actin, nuclei and cell membrane to visualize cell size differences via microscopy. Cell area was measured (50 cells per experimental group).

Results: Both GLN and AIB treatments increased HSP25, HSP32 and HSP72 after HS (P<0.05 vs. HS CT cells). MTS assays showed both GLN and AIB increased survival (P<0.05 vs. HS CT cells). Microscopy showed both GLN and AIB increased cell size >40% at 15 and 30min HS (p<0.001 vs. HS CT).

Conclusions: Both GLN and AIB, increased cell survival and HSP25, HSP32, and HSP70 in HS intestinal cells. Both GLN and AIB also increased cell size in HS injury. These data imply that GLN’s mechanism may be due to its osmotically active properties in addition to its metabolism. It is possible that osmotic changes play a role in GLN mediated HSP expression and cellular protection.

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J. Mella1, 2, R. Moitra2, E. Duffy3, D. Remick*3. 1Boston University, Boston, MA, 2Boston Medical Center, Department of General Surgery, Boston, MA, 3Boston Medical Center, Department of Pathology and Laboratory Medicine, Boston, MA

Introduction: Substance P (SP) is a neuropeptide that plays a critical role in the inflammatory cascade when bound to the neurokinin-1 receptor. This cascade triggers inflammatory cell proliferation with subsequent release of cytokines and other inflammatory mediators causing tissue injury. A neurokinin-1 receptor antagonist (NK1RA) has been shown to reduce inflammation by inhibiting SP in a non-septic rat model. This study investigated whether NK1RA impacts survival in the cecal ligation and puncture (CLP) murine sepsis model.

Methods: Mice were prospectively stratified by means of a plasma enhanced killing (PEK) assay into predicted to live (Live-P) and predicted to die (Die-P) groups. This assay utilizes a mouse’s naive plasma to forecast its ability to survive sepsis. Mice were then subjected to CLP and treated with an intraperitoneal injection of NK1RA or normal saline (NS) at the time of CLP based on their preoperative stratification.

Results: The PEK assay accurately demonstrated a difference in mortality; the Live-P group had improved survival (69%) compared to the Die-P group (44%), regardless of treatment. However, when treated with NK1RA, both the Live-P and Die-P groups trended towards lower survival (12% and 10% reduction respectively) compared to those treated with NS. Initial studies suggest that plasma pro-inflammatory cytokines are increased and anti-inflammatory cytokines are reduced in the NK1RA treated groups. These findings correlate with a decrease in circulating neutrophils and a concomitant increase in lymphocytes, platelets, and hematocrit post CLP when treated with NK1RA.

Conclusion: Mice treated with NK1RA at CLP demonstrated a paradoxical increase in inflammatory response with a subsequent increase in mortality.

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I. Nassour1, H. Gomez1, P. Loughran1, J. Brumfield1, L. Otterbein2, B. Zuckerbraun*1. 1University of Pittsburgh Medical Center, Pittsburgh, PA, 2Harvard Medical School, Boston, MA

Introduction: The purpose of this study is to evaluate the effects of carbon monoxide (CO) as an adjunct to resuscitation on hepatic sinusoidal endothelial integrity in a murine model of hemorrhagic shock and resuscitation (HSR). These investigations test the hypothesis that CO prevents hepatic injury/hypoxia by maintaining endothelial integrity and the hepatic microvascular circulation.

Methods: Male C57BL/6 mice underwent sham operation or hemorrhage to a target MAP of 25 mmHg. Mice were maintained at this pressure for 120 minutes and then resuscitated with Ringer’s lactate (2X volume of total shed blood). Mice were randomized to receive vehicle or CO-releasing molecule (CORM;10mg/kg) starting in the shock period (n=6-8/group). Relative hypoxia was determined using EF5 immunofluoresence. Sinusoidal integrity was determined by scanning EM and Evans blue tissue levels. Leukocyte stasis, rolling, and adhesion were determined using intravital microscopy. Statistical analysis was determined by ANOVA.

Results: EF5 staining demonstrated that hemorrhagic shock induced liver hypoxia, which was prevented by CORM treatment. Scanning EM imaging illustrated that HSR results destruction of normal endothelial architecture, while CORM therapy prevented these changes. Relative hepatic levels of Evans blue, suggesting endothelial leak were increased in HSR compared to sham operated mice (P<0.05). This endothelial leak was significantly reduced by CORM (P<0.05). In addition, leukocyte rolling and adhesion were significantly diminished by CORM. CORM treatment also reduced hepatic adhesion molecule expression.

Conclusion: CO protected the hepatic sinusoidal endothelium from HSR-induced injury. Further investigations into the mechanisms of action are necessary.

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S. Niederlechner, C. Aherne, A. Riegel, A. Kallweit, H. Eltzschig, P.E. Wischmeyer*. University of Colorado, Aurora, CO

Background: L-Glutamine (GLN) protects the gut and improves outcome in critical illness. However, its’ molecular mechanism of benefit remains unclear. The neuronal guidance molecule Netrin-1 (Ntr-1) has been implicated as a potent survival factor in the intestine. As such, our specific aim was to examine if injury (heat stress (HS)) has an effect on Ntr-1 expression and if Ntr-1 plays a role in GLN’s protection in intestinal epithelial-6 cells (IEC-6) following injury.

Methods: Small interfering RNA (siRNA) was used to block Ntr-1 expression in IEC-6 cells. Cells transfected +/− 20nM Ntr-1 siRNA (Ntr-1si) or non-coding control oligos (NC) (48h) were treated (15min) with 0 (CT), 0.5, 2 or 8mM GLN, and subjected to lethal HS (44°C/50min) for cell survival or non-lethal HS (43°C/45min) for protein expression. Ntr-1 and cleaved caspase-3 (CC-3) levels were measured via Western blot. Survival was determined via MTS assay.

Results: Ntr-1 levels were decreased by 80% after HS in cells exposed to 0mM GLN (p<0.05 vs. CT). GLN (0.5mM, 2mM, 8mM) prevented this decrease in Ntr-1 expression after HS (p<0.05 vs. HS CT). CC-3 levels were decreased in GLN-treated cells after HS (p<0.05 vs. HS CT). Ntr-1si (70% Ntr-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 vs. HS GLN without Ntr-1si). MTS assays revealed that GLN increased survival 2–3 fold after lethal HS (p<0.05 vs. HS CT). Ntr-1si attenuated GLN’s protection by 91% after HS (p<0.05 vs. HS CT).

Conclusion: This is the first data showing HS decreases Ntr-1 levels. Further, Ntr-1 appears to be a novel survival factor in GLN’s protective mechanism in intestinal cells after injury. In summary, GLN’s attenuation of cell death in the intestine is related to prevention of decreased Ntr-1 levels post-injury.

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J.R. Fritz, R.P. Gersch, J.M. Huston*. Stony Brook University Medical Center, Stony Brook, NY

Introduction: Uncontrolled hemorrhage is the most common preventable cause of death following civilian trauma. There are no systemic therapies available to augment normal hemostatic pathways and attenuate traumatic hemorrhage. The cholinergic anti-inflammatory pathway prevents tissue injury during lethal shock and sepsis. Recently, cholinergic stimulation was found to improve hemostasis following experimental ear trauma and hemorrhage in swine. Here we hypothesized that cholinergic enhanced hemostasis could reduce blood loss during large volume, uncontrolled lethal visceral hemorrhage.

Methods: Male aged 6 to 8 week old Lewis rats underwent laparotomy and laceration of the liver using a 4mm punch biopsy. Bleeding time, rate, and total blood loss were recorded. Thrombin activity was measured systemically and locally at the site of injury via TAT ELISA. Nicotine (0.2, 1, 2 mg/kg, i.p.) or vehicle was given 30 m, 72 h, or 14 d before injury. Other animals underwent splenectomy or sham surgery 24 h before injury.

Results: Nicotine significantly and dose-dependently reduces blood loss during liver trauma by as much as 70% versus controls (n=4–8, p<0.05). Pre-treatment with nicotine 72 h before injury also significantly attenuates hemorrhage. Nicotine treatment significantly and specifically increases thrombin activation at the site of injury, but not systemically. Nicotine treatment fails to reduce hemorrhage or regulate thrombin activity following splenectomy.

Conclusions: These findings suggest that cholinergic enhanced hemostasis can prevent life-threatening, uncontrolled traumatic hemorrhage by augmenting local thrombin activity via spleen. Further studies are needed to elucidate how cholinergic signaling in spleen can regulate hemostatic pathways at specific and distant sites of bodily trauma.

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H. Xue, D. Slavov, P.E. Wischmeyer*. University of Colorado Denver, Aurora, CO

Objective: We have demonstrated that glutamine (GLN) enhances cytoprotective heat shock protein (HSP) expression. Heat shock transcription factor-1 (HSF1) is the master regulator for HSP gene expression. The main point of control of HSF1 is widely considered to be the regulation of its transactivation activity. How HSF1 expression as a transcription factor per se is regulated remains unknown. We are aimed to study whether GLN could affect HSF1 expression in addition to its activity.

Methods: Non-stressed or heat-stressed YAMC colonic epithelial cells were exposed to GLN at 0, 0.5 or 2mM. HSF1 expression, nuclear localization, phosphorylation, trimerization and DNA binding were determined. HSF1 promoter-driven reporter assay and deletion analysis of HSF1 gene promoter were conducted.

Results: GLN upregulated HSF1’s transactivation activity by increasing its trimerization, nuclear localization, activating phosphorylation and DNA binding. Intriguingly, GLN also enhanced HSF1 protein and mRNA expression and HSF1 promoter activity. Deletion analysis identified that the −281/−200 region of HSF1 promoter serves as GLN response element. Within this region, deletion of the putative CCAAT enhancer binding protein (C/EBP) binding site abolished GLN response. C/EBPβ was proved to bind to this −281/−200 GLN response element. In absence of GLN, HSF1 expression was de-repressed in C/EBPβ-silenced cells. Over-expression of the inhibitory dominant-negative isoform of C/EBPβ repressed HSF1 expression in presence of adequate GLN.

Conclusions: We for the first time show that GLN not only activates HSF1’s transactivation activity, but also can induce HSF1 expression by activating its transcription per se in a C/EBPβ-dependent manner. This forms a dual control machinery in mobilizing HSF1, which ultimately leads to a robust HSP induction.

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N.A. Rodriguez*, E. Diaz, R. Przkora, C.C. Finnerty*, D. Herndon*. University of Texas Medical Branch/Shriners Hospitals for Children, Galveston, TX

Introduction: The anabolic steroid Oxandrolone has been shown to be beneficial in the treatment of severe pediatric burns. However, there have been concerns regarding its potential negative effects on renal function. Acute kidney injury (AKI) complicates the outcomes of children admitted to the ICU. The purpose of this study was to evaluate the impact of Oxandrolone in the development of AKI (defined as a pRIFLE classification of risk, injury, or failure) during acute hospitalization.

Methods: Patients younger than 19 years old with burns covering >30% of the total body surface area were randomized to receive oxandrolone twice daily (n=70) or placebo (n=152). Renal function was assessed by applying the pRIFLE criteria during acute hospitalization. The probability of developing AKI during the acute stay was examined using Fisher’s exact test. Alpha level of significance was set at p-value = 0.05.

Results: The Oxandrolone and Control groups were no different in Age, Gender, Ethnicity, % TBSA burned, % Third degree, presence of inhalation injury. Oxandrolone significantly decreased the incidence of AKI (20% vs. 39%, p=0.006).

Conclusion: Oxandrolone administered to severely burned children has a beneficial effect on renal function. Further research is necessary to characterize the mechanism of this effect. Oxandrolone has in the past been shown to increase levels of IGF-1, and improve muscle catabolism. The effect of Oxandrolone on cardiovascular function is currently being elucidated.

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Y. Li, P. Wang, Q. Mao, J. Wang, J. Li. Jinling Hospital, Nanjing, China

Objective: To compare the intervention of damage control surgery and the traditional surgery for systemic inflammatory response in a pig model of severe abdominal gunshot injury.

Methods: Thirty-two female domestic outbred pigs following multiple bowel injury associated with lethal triad (low temperature, acidosis, and coagulopathy) were randomly divided into two groups (n=16): conventional surgery (CS) and damage control surgery (DCS) groups. After appropriate autologous blood and fluid transfusion, laparotomy was performed, the CS group underwent end-to-end intestinal anastomosis, peritoneal lavage and definitive abdominal closure; the DCS group underwent intestinal clipping, ligation, peritoneal lavage, temporary abdominal closure. The operation time, blood loss, and volume of fluid infusion were recorded. The hemodynamic parameters, arterial blood gases, and coagulation parameters were examined. Postoperative 6 hours, six pigs per group were sacrificed, myocardium, lung, small intestine and liver were harvested for observation of histology. Ten pigs were used to assess survival at 24h after surgery.

Results: There was no significant difference in blood loss, MAP between two groups. Compared the CS group, the DCS group had a shorter operation time (1.1 ± 0.2h vs 2.4±0.3h, p<0.01), and less infusion (3204±254ml vs 3756±313ml, p<0.05). The DCS group had higher urine volume, lower heart rate, and less acidosis and coagulopathy. The tissue injuries and neutrophil infiltration were lower in the DCS group than that of CS group.

Conclusion: DCS, compared with traditional surgery, could shorten operation time, reduce fluid infusion, speed up to correct metabolic acidosis, coagulopathy, and low body temperature, avoid increasing injuries and systemic inflammatory response induced by reperfusion, and prevent MODS.

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M.A. Dubick*, J.L. Barr, D. Prince, D.L. Grubbs, J. Sondeen. US Army Institute of Surgical Research, San Antonio, TX

Hemorrhagic shock is associated with oxidative stress that contributes to the inflammatory response leading to secondary sequelae. The present study investigated whether initial resuscitation with blood products would reduce this oxidative stress better than conventional resuscitation fluids in a swine uncontrolled hemorrhage model. Anesthetized, instrumented pigs (n=8/gp) were subjected to controlled hemorrhage of 24 ml/kg, a 20 min shock period, splenic injury with 15 min of uncontrolled hemorrhage, followed by fluid administration. Plasma (FFP), whole blood (FWB), a 1:1 or 1:4 ratio of FFP:RBC and Hextend were infused to 15ml/kg, while LR was infused to 45 ml/kg. Pigs were monitored for up to 5 hr after spleen injury. At the time of death or euthanasia, lung, liver, heart, duodenum and kidney were removed and quickly frozen for analysis of antioxidant status. Hemorrhage in the current study reduced the total antioxidant potential and glutathione peroxidase activity in all tissues analyzed and increased myeloperoxidase (MPO) activity at least 50% in lung and duodenum compared with control tissue, implying an oxidant stress. At the level of resuscitation infused, all fluids produced similar changes in hemodynamics, oxygen delivery and oxygen demand, despite significant reductions in hematocrit in the LR and Hextend groups. Similarly, there were no significant differences among resuscitation groups in antioxidant potential, thiobarbituric acid reactive substances, glutathione or nitric oxide levels, or activities of antioxidant enzymes in any of the tissues examined. Also, MPO activities in lung and duodenum were similar among resuscitation groups. These data suggest that the oxidative stress induced by hemorrhage was not improved by initial hypotensive resuscitation with blood products compared with LR or Hextend.

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M. Weuster1, F. Hildebrand2, P. Mommsen2, I. Witte3, J. Mohr3, A. Seekamp*1, M. van Griensven*5. 1University of Schleswig-Holstein, Kiel, Germany, 2Medical School, Hannover, Germany, 3University Hospital, Duesseldorf, Germany, 4University Hospital, Marburg, Germany, 5Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria

Introduction: Hemorrhagic shock in conjunction with abdominal trauma and chest trauma display major reasons of early death after multiple-trauma. Despite the high clinical incidence of abdominal and chest trauma, an animal model with a combination of these entities is lacking. Furthermore, the potential effects of induced hypothermia in a pig model prone to bleeding are unknown.

Material and Methods: In this newly established model, pigs received blunt chest trauma (bolt shot to the right chest), followed by laparotomy with two incisions in the right upper liver lobe (four edges scalpel) and subsequent liver packing. Hemorrhagic shock then was induced by blood withdrawal (max. of 45% of total blood volume from femoral artery) to a MAP of 30mmHg ±5 mmHg. Resuscitation and intensive care monitoring followed afterwards.In addition, the possibility of endovascular hypothermia induction to 34°C and subsequent re-warming within the above described model was observed.

Results: Mortality rate within the establishing process was 0%. Combined trauma resulted in a severe signs of hemorrhagic shock as well as significant signs of impaired pulmonary function. Hemodynamic parameters showed significant results, lactate level and base excess attested adequate depth of shock. By endovascular cooling a cooling rate of 2°C/h was achieved.

Conclusion: A consistent, reproducible and clinically relevant porcine model of multi-system injury (pulmonary contusion, liver laceration) with both controlled as well as uncontrolled components of hemorrhage and the potential for re-bleeding to begin with induction of hypothermia was established. Further research using this model will include investigations to evaluate the optimal time point and velocity of induction of hypothermia as well as duration and severity of hypothermic therapy.

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M. Kitadate, T. Shibamoto, H. Tonami. Kanazawa Medical University, Uchinada, Ishikawa, Japan

Rat systemic anaphylaxis is characterized by hepatic venoconstriction, which partly accounts for anaphylactic hypotension. We determined the primary site of anaphylactic hepatic venoconstriction in anesthetized rats by using angiographic techniques. Anaphylactic hypotension was induced by administration of the ovalbumin antigen (0.6 mg, iv) in anaesthetized SD rats sensitized with the antigen (1 mg, sc). Liver angiography was performed anterogradely from the main portal vein (n=8) and retrogradely from the hepatic vein of the left lateral lobe (n=6) at baseline and maximal hepatic venoconstriction when portal venous pressure (Ppv) reached the peak levels after antigen. The systemic arterial pressure decreased profoundly from 115±7 (SE) to 71±9 mmHg, and Ppv increased to the peak levels of 28±3 cm H2O at 109±12 sec after antigen. Anterograde angiography performed after antigen revealed that portal venules (76 vessels) with a diameter of 228±39 μm (from 160 to 300 μm) found not visualized, suggesting the stenosis occurred distal to these levels. The corresponding visualized upstream portal vessels were markedly enlarged in diameter as compared with the baseline. However, there are some portal branches (17 vessels) with the diameter of 284±69 μm (from 176 to 416 μm), which showed apparent stenosis. In contrast, retrograde angiography showed similar images of the hepatic veins of the left lateral lobe before and after antigen, suggesting absence of constriction of these vessels. In conclusion, hepatic venoconstriction associated with anaphylactic hypotension may occur mainly at portal venules with a diameter less than 228 μm, although the larger portal veins (<416 μm), but not hepatic veins, can constrict in anesthetized rats.

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T. Doi, O. Kozawa, Y. Enomoto, S. Ogura*. Gifu University Hospital, Gifu, Japan

Objective: Disseminated intravascular coagulation (DIC) associated with shock is a critical condition with an unfavorable prognosis. Antithrombin III (AT-III) is clinically used as a therapeutic agent for DIC observed in acute severe cases such as septic shock. We have previously reported that ristocetin, an activator of glycoprotein Ib/IX/V, promotes the release of soluble CD40 ligand (sCD40L), which is an inflammatory mediator, via the production of thromboxane (TX) A2 in human platelets. In this study, we investigated the effects of AT-III on ristocetin-stimulated platelet activation.

Methods: Platelet-rich plasma was obtained from healthy volunteers, was pretreated with AT-III, and then stimulated by ristocetin. The levels of platelet-derived growth factor (PDGF) -AB, sCD40L, and TXB2 (a stable TXA2 metabolite) were analyzed by each ELISA.

Results: The secretion of PDGF-AB, the production of TXB2, and the release of sCD40L from platelets caused by ristocetin-stimulation were significantly suppressed by AT-III. Meanwhile, AT-III did not suppressed platelet aggregation or sCD40L release stimulated by U46619, which is the TXA2 receptor agonist.

Conclusion: It was suggested that AT-III hinders sCD40L release due to ristocetin stimulation by suppressing the production of TXA2. Our present study provides an evidence that AT-III might exert anti-inflammatory effects in patients with DIC associated with septic shock and other severe conditions.

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A.E. Altshuler, A.H. Penn, I. Lamadrid, D. Li, S. Ma, J.A. Yang, L. Kurre, L. Han, G.W. Schmid-Schonbein. University of California, San Diego, La Jolla, CA

Objective: During hypotension, the intestine becomes ischemic resulting in hemorrhagic necrosis, especially in the distal ileum, and inflammatory mediators are released into the peritoneal space, lymph, and circulation. Digestive serine proteases and lipases may contribute to the increased intestinal permeability and inhibitors to these enzymes serve to preserve the structure of the intestine in experimental shock. However, little is known about the effect of serine protease or lipase inhibition in the lumen of the intestine on intestinal permeability.

Methods: To produce complete intestinal ischemia, the proximal jejunum to the distal ileum was excised from male Wistar rats, flushed with saline, and sectioned into 8 equal segments. Segments were cannulated with tubing connectors, filled with 20 μg/ml fluorescein mixed with either saline, the serine protease inhibitors ANGD, aprotinin, or cyclokapron, and the lipase inhibitors orlistat or ANGD, and sealed before placing in conical tubes filled with saline (N=6/group). The fluorescence intensity in the exterior fluid was determined at times 0, 30, 60, 90, and 120 minutes.

Results: Intestinal permeability increased during ischemia and reached higher levels in the distal ileum than the jejunum. ANGD and cyclokapron administration significantly reduced the rate of permeability compared to saline treatment, while orlistat and aprotinin, alone or in combination, were not effective.

Conclusion: During ischemia, permeability across the intestinal wall is increased in the distal ileum where hemorrhagic necrosis develops during ischemia. The rise of permeability was attenuated with ANGD and cyclokapron in the lumen of the intestine. These inhibitors may reduce the transport of inflammatory mediators from the lumen of an ischemic intestine. Supported by GM-85072.

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P. Sharma, B. Benford, M. Bodo. Uniformed Services University, Bethesda, MD

Background: We and others have previously reported low volume hypertonic sodium pyruvate (HSP) as an effective resuscitative fluid in the treatment of hemorrhagic shock (HS). However, the optimum pyruvate dose in the treatment of HS is not known. Our objective was to perform a dose response study with sodium pyruvate as adjuvant resuscitation fluid on systemic physiological response and organ specific parameters in rats with HS.

Methods: Male Sprague-Dawley rats were either treated as sham or subjected to femoral arterial hemorrhage at mean arterial pressure of 40 mmHg for 30 min followed by resuscitation with hypertonic saline (HTS), 10%, 20% or 40% sodium pyruvate (5ml/kg/h) in 60 min, and subsequent reinfusion of shed blood before death. Throughout the experiment, animals were continuously monitored for physiological, hemodynamic, acid- base and metabolic parameters, and protein biomarkers of liver, kidney and muscle damage.

Results: Compared with HTS, both 10% and 20% pyruvate solutions were effective in restoring the HS induced physiological and biochemical variables. However, compared with 20% pyruvate, 10 % pyruvate was more effective in reducing the base deficit, serum Na, K, glucose, LDH and lactate levels. In addition, 10% pyruvate was more effective than 20% pyruvate in preventing the liver, kidney and muscle damage as evidenced by low serum alanine aminotransferase (AST), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN) and creatine phosphokinase (CPK) levels. In contrast 40% pyruvate immediately increased the heart rate and serum lactate, followed by death within 10 min of 40% pyruvate infusion.

Conclusions: These data suggest that 10% sodium pyruvate may be a better choice than 20% or 40% hypertonic sodium pyruvate solution in the treatment of HS.

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R.M. Corrick1, L. Li1, J.L. Messina*1, 2. 1University of Alabama Birmingham, Birmingham, AL, 2Veterans Affairs, Birmingham, AL

Severe injury and infection are frequently accompanied by muscle protein wasting, contributing to high morbidity and mortality during critical illness. Studies in humans and rodents have demonstrated the development of acute growth hormone (GH) resistance in several inflammatory models, providing a potential explanation for the profound erosion of lean body mass that often follows injury and infection. However, the underlying mechanisms of acute GH resistance remain ill-defined. In order to study the effects of injury on growth hormone signaling, we subjected 12-week old male mice to soft-tissue trauma, alone or in combination with hemorrhage, followed by intravenous injection with GH or saline. Livers were harvested 10 min later. Mice subjected to trauma alone exhibited a decline in GH-induced STAT5 tyrosine phosphorylation, with reductions occurring at 0 and 90 min. However, mice subjected to trauma combined with hemorrhage displayed a more severe decrease in GH-induced STAT5 phosphorylation that was evident within 30 min and was more pronounced than the signaling defect observed following trauma alone. Western analysis indicated that GHR abundance was unchanged following hemorrhage. However, there was an apparent 10 kDa decrease in molecular weight of a fraction of hepatic growth hormone receptors (GHR) following hemorrhage, independent of duration. Analysis of N-linked oligosaccharides determined that the decreased size of GHR was not due to changes in glycosylation pattern, suggesting a hemorrhage-dependent cleavage of the GHR. These results suggest that 1) acute injury results in rapid changes in hepatic GH sensitivity, and 2) GHR is rapidly altered following hemorrhage, likely contributing to the more severe growth hormone signaling defects observed in hemorrhaged mice.

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L. Liu*, T. Li, G. Yang, J. Xu, R. Zhou. Research Institute of Surgery, Daping Hospital, Research Institute of Surgery, Chongqing, China

Previous opinion thought that the mechanisms responsible for the incidence of vascular hyporeactivity following shock included adrenergic receptor desensitization and membrane hyperpolarization of vascular smooth muscle cell (VSMC). Based on the basic theory that the contraction efficiency of smooth muscle depends on the ratio of force and calcium (Ca2+ sensitivity), we proposed that VSMC may exist calcium desensitization after shock, which may play important role in the incidence of vascular hyporeactivity. With hemorrhagic and endotoxic shock rats, rabbits and hypoxia, LPS-treated VSMC, we confirmed calcium desensitization existed in blood vessel and VSMC after shock or after hypoxia/ LPS-treatment, the decreased calcium sensitivity played an important role in the incidence of vascular reactivity during shock. The calcium sensitivity of VSMC was regulated by PKC, PKG and Rho kinase following shock. PKC and Rho kinase are mainly through inhibition of myosin light chain phosphatase (MLCP), while PKG is mainly via activation of MLCP to regulate the calcium sensitivity. The interactions among PKC, Rho kinase, integrin linked-kinase (ILK), zipper kinase (ZIPK) and PKC dependent phosphatase inhibitor (CPI-17) played very important roles in vascular hyporeactivity. Small dosage of arginine vasopressor (AVP, with Rho kinase activation effect) and phorbol-12-myristate-13-acetate (PMA, PKC agonist) obviously restored the decreased vascular reactivity. Pinacidil pretreatment (25μg/kg) and ischemia preconditioning implemented 30 min before shock played anti-shock effect via improving vascular reactivity through activation of Rho A-Rho kinase and adenosine -PKC pathway.

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T. Li, L. Liu*. Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing, China

Aim: To investigate whether hypothermia can increase the beneficial effect of hypotensive resuscitation on hemorrhagic shock is not known.

Methods: Before bleeding was controlled, uncontrolled hemorrhagic shock rats received normotensive or hypotensive resuscitation (at 80 or 50 mmHg) in combination with normal (37°C) or mild hypothermia (34°C) (phase II). After bleeding was controlled, rats received whole blood + lactated Ringer’s solution resuscitation for 2 h (phase III).

Results: Short-term, mild hypothermia before bleeding was controlled increased the beneficial effect of hypotensive resuscitation. Hypothermia further decreased blood loss, oxygen consumption, and functional damage to the liver, kidney, and intestines during hypotensive resuscitation, protected mitochondrial function and energy metabolism (activity of Na+-K+-ATPase), and further improved survival time and survival rate (hypothermic/hypotensive combined group: survival rate, 9/10; survival time, 616 min; normothermic/normotensive group: 1/10, 256 min; hypothermic/normotensive group: 4/10, 293 min). Hypothermia slightly inhibited coagulation function.

Conclusion: Mild hypothermia before bleeding is controlled can increase the beneficial effect of hypotensive resuscitation on uncontrolled hemorrhagic shock. The mechanism underlying the benefits of short-term hypothermia may be related to the decrease in oxygen consumption and metabolism, and protection of mitochondrial and organ functions.

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K.K. Nandra1, N.S. Patel1, S. Harwood1, M. Collino2, M. Rogazzo2, M. Yaqoob1, C. Thiemermann1. 1Queen Mary University of London, The William Harvey Research Institute, Barts and The London School of Medicine & Dentistry, London, United Kingdom, 2University of Turin, Department of Drug Science and Technology, Turin, Italy

Pharmacological pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue protective effects against ‘ischemia-resuscitation’ type injuries. This protection may be mediated by mobilisation of endothelial progenitor cells (EPCs) from the bone marrow which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, such as in military conflicts or prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). EPO was administered as a 3 day pre-treatment (1000 u/kg/day i.p.) and rats were subjected to HS on day 4. Mean arterial pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 ml/kg Ringer’s lactate for 10 min and 50% of the shed blood for 50 min. Rats were sacrificed 4 h after the onset of resuscitation. EPC mobilisation was measured following 3 day pre-treatment with EPO in rats and was found to be unchanged when compared to rats pre-treated with PBS. Despite a lack in EPC mobilisation EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic, neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3β (GSK-3β; inhibition), and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing of EPO may protect against the organ injury and dysfunction induced by HS by a mechanism that may involve activation of the Akt/eNOS survival pathway and inhibition of activation of GSK-3β and NF-κB.

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T. Inoue1, 2, S. Nishino1, M. Aoyama1, 3, M. Sasano1, N. Oka1, T. Ueda3, S. Kamoshida1, M. Usami1, J. Kotani*3. 1Kobe University Graduate School of Heath Sciences, Kobe, Japan, 2Ono Ladies Clinic, Ono, Japan, 3Hyogo College of Medicine, Nishinomiya, Japan

Introduction: During testicular inflammation, which often occurs in systemic inflammation, apoptosis in germ cell induced via death receptor pathways and mitochondrial pathways leads to spermatogenesis failure and male infertility. Although IL-18 has been known to associate with apoptosis, the relationship between endogenous IL-18 and testicular germ cell apoptosis is still unclear.

Methods: C57BL/6J mice and B6.129P2-IL-18<tm1Aki>/J (IL-18 KO) mice were injected intraperitoneally with 40 mg/kg LPS. Twelve hours later, testes of each mouse were removed and then one testis was used for gene expression assay and the other testis was used for immunohistochemistry of cleaved-caspase (CC) 3, 8 or 9. The expression of TNF-α, TNFR1, Fas, FasL, FADD, iNOS, Bax, and Mcl-1 mRNA were detected by real-time PCR.

Results: In WT, the CC3 and CC8 or CC3 and CC9 positive germ cells were significantly higher in LPS than in PBS group. In IL-18 KO mice, the rates of those positive cells in LPS group were significantly lower than in WT LPS group. In WT mice, the mRNA expression of TNF-α, Fas and iNOS were significantly higher in LPS than in PBS group. In IL-18 KO mice, the expression of Fas was significantly higher but FasL was significantly lower in LPS than in PBS group. Especially, the expression of Fas and FADD was significantly higher in IL-18 KO LPS group than in WT LPS group. The expressions of TNFR1, Bax and Mcl-1 showed no obvious difference in any groups. These results suggested that IL-18 induced germ cells apoptosis via caspase-dependent death receptor and caspase-dependent mitochondrial pathways.

Conclusion: Our results suggested that endogenous IL-18 is one of the important mediators in testicular germ cell apoptosis during systemic inflammation.

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L. Eid1, Z. Bromberg2, E. Spokoiny2, Y.G. Weiss*1, 3. 1Hadassah Hebrew University Medical Center, Jerusalem, Israel, 2Goldyne Savad Gene Therapy Institute, Jerusalem, Israel, 3University of Pennsylvania School of Medicine, Philadelphia, PA

Objective: The purpose of this study was to characterize IL-6 dynamics during sepsis. We hypothesized that IL-6 classical and trans signaling pathways have divergent roles in sepsis and that their therapeutic modulation, (i.e. preserving protective IL-6 functions transmitted via membrane bound receptor and blocking detrimental effects through the soluble receptor), may be useful for increasing survival after sepsis.

Methods: Sepsis was induced in Balb-C mice using the cecal ligation double puncture method. At 6, 24 and 48 hours the animals were sacrificed and internal organs (lungs, liver, kidneys) harvested. Unoperated and sham operated mice were used as controls. Pathology, immunostaining and immunoblotting studies were performed.

Results: Both the IL-6 and IL-6 mRNA amounts were higher in the kidneys and lungs at 6 hours, followed by decline towards 48 hours, while in the liver a steady increase, peaking at 48 hours was seen. In all three organs pSTAT3 was highest at 6 hours, disappearing at 48 hours. In the lung and liver, the IL-6R receptor was found in the 80 kDa band, while in the kidney, it was found in the 75 kDa band, most likely indicating the soluble receptor.

Conclusion: Since only the soluble IL-6R receptor was found in the kidney, we demonstrated not only that IL-6 is locally produced in solid organs but that it also exerts its actions through an auto/juxta/paracrine mechanism. Trans signaling blockade may thus be used as a potential tool for kidney protection during sepsis. pSTAT3 as a marker of inflammation reflects the dynamics of sepsis: a surge of IL-6 during early sepsis corresponding to a hyper inflammatory state followed by a hypo inflammatory state matching the anergy seen in late sepsis.

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G. Bauza, R. Moitra*, D. Remick*. Boston University Medical Center, Boston, MA

Previous studies demonstrated that naïve plasma has inherent capabilities to enhance bacterial killing and that not all plasma is equally effective. Adenosine receptor antagonists have been shown to modulate cytokine response and survival in mice after a bacterial challenge. We investigated whether selective adenosine receptor blockade would influence the ability of naïve plasma to effectively control bacterial growth.

Colonic bacteria, plasma and Thioglycolate elicited peritoneal macrophages were obtained from naïve mice. Bacteria and plasma were incubated in order to allow for opsonization and then added to macrophages previously exposed to selected adenosine receptor antagonists; Theophylline: a non-selective antagonist, ZM 241385: A2α and A2β receptor antagonist, and DPCPX: A1 receptor antagonist. Final mixture was plated in blood agar plates in aerobic and anaerobic conditions and allowed to grow for 24 hours. After this timeframe bacterial colonies were counted and compared to a control culture of bacteria without plasma or macrophages.

Previous work in our lab resulted in developing an assay that could differentiate plasma that benefited bacterial killing against plasma that was not beneficial or even detrimental to this process. Blocking adenosine receptors with non-selective and selective antagonists did not alter the bacterial killing capacity of plasma. Plasma alone 5.9x105 cfu vs Antagonists combined 4.5x105 cfu (P=0.95). There was also no significant difference between the effects of the different receptor antagonists compared between each other.

Our results indicate that blocking adenosine receptors in macrophages does not change the bacterial killing capacity of plasma previously determined to improve bacterial killing pointing to other mechanisms by which plasma enhances bacterial killing.

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M. Chang, P. Cabrales*, G.W. Schmid-Schonbein*. UC San Diego, La Jolla, CA

Intestinal ischemia encountered in hemorrhagic shock, sepsis or cardiovascular surgery results in systemic responses leading to multi-organ failure; but the mechanisms are still not well characterized. We have shown that in early periods of gut ischemia disruption of mucin isoforms in the mucosal barrier is accompanied by entry of digestive enzymes into the intestinal wall, a process that starts intestinal injury. We hypothesize that depletion of oxygen and ATP in the ischemic intestine results in disruption of mucin. Accordingly we investigate the use of enteral ATP-Mg salt or oxygenated perfluorocarbons during intestinal ischemia as potential treatments for mucin breakdown. We used a rat model of ischemia by occlusion of the superior mesenteric and celiac arteries (SAO) for 30min; animals with SAO had either an injection of oxygenated perfluorocarbon or ATP-Mg salt solution into the intestinal lumen; sham animals had treatments but without SAO. After the surgery, jejunal sectors were dissected and processed for analysis. Western blot shows that during SAO mucin 2 (an isoform covering the epithelial cells) is degraded and mucin 13 (a membrane bound isoform) is fragmented with appearance of bands not observed in the sham group. Enteral treatment with either ATP-Mg salt or oxygenated perfluorocarbon significantly attenuated mucin 2 and mucin 13 disruption. We conclude that during early periods of ischemia the mucin barrier is disrupted as result of oxygen and ATP depletion, this event allows entry of mediators of shock such as digestive enzymes into the intestinal wall. Enteral treatment with supplemental oxygen or ATP during intestinal ischemia serves to attenuate intestinal ischemic injury and sheds light into the possible mechanism leading to the breakdown of the intestinal mucosal barrier. Supported by GM85072.

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K. Waldrep, J. Slack, M. Simovic, J. Dalle Lucca. United States Army Institute of Surgical Research, Fort Sam Houston, TX

Traumatic hemorrhage is the leading cause of death among military casualties, also working population of industrialized countries. Recent evidence indicates the innate immune system playing a major role in the pathogenesis of trauma and adverse outcomes. The complement system appears to represent the crucial effector of this innate immune response in the early phases after major trauma. Despite its generic beneficial functions, including pathogen elimination and immediate response to danger signals, complement activation appears to be exerting detrimental effects after trauma, mounting innocent bystander attack on host tissues. Mortality in trauma has remained unchanged over the last decade despite the recent advances in treatment protocols. In addition to high mortality rates, patients with hemorrhagic shock after critical trauma involving hypovolemia and subsequent tissue malperfusion also suffer from injury-induced morbidity stemming from a second hit insult due to subsequent immunological reactions. Conventional fluid resuscitation is designed to reestablish tissue perfusion but it fails to prevent inflammatory responses and coagulopathy. Conversely, fluid resuscitation can further boost inflammatory responses and worsen coagulopathy which could be more dangerous than the original injury. Identification of novel pharmacologic agents that can maintain homeostasis and decrease the excessively generated systemic inflammatory response after major trauma is of upmost importance. In this study we evaluate the use of early complement inhibition in the amelioration of death-associated trauma. Specifically we investigate the use of C1-Inhibitor to attenuate mortality and morbidity in a swine trauma hemorrhage model. Initial results indicate systemic complement inhibition attenuates tissue damage after injury.

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D.F. Niño2, 1, D.M. Cauvi1, A. De Maio*1. 1Department of Surgery and Center for the Investigation of Health and Educational Disparities, University of California - San Diego, La Jolla, CA, 2Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, CA

Itraconazole (ICZ) is commonly used in the treatment of fungal infections, particularly in immunocompromised patients. In addition, ICZ has been recently found to have anti-angiogenic effects and is currently being tested as a new chemotherapeutic agent in several clinical trials. We have shown that ICZ impairs complex N-linked glycosylation processing, leading to the accumulation of high-mannose glycoproteins on the surface of macrophages (Mø). This investigation was directed at determining the implications of the glycosylation defect on a major function of Mø, phagocytosis. Murine Mø (J774.1 cells) were cultured in suspension for 24 h and treated with ICZ (1 μM) for 16 h at 37°C. The phagocytic index and Fcγ III/II receptor (Fcγ III/IIR) expression (total and cell surface) were determined by flow cytometry. Gene expression was evaluated by quantitative RT-PCR. We found a statistically significant decrease in the phagocytosis of opsonized bacterial particles by ICZ treated Mø (80%) in comparison with non-treated Mø. Furthermore, this impairment was paralleled by a decrease in cell surface and total Fcγ III/IIR expression (50%) as well as a reduction in mRNA levels (60%) of all three isoforms of the Fcγ receptor family (i.e. Fcgr1, Fcgr2 and Fcgr3). The electrophoretic mobility of Fcγ III/IIR in SDS-PAGE was altered after ICZ treatment, indicating that there was, indeed, deficient glycoprotein processing. Therefore, these studies indicate that ICZ treatment certainly has a dramatic effect on Mø function, which could result in a potential impairment of the immune system’s ability to respond to bacterial infections. Finally, the proposed use of ICZ as a chemotherapeutic agent warrants further investigation to determine the potentially detrimental effects of this drug on the immune response in patients.

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F. Zarnani2, D. Maass*1, J. Wigginton*1, L. Ma1, R. Glosser2, A.H. Idris*1. 1University of Texas Southwestern Medical Center, Dallas, TX, 2University of Texas at Dallas, Richardson, TX

Introduction: Raman spectroscopy has many applications and potential for the medical diagnosis of diseased tissue and cells.

Objective: To identify the unique Raman spectra of skin, blood, and cerebral spinal fluid (CSF) from unburned and burned rats at different time points, and to identify unique spectral characteristics.

Methods: A Horiba Jobin-Yvon Raman micro-spectrometer (LabRam HR800) was used to collect the Raman spectra of ex vivo skin, blood and CSF of unburned and burned skin (Sprague Dawley rats, N=31, 40% TBSA) at 1, 6, 12, and 24 hours post burn. Samples were excited by a 633 nm laser, focused by the 50x objective (skin) and 100x (fluids) of a confocal microscope (Olympus). Spectra of 5 random points on each sample were collected and averaged in the 100 - 3500 cm-1 spectral region.

Results: We identified the spectral bands of each sample group, and found that Raman spectra can distinguish samples collected from unburned and burned rats. Multivariate statistical techniques were used for quantitative analysis and classification of samples from each group. We also observed changes in Raman spectra of skin samples that correlated with the post-burn time. We also found an increase of fluorescing constituents in skin, as a result of the burn injury.

Conclusion: Qualitative and quantitative analysis of Raman spectra show that Raman spectroscopy can be used to distinguish unburned from burned rat skin, blood, and CSF.

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S.A. Thacker2, D. Tweardy*1. 1Baylor College of Medicine, Department of Medicine, Section of Infectious Diseases, Houston, TX, 2Baylor College of Medicine, Department of Pediatrics, Section of Infectious Diseases, Houston, TX

Trauma is a major cause of mortality in the U.S. Those surviving initial trauma often succumb to multiple organ failure involving the liver, heart, and lung. Trauma with hemorrhagic shock (T/HS) has been shown to cause hepatocyte, cardiomyocyte, and alveolar epithelial cell apoptosis. IL-6 given at resuscitation was shown to prevent T/HS-induced apoptosis in these cells, and Stat3 largely mediated this. Specific genes contributing to apoptosis and its prevention; however, were not clearly delineated. Endoplasmic reticulum stress elicits the unfolded protein response (UPR), which, with marked activation, can lead to apoptosis. Prior studies of hepatic, cardiac, and pulmonary injury have examined a limited repertoire of the UPR, making it difficult to assess the maladaptive or inadequately adaptive role of the UPR in T/HS in these organs.

Global UPR transcriptome analysis was performed using Affymetrix chips of 4 groups of rats: sham, T/HS, T/HS+IL-6, and T/HS+IL-6 pretreated with a Stat3 inhibitor. Ingenuity Pathway Analysis and GO were used to create a UPR-associated gene list. Genespring was used to interpret the impact of T/HS and interventions on those UPR mediators.

T/HS significantly altered 12% of the UPR transcripts across all organs, with the most impacted canonical UPR members being DDIT3, GADD34, and ATF4 (p<0.05, ANOVA). T/HS also induced organ-specific UPR transcriptome changes, identifying the chaperone Hsp70 as the most dysregulated in the liver and heart following T/HS (25 and 10 fold, respectively; p<0.05, ANOVA) and Eif2ak2 identified as the most dysregulated in the lung (4.7 fold; p<0.05, ANOVA). Hsp70 may contribute to hepatocyte protection via an IL-6-mediated Stat3-dependent pathway, identifying a potential novel therapeutic strategy to prevent hepatocyte death and organ dysfunction in T/HS.

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F. Allantaz Frager1, F. Turrel Davin1, V. Barbalat1, F. Venet2, E. Cerrato1, A. Pachot1, G. Monneret2, A. Lepape3. 1bioMerieux, Marcy l’Etoile, France, 2Hopital E. Herriot, Lyon, France, 3Hopital Lyon Sud, Pierre-Benite, France

Monocytes/macrophages that have been exposed to minute amounts of endotoxin become refractory to further endotoxin challenge, a phenomenon called endotoxin tolerance (ET). A similar loss of LPS-reactivity has been reported in leukocytes from septic patients in whom a pronounced immunosuppressive state is observed that leads to increased mortality and susceptibility to nosocomial infections. New therapeutic strategies aim at restoring a functional immune response by treating septic patients with appropriate immunostimulatory drugs.

We have established a robust ex vivo model of ET allowing us to test different immunostimulatory drugs such as IFNg that septic patients could benefit from. In this two-hit model of ET with healthy PBMCs, using gene expression profiling, we identified a panel of transcripts that we classified as either “tolerizable” or “non tolerizable” depending on their expression profile upon LPS stimulation. We then evaluated the effect of adding IFNg on their expression and identified 20 transcripts whose expression was restored upon IFNg addition. Finally, we confirmed that those transcripts are differentially expressed in septic shock patients’ blood compared to healthy blood upon ex-vivo LPS stimulation but were restored when IFNg was added to the culture.

We therefore identified a panel of genes that will allow us to identify septic shock patients that could benefit from an immunostimulatory treatment and to monitor drug efficacy.

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Y. Nagura, T. Oshiro, T. Harada, M. Takeda, A. Yaguchi*. Tokyo Women’s Medical University, Tokyo, Japan

In the previous study, we showed the initial PAC data reflected each shock status. The purpose of the present study is to know how PAC data will change after intervention in septic shock patients and cardiogenic shock. Of 220 patients received PAC in our ICU from 1996 to 2010, 112 septic shock patients and 43 cardiogenic shock patients were included in this retrospective study. The initial, the second and the last data of PAC, including mean arterial pressure (MAP) (mmHg), pulmonary capillary wedge pressure (PCWP) (mmHg), cardiac index (CI) (l/min/m2), mixed venous oxygen saturation (SvO2) (%), oxygen delivery and consumption (DO2 and VO2) (mL/min) were compared. Values are expressed as median. Data was analyzed by Kruskal-Wallis test, Mann-Whitney U test, and Wilcoxon signed-rank test. P values less than 0.05 were considered significant. In septic shock, MAP was significantly different between the initial, the second and the last measurements (77 vs. 78 vs. 89 mmHg, p<0.05). There were significant differences in SvO2 between the initial and the second measurements (72 vs. 75 %, p<0.05). There were no significant differences in PCWP, CI, DO2 and VO2 between each measurement. In cardiogenic shock, MAP had also significant differences between each measurement (80 vs. 86 vs. 95 mmHg, p<0.05). CI and SvO2 more increased at the last than at the initial measurements (2.3 vs. 2.8 l/min/m2, 67 vs. 70 %, p<0.05, respectively). PCWP, DO2 and VO2 did not show any differences. SvO2 were increased both in septic and in cardiogenic shock patients, while CI was improved in cardiogenic shock after treatment. The changes of PAC variables could reflect as a therapeutic.

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E.C. Carnio, L.G. Branco. University of Sao Paulo, Ribeirao Preto, Brazil

This study was designed to evaluate the effects of neonatal immune challenge induced by low doses of lipopolysaccharide (LPS) on adult rats during endotoxemic shock. We have assessed mean arterial pressure (MAP), heart rate (HR), plasma vasopressin (AVP) concentration, body temperature (Tb) (Tb), and plasma nitric oxide (NO) concentration. Animal care was carried out in compliance with the guidelines set by the Brazilian College of Animal Experimentation (COBEA), and with the approval of the Animal Care and Use Ethics Committee of the University of São Paulo. Male Wistar rats were exposed to LPS (100 μg/ kg i.p.; nLPS) or saline administration (nSal) 14 days after birth (P14). On day 50 after birth, endotoxemic shock was induced by intraperitoneal injection of 10 mg/kg LPS, on rats previously implanted with polyethylene catheters in the femoral artery and loggers for Tb measurements. In nSal rats, LPS injection induced a transitory increase in AVP plasma concentration, a decrease in MAP with a concomitant increase in HR, which was statistically significant from 1 hour (p <0.01) up to 6 hours (p <0.001) after treatment. LPS-induced hypothermia (p <0.05) was observed for 2 hours after LPS administration, and was followed by an increased Tb (p <0.01). In nLPS rats we observed attenuation on the development of hypotension, no significant change in HR (p<0.05), an increased hypothermia, a decreased febrile response, a further increased (p<0.01) in plasma AVP levels and increase in NO (p<0.01) concentration were observed, in response to LPS administration.

In conclusion our data support the notion that neonatal exposure to LPS improves the shock induced by endotoxemia, and this response may involve an increased circulation of AVP and NO.

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E. Sherwood*1, 2, Y. Guo1, G. Fang1, D.S. Herzig1. 1UTMB, Galveston, TX, 2Shriners Hospital for Children, Galveston, TX

Introduction: Our previous studies show that natural killer (NK) cells facilitate systemic inflammation during septic shock caused by cecal ligation and puncture (CLP). The chemokine receptor CXCR3 is expressed by NK cells and is activated by CXCL9 and CXCL10. However, the role of CXCL9 and 10 in modulating NK cell functions during sepsis is unknown. This study was undertaken to test the hypotheses that CXCL9 and CXCL10 are important regulators of NK cell function during sepsis and that neutralization will attenuate CLP-induced pathobiology.

Methods: Bacterial clearance, core temperature, cytokine production, survival and NK cell trafficking were evaluated in CXCL10 knockout mice and in wild type mice treated with anti-CXCL9, anti-CXCL10 or a combination of anti-CXCL9 and 10. Isotype-matched, nonspecific IgG served as a control. In some experiments mice were also treated with antibiotics (Primaxin 12.5 mg/kg).

Results: Our study shows that NK cell trafficking into the peritoneal cavity was attenuated in CXCL10-deficent mice. Survival and core body temperature were significantly improved in mice treated with anti-CXCL9, anti-CXCL10 and anti-CXCL9/10 plus Primaxin compared to mice receiving non-specific IgG plus Primaxin whereas antibody treatment without antibiotics did not improve the functional outcome compared to mice treated with IgG. However, bacterial clearance was improved and systemic cytokine production attenuated in CXCL10 knockout mice that did not receive antibiotics.

Conclusion: These results indicate that CXCL9 and CXCL10 participate in regulating NK cell trafficking during septic shock caused by CLP and that neutralization of these receptors attenuates sepsis-associated immunopathology, particularly when co-administered with antibiotics.

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B. Glawe1, P. Wall*1, 2, S. Sahr1, J. Baker1, C. Leib1, C. Renner1, M. Arends1, C. Buising2. 1Iowa Methodist Medical Center, Des Moines, IA, 2Drake University, Des Moines, IA

Objective: To explain more frequent blood administration to females despite admitting more males and administering more units per patient to males.

Methods: Review trauma registry data Oct 2007-Sept 2010.

Results: Falls were the most common trauma (Table), were frequently older females (Fig, Fall ages p<0.001), and were more likely to receive blood than vehicular trauma (p<0.001). Of blood recipients who fell, males more frequently received more units (p=0.03). Older recipients had lower Injury Severity Scores and higher blood pressures which were associated with fewer units administered.

Conclusions: With an aging population, falls are likely to be an increasing portion of all traumas, and older women are likely to be an increasing fraction of blood recipients even with lower injury scores.

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E. Brandon-Warner1, A.M. Lakner1, J.H. Swet1, I.H. McKillop1, S.L. Evans*2. 1Carolinas Medical Center, Charlotte, NC, 2Ross Trauma Center, Carolinas Medical Center, Charlotte, NC

Trauma related hemorrhage is a significant cause of mortality with acute alcohol intoxication a mitigating factor in half of all trauma admissions. Injury associated with hemorrhagic-shock/resuscitation (HSR) results from hypoxia, reactive oxygen species (ROS), and oxidative stress that alters cell energy metabolism leading to cellular, individual, then multiple organ dysfunction syndrome, and death. An imbalance between ROS and antioxidant capabilities precipitates mitochondrial dysfunction, and cytochrome C release initiating apoptosis, effects exacerbated by alcohol/alcohol metabolism. In the absence of sufficient ATP, necrotic cell death prevails. These changes are evident in a rat model of HSR with necrosis identified in H&E stained tissues, and significantly (p<0.05, 93.2%) increased cytochrome C in HSR vs. Sham animals by Western blot of liver lysates. To determine if antioxidant Resveratrol (R) could blunt similar pathological events in vitro, GP7 cells, ± ethanol (E, 25 mM) pretreatment (2 hrs), were subjected to hypoxia (24 hrs), ± R(75 μM), on reversal of hypoxia. A 79.3% increase in survival was measured in cells that received R after hypoxia, and 83.3% in R treated cells +E. Measures of lipid peroxidation by TBARS for malondialdehyde (MDA), and cell lysis by lactate dehydrogenase (LDH) were significantly (p<0.05) decreased by R after hypoxia alone(45.8%-MDA, 46.6%-LDH), and after E (35.2%-MDA, 52.1%-LDH). Western blot of cell lysates confirmed increased cytochrome C. Mitochondrial superoxide anion production was also reduced by R after hypoxia alone, and after E. These data indicate that R may offer protection from hypoxic injury, ROS, and mitochondrial dysfunction thereby inhibiting necrotic cell death and preventing injury cascades and progression of HSR following alcohol exposure.

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A.V. Kozlov*, B. Faulhammer, P. Dungel*. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria

Prolonged ischemia of the adrenal glands leads to focal infarcts which compromise normal function of the glands. Nitrite was shown to protect various organs from hypoxia/reoxygenation (H/R) injury. We aimed to establish an in vitro model of H/R to investigate cytoprotection of the adrenal glands by nitrite.

PC12 were incubated at <1% oxygen and various concentrations of nitrite. Reoxygenation was performed by medium exchange and incubation at 21% oxygen. Cellular injury was analyzed by Annexin-V/propidium iodide staining and determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Effects on gene expression were determined by real-time PCR. Effects of nitrite were compared to those of nitric oxide (NO) using the NO-donor SNAP.

Under hypoxic conditions PC12 cells were able to reduce nitrite to NO. 1 hour of hypoxia followed by 2 hours of reperfusion led to distinct injury of PC12 cells confirmed by significantly elevated levels of AST and ALT and an increase in the amount of necrotic cells by 60%. Apoptosis rate was not affected by hypoxia. Effects of SNAP were similar to those of nitrite, suggesting that nitrite effects are indeed mediated by NO. Low concentrations of nitrite (5μM) decreased HO-1 expression compared to untreated controls. Nitrite had no positive effect on cell survival but increased necrosis at high concentrations.

Our data show that under hypoxic conditions PC12 cells were able to convert nitrite to NO, but this did not prevent hypoxic injury in our model. Moreover, we observed further damage of cells in the presence of nitrite. This suggests that the protective effects of nitrite are systemic processes rather than local effects in a specific cell type. Supported by FWF grant P21121.

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J. Fishman, G. Levy, V. Alli, E. Deitch*. UMDNJ - NJMS, Newark, NJ

Objective: Gut barrier failure has been shown to be important in the pathogensis of pancreatitis induced infection as well as SIRS, ARDS, and MODS. However, the mechanisms by which pancreatitis leads to early gut injury and loss of barrier function are largely unknown. Thus we tested the hypothesis that injury to the gut mucus layer is a critical factor in pancreatitis induced gut injury and dysfunction, as the mucus layer has been shown to be a major component of the gut barrier.

Methods: Male Sprauge-Dawley rats underwent laparotomy with pancreatic duct ligation (PDL) induced pancreatitis for 4.5 hours. Sham rats were subjected laparatomy without PDL. Gut permeability was measured by the passage of intraluminaly injected FD4 across the gut wall into the bloodstream. Segments of distal ileum were fixed and stained to visualize and quantify the mucus layer and intestinal villous injury. Mucus from the terminal 30cm of the distal ileum was collected, processed, and analyzed. Reactive nitrogen intermediate (RNI) mediated damage was measured using an ELISA for nitrated tyrosine residues. Reactive oxygen species (ROS) mediated damage was measured via ELISA for oxidized carbonyl derivatives. Total antioxidant capacity (capacity to respond to oxidant mediated damage) was measured via ELISA.

Results: Pancreatitis increased intestinal permeability (FD4) and this loss of gut barrier function correlated with histologic evidence of loss of mucus layer but not villous injury (see Table 1). Loss of gut barrier function and histologic damage correlated with an increase in ROS and RNI mediated damage as well as loss of mucus antioxidant capacity (Table 1).

Conclusion: These results support the conclusion that the major pathway by which pancreatitis induces gut barrier failure is damage to the mucus layer.

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M. Bohan Brown, L. Li, J.L. Messina*. University of Alabama at Birmingham, Birmingham, AL

Hepatic ER stress has been associated with the chronic development of insulin resistance. Hyperglycemia and insulin resistance induced by acute injury or infection, referred to as critical illness diabetes, increases mortality and morbidity. However, it is not known whether the ER stress response plays a role in the acute development of hepatic insulin resistance following injury. A rat model of trauma and hemorrhage was used where rats were maintained at a mean arterial pressure of 35-40 mmHg for 15 - 90 minutes. Rats became insulin resistant by 15 minutes following hemorrhage, and remained so up to 90 minutes, as evidenced by decreased insulin-induced phosphorylation of the insulin receptor, insulin receptor substrate 1 (IRS-1), and Akt. Phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) and protein kinase-like endoplasmic reticulum kinase (PERK) were measured as markers of ER stress, as well as the cellular-stress activated c-Jun NH(2)-terminal kinase (JNK). Activation of eIF2α and JNK were observed by 15 minutes, and continued to be elevated at 30, 60 and 90 minutes, after hemorrhage. The activation of PERK was significantly increased at 30, 60, and 90 minutes after hemorrhage. Therefore, the ER stress response rapidly develops, and with the same time course as insulin resistance, following trauma and hemorrhage. However, the specific role of the ER stress response in the development of acute insulin resistance is not known, but suggests that treatments to alleviate ER stress following acute injury may decrease insulin resistance and normalize metabolism.

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R.C. Perez*, M.E. Kwiatt, J. Lachant, S. Zanotti, S.M. Hollenberg. Cooper University Hospital, Camden, NJ

Introduction: Nonlinear analysis of hemodynamic parameters may reveal insights undetected by standard linear measures such as heart rate (HR). We have previously shown decreased heart rate variability (HRV) during sepsis. We hypothesized that surgical stress decreases HRV in the post-op period.

Methods: Radiotransmitters were implanted into the ascending aorta of C57/BL6 mice (n=18) and continuous HR measurements were taken for 24hr (Telemeter group). Following at least 3 days of recovery, recordings were taken for 24hr (Baseline). 3 groups were tested, each recorded for 24 hr: Anesthesia alone (Anesthesia); sepsis induction by laparotomy, cecal ligation and puncture (Sepsis); and exploratory laparotomy and closure (Sham). Beat to beat intervals were measured, and HRV (% of 5-minute standard deviations < cutoff, defined as lowest 5% during baseline) was compared among groups.

Results: Mean HR did not differ among groups: Baseline 529 bpm, Telemeter 522 bpm, Anesthesia 532 bpm, Sham 580 bpm (p=NS) but was lower for Sepsis 397 bpm (p<0.001) as previously shown. Low HRV was found in more post-op intervals for the 24hr following each surgical intervention: Sham 30.2% at 8hr, Telemeter 42.2% at 8hr, Sepsis 88.2% at 4hr post-op (p<0.05 vs Baseline for all). Sham and Telemeter groups had a gradual return close to Baseline at 24hr, while Sepsis continued to have low HRV (68.1% of intervals at 24hr). The Anesthesia group had minimal change of HRV.

Conclusions: Heart rate does not change following post-op surgical stress, but hemodynamic volatility decreases significantly, albeit to a lesser degree than sepsis. These affects are not attributable to anesthesia. Nonlinear measures were more sensitive indicators of stress. Measurement of HRV in the post-op period may be a useful method to assess recovery from surgical stress.

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L.N. Torres, J. Sondeen*, I. Polykratis, M.A. Dubick*, I. Torres Filho. US Army Institute of Surgical Research, San Antonio, TX

Hemorrhage is responsible for a large percentage of trauma-related deaths but the mechanisms underlying tissue ischemia are complex and not well understood. The vascular endothelium plays a central role in regulation of inflammatory and coagulation responses, local blood flow, and barrier function. We hypothesize that hemorrhage causes endothelial glycocalyx degradation in cremaster muscle and mesentery microvessels. We utilized intravital microscopy to estimate glycocalyx thickness in single microvessels while other microvascular parameters were measured using non-invasive optical techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. We studied post-capillary venules (< 20 μm diameter) of anesthetized rats subjected to hemorrhage (40% of total blood volume). Control rats were equally instrumented but not bled. Dextrans of different molecular weights labeled with FITC or Texas Red were injected. Glycocalyx thickness was estimated from the widths of the fluorescence columns and from anatomical diameter. While control rats did not show remarkable responses, a statistically significant decrease of 64 % in glycocalyx thickness was measured in venules after hemorrhagic shock which may have significant impact in its pathophysiology. Integration between intravital microscopy assessments and measurements of pertinent systemic parameters may be important tools to identify mechanisms by which resuscitation fluids may improve tissue recovery and outcome following hemorrhage.

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N. Habashi1, S.K. Roy2, G.F. Nieman*2, P. Andrews1, J. Statlin2, L. Gatto2. 1University of Maryland, Baltimore, MD, 2SUNY Upstate, Syracuse, NY

Airway Pressure Release Ventilation (APRV) is a mechanical ventilation strategy that has shown promise in prevention and treatment of the acute respiratory distress syndrome (ARDS). Appropriate technique in APRV application and adjustment of release time (T Low) is essential for minimization of alveolar collapse and maintenance of alveolar stability, critical to protecting the lung. We hypothesized that optimization of T Low to limit lung volume loss during the release phase would occur at a specific point during Termination of the Peak Expiratory Flow Rate: specifically at a Peak Expiratory Flow Rate (T-PEFR/PEFR) of 75% and lower ratios would not achieve alveolar stability throughout the respiratory cycle.

Methods: Alveolar stability was measured by alveolar microscopy. In-vivo microscopic fields (n=9) were prepared in anesthetized, male Sprague-Dawley rats. ARDS was induced by instilling 0.2% Tween-20 via tracheostomy. T-PEFR:PEFR was set at 10% and video in-vivo alveolar microscopy performed for multiple respiratory cycles. This procedure was repeated at T-PEFR:PEFR of 25%, 50%, and 75% by decreasing the T Low respectively. Quantification of alveolar stability was measured using image analysis software to determine the percent of inflated alveoli occupying the microscopic field at inspiration and at expiration.

Results: T-PEFR:PEFR of 75% had the least alveolar volume change at expiration (10.0%) while 10%, 25%, and 50% had significantly more alveolar collapse at expiration (54.5%, 36.4%, and 29.4% respectively p<0.001 vs T-PEFR 75%).

Conclusion: APRV as a treatment or prevention of ALI/ARDS is contingent on the appropriate technique which optimizes the Expiratory Flow Rate to maximize alveolar stability. These data confirm that a T-PEFR:PEFR of 75% is necessary to achieve maximum alveolar stability.

In vivo

In vivo

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S.K. Roy, A.J. Ghosh, J.M. Black, K. Hojnowski, M. Kuhn, B. Shapiro, J. Satalin, K. Snyder, L. Gatto, G.F. Nieman*. SUNY Upstate Medical University, Syracuse, NY

Background: Fluid therapy is standard treatment used to correct the hemodynamic compromise associated with severe sepsis. Hemodynamic compromise in early sepsis can be corrected by fluid therapy. In late sepsis, hemodynamic compromise is not responsive to fluid resuscitation; the postulated mechanism for compromise is mediated by mitochondrial dysfunction and microvascular heterogeneity.

Hypothesis: We hypothesize that there is a distinct point of inflection in fluid responsiveness that would identify the transition in fluid responsiveness from early to late sepsis.

Methods: Yorkshire pigs (30-40kg) were anesthetized & subjected to 2-hit injury: 1) Ischemia-Reperfusion- superior mesenteric artery was clamped for 30 min and 2) Peritoneal Sepsis- a fecal clot was placed in the peritoneal cavity. Animals received continuous monitoring and support per Surviving Sepsis Campaign guidelines; experimental duration was 48 hours. Data were taken before bolus initiation and 10 min after bolus termination.

Results: The first 8 fluid boluses (B1-B8) resulted in significant changes in SvO2, mixed venous oxygenation, 0.016%/mL bolus volume) and MAP, mean arterial pressure, (0.041 mmHg/mL bolus volume). Subsequent fluid boluses (B9-B16) resulted in minimal responses in SvO2 and MAP (0.011%/mL bolus volume and 0.0083 mmHg/mL bolus volume). The difference in change in MAP is statistically significant (p = 0.01) but the difference in SvO2 is not (p = 0.20). There is a distinct point of inflection between early and late sepsis, about halfway through the course of fluid therapy, after which the effect of fluid resuscitation on changes in hemodynamic parameters is greatly diminished (Figure 1).

Conclusion: Our findings suggest that we can identify the transition from early to late sepsis based on the hemodynamic responses to fluid therapy.

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Y. Yao. Burns Institute, First Hospital Affiliated to the Chinese PLA General Hospital, Beijing, China

The recent discovery of high mobility group box-1 protein (HMGB1) as a critical mediator of inflammatory diseases has stimulated tremendous interest in the field of inflammation. However, it is not clear whether HMGB1 can induce the activation of regulatory T cells as well as regulatory dendritic cells, and subsequently effector T cells, after the burn injury in vivo. With both animal experiments and clinical investigation, the present study was performed to explore the potential role and its regulatory mechanism underlying HMGB1 in host immune dysfunction in burn shock. It was found that HMGB1 formation could markedly influence cell-mediated immunity, including T lymphocytes, regulatory T cells and dendritic cells. Excessive release of HMGB1 could stimulate CD4+CD25+ regulatory T cells to maturation, and induce the marked differentiation of IL-10-producing CD11clowCD45RBhigh dendritic cells, thereby mediating immunosuppression of T lymphocytes in the setting of burn shock. While treatment with HMGB1 inhibitors, the proliferative activation of splenic T lymphocytes increased from the 1st day to the 7th day in animals after severe thermal injury, and the mortality rate was significantly reduced compared to those without treatment with HMGB1 inhibitors. Clinical data showed that plasma HMGB1 levels were negatively correlated with T cell immune function, including lymphproliferation response, IL-2 release, and the ratio of CD4+/CD8+ T lymphocytes in patients suffering from severe burns. These data proved that HMGB1 is not only identified as a novel “late” inflammatory mediator but also closely associated with immune depression after burn shock.

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T. Shibamoto1, W. Zhang1, 2, Y. Kuda1, Y. Kurata1. 1Kanazawa Medical University, Uchinada, Japan, 2Research Center for High Altitude Medicine, Xining, China

To determine fluid extravasation in the splanchnic vascular bed during anaphylactic hypotension, the mesenteric lymph flow (Qlym) was measured in anesthetized rats sensitized with ovalbumin, along with the systemic arterial pressure (Psa) and portal venous pressure (Ppv). When the antigen was injected into the sensitized rats (n=10), Psa decreased by 88 mmHg at 10 min with a gradual recovery, while Ppv increased by 16 mmHg at 2 min and returned to the baseline at 10 min. Qlym increased 3.3-fold from the baseline of 0.023±0.002 g/min to the peak levels of 0.075±0.009 g/min at 2 min, and returned to the baseline within 12 min. The lymph protein concentrations increased after antigen, which indicated increased vascular permeability. To determine roles of the Ppv elevation in the increase in Qlym, Ppv of the non-sensitized rats (n=10) was mechanically increased in a manner similar to that of the sensitized rats by compressing the portal vein near the hepatic hilus. Unexpectedly, Ppv elevation alone produced a similar increase in Qlym, with the peak comparable to that of the sensitized rats. This finding aroused a question why the antigen-induced increase in Qlym was limited despite the presence of increased vascular permeability. Thus, the changes in splanchnic vascular surface area were assessed by measuring the mesenteric arterial flow. The mesenteric arterial flow was decreased much more in the sensitized rats (75%; n=5) than the non-sensitized Ppv elevated rats (50%; n=5). In conclusion, mesenteric lymph flow increases transiently only soon after antigen presumably due to increased capillary pressure of the splanchnic vascular bed via downstream Ppv elevation, and increased vascular permeability in anesthetized rats. However, this increased extravasation is limited by decreased vascular surface area.

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B. Relja, J.P. Horstmann, K. Wilhelm, T. Lustenberger, I. Marzi*. Hospital of the Goethe-University Frankfurt am Main, Department of Trauma, Hand and Reconstructive Surgery, Frankfurt am Main, Germany

Objectives: Monocytes play a key role in the development of post-traumatic complications due to their central role in the initiation of the inflammatory response by inflammasome activation. Functional capacity of monocytes after trauma is reduced and delayed recovery critically influences the clinical course. We investigated whether the monocytes from patients early after trauma demonstrate alterations in mRNAs for inflammasome compounds in parallel to the reduced monocyte function.

Methods: Severely injured trauma patients [ISS≥16, n=10] and healthy volunteers [n=10] were enrolled. Buffy coats were used for the method verification [n=10]. After isolation of monocytes by CD14+ microbeads, the cells were assayed for ex vivo interleukin-1beta (IL-1beta) production after LPS-stimulation (10 μg/ml, 24 h) by ELISA measurements. Fresh monocyte mRNA was analyzed by qRT-PCR for caspase-1, NALP-1, NALP-3 and IPAF.

Results: LPS-stimulation increased significantly the IL-1beta release in isolated monocytes from healthy volunteers and buffy coats compared to unstimulated controls (p<0.05). The LPS-response was markedly reduced after trauma compared to healthy volunteers after stimulation. Relative copy numbers for the inflammasome mRNAs for caspase-1 were not markedly enhanced in LPS-stimulated samples. NALP-1 mRNA was significantly enhanced in LPS-stimulated samples from healthy volunteers (p<0.05) and buffy coats (p<0.05), whereas the NALP-1 mRNA expression remained nearly undetectable in LPS-stimulated monocytes isolated after trauma. NALP-3 and IPAF mRNAs showed no significant changes either after LPS-stimulation or after trauma.

Conclusions: Monocyte deactivation occurs early after trauma. Reduced monocytic function is paralleled with profound changes in NALP-1 inflammasome mRNA expression, maybe as part of this process.

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W. Blogowski1, B. Dolegowska1, M. Budkowska2, D. Salata2, L. Domanski3, M.Z. Ratajczak2. 1Pomeranian Medical University, Szczecin, Poland, 2Pomeranian Medical University, Department of Physiology, Szczecin, Poland, 3Pomeranian Medical University, Department of Nephrology, Transplantation and Internal Medicine, Szczecin, Poland

Background: Recently we reported that complement (CC) activation during organ injury may lead to stimulation of regeneration of injured organs due to mobilization of bone marrow-derived stem cells (BMSCs), via release of sphingosine-1-phosphate (S1P) from erythrocytes (Leukemia 2010). Here we examined peri-operative CC activation during ischemia-reperfusion (I/R) following kidney transplantation, and established whether CC activation creates a pro-regenerative environment, as well as, is associated with post-transplant graft function.

Methods: Renal transplant recipients (n=69) were divided into early, slow and delayed graft function groups (EGF, SGF, DGF). Blood samples were collected intra-operatively directly before, and in the 1st and 5th minute of graft reperfusion from the renal vein. CC (C3a,C5a,C5b-9), extracellular hemoglobin, and S1P levels were measured.

Results: Significantly higher C5b-9 levels were observed in SGF/DGF patients comparing to EGF individuals (P<0.001). No significant differences in C3a/C5a levels were observed in analyzed groups. Our results indicate that peri-operative CC activation does not correlate with circulating extracellular hemoglobin nor S1P levels. Enhanced peri-operative CC activation was associated with worst early and long-term (1 year) allograft function. Perioperative levels of selected CC molecules presented high diagnostic value for prediction of SGF/DGF (sensitivity/specificity of 75-89%).

Conclusion: During human renal transplantation selective activation of CC occurs, and is more evidently pronounced in SGF/DGF patients. Peri-operative CC levels are associated with post-transplant kidney graft function, but do not seem to create a biochemical environment that could result in release of pro-regenerative signaling from kidney graft to stimulate systemic BMSCs mobilization. (NCN 2011/01/N/NZ5/01398)

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J.R. Stringham2, 1, E.E. Moore*2, J.N. Harr2, 1, T. Chin2, 1, F. Gamboni1, M. Fragoso2, A. Banerjee1. 1University of Colorado-Denver, Aurora, CO, 2Denver Health Medical Center, Denver, CO

Background: Leukotrienes produced in lung tissue are linked to lung injury after trauma and hemorrhagic shock (T/HS). The creation of these bioactive eicosanoids requires 5-lipoxygenase (5-LO) and 5-lipoxygenase activating protein (FLAP). Prior work shows that mesenteric lymph diversion (MLD) reduces lung injury. It is unknown how MLD affects the association of FLAP and 5-LO.

Hypothesis: MLD will decrease the co-localization of pulmonary FLAP and 5-LO induced by T/HS.

Methods: Rats underwent one of five procedures (n=3 for all groups): Control, Anesthesia, Trauma (anesthesia and laparotomy), T/HS (laparotomy, followed by hemorrhagic shock to a MAP of 30 mmHg for 45 min, and resuscitation) and MLD (T/HS with cannulation/diversion of the mesenteric duct). Lung tissue sections were immunostained for 5-LO and FLAP. Confocal images were taken. FRET was calculated and normalized to the donor signal. FRET signal intensity was analyzed by one-way ANOVA with Tukey post-hoc analysis.

Results: T/HS demonstrated perinuclear co-localization of FLAP and 5-LO in a background of damaged lung. MLD revealed profound FRET signaling in large intra-cellular bodies distinct from the nuclei in otherwise normal lung. MLD had a 2.6-fold increase in FRET signaling over Control, Anesthesia and Trauma (p<0.01 for all). MLD had a 1.5-fold increase in signaling over T/HS, but this did not reach significance.

Conclusion: While T/HS induces the co-localization of FLAP and 5-LO, MLD causes sequestration of these complexes into large cytoplasmic bodies in lung tissue. The results show that both trauma and shock are necessary to induce FLAP and 5-LO to associate. Moreover, the data indicate that mesenteric lymph is necessary to release any synthesized products from these bodies.

FIG. 1

FIG. 1

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C. Li*, T. Ha, X. Wang, X. Zhang, M. Gao, J.L. Kelley, J. Kalbfleisch, R. Kao, D.L. Williams*. East Tennessee State University, Johnson City, TN

We have previously reported that TLR4 deficiency (TLR4-/-) attenuates myocardial ischemia/reperfusion (I/R) injury via down regulation of I/R induced NFκB activation and cardiac apoptosis. We also found that the levels of microRNA-146a (miR-146a) were significantly higher in TLR4-/- hearts than in wild type hearts after I/R. We hypothesized that miR-146a plays a protective role during myocardial I/R injury. To critically evaluate our hypothesis, we transfected mouse hearts (n=6) with lentivirus expressing miR-146a (LmiR-146a). Lentivirus vector (L-vector) served as control (n=5). Seven days after transfection, the hearts were subjected to ischemia (45 min) followed by reperfusion (4 hrs). Infarct size was measured by TTC staining. We also examined cardiac function by echocardiography 3 and 7 days after myocardial I/R. LmiR-146a transfection reduced infarct size by 55% compared with the L-vector control group. Overexpression of miR146a also significantly attenuated cardiac dysfunction at 3 and 7 days after I/R. The levels of IRAK and TRAF6 in the LmiR-146a group were significantly lower than in the L-vector control group. I/R induced NFκB activation was markedly attenuated by LmiR-146a transfection. Overexpression of miR-146a also attenuated I/R induced cardiac apoptosis. In vitro studies demonstrated that transfection of miR-146a into H9C2 myoblast cells significantly attenuated hypoxia/reoxygenation (H/R) induced cell injury, apoptosis and TNFα production. We conclude that myocardial overexpression of miR146a decreases myocardial infarction and attenuates cardiac dysfunction associated with I/R. The protective mechanisms of miR-146a are mediated, in part, by attenuation of myocardial IRAK/TRAF6/NFκB activation and cardiac apoptosis in I/R.

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E. Watanabe*2, 1, B.A. Zehnbauer2, 4, T.G. Buchman*2, 3, H. Hirasawa*1, S. Oda*1. 1Graduate School of Medicine, Chiba University, Chiba City, Japan, 2Washington University, St. Louis, MO, 3Emory Center for Critical Care, Atlanta, GA, 4Centers for Disease Control and Prevention, Atlanta, GA

Purpose: Management of sepsis in critically ill patients remains difficult and requires prolonged intensive care. Genetic testing has been proposed as a strategy to identify patients at risk for adverse outcome of critical illnesses. Therefore, we wished to determine the influence of heredity on predisposition to poor outcome and on duration of ventilator support of intensive care unit (ICU) patients.

Methods: A study was conducted from July 2001 to December 2005 in heterogeneous population of patients from 12 US ICUs represented by the Genetic Predisposition to Severe Sepsis (GenPSS) archive. In 1057 Caucasian critically ill patients with SAPS2 probability of survival of >.2 in the US, 6 functional single nucleotide polymorphisms in relation to inflammatory cytokines and innate immunity (rs1800629, rs16944, rs1800795, rs1800871, rs2569190, and rs909253) were evaluated in terms of mortality and ventilator free days.

Results: The AA homozygote of TNF(-308) (rs1800629) was most over-represented in the deceased patient group (P=.015). The carriage of the TNF(-308)*AA genotype showed significantly higher odds ratio of 2.67(1.29-5.55) (P= .008) after adjustment with the covariates. However, the presence of 1, 2, or 3 acute organ dysfunctions was larger prognostic factors for the adverse outcome (OR(95%CI) = 2.98(2.00-4.45), 4.01(2.07-7.77),or 19.95(4.99-79.72), P<.001 for all). Kaplan-Mayer plot on ventilator duration of TNF(-308)*AA patient significantly diverged from that of TNF(-308)*(GG+GA) ((AA v GG+GA), Adjusted HR(95%CI) = 2.53(1.11-5.79) with Cox regression, P= .028).

Conclusions: TNF(-308)*AA is significantly associated with susceptibility to poor outcome and to longer ventilator duration. Therefore, heredity likely affects both predisposition to ICU prognosis as well as the resource utilization.

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M.H. van Lieshout, D.C. Blok, T. van der Poll, C.W. Wieland, A.F. de Vos, C. van ’t Veer*. Academic Medical Center, Amsterdam, Netherlands

Background & Objective: Pneumonia is frequently caused by gram-negative pathogens, among which Klebsiella pneumoniae prominently features. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is important for an appropriate immune response during infection. TLR signaling can proceed via two distinct routes which are dependent on the adaptor proteins Myeloid differentiation primary response gene (88) (MyD88) and TIR-domain-containing adaptor-inducing interferon-β (TRIF) respectively. Aim of the study was to determine the relative contribution of MyD88 and TRIF signaling in resident and hematopoietic cells to host defense during pneumonia caused by K. pneumoniae.

Methods: Bone marrow chimeras of MyD88 deficient/wild type and TRIF mutant/wild type mice were created and infected with K. pneumoniae via the airways. Bacterial loads and inflammatory responses were determined after 6 and 24 (MyD88 chimeras) or 48 hours (TRIF chimeras) and survival rates were monitored.

Results: MyD88 in both resident and hematopoietic cells contributed to survival and antibacterial defense in late stage infection, whereas only TRIF in hematopoietic cells was protective. On the other hand, resident MyD88 and hematopoietic TRIF contributed to distant cellular injury. Resident MyD88 was pivotal for early chemokine release and neutrophil recruitment in the bronchoalveolar space.

Conclusion: MyD88 and TRIF dependent signaling have a differential contribution to host defense in different cell types that changes from early to late stage gram-negative pneumonia.

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D. Kudo1, 2, K. Ishii2, S. Kushimoto1, K. Kawakami2. 1Division of Emergency Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan, 2Division of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan

Objectives: Due to the severity of symptoms, therapeutic strategies for acute respiratory distress syndrome (ARDS) have been insufficient. High-mobility group box 1 (HMGB1), originally identified as a DNA binding protein, has potent pro-inflammatory properties. Some reports have suggested HMGB1 as a mediator of acute lung injury. Here, we investigate the role of HMGB1 in fulminant ARDS using a mouse model.

Methods: Fulminant ARDS was induced in C57BL/6 mice by sequential intratracheal administration of α-galactosylceramide (α-GalCer), a potent activator of natural killer T (NKT) cells, and LPS (α-GalCer/LPS); this results in diffuse alveolar damage and fatal outcome 3 to 4 days after LPS challenge (Int. Immunol. 23:97-108, 2011). PBS was used as a control. Concentrations of HMGB1 in bronchoalveolar lavage fluids were measured at 0, 6, 12, and 24 h, and immunohistochemical staining of HMGB1 was performed at 12 h after LPS challenge.

Results: At 6 h after LPS challenge, HMGB1 levels were higher in the α-GalCer/LPS and α-GalCer/PBS groups than in the PBS/LPS and PBS/PBS groups (Fig.1). At 12 h, HMGB1 levels were increased, and peaked in the α-GalCer/LPS group, which consistently showed higher levels than other groups up to 24 h. In immunohistochemical analysis, HMGB1 expression was seen in the cytoplasm of bronchial epithelial cells in the PBS/PBS group; in striking contrast, in the α-GalCer/LPS group, bronchial epithelial cells were only marginally stained, but abundant HMGB1-expressing neutrophils and macrophages were observed in the cytoplasm (Fig.2).

Conclusions: Prior activation of NKT cells may sensitize the lungs to LPS-induced injury through increased secretion of HMGB1 by inflammatory leukocytes rather than by bronchial epithelial cells; this may be involved in the core pathogenic mechanism of fulminant ARDS.

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W.L. Jones1, E.E. Moore*1, 2, M. Sumner1, J.N. Harr1, M. Kelher1, 3, J.R. Stringham1, T. Chin1, K. Lo1, L. Lee1, J. DiPaola1, C.C. Silliman1, 3, A. Banerjee*1. 1University of Colorado Denver, Aurora, CO, 2Denver Health Hospital, Denver, CO, 3Bonfils Blood Center, Denver, CO

Background: The role of platelet dysfunction in the acute coagulopathy of trauma has recently been documented, but the mechanism remains unclear. Proteomic analysis has shown an increase in apolipoprotein A1 (APOA1) in post-shock plasma. APOA1 is the primary protein component for high-density lipoprotein (HDL). Epidemiologic studies have shown that higher concentrations of HDL are associated with decreased risk of thromboembolic events, and HDL itself has been shown to inhibit platelet aggregation.

Hypothesis: APOA1 inhibits collagen-induced platelet aggregation via decreased platelet activation.

Methods: Apheresis platelet concentrates (PCs) were obtained from healthy volunteers (n=4) and used for platelet aggregations. HDL levels were assumed to be in the normal range for the volunteers. PCs were incubated in varying additional concentrations of APOA1 (control, 50, 100, 200, and 300 μg/ml) for 5 minutes at 37°C and then platelet aggregation (%) to collagen (1 μg/ml) was measured and platelet activation was completed by ATP release (nm). Collagen was chosen because it elicits maximal aggregation. Results are given as mean ± SEM. Statistical analysis was done using ANOVA followed by a Newman Keuls test for multiple comparisons with p<.05 considered significant.

Results: APOA1 inhibits platelet aggregation in a concentration-dependent manner with higher doses effecting a greater inhibition (p= 0.01). Platelet activation was decreased by APOA1 in a concentration-dependent manner (p= 0.01).

Conclusion: The addition of APOA1 to platelets in donor plasma with HDL levels in the normal range inhibits the platelet component of coagulation. APOA1’s effect on membrane cholesterol concentration could ultimately prove to be important in the anti-coagulation effect, and warrants further investigation.

APOA1 Inhibits Collagen Mediated Platelet Aggregation and Platelet Activation

APOA1 Inhibits Collagen Mediated Platelet Aggregation and Platelet Activation

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H.K. de Jong1, G.C. Koh2, 3, J.T. van Dissel4, J.J. Roelofs5, T. van der Poll*1, J. Wiersinga1. 1Center for Infection and Immunity Amsterdam (CINIMA), Center of Experimental and Molecular Medicine (CEMM) and Division of Infectious Diseases, Academic Medical Center, Amsterdam, Netherlands, 2Addenbrooke’s Hospital, University of Cambridge, Cambridge, United Kingdom, 3Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 4Department of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands, 5Department of Pathology, Academic Medical Center, Amsterdam, Netherlands

Background: Caspase-1 activating adaptor protein (ASC) and NLRP3 are key inflammasome proteins, which have emerged as central cytosolic immune response regulators against pathogens by triggering IL-1β and IL-18 maturation. In vitro studies have suggested a key protective role for the inflammasome during S. typhimurium infection, which can cause sepsis and death in immunocompromised patients. Here we aim to study the in vivo relevance of ASC and NLRP-3 during murine Salmonella infection.

Methods: We made use of wild-type (WT), ASC-/- and NLRP3-/- knockout (KO) mice and two different Salmonella infection models. Mice were starved for 12 h and inoculated orally with S. typhimurium (typhoid fever model) or pretreated with streptomycin (colitis model) before infection and sacrificed at 2 and 5 days post-infection to assess bacterial loads, inflammation, multi-organ failure and pathology.

Results: Corresponding with expected loss of caspase-1 activation, ASC-/- and NLRP3-/- KO mice showed completely abolished respectively partially reduced IL-18 serum levels after S. typhimurium infection. However, neither ASC nor NLRP3 seemed to play a role in the host defense against S. typhimurium infection: equal bacterial loads were seen in WT, ASC-/- and NLRP3-/- KO mice after infection in all organs (lymph nodes, liver, spleen, blood and feces) and in both infection models. In line, proinflammatory cytokine levels (TNF-α, IL-6, IL-10, INF-γ), markers for liver (AST, ALT, LDH) and organ pathology did not differ between groups.

Conclusions: Our results reveal an unexpected limited role for ASC and NLRP3 during in vivo S. typhimurium infection which is surprising given the central role attributed to the inflammasome in the host defense against various - and certainly intracellular - bacteria.

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H. Chen, H. Huang, A. Tsung. University of Pittsburgh Medical Center, Department of Surgery, Pittsburgh, PA

Introduction: The initiating events that account for organ damage after hepatic ischemia/reperfusion (I/R) injury are partially understood. IL-33 is a novel member of the IL-1 cytokine family recently been suggested to function as an alarmin that is released upon stressful conditions. As a soluble mediator, IL-33 plays either a pro- or anti-inflammatory role, but its function in liver I/R is unknown. We hypothesize that IL-33 is released during oxidative stress and contributes to immune response following hepatic I/R via its interaction with the receptor ST2.

Methods: In vivo, WT and ST2-KO mice underwent partial (70%) warm hepatic I/R. IL-33 siRNA or recombinant IL-33 (rIL-33) were administered in select groups. In vitro, primary hepatocytes (HCs) and nonparenchymal cells (NPCs) were isolated and exposed to hypoxia, H2O2, or rIL-33.

Results: Following liver I/R, mice treated with rIL-33 experienced significantly increased ALT levels when compared to controls. However, both the ST2-KO mice and IL-33 siRNA-treated mice showed significant protection compared to controls suggesting a pro-inflammatory role of IL-33/ST2 signaling. In vitro, both hypoxia and H2O2 treatment led to the release of IL-33 from NPC nuclei to the cytoplasm and supernatant in a time- and dose-dependent manner. Interestingly, ST2 receptors were constitutively expressed on HCs and increased in response to hypoxic stress. In addition, rIL-33 treatment increased activation of MAP kinases in HCs and production of TNF-α in NPCs.

Conclusion: We demonstrate that oxidative stress induces release of IL-33 from NPCs and that IL-33/ST2 contributes to liver inflammation and organ damage after liver I/R injury. Furthermore, ST2 signaling in HCs provides evidence that parenchymal cells, together with immune cells, may play an important role in responding to IL-33.

Fig. 1

Fig. 1

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M. Deng1, M.J. Scott*1, P. Loughran2, G. Gibson2, C.P. Sohdi3, D.J. Hackam3, S.C. Watkins2, T.R. Billiar*1. 1UPMC, Pittsburgh, PA, 2UPMC CBI, Pittsburgh, PA, 3UPMC Division of Pediatric Surgery, Pittsburgh, PA

High circulating lipopolysaccharide (LPS) levels in sepsis correlate with adverse outcome. LPS signaling via TLR4 induces cytokine production in macrophages (MΦ), and we have shown that hepatocytes (HC) take up and clear LPS via novel TLR4-dependent pathway. However, cell-specific roles for TLR4 in sepsis are unclear. Here we hypothesized that TLR4 plays multiple roles in endotoxin homeostasis during cecal ligation and puncture (CLP) in mice. WT (C57BL/6), global TLR4ko, TLR4flox (cell-specific controls), HC-specific TLR4KO (HCTLR4ko), and MΦ-specific TLR4ko (MΦTLR4ko) mice underwent CLP. As expected MΦTLRko and TLR4ko mice had significantly higher bacterial loads at 18h compared with other strains. Interestingly, HCTLR4ko mice had significantly lower bacterial loads but slightly higher endotoxin level than WT/flox mice at 18h. Furthermore, live-cell uptake, immunofluorescence and LPS-detection confirmed our previous finding that HCTLR4 was required for LPS uptake in vitro and clearance in vivo. Treatment of mice with 25mg/kg imipenem equalized bacterial loads between mice and revealed higher plasma endotoxin in HCTLR4ko and TLR4ko mice as hypothesized. We further investigated if increased plasma LPS in HCTLR4ko lead to MΦ priming, decreased bacterial & increased cytokine levels after CLP. We found that low-dose LPS given 1h after CLP in WT mice decreased bacterial levels similar to HCTLR4ko. In vitro and in vivo phagocytosis assays also showed increased phagocytosis in WT and HCTLR4ko peritoneal MΦ after LPS treatment. Additionally, neutrophil MPO levels were not different between mouse strains, all suggesting differences in bacterial clearance were due to priming of MΦ by increased LPS in HCTLR4. Our data suggest previously unrecognized roles for cell-specific TLR4 in endotoxin homeostasis and inflammation during sepsis.

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J. Song*1, P. Hornsby2, S.E. Wolf*1. 1UT Southwestern Medical Center, Dallas, TX, 2UT Health Science Center at San Antonio, San Antonio, TX

Objectives: Acceleration of muscle regeneration is highly desirable in trauma patients with severe muscle injury. Extracellular matrix extracted from porcine (UBM) has shown its benefits on tissue regeneration, however, the mechanism of UBM in inducing myogenesis is not clear. The objective of this study was to investigate whether UBM improves muscle regeneration by enhancing satellite cell activation and differentiation after local cryoinjury.

Methods: Under general anesthesia, RAG2/γC knockout mice received cryoinjury on both sides of the gluteus maximum. Following injury, 200μg of UBM was implanted on one side of wound into the muscle; 20μl of vehicle solution was delivered to the contralateral side as sham treatment. Five animals without any operation served as baseline control. Animals were sacrificed at day 1, 3, 7 and 14 following injury for muscle tissue harvest. The area of regenerating myofiber was measured with H&E histologic staining. Total RNA samples were extracted and myogenesis makers were detected by real time PCR.

Results: H&E stained slides showed that damaged muscle tissue was infiltrated with immune cells at day 1 after injury. The area of regenerating myofiber increased from day 7 after injury (1.45±0.41 mm2 in sham group; 1.77±0.33mm2 in UBM group). There was no significant difference between two groups. Myogenic markers MyoD1 and myogenin significantly increased at day 3 and 7 after injury respectively (p<0.05), macrophage markers CD68 and F4/80 were elevated as well (p<0.05). No differences were observed between UBM and vehicle treatment group.

Conclusion: Skeletal satellite cells were activated and differentiated with macrophages infiltration after injury. UBM application did not improve myogenesis in the process of muscle repair after cryoinjury.

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S. Nakamura, S. Oda*, S. Tomohito, M. Nakamura, E. Watanabe*, R. Abe, Y. Morita, K. Shinozaki, M. Maganuma, H. Hirasawa*. Chiba University Graduate School of Medicine, Chiba, Japan

Recent studies have suggested that functions of toll-like receptor 4 (TLR4) are influenced not only by its well-known ligand lipopolysaccharide (LPS) but also by neutrophil elastase (NE). However, few investigations regarding TLR4 have been conducted using lung microvascular endothelial cells, an important site of action of NE. The present in vitro study using human lung microvascular endothelial cells investigated the effect of NE on TLR4 expression and IL-8 production in human lung microvascular endothelial cells and whether Sivelestat, a neutrophil elastase inhibitor (NEI), could suppress it. Expression of TLR4 increased immediately after the start of NE stimulation and reached the peak level at 30 min after the initiation of NE stimulation. Expression of IRAK-1 (Interleukin-1 receptor-associated protein kinase 1) and p65 (NF-kappa B subunit) proteins, components of an intracellular signal transduction pathway, was also increased. Furthermore, production of IL-8 protein increased from 1 h after the start of NE stimulation. NEI suppressed increase in both expression of TLR4 protein and production of IL-8 protein. Thus, the present in vitro study demonstrated that NE increased TLR4 expression and promoted IL-8 production in human lung microvascular endothelial cells as well. These reactions are likely to be closely involved in the pathophysiology of ARDS. An inhibitory effect of Sivelestat on a vicious circle of cytokines and NE might explain a mechanism of its known clinical efficacy in ARDS.

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Q.S. Zang*, X. Yao, B. Martinez, D. Maass, S. Wolf*, J. Minei. UT Southwestern Medical Center, Dallas, TX

Objective: Our previous studies identified sepsis-induced mitochondrial dysfunction and loss of mitochondrial defense against ROS in the heart. This study examined the protective effects of mitochondria-targeted vitamin E (Mito-Vit-E) in the heart of a pneumonia-related sepsis rat model.

Methods: Sepsis was produced in adult male SD rats by intratracheal injection of S. pneumonia (4000,000 CFU/rat). Mito-Vit-E (21.5 micro moles/kg) or control vehicle was administered 30 minutes post-inoculation and heart tissue was harvested 24 hours post-inoculation. Mitochondrial DNA (mtDNA) content was quantified by long polymerase chain reaction (LPCR) using genomic DNA and mtDNA-specific primers. MtDNA damage was determined by measurements of apurinic/apyrimidinic (AP) sites and 8-hydroxy-2-deoxy guanosine levels in mtDNA. Expression of mtDNA repair enzymes was analyzed by western blots.

Results: Mito-Vit-E treatment inhibited sepsis-induced mtDNA damage, shown by changes of mtDNA content and oxidative damage. This intervention further prevented sepsis-induced down regulation of mitochondria-specific DNA repair enzymes that include DNA polymerase gamma, AP endonuclease, 8-oxoguanine glycosylase (OGG1), and uracil-DNA glycosylase (UNG1).

Conclusion: Our data suggest that imbalanced mtROS is a critical causative factor to induce mtDNA damage in the heart, contributing to cardiac dysfunction during sepsis. These results also suggest that mitochondria-targeted antioxidants are a potentially effective therapeutic approach for sepsis. (This project was supported by AHA South Central Affiliate Beginning-in-Aid Grant 09BGIA2220114 and UTSW Department of Surgery internal funding to Q. S. Zang.)

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L.E. White1, Y. Cui1, H.T. Hassoun*1, 2. 1The Methodist Hospital and Research Institute, Houston, TX, 2The Methodist DeBakey Heart & Vascular Center, Houston, TX

Objectives: We previously identified TNFR1-mediated lung apoptosis in vivo during ischemic acute kidney injury (AKI). Hypothesizing that lung endothelial cells (ECs) are key targets for microvascular barrier dysfunction, we developed an in vitro model to investigate lung EC-specific inflammation and apoptosis mediated by TNFR1 during ischemic AKI.

Methods: Male Sprague-Dawley rats (∼250g) underwent 60min of bilateral renal pedicle occlusion (IRI) or sham laparotomy (sham) and were sacrificed at 24hrs for serum. Rat lung microvascular ECs (RLMVECs) grown to confluence were treated with serum from rats after sham or IRI +/- Etanercept, a TNFα/TNFR1 signaling inhibitor, or vehicle. At 2, 4, and 24hrs, we performed custom RT-PCR analysis for pro-apoptotic, inflammatory, and TNF superfamily candidate genes and caspase-3 and PARP activity assays to correlate transcriptional changes with phenotypic apoptosis. *p<0.05 vs. sham, paired t-test; n≥4/group.

Results: Treatment with IRI serum induced a pro-apoptotic RLMVEC transcriptome with activation of TNF superfamily and apoptosis genes (Figure 1). RLMVECs demonstrated phenotypic apoptosis with increased caspase-3 (100±1.12 vs. 106.5±0.79*) and PARP activity (10±3.47 vs. 61.75±20.45*) during ischemic AKI. Treatment with Etanercept attenuated RLMVEC inflammatory gene expression (Figure 2) and apoptosis with no increase in PARP activity (10±2.33 vs. 10.91±2.15, p=0.783) vs. vehicle during ischemic AKI.

Conclusions: Ischemic AKI incites a distinct apoptotic transcriptome in lung ECs with the induction of TNFR1-mediated inflammation and apoptosis. Further investigation into potential mechanisms of kidney-lung crosstalk may identify potential therapeutic targets for this deadly disease.

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P.J. Jeziorczak1, 2, J.A. Rydlewicz1, 2, S. Kaul1, 2, K.A. Pritchard1, 2, K.T. Oldham*1, 2, E.R. Jacobs1, J.C. Densmore*1, 2. 1Medical College of Wisconsin, Milwaukee, WI, 2Children’s Research Institute, Milwaukee, WI

Purpose: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) carry 30% mortality, cause 72,000 US deaths annually, and consume 3.4 million extra US ICU days yearly among survivors. We modeled Endothelium-derived microparticle (EMP)-induced ALI in mice as a novel pathogenic step. The full-length receptor of advanced glycosylation end-products (fRAGE) is implicated in ALI/ARDS due to its high lung expression, activation of innate immunity, and presence of it’s cleaved form in ALI serum. We hypothesize that EMPs induce expression of lung fRAGE and activation by ligand high-mobility group box-1 (HMGB1) protein in ALI.

Methods: EMPs were isolated from murine endothelial cell culture (ATCC # CRL-2280) and stimulated with murine plasminogen activation inhibitor-1 (PAI-1, 50 ng/mL). C3H/HeOuJ mice (male, 6-8 wks, 20/grp) were treated with intravenous PBS, LPS 10 mg/kg, EMP 106/mL, or EMP + LPS for 24 hours. Evans Blue lung permeability (20 mg/kg) was measured in half of each group. Cohort lungs were homogenized and a microsomal preparation used to separate RAGE fractions. HMGB1, fRAGE and sRAGE levels were quantified and normalized to β-Actin using Western blot analysis.

Results: EMP, LPS, and combined treatment result in increase in pulmonary capillary permeability (40%, p <0.05) as compared to PBS lungs. fRAGE and sRAGE levels were increased in LPS treated animals and highest in EMP and cotreated animals (p <0.05). As a ratio, fRAGE/sRAGE levels were highest in EMP treated mice.

Conclusions: EMPs induce pulmonary capillary leak, expression of HMGB1, and upregulation of the pulmonary RAGE receptor with a greater fraction of fRAGE. As HMGB1 activates fRAGE, the cellular source of HMGB1 and the effect of RAGE blockade will be studied as therapeutic targets to inhibit EMP-induced lung injury.

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F.E. van den Boogaard, X. Brands, J.J. Roelofs, C. van ’t Veer, T. van der Poll*. AMC, Amsterdam, Netherlands

Background: S.pneumoniae is the most common causative pathogen in community-acquired pneumonia. Mast cells are mainly located at the host-environment interface where they function as sentinels and produce tryptase, amongst other pro-inflammatory mediators. We examined the role of mast cells during pneumococcal pneumonia in mice.

Methods: Pneumonia was induced by intranasal inoculation of S.pneumoniae in wild type (WT) and mast cell deficient (Kit/Kitv) mice; animals were observed in a survival study or sacrificed at 24 and 48h after induction of pneumonia for analyses. In separate experiments WT mice were treated with mast cell stabilizing agents doxantrazol, cromoglycate or vehicle.

Results: Kit/Kitv mice showed a prolonged survival until 70 hours. Bacterial counts were reduced in lung homogenates of Kit/Kitv mice at 24 and 48h (both p<0.05) and BALF at 48h (p=0.007) when compared to WT mice. Kit/Kitv mice showed less bacteremia and lower bacterial loads in spleens at 24 and 48h at both time points. Although the number and composition of cells in BALF did not differ between groups, Kit/Kitv mice showed less neutrophil infiltration into lung tissue as reflected by lower myeloperoxidase levels at 24h (p=0.01) and fewer Ly6+ neutrophils in lung tissue slides at 48h (p=0.04). Total lung histopathology scores were lower in Kit/Kitv mice compared to WT mice at 24 and 48h (p=0.06 and p<0.01 respectively), mainly due to less endothelialitis and pleuritis. Kit/Kitv mice had lower levels of cytokines and chemokines (IL-1β, IL-6 and KC) in BALF and in plasma (IL-6) at 24h. Inhibiting mast cell degranulation by mast cell stabilizers did not influence bacterial loads.

Conclusion: Mast cells, but not mast cell degranulation, exhibit an unfavorable role in host defense during pneumococcal pneumonia.

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C. Ziraldo, Q. Mi, D. Barclay, B. Jefferson, R.A. Namas, Y. Vodovotz*, R. Zamora*. University of Pittsburgh, Pittsburgh, PA

Objective: Using multiplexing mediator analysis and data-driven modeling, we had previously studied the response of wild-type (wt) mouse hepatocytes (HC) to both normoxia (N) and hypoxia (H), and suggested the chemokine MCP-1 as a central network node. Therefore, we investigated the impact of MCP-1 on the dynamics of inflammation, using HC from MCP-1-/- (ko) mice.

Methods: Freshly isolated HC from either wt or ko mice were cultured either under N (21% O2) or H (1% O2) for 1-72 h. Cell supernatants and protein lysates were assayed for 18 mediators by Luminex™ technology. Principal Component Analysis (PCA) was employed to characterize the main components of the cellular response. Dynamic Network Analysis (DyNA) was utilized in order to define the principal (most connected and thereby possibly controlling) nodes as a function of time, O2, and MCP-1 expression.

Results: The greatest inflammatory coordination was found in H-wt cells. In contrast, N-ko cells showed very low network connectivity across all time points. The H-wt response depended on MCP-1 across every time point, but involved many cytokines (as indicated by high network densities). In N-wt, by contrast, there were no significantly correlated mediators until 24 h. This response, though occurring later, was also dominated by MCP-1 and KC. In lysates of H-ko, there were no correlations before 6 h, and thereafter the response was dominated by IL-6 and IL-10. In H-ko supernatants, DyNA revealed mediators correlating strongly with KC and MIG. PCA identified two additional coordinated responses dominated by IL-6 and IFN-γ, respectively.

Conclusion: This study suggests that MCP-1 controls HC inflammation as a function of O2.

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E. Okeke*, I. Okwor, P. Jia, J. Uzonna. University of Manitoba, Winnipeg, MB, Canada

Introduction: Sepsis, a systemic immune response to severe bacterial infection, is a leading cause of death in critical care medicine. It has a high mortality rate and presents a huge fiscal burden in health care expenditure. Conventional Regulatory T cells (Tregs) defined by the constitutive expression of CD4, CD25 and FoxP3 have been shown to express the pattern recognition receptor, toll-like receptor 4 (TLR4), and are thus able to respond to lipopolysaccharide (LPS) stimulation. Tregs play an important role in down-regulating inflammatory immune response. However, their role in sepsis is unclear. Here, we investigated the role played by these cells in pathogenesis of septic shock.

Methods: Wild type C57BL/6 mice were injected with PBS or with anti-CD4 and anti-CD25 monoclonal antibody (mAb) to deplete CD4+ T cells and CD4+CD25+ Tregs, respectively. The mice were then subjected to endotoxic shock by intraperitoneal injection of sub-lethal dose of LPS 24 hr post antibody injection. In another experiment, CD25 knockout (CD25 KO) mice received sub-lethal dose of LPS or CD4+CD25+ Tregs and LPS. Mice were monitored for survival and at sacrifice samples were collected and assessed for cytokines by ELISA.

Results: Groups of mice depleted of Tregs or both CD4+ T cells and Tregs showed enhanced susceptibility and mortality to an otherwise sub-lethal dose of LPS and had elevated serum levels of pro-inflammatory cytokines compared to those depleted of only CD4+ T cells or given LPS alone. Also CD25 KO mice showed 100% mortality to a non-lethal dose of LPS, which was abrogated by the adoptive transfer of WT CD4+CD25+ Tregs.

Conclusion: These results suggest that CD4+CD25+ Tregs play a critical role in amelioration of LPS-induced septic shock.

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Q. Li1, 2, H. Li2, G. Leikauf4, B. Pitt4, T. Billiar3, L. Zhang2. 1Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China, 2Departments of Anesthesiology, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, 3Departments of Surgery, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, 4Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA

Our previous studies showed that the TLR4-MyD88 signaling pathway and WISP1 play an important role in VILI. The current study explored the mechanisms of WISP1 induced macrophage activation and its relation to TLR signaling pathway in VILI.

Methods: A/J, C57B6 and TLR4 null mice were ventilated with high tidal volume (HTV, 20 ml/kg) for 4 h after tracheotomy. VILI was evaluated by histopathology and permeability to Evans blue albumin. WISP1 expression was measured by Western blot and immunohistochemistry. Peritoneal macrophages (PMs) obtained from C57B6, TLR4, MyD88 and TRIF null mice were treated with WISP1 and TLR1-9 agonists including HMGB1. TNF-α release was measured by ELISA.

Results: Lung WISP1 protein expression and permeability increased with HTV in WT (A/J) mice. In contrast, there was neither WISP1 expression nor permeability change in TLR4 null mice after HTV. HTV enhanced HMGB1-induced TNF-α release from PMs in A/J mice, but not from TLR4 null mice. WISP1 alone induced little TNF-α release from PMs, but significantly amplified TNF release when added with TLR 1-9 agonists. The synergy between WISP1 and the TLR agonists was absent in PMs obtained from TLR4 or MyD88 null mice while PMs from TRIF null mice responded similar to WT cells. WISP1 co-immunoprecipitated with TLR4 in lung homogenates.

Conclusions: WISP1 induces susceptibility of mice to VILI by increasing lung permeability and inflammation probably through amplifying inflammation of macrophages to TLR agonists. WISP1 may act as a synergistic signaling molecule or amplifier to modulate immune response to other TLR agonists including PAMPs (LPS) and DAMPs (HMGB1). Such a modulation by WISP1 mainly depends on TLR4-MyD88 signaling pathway. These findings are potentially clinically relevant for the development of new strategies in the treatment of VILI.

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M. Aoyama1, 2, J. Kotani*2, T. Ueda2, N. Maeshige1, M. Miyoshi1, N. Oka1, T. Inoue1, M. Usami1. 1Kobe University Graduate School of Health Sciences, Kobe, Japan, 2Department of Emergency and Critical Care Medicine, Hyogo College of Medicine, Nishinomiya, Japan

Introduction: The plasma DNA concentration has been known to be increased in the circulation of patients after trauma and may have prognostic potential. We examined the relevance of the plasma DNA concentration in ICU patients and their mortality.

Methods: Approval for this study was granted by the ethics committees at Hyogo College of Medicine and Kobe University School of Medicine. We collected blood samples from 85 ICU patients for 5 consecutive days. Plasma DNA was detected by quantitative real time PCR using primers for the human beta-globin gene. The generation of plasma DNA standard curve was accomplished by using human genomic DNA. Results are expressed as genome equivalents (GE) /mL in the following experiments. One kilo- genome equivalents corresponds to six point six pg DNA.

Results: The 45 patients were sepsis or septic shock and the 36 patients were non-septic patients (non SIRS; 5, hemorrhagic shock; 11, pancreatitis; 17 and burn; 3 patients). The mortality of septic patients was 35.6% (16 out of 45) and that of non-septic patients was 19.4% (7 out of 36). The patients who needed emergency operation were 46.7% (21 out of 45) in sepsis and 44.4% (16 out of 36) in non-sepsis. The plasma DNA concentrations of operated septic patients in 5 days were lower than that of non-operated patients (p<0.05). When the average of plasma DNA concentrations for 3 days was higher than 16 kGE/mL, the mortality tended to be high in sepsis (p=0.08). In the non-septic patients (36 patients with 19.4% mortality), the plasma DNA concentrations of non-surviving patients were higher than that of surviving patients (p<0.05) for 5 consecutive days.

Conclusion: Plasma DNA concentration could be a useful prognostic marker of mortality in ICU patients.

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T. Suzuki1, N. Matsuda2, H. Teramae1, N. Sato*1, K. Koike*1. 1Kyoto University Graduate School of Medicine, Kyoto, Japan, 2Nagoya University Graduate School of Medicine, Nagoya, Japan

Introduction: Although studies have shown that endoplasmic reticulum (ER) stress is involved in the pathophysiology of inflammation, it remains unclear in which cell types ER stress has a critical role under septic conditions.

Hypothesis: ER stress implication in the pathophysiology of sepsis depends on cell types in pancreas and adrenal glands.

Methods: Male mice underwent cecal ligation and puncture (CLP). The pancreas and the adrenal gland samples were collected at 24 hours after CLP and immunohistochemical staining was performed.

Results: Three ER stress sensor proteins, IRE1α, PERK and ATF6, expressed stronger in the endocrine pancreas than in the exocrine. Moreover, PERK strongly expressed especially in glucagon-secreting cells in the endocrine pancreas. ATF6 expression in the exocrine pancreas was greatly increased following CLP. In adrenal glands, IRE1α, PERK and ATF6 expressed stronger in the cortex where steroid hormone is released than in the medulla where catecholamine is produced. However, the expression of all these proteins in the adrenal medulla was greatly increased following CLP. Moreover, phosphorylation of PERK was also augmented by polymicrobial sepsis in the zona glomerulosa of the cortex, in which mineral corticoids were produced.

Conclusions: The present results suggest that the expression of ER stress sensor proteins is different in each cell type in pancreas and adrenal glands. Furthermore, it is likely that the influence of sepsis on these proteins expression also depends on cell types.

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A. De Vos1, M.C. Dessing1, J.J. Lammers1, A. De Porto1, H. Bootsma2, P.W. Hermans2, S. Florquin1, C. Van ’t Veer1, T. Van der Poll*1. 1Academic Medical Center, Amsterdam, Netherlands, 2Radboud Nijmegen Medical Center, Nijmegen, Netherlands

Background & Aim: Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. S.pneumoniae is a human respiratory pathogen; >90 different serotypes exist which vary in the composition of capsular polysaccharides. Serotype 3 pneumococcal pneumonia is associated with a high mortality in humans. MyD88 has been found essential for host defense during serotype 4 pneumococcal pneumonia. We here sought to determine the role of MyD88 in innate immunity against serotype 2 and 3 S.pneumoniae.

Methods: WT and MyD88-/- mice were inoculated intranasally with serotype 2 or 3 S.pneumoniae or with an unencapsulated serotype 2 mutant, and analysed for bacterial outgrowth and inflammatory responses in the lung.

Results: In line with previous results with serotype 4 S.pneumoniae, MyD88-/- mice demonstrated enhanced bacterial outgrowth during serotype 2 pneumococcal pneumonia accompanied by decreased inflammatory responses as revealed by reduced cytokine levels and pathology in the lung. These results were not dependent on the capsule, since MyD88 was also essential for the clearance of unencapsulated serotype 2 pneumococci. Strikingly, MyD88 deficiency did not alter survival, bacterial growth or lung pathology during serotype 3 pneumococcal pneumonia, despite attenuated cytokine responses. Similar results were obtained with TLR2-/-xTLR4-/- mice.

Conclusion: These data demonstrate that MyD88-dependent signaling is essential for defense against serotype 2 pneumococci. In contrast, serotype 3 S.pneumoniae, a strain more relevant for human disease, is resistant against MyD88-mediated innate immune responses. These results thus indicate that TLR and MyD88 differently contribute to host defense against distinct serotypes of S.pneumoniae.

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C.R. Sabbag, A.M. Liberatore, J.L. Menchaca-Diaz, I.B. Casella, I.H. Koh. Federal University of São Paulo, São Paulo, Brazil

The bacterial translocation (BT) has been implicated as an amplifying factor of the systemic inflammation in hemorrhagic shock (HS) by activating gut-associated lymphoid tissue (GALT) and releasing pro-inflammatory products into the systemic circulation through the mesenteric lymph. Herein we examined the role of the mesenteric lymph exclusion (MLE) and BT in HS.

Methods: Wistar-EPM rats were distributed in 4 groups: HS and fluid resuscitation (SR); HS and fluid resuscitation with MLE (SRL); HS and fluid resuscitation with BT (SRTB); HS and fluid resuscitation with MLE and BT (SRLTB). HS was induced by withdrawing blood from jugular vein up to 50% of mean arterial pressure (MAP) and maintaining per 60 minutes. After, animals were treated with total blood volume (TBV) replacement plus Ringer lactate (3 times TBV during 20 minutes). The BT was induced by confinement of 10mL of inoculum (10 to 10CFU/mL E. coli R6) per 2 hours in the small bowel by oroduodenal catheterization under laparotomy. The MLE was made 5 days prior experiments by duct ligation under laparotomy. Splanchnic organs and kidney tissue perfusion were monitored by laser Doppler before the onset of shock (T0), immediately after HS (T20), at the end of HS (T60) and before and after volume restoration (T80, T100). Mortality was followed per 30 days.

Results: The HS mortality was 40%, with or without BT. The lymph exclusion promoted 100% survival (Fig 1), faster MAP recovery and minimized HS related splanchnic and kidney hypoperfusion. (Fig 2), showing that gut origin lymph pro-inflammatory factors have an important role in the abdominal organ’s microhemodynamic dysfunction and death in HS. In conclusion, mesenteric lymph has a key role in organ dysfunction and death in HS, besides, BT demonstrated to have a potent role in the lethality when associated to HS.

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G. Wang*, Z. Tang, F. Hu, R. Cooney*. SUNY Upstate Medical University, Syracuse, NY

Autophagy is a protective cellular response to various stresses including sepsis. Surfactant proteins A and D (SP-A, SP-D) play crucial roles in immunity and homeostasis. SP-A/D deficient mice demonstrate better survival in CLP septic model.

Objective: Study functions of SP-A and SP-D in the regulation of autophagy and apoptotic pathways in CLP sepsis.

Methods: SP-A and SP-D double knockout (KO) and matched wild-type (WT) C57BL/6 male mice (9-12 weeks) were studied. Mice underwent CLP surgery or sham surgery (No manipulation of cecum). Autophagic activity and autophagy-related gene expression in the liver of KO and WT mice were studied by Western blot, light and electron microscopies, and Real-time PCR Array system. Data are means+SE, p<0.05 by ANOVA or t-test is significant