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doi: 10.1097/SHK.0b013e3181e2f08b
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We evenly allocated 60 Sprague-Dawley rats to four groups: one no hemorrhage group, and three groups with hemorrhage at a fixed volume of 2, 2.5, or 3 mL/100 g/15 min. Survival and non-survival was determined two hours after hemorrhage ended. We analyzed heart rate, systolic and diastolic blood pressure, respiration, and body temperature, and then obtained a logistic regression equation as below predicting survival rates between 0 for death (n = 26) and 1 for survival (n = 19). We initially performed forward selection logistic regression with five parameters. HR and SBP were identified as significantly associated with survival rate. The area under the receiver operating characteristic curves was 0.986. The optimal boundary value between survival and death was determined to be 0.37. With this boundary value, the sensitivity, specificity, and accuracy of survival prediction by the regression equation were 96.8, 95.4, and 96.0%, respectively. We also found several important qualitative changes in parameters with survival or non-survival group. First, the simple parameters such as SBP and HR contributed the most to the prediction of survival or death of rats, although DBP and RR were also contributing parameters. Second, no increase in SBP, DBP, or pulse pressure after the end of bleeding, despite increasing HR, could be a sign of shock. Third, another sign of shock might be a considerable and continuous decrease in RR during hemorrhage and no increase even after the end of bleeding. Future study should be focused on how the vital signs investigated in this study would change with survival or death in animals, and then humans with uncontrolled hemorrhage. (Supported by NRF R01-2007-000-20819-0).


PREDICTION OF BLOOD LOSS IN RATS DURING FIXED-VOLUME HEMORRHAGE USING A REGRESSION MODEL.J.L. Choi,* D.W. Kim,* Y.S. Park,* and (J-M Kim). Yonsei University College of Medicine, Seoul, Korea

The purpose of this study was to accurately predict percent blood loss for fixed-volume hemorrhagic shock in rats using a stepwise linear regression (SLR) model. We equally allocated 30 Sprague-Dawley rats to two groups: no-hemorrhage group as a reference and another group with hemorrhage at a fixed volume of 3 mL/100 g/15 min which clinically corresponded to class IV hemorrhage. We analyzed the heart rate (HR), systolic (SBP) and diastolic blood pressure (DBP), respiration rate (RESP), and body temperature (TEMP) in relation to the percent blood loss. We generated a SLR predicting the percent blood loss using a 120 randomly chosen data set. The SLR selected SBP, DBP, RESP, and TEMP. SBP was the most contributing predictor. The R-squared and root mean square error of the tested 120 data set using the SLR were 0.874 and 5.6%, respectively. Even though the SLR equation is not directly applicable to clinical practice, our method of predicting a percent blood loss could be helpful in determining the necessary fluid volume for resuscitation in the future. (Supported by NRF R01-2007-000-20819-0).




Bacterial superantigen (BSAg)-induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)-induced shock are characterized by severe systemic inflammation. It is known that NFκB plays an important role in these above inflammatory conditions. Bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NFκB activation. Therefore, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using HLA class II transgenic mouse models. Bortezomib significantly blocked BSAg-induced T cell activation and cytokine production in vitro. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, serum levels of TNF-α, an important cytokine implicated in TSS was reduced but not abolished at 3 hours and at 6 hours, there was no difference in the serum TNF-α levels between bortezomib treated and untreated mice challenged with SEB. Paradoxically, all mice treated with bortezomib either before or after BSAg or LPS challenge succumbed to TSS. Bortezomib sensitized mice to death even at non-lethal concentrations of BSAg (2 μg/mouse) or LPS (10 μg/mouse). Bortezomib by itself was never lethal at the doses tested. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB challenged mice treated with bortezomib showed a significant reduction in NFκB activation compared to SEB challenge alone. Since NFκB-dependent antiapoptotic pathways protect hepatocytes from TNF-α-induced cell death, inhibition of NFκB brought forth by bortezomib in the face of elevated TNF-α levels caused by BSAg or LPS is detrimental. This could have clinical relevance.


BRAIN INJURY AND CONTROLLED HEMORRHAGIC SHOCK (CHS) MODEL: THE EFFECT OF REDUCED INJURY AND MECHANICAL VENTILATION.M. Krausz* M. Okun-Gurevich* J. Soustiel* M. Zaaroor* L. Semenikhina* A. Solopov* V. Cohen-Kaplan* E. Brotfain* A. Leibowitz* Y. Shapira* and D. Dar. Department of Surgery A', the Laboratory for Shock and Trauma Research, Rambam Health Care Campus, Technion, Haifa 31096, Israel

We have previously observed only minor functional differences between traumatic brain injury (TBI) and CHS compared to TBI alone (model 1). In model 2, the severity of brain injury was reduced and ventilation added in order to better simulate human trauma.

Methods: Anesthetized rats were divided into eight groups: Gr-I was sham operated, Gr-II had severe brain injury (SBI, 400 millibar negative pressure induced for 10 sec), G-III had CHS, (withdrawal of 30% of blood volume), Gr-IV had SBI and CHS (model 1), Gr-V was ventilated and sham operated, Gr-VI was ventilated and had less severe brain injury (LSBI- 300 millibar), Gr-VII was ventilated and had CHS, Gr-VIII was ventilated and had LSBI and CHS (model 2).

Results: CHS in Gr-IV resulted in fall of MAP to 28 ± 1.6 mmHg, hematocrit dropped to 35.7 ± 0.8%, lactate level was elevated to 4.1 ± 0.4 mmol/L and base excess dropped to −2 ± 0.7 mmol/L. CHS in Gr-VIII resulted in fall of MAP to 50.7 ± 2.9 mmHg, hematocrit dropped to 34.4 ± 0.6%, lactate level was elevated to 4.5 ± 0.4, and base excess dropped to −3 ± 0.9 mmol/L. 24 hours after injury, the neurological severity score (NSS) of Gr-VIII was significantly (p < 0.05) higher than that of Gr-VI, while NSS of Gr-IV was similar to Gr-II. Body weight of Gr-VIII was significantly (p < 0.05) lower than in Gr-VI, whereas the body weight of Gr-IV was similar to Gr-II. The number of surviving neurons in Gr-VIII was similar to that of Gr-VI, but significantly higher than that of Gr-IV and Gr-II.

Conclusions: CHS in model 2 significantly increased NSS following LSBI, while in model 1 no was observed. Model 2 better simulates clinical combined LSBI and CHS.


EFFECTS OF CEFTAZIDIME ON CARDIAC CYTOKINE LEVELS IN OVINE SEPTIC SHOCK.D.M. Maybauer, D. Carlson, M.O. Maybauer, J.F. Fraser, L.D. Traber, and D.L. Traber. Dept. of Anesthesiology, UTMB Galveston, TX

Objective: We have recently shown that ceftazidime (CEF) improved cardiac dysfunction in ovine septic shock (1) and aimed to further investigate these effects measuring cardiac cytokine levels of interleukin 1 beta (IL-1b), IL-10, and macrophage migration inhibitory factor (MIF) involved with this process.

Methods: Eighteen sheep were chronically instrumented, and randomly allocated to the sham, control or CEF group. Septic shock was produced in control and CEF groups according to an established protocol (1). Shams received the vehicle. Sheep were ventilated (FiO2 1.0) for 24h and CEF was given as 3g IV bolus at 1h and 13h post injury in the CEF group (1). Heart tissue was obtained during necropsy, total RNA was extracted and processed using reverse transcription PCR. Statistics: two-way ANOVA and Student-Newman-Keuls test. Data: Mean±SD (*P < 0.05). Preliminary data of n = 4 per group has presently been analyzed.

Results: After 24h, normalized mRNA levels of IL-1B were 1.06 ± 0.07 in sham, 1.06±0.03 in the control group, and 0.74±0.25 in the CEF group (n.s., respectively). IL-10 levels were 1.28±0.15 in sham, 1.28±0.22 in the control group, and 1.56±0.34 in the CEF group (n.s., respectively). MIF levels were 0.46±0.12 in sham, and significantly increased in the control group (0.94±0.18*). In the CEF group, MIF levels were significantly lower than in controls (0.42±0.22*).

Conclusion: Cardiac IL-1b and IL-10 levels were not affected in this model of septic shock, and did not change using CEF as treatment. Cardiac MIF levels significantly increased due to the injury and were significantly decreased by treatment with CEF, this may contribute to the cardiovascular improvement seen in CEF treated animals. These findings may lead to further investigations on the effects of CEF on cardiovascular function in septic shock. Reference: (1) Maybauer et al., Intensive Care Med Jul; 33(7):1219-1227, 2007.


EFFECT OF A NOVEL PEPTIDE ANTAGONIZING NF-κB P65 ON MATURATION OF DENDRITIC CELLS AND COLLAGEN-INDUCED ARTHRITIS IN MICE.H. Liang,* X. Fan,* X. Yang,* X. Wang,* Q. Wei,* and (Spon: L. Zhou). Research Institute of Surgery & Daping Hospital, Third Military Medical University, Chongqing 400042, PR China

Objective: We have successfully designed and synthesized a membrane-permeable hexapeptide, which can effectively interfere with DNA binding of NF-κB p65 subunit. The influence of the peptide on maturation of dendritic cells (DC) and on collagen-induced arthritis (CIA) in C57BL/6 mice was investigated in this study.

Methods: Bone marrow-derived DC were cultured and induced to maturation with lipopolysaccharide (LPS) in the presence or absence of the peptide. 24 hours later, DC functions were determined. In vivo CIA experiment, two hind ankle joints were given intra-articular injection of 5 μg peptide, 2 days after the second immunization, daily for consecutive three times in mice. Two weeks later, the incidence of CIA and histopathological changes were determined, Cytokine mRNA expression was detected in synovial tissues by real-time polymerase chain reaction.

Results: In the presence of the peptide, MHC II, CD40, CD80, CD86 expression on LPS induced DC were significantly suppressed, secretion of IL-12p40, IL-12p70, TNF-alpha were reduced while IL-10 production had no obvious change, priming effect of DC on naïve allogeneic T cells in vitro was also inhibited. The negative control peptide had no significant effect on DC functions. As compared with vehicle or negative control peptide group, the incidence of CIA, clinical scores and magnitude of joints erosion were significantly reduced in peptide treatment group. Synovial iNOS, COX-2, IL-1β, IL-6, and TNF-α mRNA was also lower than that in control group. In contrast, no difference was observed in IL-10 mRNA between these groups.

Conclusion: These findings suggest that the peptide antagonizing NF-κB can inhibit DC maturation and possess effective anti-inflammatory activity for treating rheumatoid arthritis.


SPLENECTOMY DOES NOT AFFECT ENDOTOXIN-INDUCED CIRCULATING TNF-ALPHA IN PIGS.A. Schultz, T. Hausner, M. van Griensven, C. Keibl, M. Jafarmadar, C. Czura,* K. Tracey,* H. Redl, and S. Bahrami. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna Austria; *The Feinstein Institute for Medical Research, NY

Splenectomy has been shown to reduce inflammatory response in both rodents and humans and the splenic macrophages have been considered as the main TNF source. Close human resemblance of immune-inflammatory makeup warrants the increasing use of pigs in preclinical studies. The present study investigates whether spleen is involved in increase of circulating TNF-alpha by endotoxin in pigs. Anesthetized domestic pigs were randomly assigned to undergo splenectomy or sham operation (n = 9 each group). After hemodynamic stabilization animals were subjected to endotoxin challenge (10 μg/kg, PAP controlled infusion of increasing dose over 60 min). Blood samples were obtained hourly and the experiment was terminated 5h post-endotoxin. Plasma TNF-alpha level did not differ between splenectomized and sham operated groups after endotoxin challenge (28701 ± 5664 and 35640 ± 8450 pg/mL). Similarly, TNF-alpha concentrations did not differ in the pre and post splenic vascular bed in sham operated animals (9128 ± 3801 and 9193 ± 3968 pg/mL). In summary, in contrast to data reported in rodents, our results indicate that in pigs, splenectomy does not modulate the systemic TNF-alpha response induced by endotoxin. Thus, preclinical studies, investigating the spleen related inflammatory response in pig call for re-evaluation.


COMPARISON OF HEMODYNAMIC CHANGES AFTER HEMORRHAGE AND TREATMENT WITH HYPERVISCOUS FLUIDS.F. Nunez, S. Trach, V. Kislukin, M. Van Dyke, M. Callahan, T. Smith, and L.A. Koman. Wake Forest University Baptist Medical Center, Winston Salem, NC 27157

Introduction: Hemorrhage is a life-threatening problem. No plasma expander has proven to be more beneficial or as effective as whole blood. Keratose Fluid (KRF) may potentiate the release of vasodilators through vascular wall sheer stress and increase circulating volume by promoting water transport into the vascular space. The net effects would be better tissue perfusion and increased cardiac contractility due to increased venous return.

Methods: Sprague-Dawley rats were anesthetized with isoflurane, instrumented with an arterial pressure catheter and an extracorporeal loop (∼1.0ml) between carotid artery and jugular vein. Loop flow probes provided a signal of volumetric flow and blood density. After activation of the extracorporeal pump (6ml/m), venous injection of warm saline triggered automatic determination of cardiac output (CO). Animals were hemorrhaged (37%) and given 6% Hetastarch or KRF. Blood density measures were obtained and CO measures taken at baseline, after hemorrhage/resuscitation and up to 2 hours later.

Results: Hemorrhage decreased blood density. Resuscitation with Hetastarch or KRF further decreased blood density. While the blood density remained low in the Hetastarch group, it rapidly increased with KRF to a new level by ∼10 minutes (p=.04), suggesting an increase in the hematocrit in the arterial blood. Hetastarch increased CO post-resuscitation better than KRF.

Discussion: The rapid increase in blood density with KRF suggests it can mobilize RBCs from the systemic microcirculation. In another work we demonstrated KRF's vasodilatory effect in the microvasculature. The use of KRF in hemorrhage could reduce cardiac work while also improving perfusion of a higher hematocrit blood to ischemic tissues.


INTRAVENOUS INFUSION OF KERATOSE-BASED FLUID INDUCES ARTERIOLAR VASODILATION IN CREMASTER MUSCLE OF RATS.F. Nunez, S. Trach, M. Van Dyke, M. Callahan, and T. Smith. Wake Forest University Baptist Medical Center, Winston Salem, NC 27157

Introduction: Synthetic plasma expanders have been investigated for treatment of hypovolemic shock. None have proven to be more beneficial than another or as effective as whole blood. Hyperosmotic hyperviscous Keratose is proposed as a possible plasma expander. This type of compound potentiates the release of strong vasodilators and induces net intravascular transport of water, increasing circulating volume. This results in peripheral vasodilation and increased cardiac contractility which translates into lower cardiac work and better tissue perfusion. The null hypothesis was tested that a Keratose resuscitation fluid (KRF) would not induce more arteriolar vasodilation than a current plasma expander, Hetastarch (HS).

Methods: A topload of KRF, HS, or PBS was infused into euvolemic rats. Changes in arteriolar diameters were measured using intravital microscopy.

Results: ANOVA showed significant differences (p <0.05) between the three treatments. Post-hoc comparisons showed: KRF induced greater vasodilation than PBS or HS. HS vasodilatilatory effects were not significant when compared to PBS.

Discussion: KRF induced significant vasodilation in muscle microvasculature compared to HS or PBS. The use of KRF in a hemorrhage model could improve functional capillary density, a reliable index of tissue perfusion highly correlated with survival in severe hemorrhagic shock.


LAPAROSCOPIC MODEL OF NON-COMPRESSIBLE LIVER HEMORRHAGE IN NON-HUMAN PRIMATES.P. Hwang,* D. Fryer,* E. Elster,* N. Aziz,* D. Tadaki,* and F. Sheppard. Naval Medical Research Center, Silver Spring, MD 20910

Background: Current animal models used to investigate infusible hemostatics in non-compressible intra-abdominal hemorrhage are of uncertain clinical relevance. They disrupt abdominal wall integrity and do not consider xenogeneity in the evaluation of human derived hemostatic agents when used in animals such as swine. To overcome these limitations, a more clinically relevant model was developed.

Methods: Rhesus Macaques were anesthetized and the abdomen entered laparoscopically to perform a left hepatectomy transversely at 25 or 50% with no attempt at hemorrhage control. Immediately, CO2 was vented from the abdomen and operative ports were removed and all incisions closed. ATLS guided resuscitation commenced at 15 min post-injury, simulating the pre-hospital phase. The hospital phase commenced at 2 hrs, at which point a laparotomy was performed and operative hemostasis was achieved after the quantification of intraabdominal blood loss. Physiologic parameters and labs were recorded/performed 15 min prior to and for 4 hrs post injury. Results reported as mean (SEM).

Results: A 50% hepatectomy was required to induce significant grade 3 hemorrhagic shock (fig 1). Measured blood loss: 11.3ml (0.9), 2.4% (0.2) and 119.7ml (34.9), 27.9% (7.4) for 25 & 50% hepatectomies, respectively. 50% hepatectomy resulted in significant reductions in Hct (p<0.05) with 100% survival at 4hrs.

Conclusion: This laparoscopic 50% hepatectomy model in a Rhesus Macaque consistently demonstrates inducible grade 3 hemorrhagic shock within 5 min and 100% survival at 4 hrs. This model allows for true investigations of non-compressible intra-abdominal hemorrhage that is more consistent with clinical injuries.




Objective: Sepsis induces robust apoptosis in T cells and the loss of immune cells could be one explanation for the profound immunosuppression observed in this illness. The mechanisms responsible for lymphocyte apoptosis in sepsis remain unknown. In T cells, apoptosis can occur through activation-induced cell death (AICD) which is initiated by T cell receptor (TCR) restimulation of already activated and expanded peripheral T cells. Adenosine is a purine nucleoside signaling molecule and its immunomodulatory effects are mediated by four G protein-coupled receptors: A1, A2A, A2B and A3. In this study, we investigated the role of A2A receptors in regulating CD4+ T lymphocyte AICD.

Methods: 7.5 murine T hybridoma cells or human Jurkat cells were incubated with various doses of selective A2A receptor agonist, CGS21680, and selective A2A receptor antagonist, ZM241385, 30 min prior to activation by ConA. 18-20 h later the cell viability, and T cell death was measured. The apoptotic markers and transcription factor levels were detected by Western blot. FAS and FASL expression was determined using quantitative PCR. NF-κB-dependent transcriptional activity was measured by luciferase assay.

Results: CGS21680 rescued mouse CD4+ hybridomas and human Jurkat cells from AICD, and that this effect was reversed by ZM241385. CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase-3, caspase-8 and PARP. In addition, CGS21680 attenuated both Fas and Fas ligand (FasL) mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF-κB, NF-ATp, Egr-1 and Egr-3. The anti-apoptotic effect of A2A receptor stimulation was mediated by protein kinase A. Together these results demonstrate that A2A receptor activation suppresses the AICD of peripheral T cells.


ACTIVATED COMPLEMENT FACTOR 5 (C5A) DELAYS NEUTROPHIL APOPTOSIS - INVOLVEMENT OF P13-KINASE?M. Huber-Lang, S. Braumüller,* F. Gebhard, and M. Perl. Dept. of Orthopedic Trauma, Univ. of Ulm, 89075 Ulm, Germany

In response to severe trauma an early increase of apoptosis-inducing factors, e.g. Fas-Ligand, can be observed. However, the lifespan of neutrophils (PMN) after trauma is not shortened, but is rather prolonged, what may be associated with end organ damage, such as acute lung injury. Because the complement system is also activated early after severe trauma, we investigated whether C3a and C5a might be able to delay PMN apoptosis. To study this, PMN were isolated from healthy donors (n=6) and were incubated with either a Fas-agonist (CH11) to induce extrinsic or hydrogen peroxide (H2O2) to induce intrinsic apoptosis in the presence or absence of C5a or C3a (10-1000 ng/ml). The effect of C5a was controlled using a specific C5a receptor antagonist (10 μg/ml). Wortmannin (250 nM) was used to block PI3-Kinase (PI3K). PMN apoptosis was assessed via flow cytometry using Annexin-V and 7-Aminoactinomycin (7-AAD) staining.

Two hours after stimulation of the death receptor Fas (CH11) or incubation with H2O2 significant apoptosis was induced in PMN. In the presence of C5a, but not C3a, a dose-dependent reduction of PMN apoptosis was detected. PMN apoptosis was fully reversible upon specific blockade of the C5a-receptor. Inhibition of PI3K markedly reduced the antiapoptotic capacity of C5a regardless how apoptosis was induced. Thus, our results indicate that C5a, but not C3a, reduced PMN apoptosis regardless whether it was induced via the intrinsic or extrinsic pathway. This correlates with clinical findings in which PMN apoptosis is delayed after trauma in spite the presence of proapoptotic molecules. This protection of PMN by C5a appears to be mediated, at least in part, through activation of PI3K signaling pathways. It remains to be elucidated whether therapeutic inhibition of the prolonged lifespan of PMN after trauma is beneficial with respect to the development of end organ damage. (Supported by DFG KFO-200 & PE 908/2).


MIDAZOLAM INFLUENCES THE INFLAMMATORY RESPONSE FOLLOWING BURN INJURY.A. Dugan, L. Hernandez,* E. Yadav,* and G. Babcock*. Shriners Hospital Cincinnati, OH 45229

Burn patients requiring hospitalization are often treated for anxiety with benzodiazepines. Benzodiazepines are reported to influence immune system function. Immune system alterations are a major cause of burn-induced mortality. In a previous study, we found that mice treated daily with an anxiolytic dose of the benzodiazepine, midazolam, had a significantly improved survival rate following s.c. Pseudomonas aeruginosa inoculation. In the current study we sought to ascertain the mechanism(s) responsible for the protective effect.

Methods: Mice received a 15% TBSA flame burn and half received midazolam 1 mg/kg i.p. daily. Blood and skin wounds were harvested 24 hours after injection on post-burn day 2, 3, 7, or 8 to measure cytokines/chemokines and inflammatory cells.

Results: Mice treated with midazolam had significantly lower serum IL-1β (p = 0.002), TNF-α (p = 0.002), IL-6 (p = 0.016), IL-10 (p = 0.009), and TGF-β (p = 0.004) than saline-treated mice, with little impact on serum chemokine levels. In the wound, TNF-α and IL-10 were the only cytokines significantly influenced by the drug, being lower (p = 0.018) and higher (p = 0.006) respectively. The chemokines in the wound influenced significantly by midazolam were MIP-1α, MIP-1β, and MIP-2 while MCP-1 and KC were not. There were more inflammatory cells at the burn wound margin in midazolam-treated mice. Although serum nitrate/nitrite was significantly increased by midazolam (p = 0.03), both eNOS and iNOS mRNA expression in the wound were similar to the saline group.

Conclusion: We found that midazolam given daily after burn injury significantly influenced the inflammatory response. The clinical implications of these findings on wound healing and shock following burn injury, especially larger burns, deserves further investigation.



Inflammatory monocytes (iMo) are increased in the periphery after burn, and they are a major source of post burn TNFα. We hypothesized that CCR2 KO mice, which cannot mobilize iMo from the marrow, would have reduced post burn inflammation leading to altered post burn susceptibility to endotoxin and bacteria. CCR2 KO and wild type (WT) mice were burned or sham burned, and on post burn day 8 (PBD8) the splenocytes were isolated and stimulated with endotoxin or peptidoglycan. In separate experiments, WT and CCR2 KO mice at PBD8 were injected with LPS or Pseudomonas Aeruginosa and mortality monitored. Thermal injury increased spleen iMo in WT but not KO mice, but PMN were increased by burn in both WT and KO mice. TNFα and IL6, but not IL-10 release correlated with the percentage of iMo in the spleen. Relative to sham WT mice, burn mice were more susceptible to death when endotoxin was injected. The absence of CCR2 had a small protective effect on the susceptibility of burn mice to endotoxin but no effect on the susceptibility of burn mice to bacteria. Our data indicate that CCR2 is required for the post burn production of IL6 and TNFα in the spleen and that this correlates with changes in iMo. In contrast, the lack of CCR2 did not effect the post burn secretion of IL-10 and the susceptibility to endotoxin or bacteria.



MULTICENTER ANALYSIS ON AGE RELATED DIFFERENCES IN MORBIDITY AND MORTALITY AFTER SEVERE BURN INJURY.N. Rodriguez Escobar,* R. Kraft, D.N. Herndon, C.C. Finnerty, and M.G. Jeschke. University of Texas Medical Branch, Galveston, TX 77550

Objective: As indicated in the Beaux-score (burn size plus age) age appears to be a central determinant for survival post-burn. However, to date no large prospective multi-center study exists that examines age differences in relation to post-burn outcomes. We thus conducted this study to determine the effect of aging in severely burned using the Glue Grant platform.

Methods: Four hundred eighty six patients were included in this study and grouped according to their age in children (0-16 yrs), adults (17-54 yrs) and elderly (>55 yrs). Patient's demographics, clinical relevant parameters and outcome were assessed by the participating hospitals. Statistical analysis was performed using student's t-test, Mann-Whitney rank sum test and stepwise logistic regression analysis. Statistical significance was set at p <0.05.

Results: Pediatric burn patients have the largest burn with the best survival and shortest Length of Stay, p < 0.05. Interestingly, incidence of burn wound infection and inflammatory responses were significantly higher in pediatric patients compared to adults and elderly, p<0.05. However, adverse events such as MOF, ARDS, cardiac, and sepsis significantly increased with age with the greatest ratio in elderly patients, p < 0.05.

Conclusion: Pediatric patients have despite a bigger burn size less clinical complications and a shorter LOS. Elderly have a significant higher mortality associated with greater MOF, infections, and sepsis.


Abstract withdrawn.



The recently developed murine model of smoke inhalation and burn (SB) injury was used to study the effect of the substance-P antagonist CP96345. C57BL/6 mice were pretreated with an i.v. dose of a specific NK-1 receptor antagonist, CP9635, or its inactive enantiomer, CP96344, (10 mg/Kg) 1 hr prior to SB injury per protocol (n=5). Mice were anesthetized and exposed to cooled cotton smoke, 2X 30 sec, followed by a 40% total body surface area (TBSA) flame burn per protocol. Mice were resuscitated with normal saline (4ml/Kg/% TBSA burn, i.p.). At 48 hr after SB injury Evans Blue (EB) dye and myeloperoxidase (MPO) were measured in lung after vascular perfusion. Lungs were also analyzed for hemoglobin (Hb) content and wet/dry weight ratio. In the current study, CP96345 pretreatment caused a significant decrease in wet/ dry weight ratio (23%, p=0.048), EB (31%, p = 0.047), Hb (46%, p=0.002) and MPO (54%, p=0.037) levels following SB injury compared to animals with SB injury alone. The inactive stereoisomer CP-96344 pretreatment caused an insignificant decrease in wet/dry weight ratio (14%, p = 0.18), EB (16%, p = 0.134), Hb (9%, p=0.39) and an insignificant increase in MPO (4%, p = 0.79) as compared to mice that received SB injury alone. As expected, levels of EB, Hb, MPO, and wet/dry weight ratios were all significantly (p < 0.05) increased 48 hr following SB injury alone compared to respective sham animals. In conclusion, the current study indicates that pretreatment with specific NK-1R antagonist CP-96345 attenuates the lung injury and inflammation induced by combined burn and smoke inhalation injury in mice.


SEX DIFFERENCES IN RECOVERY FROM BURN INJURY AND INFECTION.C. Deburghgraeve, J. Palmer, and E. Kovacs. Loyola University, Maywood, IL 60153

Objective: Clinical and laboratory evidence suggest that females fare worse after burn injuries than their male counterparts. Our earlier studies demonstrated that female mice became immunosuppressed after burn in part due to extremely high levels of injury-induced estrogen. Here, we expand upon our studies by challenging mice with an infection after burn injury.

Methods: Intact and ovariectomized (OVX) C57JBL/6 female mice were anesthetized and given a 15% total body surface area (TBSA) scald burn and a subsequent topical infection with Pseudomonas aeruginosa. The effects of female sex hormones on systemic and pulmonary cytokines and chemokines were analyzed.

Results: OVX mice exhibited prolonged survival relative to intact females. 24h post-burn and infection, 70% of intact mice died vs. only 8% of OVX mice. By day 5, both groups reached 60% mortality. At day 7, all intact mice died while 15% of OVX mice appeared to fully recover. Circulating and pulmonary IL-6 was decreased in OVX relative to intact burn and infection mice (60% and 90%, p < 0.01). Levels of the neutrophil chemoattractant KC were 85% lower in lungs of OVX compared to intact burn and infection (p < 0.01). Lung myeloperoxidase activity decreased 55% in OVX burn and infected mice compared to intact (p<0.05).

Conclusions: These experiments confirm the involvement of ovaries and/or ovarian hormones in survival after burn and infection. Moreover, they suggest that removal of ovaries/ovarian hormones may help in the clearance of opportunistic pathogens in a post-burn infection. Though OVX mice eventually "catch up" to intact mice in terms of mortality, their delay might provide time for medical intervention to improve outcomes. The less severe pulmonary and systemic inflammatory response observed in OVX mice could allow them to mount a more effective immune response to an infection after burn injury. (Supported by DoD-USAMRAA W81XWH-07-1-0673).


ANTIOXIDANT STATUS IN TISSUES FROM RATS SUBJECTED TO THERMAL INJURY, HINDLIMB UPLOADING AND INSULIN THERAPY.M. Dubick, L. Baer,* J. Barr,* X. Wu,* D. Grubbs,* H. Pidcoke,* and C. Wade. US Army Institute of Surgical Research, San Antonio, TX 78234

Oxidative stress has been associated with burn injury. We investigated whether insulin therapy and hindlimb unloading (HU) affected tissue antioxidant status in burned rats (n=6 or 8/gp). HU was used as a model of prolonged bed rest. After a 40% TBSA scald burn, rats were injected with either insulin (5 U/kg/d) or saline (control) and subjected to HU for 14 d. Insulin injections were stopped 36 hr prior to euthanasia. Rats were then euthanatized and liver, lung, muscle and pancreas were removed and flash frozen for analysis of antioxidant potential, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), nitric oxide and superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase activities. Plasma insulin (0.48 ± 0.14 vs 0.47 ± 0.08 ng/ml) and glucose (116 ± 15 vs 116 ± 3 mg/dl) levels after 36 hr insulin withdrawal were similar between insulin-treated and controls, respectively. In liver and pancreas, primarily, saline injected rats showed 20-40% lower MnSOD, catalase, GPx and GR activities, 15% lower NO levels and 25% lower antioxidant potential levels compared to HU insulin injected rats. Minor changes were noted in lung and no significant effects were observed in muscle. These data suggest that at least in liver and pancreas, daily insulin injection increased tissue antioxidant status in the presence of prolonged bed rest and burn injury.


THE EFFECTS OF RADIATION AND BURN INJURY IN A MURINE MODEL.J. Palmer, C. Deburghgraeve, M. Bird, and E. Kovacs. Loyola University, Maywood, IL 60153

Objective: Combined radiation and burn injuries are likely to occur after nuclear events, such as a meltdown at a nuclear energy plant or a nuclear attack. Little is known about the mechanisms by which combined injuries result in higher mortality than either insult alone, and few animal models exist. To examine remote organ injury and failure following combined injury, we developed a mouse model of moderate radiation and burn. We first measured parameters in the blood and lung tissue, since lungs are especially prone to irreversible damage after burn injury and irradiation due to overwhelming inflammation. We reasoned that the two injuries combined would result in more extensive lung damage.

Methods: C57JBL/6 male mice were subjected to a 0, 2, 4, 5, 6, or 9 Gray (Gy) dose of radiation, and then anesthetized and given a 15% total body surface area (TBSA) scald burn. Blood and lung tissue was analyzed for changes in leukocyte distribution and inflammatory mediators.

Results: When combined with burn injury, higher doses of radiation affected mortality. Early after injury, lungs from burn- and combined-injured groups had moderate congestion and neutrophil infiltration. Pulmonary TNFα and MCP-1 were elevated 5-fold and 3-fold in the combined-injured group compared to sham and either injury alone (p < 0.05). Radiation- and combined-injured groups had decreases in circulating white blood cell counts by 48h post-injury compared to sham (90% and 60%, p < 0.05).

Conclusions: The combined injury exacerbated the pulmonary and systemic inflammatory response and lowered peripheral leukocytes, which might contribute to an inability to survive the injury or subsequent infections. These studies give insight into potential therapeutic interventions that might diminish the extensive pulmonary inflammatory responses and resulting damage observed in this unique type of injury. (Supported by NIH R21 AI080528 and NIH F32 AA018068).


BURN SERUM CAUSES A CD-14 DEPENDENT MITOCHONDRIAL DAMAGE IN PRIMARY CARDIOMYOCYTES.Q. Zang, D. Maass, J. Wigginton, R. Barber, B. Martinez,* A. Idris, F. Nwariaku,* and J. Horton (deceased). UTSW, TX 75390-9160

Objective: An ex vivo model of primary cardiomyocyte cell culture was applied to investigate the regulatory mechanisms of burn-induced mitochondrial impairments.

Methods: Burn serum (BS) was collected 24 hr after 40% TBSA burn in adult SD rats. Cardiomyocytes were isolated from SD rats as well as WT and CD14−/− mice. Mitochondrial damage was evaluated by outer membrane integrity, lipid oxidation and antioxidant activities (glutathione peroxidase, GPx; superoxide dismutase, SOD) in the mitochondrial fractions. Mitochondrial metabolism was determined by the activity of cytochrome C oxidase (COX). MtROS was detected by fluorescent labeling with MitoSox.

Results: An 18-hour BS challenge (10% V/V) of primary cardiomyocytes impaired mitochondrial membrane integrity and metabolic function. This treatment doubled the amount of mitochondrial membrane damage and cytosolic accumulation of the mitochondrial protein cytochrome C. Mitochondrial content of lipid oxidation was increased more than 30%. Compared with control cells, mitochondrial antioxidant activities (SOD and GPx) from BS challenged cells were reduced about 30% and 50% respectively. Mitochondrial metabolic deficiency was indicated by a 30% decrease in COX, a key enzyme in mitochondrial respiratory complexes. BS challenge significantly elevated mtROS production in the myocytes, and this increase was abrogated by pre-exposing the cells with a CD-14 neutralizing antibody. Furthermore, the burn injury-induced mitochondrial impairments were not detectable in the cardiomyocytes isolated from CD14 knockout mice.

Conclusion: The studies suggest that burn injury produces mitochondrial damage in myocardium via a CD14-dependent signaling pathway.


THE INFLAMMATORY RESPONSE TO HIGH RISK GLYCEMIC VARIABILITY IN PATIENTS WITH SEVERE BURNS.A. G. Mora,* M. G. Schwacha, H. F. Pidcoke,* K. K. Chung,* M. Park,* S. E. Wolf, and C. E. Wade. USAISR, Ft. Sam Houston, TX 78234

Patients with severe burns have an increased incidence of infection and sepsis that is associated with an overwhelming inflammatory response. Glycemic control and decreased intra-patient glycemic variability improve outcomes in severely injured patients. Our previous work identified average daily risk range (ADRR), a measure of glycemic variability, to be an independent predictor of outcome. We propose inflammatory mediators are associated with the degree of glycemic dysregulation using ADRR as a measure of variability. From a prospective observational study, a post-hoc analysis was performed on patients with burns with an<3 day expected ICU stay, and enrolled within 24 hours of injury. Glucose was controlled (80-110 mg/dl) in all patients with insulin. IL-6, IL-8, IL-1β, GM-CSF, and TNF-α levels were collected on days 0, 1, 3, 5, and 7 following injury. Patients were categorized based on ADRR over 7 days. ADRR assigns risk over average daily intra-patient glycemic variability where ADRR <16 is low risk and ADRR ≥16 is high risk. Fifty-nine subjects were low risk and 21 were high risk. Glucose levels were 129±21 mg/dl and 151±21 mg/dl (p=0.03) respectively with coefficient of variation 18±7% vs. 28±17% (p=0.01). Groups did not differ in: age; glucocorticoids; vent free, ICU fee, or hospital free days; incidence of ARDS, SIRS, or sepsis (ABA guidelines). The high risk group was more critically injured, with increased mortality (32% vs. 76%; p<0.001), and had a higher incidence of septic shock. Average concentrations of IL-6, IL-1β, GM-CSF, and TNF-α were similar while IL-8 levels were increased in the high risk group. Using ADRR, high risk glycemic variability is associated with an increased risk of septic shock and subsequent mortality. However, ADRR was only related to IL-8 concentrations in the first 7 days.


NEBULIZATION OF GAMMA-TOCOPHEROL IMPROVES PULMONARY FUNCTION IN OVINE WITH COMBINED BURNS AND SMOKE INJURY.Y. Yamamoto,1 P. Enkhbaatar,1 E.R. Kraft,1 S. Rehberg,1 R.A. Cox,1 L.D. Traber,1 C. Szabo,1 M.G. Traber,2 D.N. Herndon,1 and D.L. Traber1. 1The University of Texas Medical Branch, Galveston, TX, 2Linus Pauling Institute, Oregon State University, Corvallis, OR

We have previously reported that nebulization of gamma tocopherol (gT) carried by flaxseed oil into the airway of an ovine model of acute lung injury was beneficial. In the present study, we hypothesized that nebulization of gT dissolved in ethanol would be more clinically relevant and will effectively improve pulmonary function following burn/smoke injury.

Methods: Adult ewes (n=11) were subjected to 40% TBSA, third-degree flame burn and insufflated with smoke (48 breaths of cotton smoke, <40°C) under deep anesthesia. One g of gT dissolved in 2.2 mL of ethanol was continuously delivered by customized aerosolization device for 48 hrs (gT group, n=6). Untreated animals (n=5) were nebulized with same amount of ethanol. All animals were placed on ventilator and monitored for 96 h. Weaning from the ventilator was initiated if PaO2/FiO2 was above 250 mmHg at 48 h post-injury. This experiment was carried out as a double-blind comparative study.

Results: PaO2/FiO2 (mean±SD) was 310±152 in the gT and 150±27.0 in the untreated groups at 48 h (r<0.05). At 96 h post-injury, all gT animals were weaned from ventilator, while none of untreated animals could completely weaned. The lung tissue gT levels (mean±SD) were 38.5±16.8 nmol/g in the gT and 0.39±0.46 nmol/g in the untreated groups (r<0.01). Bronchioles obstruction score were 2.0±1.1 % in the gT and 4.0±0.8 % in the untreated groups (r<0.05).

Conclusion: The nebulization of gT carried by ethanol improved pulmonary oxygenation and reduced the ventilator time in burn/smoke injured sheep. Delivery of gT into the lungs via ethanol may be a safe, novel, and efficient approach for management of ALI patients.


EFFECTS OF ACUTE ETHANOL EXPOSURE COMBINED WITH BURN INJURY ON SPLENIC IMMUNE CELL RESPONSE.J.L. Rendon,* X. Li,* and M.A. Choudhry. Loyola University Chicago Stritch School of Medicine, Maywood, IL 60153

Nearly 50% of adult burn patients have a measurable blood ethanol (EtOH) level at the time of hospital admission. These patients exhibit an increased risk of infection and higher mortality when compared to burn patients without EtOH exposure at the time of injury. Additionally, EtOH exposure prior to burn injury has been shown to exacerbate T cell suppression and enhance susceptibility to infection. This study examined whether EtOH intoxication prior to burn injury modulates dendritic cell (DC) function, as they play a role in T cell activation and differentiation. Male mice, ∼25g, were gavaged with EtOH (2.9mg/kg) 4 hours prior to receiving a 12-15% total body surface area full thickness burn. Animals were sacrificed on days 1 and 3 post injury. Spleens were collected and the number of splenic DC and T lymphocytes was determined using flow cytometry. Remaining cells were cultured with lipopolysaccharide (LPS) for 24 hrs and the supernatants were collected and analyzed for IL-10 and IL-12 levels. There was no significant decrease in DC number on day 1 post injury as compared to sham injury; however, by day 3, 40% decreases in splenic DC number and MHC II expression were noted. T lymphocyte number remained unaffected following combined insult of EtOH exposure and burn injury. Interestingly, a ∼2-fold increase in IL-10 and a ∼1.5-fold increase in IL-12 levels in supernatants after LPS stimulation was observed on day one after combined insult compared to sham. On day 3, IL-10 remained elevated. In contrast, a 50% decrease in IL-12 levels was observed on day 3 post injury as compared to sham. These results indicate that EtOH exposure prior to burn injury modulates DC effector responses which may alter ability to fight microbial pathogens and drive T cell polarization. (NIH R01AA015731 (MAC) and the Dr. Ralph & Marian C. Falk Medical Research Trust (JLR)).


THE ROLE OF γδ T-CELLS IN WOUND INFLAMMATION AFTER BURN INJURY.R.F. Oppeltz, Q. Zhang, M. Rani, and M.G. Schwacha. Dept of Surgery, University of Texas Health Science Center at San Antonio, TX 78229

Objective: Wound healing complications are common after major burn injury and the γδ T-cell subset has been shown to play a pivotal role in the healing response through the regulation of growth factor production. Inflammation, however, is also an important part of the wound healing process and the potential role of γδ T-cells in burn wound site inflammation is unknown.

Methods: Male C57Bl/6J wildtype (WT) and γδ T-cell deficient mice (γδ KO) were subjected to a major burn injury (3rd degree, 25% TBSA) or sham treatment. Three days thereafter skin samples were collected from the burn site, wound edge and uninjured skin. Skin samples were processed and skin homogenates were analyzed for inflammatory cytokine (IL-1β, IL-6, IL-10, TNF-α, KC and MCP-1) levels by Luminex multiplex bio-assay.

Results: Burn injury in WT mice resulted in significantly increased (p< 0.05) levels of IL-1β, IL-6, TNF-α, MCP-1 and KC at the burn site and/or wound edge as compared to non-injured skin and sham skin. In contrast, IL-10 levels were not significantly elevated in any skin samples. Analysis of skin samples from γδ KO mice revealed a similar inflammatory cytokine pattern that was not significantly different from that of WT mice.

Conclusions: These findings indicate that the wound inflammatory response is active and robust at 3 days after burn injury. Surprisingly, it does not appear to be regulated by γδ T-cells, since the inflammatory responses were similar in WT and γδ KO mice. The lack of a role for γδ T-cells in burn wound inflammation in our model may be related to the time after burn injury studied or the overriding contribution of other inflammatory cell populations. Further studies are required to elucidate the mechanism(s) regulating wound inflammation after burn injury. (Supported by NIH grant GM 079122).


NF-κB SPLICING VARIANTS PLAY A ROLE IN THE REGULATION OF CYTOKINE EXPRESSION.H. Phan, K. Cho, I. Lee,* T. Green,* and D. Greenhalgh. UC Davis Medical Center/Shriners Hospitals for Children, CA 95817

Objective: Nuclear factor-κB (NF-κB) regulates the expression of various genes involved in inflammation. Recently, we demonstrated that burn injury led to an up-regulation of nine NF-κB alternative splicing variants in lungs of CD 14 knockout mice. In this study, we evaluated the function of NF-κB splicing variants in respect to their ability to regulate cytokine expression.

Methods: NF-κB splicing variants were overexpressed in RAW 267 cells, which were collected at 24 hours for RT-PCR and Western analysis of cytokine expression.

Results: IL-6, IL-1β, and iNOS mRNA expression were increased when one of the four rel B splicing variants (relB.v3) was overexpressed, compared to native full-length rel B overexpression. The protein expression of Cox-2 also increased with relB.v3 overexpression. When the native full-length rel A was overexpressed, the mRNA expression of IL-6, IL-1β, and ICAM increased, while TNF-α, Cox-2, and iNOS stayed neutral. Interestingly, when the rel A variant (relA,v1) was overexpressed, the mRNA expression of IL-6, IL-1β, ICAM, TNF-α, Cox-2, and iNOS were profoundly suppressed. Similarly, the protein expression of iNOS, Cox-2, and IκBα were increased with native rel A overexpression, but decreased with relA.v1 overexpression.

Conclusions: Splicing variants of NF-κB play a role in the regulation of genes involved in inflammation. CD 14, a component of the LPS receptor, may regulate inflammatory gene expression through NF-κB alternative splicing mechanism.



POST-BURN DIFFERENTIAL ACTIVATION OF HUMAN ENDOGENOUS RETROVIRUSES AND THEIR ROLE IN BURN PATHOGENESIS.K. Lee,1* Y. Lee,1* H. Rah,1* D. Kwon,1* D. Moon,1* L. Fitzsimon,1* D.G. Greenhalgh,1 J. Nemzek,2 and K. Cho1. 1Department of Surgery, University of California, Davis and Shriners Hospitals for Children, Sacramento, CA 95817, and 2Department of Pathology, University of Michigan, Ann Arbor, MI 48109

Recent studies have shown that human endogenous retroviruses (HERVs) are associated with a range of disease processes. In this study, to investigate the role of HERVs in burn pathogenesis, we first examined changes in HERV expression in blood samples of eleven patients at six to eleven different time points, up to nine months, after injury. The expression profile of 21 different HERV families was analyzed by semi-quantitative RT-PCR using a set of time course blood samples from each patient. The post-burn HERV expression profile was patient-specific and it was variable depending on time after injury and HERV type. A total of 1,162 HERV fragments were isolated from the eleven patients and cloned for in silico mapping of relevant HERVs using the NCBI genome database. A survey of the database using the sequences from two patients identified 16 putative HERVs so far, including one full-length provirus with intact coding potential. To investigate the role of some of these putative HERVs in inflammation, their gag and env genes were cloned from the respective patient's genomic DNA. Overexpression of the env polypeptides from the HERV-K (HML2) family, which was activated in both patients at least at one time point, induced IL-1β, IL-6 and COX-2 in U937 human monocytes. The findings from this study suggest that burn-elicited stressors differentially modulate expression of certain HERVs in a time- and HERV type-specific manner. It is likely that the genetic backgrounds and ages of the patients as well as nature of injury (e.g., size and degree) contributed to the differences in the HERV expression profile. Further investigation into the role of HERVs in immune dysregulation may provide fresh insights into understanding of burn pathogenesis.



Introduction: Patients who undergo burn excision and grafting lose significant blood volume, but it is not known if appropriate replacement volumes are infused during surgery. To answer this question, we directly measured blood volume before and after excision and grafting in survivors without ongoing post-operative losses, and compared these data to standard estimates.

Methods: We collected prospective data on burned subjects (N=7, >20% TBSA burned). Blood volume calculations were made with the Daxor blood volume analyzer (BVA-100) and compared to 1. calculated blood volume based on weight and estimated blood loss and 2. calculated blood loss using hemoglobin as the tracer: (PRBC[in cc]*2)+(preop Hgb-postop Hgb)*500.

Results: Preoperative blood volume estimates were 8.0 ± 1.2 L based on body mass, while measured volume (BVA-100) was 5.5 ± 0.3 L (p=0.04). Mean estimated blood loss/cm2 excised was 0.30 ± 0.04 cc/cm2, while measured loss was 0.69 ± 0.09 cc/cm2 (p=0.001) hemoglobin-based equation was 0.65 ± 0.08 cc/cm2 (p=0.58 compared to BVA-100). When comparing measured blood volume pre and post-operatively, all but one were negative at the end of the operation compared to pre-operative values (−1067 ± 315 cc) (p=0.015).

Conclusion: Estimated blood loss due to excision and grafting is notably different when compared to measured or calculated values. Measured blood volumes are significantly lower postoperatively compared to before operation. Utilizing the measured or calculated methods above to determine blood loss may be a more prudent approach to guide blood replacement.


DENDRITIC CELL MODIFICATION OF NEUTROPHIL RESPONSES AFTER BURN INJURY.J. Bohannon,*,* W. Cui,* and T. Toliver-Kinsky. University of Texas Medical Branch, Galveston, TX 77555

Treatment of burned mice with the dendritic cell (DC)-growth factor, Fms-like tyrosine kinase-3 ligand (FL) has been shown to increase resistance to a Pseudomonas aeruginosa burn wound infection in a DC-dependent manner. The primary roles of DCs in response to infection include presentation of antigen and activation of other innate effector cells, but DCs are not effective bacterial killers. Neutrophils, which are among the first responders to burn and skin injuries and are primarily involved in bacterial killing and clearance, have been shown to interact with and engage in bidirectional activation with DCs. This study was designed to determine if FL treatments can affect neutrophil responses through modification of DCs following burn injury. To address this, neutrophil in vitro migratory capacity, MPO levels, and bacterial clearance following a burn wound infection were measured before and after treatment with FL or co-incubation with DCs. To examine the role of DCs, CD11c-DTR transgenic mice were utilized for depletion of DCs during FL treatment. Studies revealed that both in vivo and in vitro treatment with FL enhances neutrophil migratory capacity, and this enhancement was dependent upon the involvement of DCs. DCs were required for FL-mediated increase in MPO levels, suggesting DC-dependent mobilization of neutrophils in response to FL treatment. Additionally, FL-treatment enhanced neutrophil-mediated bacterial clearance in a DC-dependent manner. These results suggest that DC enhancement of neutrophil responses played a role in FL-mediated resistance to a burn wound infection.


EARLY SINGLE-DOSE ESTROGEN TREATMENT FOLLOWING SEVERE BURN INJURY REDUCES ACUTE AND SUB-ACUTE BRAIN INFLAMMATION.R. Barber, J. Simpkins,* J. Gatson, D. Maass, A. Idris, P. Pepe,* J. Minei, and J. Wigginton*. University of Texas Southwestern Medical Center, Dallas, TX 75390

Introduction: Patients with severe burn injury experience a rapid elevation in multiple circulating pro-inflammatory cytokines, with the levels correlating with both injury severity and outcome. Accumulations of these cytokines in animal models have been observed in remote organs, however data are lacking regarding early brain cytokine levels following burn injury, and the effects of estradiol on these levels. Using an experimental animal model, we studied the acute and sub-acute effects of a full-thickness third degree burn on brain levels of TNF-alpha, IL-1beta, and IL-6 and the anti-inflammatory effects of acute estrogen treatment on these levels.

Methods: In this study, 168 male rats received 3rd degree 40% total body surface area (TBSA) burns. Fifteen minutes following burn injury, the animals received a subcutaneous injection of either placebo or 17 beta-estradiol (0.5 mg/kg). Brains were harvested in 144 rats over the first 24 hours, with 24 rats harvested at 7days to determine the effect of a single dose of estrogen on inflammation in the brain. Brain cytokine levels were measured using the ELISA method.

Results: 17 beta-estradiol significantly decreased the levels of brain tissue TNF-alpha (∼25%), IL-1beta (∼60%), and IL-6 (∼90%) when compared to the placebo group at the early (0.5, 1, 2, 4, 6, 7, 8, 12, 18, 24 hours) time-points. Additionally, early estrogen treatment resulted in significant decrease in the levels of TNF-alpha (∼100%), IL-1beta (∼94%), and IL-6 (∼98%) at day 7.

Conclusion: A single early dose of estrogen decreases both acute and sub-acute brain inflammation following severe burn injury.


EARLY, SINGLE-DOSE ESTROGEN DECREASES INFLAMMATION AND APOPTOTIC SIGNALING IN THE HEART FOLLOWING SEVERE BURN INJURY.D. Maass, Q. Zang, J. Gatson, J. Minei, A. Idris, P. Pepe, and J. Wigginton. University of Texas Southwestern Medical Center, Dallas, TX 75390

Background: Patients with severe burn injury experience a rapid elevation in multiple circulating pro-inflammatory cytokines, with the levels correlating with both injury severity and outcome. Accumulations of these cytokines in animal models have been observed in remote organs, however data are lacking regarding acute and sub-acute serial heart cytokine levels following burn injury, and the therapeutic effects of estrogen on these levels. Using an animal model, we studied the acute effects of a full-thickness third degree burn on cardiac levels of IL-1beta, IL-6, IL-10, and TNF-alpha. Here, we hypothesized that single-dose acute estrogen treatment decreases inflammation in the heart up to seven days.

Methods: In this study, 144 male rats received 3rd degree 40% total body surface area (TBSA) burns. Fifteen minutes following burn injury, the animals received a subcutaneous injection of either placebo or 17β-estradiol (0.5 mg/kg). The hearts were harvested at 0.5, 1, 2, 4, 6, 8, 12, 18, and 24 hours after injury, and the heart cytokine (IL-1beta, IL-6, IL-10, TNF-alpha) levels were measured using the ELISA method.

Results: In the burned rats, 17β-estradiol significantly decreased the cardiac levels of TNF-alpha (∼95%), IL-6 (∼50%), IL-1beta (∼25%), IL-10 (∼20%), when compared to the placebo group.

Conclusions: Following severe burn injury, estrogens decrease heart inflammation and the levels of the pro-apoptotic, cytochrome C. In addition, estrogen signaling promotes cell survival, as indicated by an increase in NF-kappaB levels. Importantly, estrogen treatment following burn injury almost completely blocked the burn-induced increase in TNF-alpha in the heart, which has previously been linked to a poor outcome.



Objective: Clinical studies and animal models have shown that burn injury leads to hyperglycemia and insulin resistance. These mechanisms induce the modulation of pro-inflammatory pathways and organ dysfunction. It has been shown that tight euglycemic control achieved by insulin administration improves these conditions. However it is unknown whether restored normal glycemia or insulin administration leads to these effects. Therefore we conducted an animal study to investigate the effects of insulin, metformin and fenofibrate post burn.

Methods: Rats received a 60% full thickness scald burn and tissue was harvested at 24h, and 48h post-burn. Rats were randomized to receive saline, insulin 5 IU/kg, metformin or fenofibrate (n=4 per group per time point) to achieve tight euglucemic control. Hepatic JNK pathway activation was determined by protein expression of ATF6, ERO1α, pJNK1/2 and JNK1/2 Statistical analysis was evaluated by Student's t-test corrected with Bonferronis post-hoc test. Statistical significance is set at p<0.05.

Results: We detected a significant (p<0.05) decreased expression of cleaved ATF6, ERO1α and pJNK1in the insulin treated group compared to burned controls and the metformin and fenofibrate group. Unphosphorylated JNK1 was consecutively significantly (p<0.05) elevated whereas JNK2 and pJNK2 showed no differences between the treatment groups.

Conclusions: Insulin administration leads to the non phosphorylation of JNK1 and therefore to a down regulation of the pathway. Reduction of post burn hepatic ER stress can be shown by the downregulation of cleaved ATF6 and ERO1α. Metformin and fenofibrate did not affect the investigated proteins and therefore we suggest that insulin not only the tight glycemic control leads to these improvements.


PROPRANOLOL REDUCED POST-BURN INSULIN RESISTANCE BY RESTORING IRS-1/AKT PATHWAY.J. Song,* R. Kraft, G. Gauglitz, M. Huang,* D.F. Boehning,* D.N. Herndon, and M.G. Jeschke. Shriners Hospitals for Children, and the University of Texas Medical Branch, Galveston, TX 77550

Post-burn hyperglycemia was linked to the impairment of insulin sensitivity, and significantly contribute to adverse outcome of burn patients. Recently we found that severe burn injury induced hepatic insulin resistance associated with endoplasmic reticulum (ER) stress and unfolded protein response (UPR) which leads to a phosphorylation of Insulin receptor substrate (IRS)-1 at the tyrosine site. We have evidence that propranolol a non-selective β1/β2 receptor antagonist improves insulin resistance post-burn. We hypothesized that propranolol reduces hepatic post-burn ER stress/UPR and subsequently restores phosphorylation of IRS-1. Rats received a 60% total body surface area thermal injury with 5mg/kg/day of propranolol treatment orally. A laparotomy was performed at 72 hours post-burn. Liver was harvested before and 1 min after insulin injection (1 IU/kg) into the portal vein, and expression patterns of various proteins known to be involved in insulin and ER-stress signaling cascades were determined by Western blotting. Our results showed that severe burn injury activated ER stress and UPR by significantly increasing phospho-PKR-like ER-Kinase (PERK) and phospho-inositol requiring enzyme (IRE)-1 leading to an elevation of phospho-Jun N-terminal Kinase (JNK) (p<0.05). Phospho-PERK and phosphor-IRE-1 significantly decreased with propranolol treatment after burn injury (p<0.05). Burn induced IRS-1 phosphorylation at serine 307 and de-phosphorylation at tyrosine 612 resulting in an impaired Akt signaling (p<0.05). Propranolol treatment improved the response of insulin signal pathway by restoring IRS-1 signaling and upregulating Akt (p<0.05). We thus conclude that propranolol attenuated the ER stress response induced by severe burn which was associated with restored IRS-1/Akt signaling.



Objective: Cell swelling and osmosensing pathways can induce cellular protection. Glutamine (GLN) is known to induce the protective heat shock protein (HSP) response via metabolism by the O-glycosylation (O-GlcNAc) pathway. We hypothesize this effect may be related to GLN-mediated cell swelling. The aim of our study was to investigate whether the integrin cell volume pathway is related to GLN's cell protective mechanism.

Methods: Intestinal Epithelial Cells (IEC-6) treated for 15 min with increasing doses of GLN up to 20mM, with or without the integrin inhibitor GRGDSP (50μM) or inactive control peptide GRGESP (50μM). Cell survival via MTS assay 24h post-heat shock (HS) (44°C×50min). O-glycosylated protein levels and HSP70 determined via Western blot after non-lethal HS (43°C×50min).

Results: GLN increased cell survival in a dose dependent manner (p< 0.001 vs. HS CT; n=3). Integrin inhibition with GRGDSP completely attenuated GLN's protection in 2mM GLN group, decreased protection by 87% and 78% in 10mM and 20 mM GLN groups (p< 0.001 vs. respective GLN groups; n=3). O-GlcNAc modified proteins increased 100% post-HS after 10 mM GLN (p< 0.001 vs. HS C; n=5). GRGDSP attenuated 10 mM GLN's effect on O-GlcNAc protein modification by 84% (p< 0.05 vs. HS 10 mM GLN; n=5). HSP70 increased by 124% in the 10 mM GLN group after HS (p< 0.05 vs. HS CT; n=3). GRGDSP attenuated HSP70 expression in this group by 91% (p< 0.05 vs HS GLN; n=3). GRGESP control peptide did not have any effect on any of the GLN-mediated cellular protection pathways.

Conclusion: The integrin cell volume sensing pathway appears to be an essential component of GLN's molecular mechanism of cellular protection. This osmosensing pathway may play a key role in GLN's HSP induction via the O-GlcNAc pathway.



Hyperglycemia is common following injury, infection and critical illness. However, little is known about the mechanism of development of acute insulin resistance that causes hyperglycemia. In a mouse model of injury, we determined whether trauma and hemorrhage induced acute insulin resistance. Acute insulin resistance occurred in both liver and skeletal muscle following the hemorrhage period (90 min). Following resuscitation and recovery (3 ½ hr), skeletal muscle insulin resistance recovered more quickly than hepatic insulin resistance. Further data indicate that the hyperglycemia resulted from the defects of insulin signaling in liver and skeletal muscle during hemorrhage. IL-6 is thought to play a major role in the chronic development of insulin resistance and is also increased following injury. Therefore IL-6 knockout (KO) mice were used to investigate the role of IL-6 in trauma and hemorrhage-induced hyperglycemia. Insulin's ability to induce phosphorylation of IR and Akt, were rescued in skeletal muscle of IL-6 KO mice during hemorrhage, but not in liver. This suggests that loss of IL-6 can reduce hyperglycemia through the improvement of skeletal muscle insulin signaling during hemorrhage. To investigate the role of IL-6 in hyperglycemia after resuscitation, hepatic insulin signaling was measured in IL-6 KO mice. Following resuscitation, 3 ½ hr after hemorrhage loss of IL-6 decreased hyperglycemia and partially rescued hepatic insulin signaling. Our results indicate that the timing of the role of IL-6 following injury may differ between skeletal muscle and liver.


ANALYSIS OF NEW MOLECULAR MECHANISMS AND THE ANTI-INFLAMMATORY EFFECTS OF ANTITHROMBIN III IN HUMAN PLATELETS.T. Doi,* S. Adachi,* H. Kato,* O. Kozawa,* and S. Ogura. Advanced Critical Care Center, Gifu University Hospital, Japan

Purpose: 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. It has recently been reported that AT-III acts directly on human platelets and exhibits anti-inflammatory effects. However the exact mechanism of AT-III in platelets remains to be clarified. In the present study, we examined the effect of AT-III on the adenosine diphosphate (ADP)-induced platelet granule secretion and the exact mechanism.

Methods: Platelet-rich plasma (PRP) was obtained from healthy volunteers to analyze the secretion of platelet-derived growth factor (PDGF)-AB and serotonin stimulated by ADP as well as the release of soluble CD40 ligand (sCD40L), an anti-inflammatory marker, using ELISA. In addition, the levels of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, and heat shock protein (HSP) 27 phosphorylation using Western blot were analyzed.

Results: The secretion of PDGF-AB and serotonin, and the release of sCD40L from platelets caused by ADP-stimulation were significantly suppressed by AT-III. MAPKs and HSP27 phosphorylation were also suppressed by AT-III.

Conclusion: The results suggest that AT-III directly acts on platelets and suppresses ADP-induced platelet granule secretion due to inhibiting HSP27 phosphorylation. Our present study provides evidence that AT-III might act in patients with DIC associated with septic shock and other severe conditions.


ALTERATION OF LPS SIGNALING PATHWAYS IN DENDRITIC CELLS BY FMS-LIKE TYROSINE KINASE-3 LIGAND.T. Toliver-Kinsky, W. Cui,* and E. Sherwood. University of Texas Medical Branch, Galveston, TX 77555

Fms-like tyrosine kinase-3 ligand (FL) is a dendritic cell growth factor that has been shown to increase resistance to Pseudomonas aeruginosa infection in burned mice. The receptor for FL, flt3, is expressed on hemopoietic progenitor cells and terminally differentiated dendritic cells (DC). While the effect of flt3 activation in progenitor cells is known and results in proliferation and differentiation into DCs, the effects of flt3 activation in terminally differentiated DCs are not known, although previous studies have shown that FL enhances DC promotion of immune responses. This study was designed to determine the effects of FL on the expression and activation of proteins involved in LPS signaling pathways. DCs from burned mice were treated with or without FL in vitro or were harvested from burned mice that had been treated with or without FL in vivo. DCs were stimulated with LPS for various times and proteins were isolated for analysis by western blot. Compared to non-treated DCs, phosphorylation/activation of proteins involved in the p38 MAP kinase/NFκB pathway, which is associated with DC maturation, was decreased in DCs by FL treatment. However, FL-treated DCs had increased levels and LPS-induced phosphorylation of proteins involved in the PI3K/AKT pathway, which is associated with pro-survival responses in DCs. DCs treated with FL had decreased levels of activation-induced apoptosis in vitro. These data indicate that prior FL stimulation may shift LPS signaling responses in DCs towards pathways that promote survival and delay activation-induced apoptosis during a bacterial challenge.


PURIFICATION OF THE PROTEIN IN HUMAN MILK THAT INDUCES THE EXPRESSION OF PGP IN HUMAN ENTEROCYTE CELL LINES.A. Franklin, Y. Guner, J. Upperman, A. Grishin, and H. Ford. Children's Hospital Los Angeles, Los Angeles, CA

Background: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency that is associated with small and large intestinal perforation. Breast milk is highly protective against NEC. P-glycoprotein (Pgp) is an efflux ATP-binding cassette transmembrane transporter that protects against intestinal inflammation and it may be the protective factor in breast milk. We hypothesize that breast milk protects against NEC by inducing Pgp.

Objective: The objective is to purify the factor(s) in human breast milk that induces the expression of Pgp in HT-29 cells, a human intestinal cell line.

Methods: After IRB approval, ion exchange chromatography and ammonium sulfate precipitation were employed to fractionate soluble proteins of human milk. HT-29 cells were treated with the fractionated soluble proteins. Pgp induction was determined by immunoblotting with the C219, an anti-Pgp antibody.

Results: The Pgp-inducing protein precipitated at 40% saturation of (NH4)2SO4 (Fig. 1). Interestingly, this factor did not bind to DEAE- or CM-cellulose at physiologic pH, suggesting that it is a neutral protein.

Conclusion: Further purification and identification is needed to characterize the Pgp-inducing factor. This factor offers hope in the potential treatment of NEC through formula supplementation with this protective component.

FIG. 1
FIG. 1:
Ammonium sulfate precipitation milk in HT-29 cells


INTERMITTENT HYPOXIA ACTIVATES NF-κB AND NITRIC OXIDE PATHWAYS.V.P. Good, J.E. Baumgardner, and C.M. Otto. University of Pennsylvania, Philadelphia, PA 19104

Introduction: Intermittent hypoxia (IH), a hallmark feature of acute lung injury and sleep apnea, is associated with inflammation and oxidative stress. IH activates the NF-κB pathway in people, animals and cell culture. Nitric oxide (NO) has been implicated as an important molecule in inflammation. Hypoxic stress and NF-κB activation both result in upregulation of nitric oxide synthase (NOS). We took advantage of the short lifespan, and available genetic mutations in Drosophila to investigate the contributions of the NF-κB (Relish) and NO pathways to the response of flies exposed to IH.

Methods: Flies: In Drosophila, a single NOS gene, (dNOS), is responsible for NO production. Homozygous dNOS "knockout" flies are non-viable, however, heterozygous dNOS mutant flies are viable and have reduced levels of dNOS activity. E20 and E38 are 2 Relish mutants, these along with heterozygous dNOS mutants, and their wildtype Canton S (CS) or white 1118 (w1118) controls were exposed to IH (30 cycles/hr; 21% to 5% O2) or normoxia (21% O2) for 8 hrs during the dark (sleep) period. A group of CS or w1118 flies were fed (10 mM) L-NAME for the duration of the experiment to inhibit NO production. Survival was determined by daily counts of dead flies and was analyzed with Kaplan-Meier Survival Analysis.

Results: IH significantly decreased the lifespan of CS and w1118 flies. Elimination of the Relish gene or reduction of NOS activity (genetic or chemical) conferred resistance to IH-induced mortality. In 21% O2, chemical or genetic reduction of NOS activity increased mortality.

Conclusions: IH induces oxidative and nitrosative stress and initiates inflammatory responses in mammals. Drosophila respond to IH with increased mortality that is at least in part mediated by activation of the NF-κB (Relish) pathway and production of NO. (Supported by AHA 09GRNT2020197).



This study tested the hypothesis that LPS regulates Sp1 activity in an organ-dependent manner. Mice were injected with saline or Samonelia Enteritidis LPS (10 mg/kg, i.p.). Tissue levels of Sp1 DNA binding activity and Sp1 protein were determined using electrophoretic mobility shift assay and Western blot. LPS caused a 79%, 91% and 55% reduction in Sp1 binding activity in the lungs at 60, 120 and 240 minutes post-LPS. By contrast, LPS had no effects on Sp1 binding activity in heart and liver of the same groups of mice. Sp1 protein from lungs is heavily phosphorylated at serine and threonine residues, but from heart of the same mice is not phosphorylated. LPS dephosphorylated Sp1 protein at serine and threonine residues in the lung. Sp1 dephosphorylation was initially observed at 30 minutes, further increased thereafter and peaked at 240 minutes after LPS challenge. LPS caused no change in Sp1 phosphorylation state in the heart. Treatment of nuclear proteins from control lungs, but not from control heart, with calf intestinal phosphatase in vitro dephosphorylated Sp1 protein, and significantly reduced Sp1 DNA binding activity. At 60, 120 and 240 minutes after LPS challenge, lung tissue level of Sp1 protein was reduced by 88%, 70% and 10%, respectively. By contrast, tissue level of Sp1 protein in heart and liver of same groups of mice was not affected. Both dephosphorylation and degradation of Sp1 protein were temporally correlated to the reduced Sp1 binding activity. LPS induced a Sp1-degrading protease activity in the lungs, but not in heart and liver. These results demonstrate that LPS down-regulates Sp1 activity in vivo in the lungs, but has no effects on tissue Sp1 activity in heart and liver, demonstrating a lung-specific mechanism regulating Sp1 activity. (Supported by NIH R21AI076987 and AHA 0355609).


ACTIVATION OF Aβ-CATENIN MEDIATED WNT SIGNALING PATHWAY IN MICROVASCULAR ENDOTHELIAL CELLS.D. Sawant,* B. Tharakan, F. Hunter,* and E. Childs. Department of Surgery, Texas A&M Health Science Centre College of Medicine and Scott and White Memorial Hospital, Temple, TX 76508

Objective: Microvascular hyperpermeability occurs due to the disruption of the endothelial adherens complex. β-catenin is an integral component of the adherens junction complex that determines barrier integrity. Another important function of β-catenin is to mediate Wnt signaling. However, the role of β-catenin in regulating Wnt signaling and its relationship to endothelial cell barrier functions is not clearly known. Our objective was to determine if an active Wnt signaling pathway exist and if so, how β-catenin regulates this pathway in microvascular endothelial cells.

Methods: Rat lung microvascular endothelial cells (RLMEC) were transfected with a β-catenin gene expression construct and the Tcf-mediated transcriptional activity was determined using TOPflash-luciferase reporter assay. The role of free cytosolic β-catenin on Tcf-mediated transcriptional activity was studied following inhibition of glycogen synthase kinase (GSK3-β) using SB 216763 or lithium chloride (LiCl). Quercetin, a Tcf inhibitor was used to confirm β-catenin/Tcf-mediated transcriptional activity.

Results: Transfection of β-catenin gene induced luciferase activity indicating increased Tcf-mediated transcriptional activity (p<0.05). Inhibition of GSK activity by SB 216763 or LiCl that is known to increase free β-catenin availability increased Tcf-mediated transcriptional activity significantly (p<0.05). Tcf inhibitor quercetin decreased β-catenin/Tcf-mediated transcriptional activity significantly (p<0.05).

Conclusion: Our findings show that an active Wnt signaling pathway exist in microvascular endothelial and that β-catenin plays a significant role in regulating Tcf-mediated transcriptional activity of Wnt target genes.


INSULIN INHIBITS HEPATOCYTE INOS VIA AN AKT-DEPENDENT MECHANISM.B. Harbrecht, S. Liu,* and B. Zhang*. University of Louisville, Louisville, KY 40292

Objectives: Hepatocyte iNOS expression is a tightly controlled metabolic pathway. We have shown that PI3K signaling and protein kinase B/Akt regulate iNOS expression following cAMP stimulation. Insulin regulates hepatocyte gene expression through effects on PI3K and MAPK signaling pathways. We tested the hypothesis that insulin would regulate hepatocyte iNOS gene expression.

Methods: Primary cultured rat hepatocytes were isolated and stimulated with IL-1β+IFN to induce iNOS in the presence and absence of insulin. A dominant negative (DN) Akt plasmid (AktKD) was used to block Akt signaling. NOS expression was measured by nitrite and Western blot.

Results: Insulin inhibited nitrite production (Figure) and iNOS protein expression (not shown) in cytokine-stimulated hepatocytes in a dose-dependent manner associated with increased Akt phosphorylation (Figure). DN Akt blocked the inhibitory effect of insulin on iNOS (Figure).

Conclusion: Insulin inhibits cytokine-stimulated hepatocyte iNOS expression through effects on Akt-mediated signaling pathways.



ATTENUATION OF POST-BURN HYPERTROPHIC SCARRING WITH PROPRANOLOL.C. Finnerty, J. He,* S. Hegde,* G. Mecott,* T. Huang,* D. Herndon, and M. Jeschke. University of Texas Medical Branch, Galveston, TX 77555

Objectives: Hypertrophic scarring is a major clinical problem affecting up to 91% of severely burned patients. These hypertrophic scars (HTSs) are hyperinflammatory, pruritic, painful, and disfiguring, and can only be treated surgically. We have recently found that administration of propranolol, a non-specific β1, β2 adrenergic receptor antagonist, decreases hypertrophic scarring. This is the first agent found to reduce the severity of post-burn hypertrophic scarring. We used genomics data to identify signaling pathways likely to be affected by propranolol treatment, leading to the hypothesis that blockade of the β-ARs in HTS cells with propranolol will reduce the inflammatory response, improve cell viability, restore cell migration, and stimulate beneficial β-AR signaling.

Methods: GeneChips were run on HTS biopsies harvested 3 & 22 months post burn; the data was analyzed using BRB Array Tools (p<0.001). Primary skin & scar fibroblast cultures were established from unburned skin and burn scar from children 3-9 months post burn. Media was assayed for the expression of 17 cytokines. Cell migration was studied using a scratch assay. Cell viability was measured by MTT. Protein expression was confirmed via Western blot.

Results: Comparison between HTS harvested 3 & 22 months post burn showed differences in genes associated with β-AR, IL6, G-protein, and cAMP mediated signaling. Outcomes related to these genes were then examined in vitro. In HTS cells, propranolol treatment decreased cytokine production, increased cell viability, altered migration, and modulated expression of the β-ARs and related proteins.

Conclusion: These results provide a mechanistic basis for the decrease in hypertrophic scarring following propranolol treatment.


TOLL-LIKE RECEPTOR SIGNALING PATHWAY GENE EXPRESSION IN LPS-TOLERANT HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS.M. Mendes, M. Fernandes, G. Baggio-Zappia, M. Brunialti, and R. Salomão. Escola Paulista de Medicina, Federal University of São Paulo, SP, Brazil

Objective: To evaluate the expression of a set of 84 genes related to the Toll-like Receptor (TLR) signaling pathway, in a model of LPS tolerance in PBMC.

Methods: Tolerance was induced by incubating PBMC with 1ng/mL of LPS for 48 hours before challenge with 100 ng/mL of LPS for 2, 6 and 24h. Tolerance was confirmed by diminished TNF-alpha secretion. TLR4 and CD14 expression was evaluated on monocytes by flow cytometry, following 48 hours incubation with 1 ng/mL of LPS, in order to evaluate their expressions at the moment of LPS challenge. PCR array comprising 84 genes belonging to the TLR pathway was performed in each condition. Significant difference in gene expression was considered when fold change between groups was equal or higher than 2.

Results: No difference in the expression of TLR4 and CD14 on monocytes was observed between conditioned and non-conditioned cells after 48 of incubation. Sixty of the 84 genes were activated by LPS and among them, 27 were made tolerant to LPS (fold change>2). Genes coding for the surface receptors TLR2 and TLR4 were not tolerized, while the CD14 was. Genes belonging to the TRIF-dependent pathway were tolerized. The MyD88 dependent upstream pathway was not tolerized, but NF-kB and genes regulating its activation were inhibited in tolerized cells. MAPKs pathway included tolerant and non-tolerant genes. Among cytokines, IL-10 and IL-6 were not tolerized while TNF-alpha was.

Conclusion: The presence of tolerant and non-tolerant genes shows that tolerance is not directly controlled by the LPS binding to its receptor complex. Down-regulation probably occurs in an attempt to stop tissue damage caused by excessive inflammation, while preserving other cell functions.


HYPERTONIC SALINE INHIBITS IL-8 MEDIATED NEUTROPHIL PRIMING.W. Choi, C. Silliman, R. Shah, S. Khan, E. Moore, and A. Banerjee. University of Colorado at Denver, Aurora, CO 80045

Introduction: Our animal model of hemorrhagic shock and resuscitation with inhaled hypertonic saline (HTS) shows significant reductions in lung protein leak. This occurred without an associated decrease in chemotactic gradient (CINC-1). IL-8, the human analogue of CINC-1, is a potent PMN chemoattractant and priming agent. Its level is correlated with pulmonary neutrophilia, and is an independent marker of adverse outcomes in the critically ill. We hypothesize that HTS inhibits IL-8 mediated PMN priming, and subsequent superoxide (O2) production.

Methods: PMNs were isolated from healthy human volunteers by standard protocols. Cells were screened for pre-activation via fMLP stimulation. PMNs were pre-incubated in buffer, or hypertonic saline (180 mM) for 5 minutes at 37°C, then primed for 3 minutes with IL-8 (100 nM), or buffer. Cells were then activated with phorbol myristate acetate (PMA) and the maximal rate of O2 generation was measured.

Results: IL-8 priming increased O2 production 1.7-fold (p<0.02). HTS inhibited O2 production to control (un-primed) levels.

Conclusions: IL-8 mediated priming of PMN NADPH oxidase is completely mitigated by HTS. This corroborates animal study data in which inhaled HTS attenuates lung injury, while having no effect on alveolar chemotactic gradients (CINC-1). Thus, HTS inhibits PMN mediated lung injury by inhibiting priming, and subsequent augmentation of O2 release when activated.



ATP RELEASE VIA PANNEXIN 1 HEMI-CHANNELS CONTROLS NEUTROPHIL CHEMOTAXIS.Y. Chen, Y. Yao,* Y. Sumi,* T. Woehrle,* A. ElKhal,* and W. G. Junger. Department of Surgery, BIDMC/Harvard Medical School, Boston, MA 02215

Chemotaxis of polymorphonuclear neutrophils (PMN) is a key cell function that protects against invading microorganisms but can promote inflammation and harm trauma patients. Our previous work has shown that cellular ATP and feedback through purinergic receptors controls fMLP induced chemotaxis. Here we determined the mechanism by which ATP is released from migrating PMN. We found that pannexin 1 hemi-channels (Panx 1) are expressed in human PMN and translocate to the leading edge upon cell stimulation with fMLP. Panx 1 at the leading edge facilitates ATP release. We found that ATP release is blocked by the hemi-channel inhibitor CBX and the Panx 1 inhibitory peptide 10Panx 1. Treatment with these inhibitors or silencing of Panx 1 significantly reduces the ability of cells to undergo chemotaxis to a point source of fMLP (Fig).

We conclude that ATP release via Panx 1 hemi-channels is important for fMLP-induced PMN chemotaxis and that pharmacological approaches targeting these mechanisms might reduce PMN recruitment and PMN-induced damage of inflamed host tissues after shock and trauma. (Support: Shock Society Novo Nordisk, NIGMS, NIAID, DOD.)



PROGRESSIVE POSTINJURY THROMBOCYTOSIS (TC) IS ASSOCIATED WITH THROMBOEMBOLIC (TE) COMPLICATIONS.J. Kashuk, E. Moore, J.L. Johnson, W.L. Biffl, C.C. Burlew, C. Barnett, and A. Sauaia. Denver Health Medical Center and University of Colorado, Aurora, Denver, CO 80204

Objective: Despite routine enzymatic chemoprophylaxis, a hypercoagulable state was previously identified by thrombelastography in critically ill patients that was associated with TE. The contribution of platelet hyperactivity to the hypercoagulable state, however, remains unknown. We hypothesized that progressive post-injury TC contributes to a hypercoagulable state that is associated with TE.

Methods: 1440 injured patients surviving >48 hours were prospectively studied over 12 years. Variables associated with TC (PLT>450K) included age, ISS, RBC/12 hrs, and TE. Time frame for the development of TC was assessed at ≥ or ≤7 days post-injury. Logistic regression (LR) identified independent variables predictive of TC and to adjust the association of TC with TE for other risk factors.

Results: Mean ISS was 29.3±0.3, RBC/12 hrs 4.4±0.2units, and age 37.4±0.4years. TC occurred in 447 (31%) of patients, and occurred more often >7days post injury (440, 98%). Eighty patients (6%) developed TE and this was significantly associated with TC (p=0.01). LR indicated that higher ISS (p<0.0001) and more RBC/12 hours (p=0.03) both significantly predicted TC, while age was not associated with it. TE were significantly associated with TC (p=0.046, Odds Ratios=1.6, 95% Confidence Interval:1.1-2.6) after adjustment for other risk factors. Both the TC and TE rates increased significantly with length of stay (Chi sq. for trend p<0.0001 for both). In fact, TE increased exponentially with longer stays.

Conclusion: TC in critically injured patients receiving routine chemoprophylaxis is associated with TE which increase exponentially with ICU length of stay. Further investigation is warranted to differentiate enzymatic from platelet hypercoagulability to ascertain the role of anti-platelet therapy for prevention of TE.


DIFFERENTIATION OF ENZYMATIC FROM PLATELET HYPERCOAGULABILITY USING THE NOVEL THROMBELASTOGRAPHY (TEG) PARAMETER DELTA.E. Gonzalez, J.L. Kashuk, E.E. Moore, and C.C. Silliman. Denver Health Medical Center and University of Colorado, Aurora Denver, CO 80204

Background: TEG permits global assessment of coagulation function. We previously showed that a hypercoagulable state via TEG G value is associated with thromboembolic events despite routine chemoprophylaxis. We hypothesized that a hypercoagulable state could be differentiated into enzymatic or platelet etiology with thrombus velocity curves; specifically the time to maximum rate of thrombus generation (TMRTG) and the novel TEG parameter Delta.

Methods: Ten patients receiving thromboprophylaxis for ≥72 hrs were retrospectively studied using kaolin activated citrated TEG. Thrombus velocity curves were plotted, and Delta was calculated as the difference between the TEG parameters R and SP, corresponding to the time to maximum rate of thrombus generation (TMRTG), reflecting the enzymatic contribution to clot formation. The TEG parameter G, (G=5000 × A / 100-A) was determined. A hypercoagulable state was defined as delta < 0.6 minutes and/or G >11 dynes/cm2.

Results: A hypercoagulable state was identified via Delta in 6 patients (60%); all remained hypercoagulable following heparinase addition, suggesting chemoprophylaxis was ineffective. Of six patients with a hypercoagulable G value, 50% had a normal delta suggesting the presence of platelet hypercoagulability. Delta closely correlated with TMRTG (r=0.94). However, the varying contribution of platelets to hypercoagulability was shown by a non-linear, weak correlation of Delta and TMRTG with G (r=0.11 and 0.14 respectively).

Conclusion: Delta reflects changes in thrombin generation as measured by TMRTG, permitting differentiation of enzymatic from platelet hypercoagulability. Future studies will be required to validate these findings.


INITIAL EXPERIENCES WITH POINT OF CARE (POC) RAPID THROMBELASTOGRAPHY (TEG) FOR MANAGEMENT OF LIFE THREATENING POSTINJURY COAGULOPATHY (PIC).J. Kashuk, E. Moore, M. Wohlauer, J. Johnson, M. Pezold, J. Lawrence, W. Biffl, C. Burlew, C. Barnett, M. Sawyer, and A. Sauaia. Denver Health Medical Center and University of Colorado, Denver, CO 80204

Background: Designing the optimal massive transfusion protocol (MTP) is currently hindered by lack of real time assessment of coagulation function. TEG provides point of care analysis of clot formation. Via a pilot study we hypothesized that integration of TEG into our MTP would facilitate goal directed therapy with equivalent clinical outcomes when compared to conventional coagulation testing.

Methods: 34 patients who received >6u RBC/6 hours (placing them at risk for PIC) after TEG implementation (TEG) were compared to 34 similar patients admitted prior to TEG implementation (Pre-TEG). Data are presented as mean ±SEM.

Results: ED Pre-TEG vs. TEG shock, and coagulation indices, were not different between groups: SBP (94mm Hg vs.101 mm Hg), temp (35.3° vs. 35.9°), pH (7.16 vs.7.11), base deficit (−13.0 vs. −14.7), lactate (6.5 vs.8.1), INR (1.59 vs.1.83), and PTT (48.3 vs. 57.9). Similarly, FFP: RBC, platelets (plt): RBC, cryoprecipitate (cryo): RBC ratios were not significantly different. INR at 6hrs did not discriminate between survivors and non-survivors (p=0.10), whereas TEG "G" value was significantly associated with survival (p=0.03), as was the maximum rate of thrombin generation (MRTG; mm/min) and total thrombin generation (TG; area under the curve) (p=0.03 for both). Patients with MRTG >8 received less components of RBC, FFP, cryo, and plt (p=0.048, 0.03, and 0.04 respectively).

Conclusion: TEG guided resuscitation provides comparable results to conventional approaches. Further experience with TEG could lead to a reduction of transfusion requirements. Future studies with more robust data are required to validate the usefulness of this technique.


PRIMARY FIBRINOLYSIS IS INTEGRAL IN THE PATHOGENISIS OF ACUTE COAGULOPATHY OF TRAUMA.J. Kashuk, E. Moore, M. Sawyer, M. Wohlauer, C. Barnett, W. Biffl, C. Burlew, J. Johnson, and A. Sauaia. Denver Health Medical Center and University of Colorado, Denver Health Sciences Center, Denver, CO

Background: The existence of primary fibrinolysis (PF) and a defined mechanistic link to the "Acute Coagulopathy of Trauma" is controversial. Rapid thrombelastography (r-TEG) offers point of care comprehensive assessment of the coagulation system. We hypothesized that postinjury PF occurs early in shock, leading to postinjury coagulopathy, and ultimately hemorrhage related death.

Methods: Consecutive patients over 14 months at risk for postinjury coagulopathy were stratified by transfusion requirements into massive (MT),>10 units/6 hours (n=32), moderate (Mod), 5-9 units/6 hours (n=15) and minimal (Min), <5 units/6 hours (n=14). r-TEG was performed by adding tissue factor to uncitrated whole blood. r-TEG estimated percent lysis (EPL) was categorized as PF when >15% EPL was detected. Coagulopathy was defined as r-TEG clot strength= G< 5.3 dynes/cm2. Logistic regression was used to define independent predictors of PF.

Results: 34% of injured patients requiring MT had PF, which was associated with lower ED systolic blood pressure, temperature, and worse base deficit/pH/lactate (ANOVA, all p<0.0001). The risk of death correlated significantly with PF (p=0.026). PF occurred early (median 58 min, IQR 1.2 min-95.9 min); every one unit drop in G increased the risk of PF by 30%, and death by over 10%.

Conclusion: Our results confirm the existence of PF as detected by r-TEG in severely injured patients. It occurs early (< 1 hour), and is associated with massive transfusion requirements, coagulopathy, and hemorrhage related death. These data warrant renewed emphasis on the early diagnosis and treatment of fibrinolysis in this cohort.


PLATELET STORAGE LESION IS ASSOCIATED WITH ACQUIRED MITOCHONDRIAL DYSFUNCTION.R.J. Levy, A. Thomas,* and Y. Diab*. Children's National Medical Center, Wash., DC 20005

Platelet storage lesion (PSL) is the storage-related deterioration of platelet function that limits the shelf life of products intended for transfusion. The five day limit of viability results in > 25% of donated platelet to be wasted, costing the US health care system over $1 billion per year. Although PSL has been widely studied, the underlying cause is unknown. Because platelets are a nuclear cell fragments that utilize oxidative phosphorylation for energy production, we hypothesized that PSL may result from acquired mitochondrial dysfunction. Single donor apheresis platelet concentrates were collected from 5 healthy donors in gas permeable containers and agitated at room temperature for up to 7 days. Samples were studied at day 0, 2, 4, and 7 post collection. Platelet count, mean platelet volume, platelet aggregation and degree of activation were assessed and plasma pH, pCO2, pO2, lactate, and glucose were measured. Steady-state cytochrome oxidase kinetic activity was determined in isolated platelet mitochondria at the designated time points. Statistical significance was assessed with ANOVA and post-hoc Tukey's test. Platelet count, mean platelet volume, and pO2 were unchanged over time. Aggregation amplitude and slope and platelet ATP release decreased significantly at day 4 and diminished further at day 7 consistent with PSL. Plasma pH decreased significantly at 7 days, pCO2 decreased in a stepwise fashion over time, and lactate levels increased significantly over time suggesting impaired oxidative phosphorylation. Cytochrome oxidase activity decreased significantly at day 4 and decreased further day 7 post collection. Thus, mitochondrial dysfunction was associated with the biochemical features of PSL. Bioenergetic failure may underlie PSL and may be amenable to therapeutic intervention to enhance the shelf life of donated platelets.


DEFINING THE EFFECTS OF TISSUE HYPOPERFUSION ON CLOT STRENGTH IN A SWINE MODEL OF TRAUMATIC SHOCK.N. White, E. Martin,* P. Reynolds, R. Barbee, D. Brophy,* and K. Ward. Virginia Commonwealth University, Richmond, VA 23298

Objective: Tissue hypoperfusion has been implicated in the acute coagulopathy of trauma. However, the relationship between tissue oxygen utilization and hemostasis in trauma remains undefined. We used a swine traumatic shock model to further define the relationship between whole body tissue oxygen consumption (VO2) by indirect calorimetry and clot strength (MA) by thrombelastography (TEG).

Methods: Immature male swine were anesthetized, injured, hemorrhaged, and maintained in shock until oxygen debt=80 ml/kg. Metabolic and hemostatic variables were measured at baseline and during shock when OD=40 and 80 ml/kg.

Results: N=17 subjects achieved goal OD after being hemorrhaged 38% (SD 1%) of total blood volume. VO2, MA, fibrinogen, base excess, and pH all decreased during shock (all p<0.01). Univariate analysis revealed positive correlations between MA and VO2, fibrinogen, platelet count, and pH during shock (all p=<0.001). However, the effect of VO2 on MA was lost after adjusting for covariates (platelet count, fibrinogen, & blood pH) using multivariate regression. Only fibrinogen (p<0.001) and pH (p=0.02) maintained a significant effect on MA in the multivariate model. (Whole model R2=0.83, p<0.001).

Conclusion: In this swine model, we found that the association between tissue oxygen utilization and clot strength during traumatic shock may be explained by the effects of fibrinogen consumption and acidosis. (N.W. supported by NIH T32 GM008695).


ALTERED FIBRINOGEN FUNCTION DURING TRAUMATIC SHOCK OBSERVED IN A SWINE POLYTRAUMA MODEL.J. Newton,* N. White, E. Martin,* R. Diegelmann,* P. Reynolds, R. Barbee, D. Brophy,* and K. Ward. Virginia Commonwealth University, Richmond, VA 23298

Objective: Fibrinogen is a critical clotting protein whose role remains undefined in the acute coagulopathy of trauma (ACT). We hypothesize that fibrinogen function becomes altered under pathologic conditions consistent with ACT.

Methods: Functional fibrinogen concentration, (by the method of Clauss) and total fibrinogen protein, (by swine-specific enzyme linked immunoassay), were simultaneously measured in a swine model of traumatic hemorrhagic shock (Injury Severity Score=17) at baseline prior to injury and again during shock after meeting metabolic conditions for ACT with acidosis (base deficit>6, pH< 7.35). Prior to analysis, fibrinogen values were standardized to total protein concentration to control for the effects of plasma dilution from auto-resuscitation.

Results: N= 6/9 male swine weighing 45(SD 7)kg achieved metabolic ACT criteria after bleeding an average of 20(SD 6) ml/kg and a shock duration of 91(SD 33) minutes. Plasma concentration of functional fibrinogen was reduced during shock when meeting metabolic conditions of ACT (25.4 vs. 20.4 mg/dl/g tot. prot., p=0.04) while total fibrinogen protein concentration was unchanged (26.9 vs. 26.6 mg/dl/g tot. prot., p=0.98). There was a strong trend towards a reduced average functional/total fibrinogen ratio during shock (functional/total ratio baseline=1.05 vs. shock=0.77, p=0.064).

Conclusion: This swine model further supports the hypothesis that fibrinogen function is altered under physiologic conditions consistent with ACT. (J.N supported by Dept. of Army GI Bill.) (N.W. supported by NIH T32GM008695).


COMPARISON OF LAMINAR VS DISTURBED FLOW ON TISSUE FACTOR (TF) EXPRESSION IN HUVEC.R. Abe,* N. Yamashita,* A. Rochier,* A. Nixon,* R. Abe,* S. Oda, H. Hirasawa, and B. Sumpio. Department of Surgery, Yale University School of Medicine, New Haven, CT 06510

Objective: TF is expressed on vascular endothelial cells (EC) under pathological conditions, such as septic shock, and is an initiator of thrombosis during sepsis. However, the stimulus for TF expression during sepsis has not been fully clarified. The objective of this study is to investigate the effect of different types of hemodynamic forces on TF expression by EC. We also determined the effects of combined chemical stimuli with these mechanical forces.

Methods: HUVEC seeded on glass slides were exposed to pulsatile forward flow shear or To&Fro shear, a novel model of disturbed flow, in a parallel flow chamber in the presence or absence of 4U/mL of Thrombin (Th), for 2hr, 4hr and 6hr. TF RNA expression was determined using real-time quantitative RT-PCR. RNA expression level was assessed by fold changes compared to that in static control at 2hr.

Results: TF RNA expression in HUVEC stimulated with Th for 2hr was 3.88 ±0.3 fold higher than static control, while exposure to pulsatile flow or To&Fro shear for 2hr showed 4.28 ±1.2 or 6.63 ± 0.8 fold increase, respectively (ANOVA, p<0.05). In the presence of Th, TF expression in HUVEC stimulated with pulsatile forward shear for 2, 4, 6 hrs showed 9.47 ±1.5, 7.44 ±1.3, 6.21 ±1.6 fold increases, respectively, while HUVEC exposed to Th and To&Fro shear for 2, 4, 6 hrs demonstrated 18.68 ±5.9, 15.12 ±6.9 15.35 ±8.2 fold increases, respectively. TF expression in HUVEC exposed to To&Fro shear in the presence of Th at 4 and 6hrs was significantly higher than HUVEC exposed to Pulsatile forward flow and Th. (ANOVA, p<0.05).

Conclusion: Disturbed fluid flow induced a greater and sustained amplification of TF expression in both the presence or absence of chemical stimuli. This result suggests a therapeutic value of hemodynamic stabilization to prevent excessive activation of the coagulation cascade.


DIMINISHED HEMOSTATIC POTENTIAL OF THAWED PLASMA MEASURED BY THE TWO NOVEL ASSAYS.W. Wang,* J.B. Holcomb, V. Kostousov,* and N. Matijevic*. *UTHSC-H, Center for Translational Injury Research, Houston, TX 77030

Background: Following major traumatic injury, severe bleeding requires massive transfusion of blood products including fresh frozen plasma (FFP). According to AABB protocols, thawed plasma (TP) is approved for transfusion up to five days after thawing, when stored at 1-6°C.

Hypothesis: We hypothesized that TP stored for 5 days has decreased hemostatic potential when compared to newly thawed plasma (day 0).

Methods: FFP was obtained from 18 single donor commercial units, thawed at 37°C, and kept refrigerated at 4°C for 5 days. Hemostatic properties of TP were evaluated at day 0 and 5 by two functional tests of global hemostasis: the Calibrated Automated Thrombogram (CAT), measuring plasma capacity to generate thrombin, and a modified thromboelastography (TEG), which measures kinetics of clot formation, clot strength and stability.

Results: CAT measures of thrombin generation (TG) decreased by 30% at day 5, and hemostatic potential decreased by 51% at day 5 (14.7 nM/min vs. 7.2 nM/min, p<0.000001). TEG values revealed a longer reaction time (R, 2.6 min vs. 3.7 min, p<0.00001), prolonged maximal amplitude (MA, 13.5 vs. 15.5 min, p<0.01), and a longer time to maximal rate of thrombus generation (MRTG, 3.8 min vs. 4.8 min, p=0.001), indicating that day 5 plasma clots form slower and take longer to achieve MRTG.

Conclusions: These two functional assays demonstrated decreased hemostatic potential and delayed clot propagation in plasma stored for 5 days. The clinical effectiveness of day five thawed plasma is uncertain, as outcome data with this product are scarce, especially in massively transfused patients.


GOAL-DIRECTED INTRAVENOUS HEPARIN, BUT NOT SUBCUTANEOUS HEPARIN, NORMALIZES COAGULATION STATUS IN SICU PATIENTS.A. Baer,* C. Hamiel, S. Cheng,* E. Luzier,* N. Pearlman, and P.E. Wischmeyer. University of Colorado, Aurora, CO 80045

Macro- and micro-thromboses contribute significantly to morbidity and mortality in the surgical intensive care unit (SICU). Our objective was to compare the coagulation status of post-operative SICU patients receiving current standard of care for thromboembolism (TE) prophylaxis, subcutaneous (SC) heparin, and that of patients receiving goal-directed low-dose intravenous (IV) heparin to normal coagulation profiles.

Methods: Patients (n=31) were randomized to TE prophylaxis of SC heparin (5000u 3×/day) or IV heparin (titrated to aPTT goal of 40-45 sec.) following major abdominal surgery requiring SICU admission. Blood collected daily and analyzed for clotting time (ACT) and clot rate (CR, the rate of fibrin polymerization) using Sonoclot Analyzer. Blood collected at day 0 just prior to surgery, day 1 prior to heparin initiation, and then daily for 6 days.

Results: IV heparin patients had ACTs similar to normal volunteers, 181±7 sec. (p=0.62 vs normal) while SC heparin patients were significantly hypercoagulable, 158±3 sec. (p=0.006 vs normal). CR in the SC group was twice that of normal volunteers (39±1 vs 20±0.6, p<0.001) but in the IV group it was only 50% higher than normal (30±1, p<0.001). Anti-Xa levels showed detectable heparin in the IV group only. These results are independent of age, sex, and type of surgery performed.

Conclusions: Our data shows major surgery leads to acute post-operative hypercoagulability. SC heparin demonstrated no measureable anticoagulant effect (as measured by ACT and CR) in post-operative SICU patients. Goal-directed low-dose IV heparin normalized SICU patients' post-operative coagulation state, and thus may be more effective in preventing TE-related complications in SICU patients. Larger clinical trials of goal-directed low-dose IV heparin appear warranted.


MECHANISM OF LPS-INDUCED DOWN-REGULATION OF PROTEIN C ANTICOAGULANT PATHWAY.D. Song, X. Ye, and S.F. Liu. Feinstein Institute for Medical Research, New Hyde Park, NY

This study examined a causative role of endothelial NF-κB signaling in LPS-induced down-regulation of the thrombomodulin, protein C and endothelial protein C receptor (TM-PC-EPCR) anticoagulation pathway. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I-κBα selectively on endothelium were injected with saline or E Coli LPS (5 mg/kg, i.p.). Tissue levels of TM and EPCR mRNA and protein, plasma level of EPCR and PC and tissue level of TNF-α converting enzyme (TACE) activity were measured at 6 hours after LPS injection. Plasma levels of total and activated PC (mO.D. units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were: 73.4±2.4, 72.0±3.2, 26.0±6.2 and 58.5±4.7, and 45.6±1.6, 43.0±1.2, 2.8±1.8 and 29.9±4.1 (p < 0.01, WT-LPS vs other 3 groups). LPS reduced tissue TM and EPRC protein level in heart, lungs and kidney of WT-LPS, but not TG-LPS mice. In parallel with the reduced tissue EPCR level, plasma EPRC level was significantly elevated in WT-LPS, but not in TG-LPS mice. Kidney and liver from WT-LPS, but not TG-LPS mice, showed a significantly increased tissue level of TACE activity, an enzyme that is responsible for EPRC shedding from endothelial cells. LPS increased TM and EPCR mRNA expression in WT mice, which was prevented in TG mice. Stimulation of WT lung endothelial cells (ECs) with TNF-α activated NF-κB and concomitantly reduced cellular levels of EPCR and TM proteins. The TNF-α induced NF-κB activation and EPCR and TM protein reduction were prevented in TG ECs. These results establish a causal link between endothelial NF-κB activation and LPS-induced down-regulation of the TM-PC-EPCR pathway, and suggest that LPS down-regulates TM-PC-EPCR system by promoting by TACE-mediated endothelial EPCR and TM shedding. (Supported by NIH R01GM063907).



Introduction: Liquid plasma stored at 4°C for up to 5 days is considered equal to freshly thawed plasma for transfusion in order to correct trauma coagulopathy. Storage of plasma at 4°C could lead to "cold activation" of coagulation due to factor XII autoactivation and kallikrein generation. Generated kallikrein inactivates protein C inhibitor (PCI) by cleavage or complex formation. PCI is the main regulator of the protein C anticoagulant system which was recently implied in pathogenesis of trauma coagulopathy.

Hypothesis: We hypothesized that PCI activity was decreased in plasma stored at 4°C for 5 days.

Methods: PCI activity was measured in 24 single donor samples (7 males) stored for 5 days (Day 5) and compared with initial levels (Day 0) using an in-house ELISA assay.

Results: We found that 22 samples had preserved PCI activity during the storage period: 101±21%, range 62-146% on Day 0 compared with Day 5 (100±25%, range 52-152%). In 2 female samples on Day 5 PCI activity dropped to less than 5%.

Conclusion: PCI consumption due to "cold activation" rarely happens (8%) in liquid plasma stored at 4°C for 5 days. Nevertheless, "cold activated" liquid plasma with inactivated PCI might not be equally efficient as freshly thawed plasma for trauma coagulopathy correction. The impact of such plasma infusion on the hemostasis in trauma patients remains to be established.


VULNERABILITY TO SEPSIS IN THE AGED IS LINKED TO REDUCED PROTEIN C PATHWAY ACTIVATION.M. Starr,* H. Takahashi,* C.T. Esmon, B.M. Evers,* and H. Saito. University of Kentucky, Lexington, KY 40536

The protein C (PC) pathway is an important anti-coagulant mechanism that prevents disseminated intravascular coagulation during sepsis. Thrombomodulin (TM), an endothelial cell membrane receptor, accelerates the conversion of PC to activated protein C (APC) which leads to down-regulation of pro-coagulant factors. The purpose of the present study was to understand why mortality to sepsis increases with advancing age and how coagulation plays a major role in age-associated mortality. Two mouse models of sepsis were utilized in this study: 1) acute endotoxemia by injection with bacterial endotoxin lipopolysaccharide (LPS); and acute peritonitis by cecal ligation and puncture (CLP). Induction of acute endotoxemia in young and aged mice with a low dose of LPS (2.5 mg/kg) caused high mortality in aged (80%) but not young (0%) mice. Fibrin formation was significantly elevated in several tissues from aged mice only; plasma APC levels were increased in young but failed to increase in aged mice; and TM expression was down-regulated in both young and aged mice but more profoundly depressed in the aged. The increased thrombosis, suppressed APC and profoundly decreased TM expression were not observed in young mice receiving a high dose of LPS (20 mg/kg) which resulted in a mortality rate (78%) equivalent to that seen in aged mice (2.5 mg/kg). These findings of age-associated alterations were all confirmed by the CLP model. Mutant mice with reduced TM expression and deficient protein C activation showed significantly increased fibrin formation compared to wild-type mice after LPS injection or CLP, suggesting that TM deficiency, as seen in aged mice during sepsis, contributes to increased thrombosis. Taken together, these results demonstrate that PC pathway activation is suppressed with aging and is responsible, in part, for increased coagulation and high mortality to sepsis in the elderly.


TUMOR NECROSIS FACTOR-ALPHA AND MORTALITY IN PATIENTS WITHVIBRIO VULNIFICUSINFECTION.D. Kim,1 and S. Lee2. 1(Spon: Chosun University) School of Medicine Chosun University, City of Gwangju, Republic of Korea; 2(Spon: Seonam University) Seonam University, City of Namwon, Republic of Korea

Objective:Vibrio vulnifcus (V. vulnificus) is a halophilic gram-negative bacillus that can cause fulminant sepsis in patients with chronic liver disease or immunocompromised conditions. The aim of this study was to examine the association between serum tumor necrosis factor-α concentration and mortality in patients with V. vulnificus infection.

Methods: This study included 25 adult patients aged ≥18 years who were diagnosed with skin and soft tissue infections at 4 university hospitals between 2006 and 2008. Serum TNF-α concentration was measured with blood samples taken at presentation. Thirteen healthy adults were used as controls.

Results: The median TNF-α level was 261.0 pg/mL (IQR, 101.0-376.0 pg/mL) in the non-survivor group and 69.5 pg/mL (IQR, 17.5-103.5 pg/mL) in the survivor group, and the difference was statistically significant (P=0.001). The serum TNF-α level measured 6 to 48 hours after antibiotics administration was also significantly higher in non-survivors than in survivors (P=0.044). The median TNF-α level was significantly higher in patients with Vibrio infection than in control subjects (P<0.001).

Conclusion: From the results of this study, it is concluded that serum TNF-α concentration at presentation may be a useful early predictor for mortality of patients with Vibrio septicemia.



Previous work demonstrated that the addition of the anti-oxidant dimethyl sulphoxide (DMSO) to human whole blood at the time of stimulus by lipopolysaccharide (LPS) only altered IL-8 and IL-1β. For a more clinically relevant treatment, we decided to delay the addition of 1% DMSO until six hours after stimulus. Briefly, we drew blood from healthy donors into hepranized tubes and separated the whole blood into four groups, LPS alone (50ng/ml), LPS + 1% DMSO @ 0h, LPS + 1% DMSO @ 6h, and Vehicle (no LPS). After 6 hours of stimulation at 37°C in 5% CO2, DMSO was added and plasma harvested 24 hours after LPS stimulation. Plasma was collected and stored at −20°C until analysis. The samples were run on a cytokine multiplex assay. The results confirm that while IL-8 is significantly decreased and IL-1β is significantly increased when DMSO is added at 0h, DMSO loses the ability to modulate cytokine production when the addition is delayed by 6h. All other cytokines and chemokines measured were not altered significantly either when DMSO was added at 0h or at 6h. These data demonstrate that the anti-inflammatory effects of DMSO when given after the onset of inflammation are not mediated by reduced production of cytokines.



LYOPHILIZED PLASMA DECREASES PRO-INFLAMMATORY CYTOKINE SYNTHESIS IN SEVERE HEMORRHAGIC SHOCK.G. Hamilton,* N. Spoerke,* A. Alexandridis,* I. Kremenevskiy,* S. Cho,* K. Zink,* J. Differding,* A. Karahan,* J. Watters,* J. Sondeen, J. Holcomb, and M. Schreiber. Oregon Health & Science University, Portland, OR 97239

In trauma patients requiring massive transfusion, high ratios of plasma to packed red blood cells (PRBC) reduce mortality. Hemorrhagic shock increases pro-inflammatory cytokines and the risk of multiple organ dysfunction syndrome. Lyophilized plasma (LP) facilitates high ratios and reconstitution with ascorbic acid could decrease dysfunctional inflammation. To test this hypothesis, 32 female swine underwent severe trauma consisting of a femur fracture, hemorrhage, hypothermia, acidosis, and grade V liver injury. Animals were randomized to 4 groups: FFP, 1:1 ratio of FFP to PRBC, LP, and 1:1 ratio of LP to PRBC. Blood was collected at baseline, 2 and 4 hours for ELISA. Lung tissue was collected at 4 hours for qRT-PCR. We measured pro-inflammatory cytokines IL-6, IL-8, and TNF-α and found significant differences in IL-6 and TNF-α serum values (Table 1). qRT-PCR showed no significant differences (Fig. 1). Resuscitation with LP and ascorbic acid may suppress the pro-inflammatory response. While promising, longer observation, additional time points and greater power are needed to confirm the influence of LP on cytokine gene expression and plasma concentration in this model. (Supported by Army MRMC Award W81XWH-04-1-0104).

Fig. 1
Fig. 1:
Box-plot of median fold-change cytokine RNA expression from qRT-PCR. P-values are between FFP/LP:PRBC for IL-6 and TNF-α and both FFP/LP and FFP:PRBC/LP for IL-8.
Statistically significant differences in ELISA serum cytokine concentrations.



Introduction: Discovery of a biomarker(s) that can predict death or infection in the ICU, and be measured within 24 hours of admission could greatly improve the potential of anti-inflammatory therapies to show benefit, while minimizing risk to patients unlikely to benefit from these interventions. Biomarkers such as these are needed to help guide clinical trial enrollment. Our group has evaluated a number of potential biomarkers that could identify those most likely to benefit from anti-inflammatory intervention in an adult ICU population.

Methods: 200 patients had blood drawn an Day 1 of their ICU stay as part of a prospective observational ICU trial. Two non-selected sub-groups were analyzed for cytokine biomarkers IL-12p70, INF-gamma, IL-10, IL-13, IL-2, IL-4, and IL-5 (n=123) via Mesoscale® and IL-8 using ELISA (n=115). Statistical analyses were carried out using SAS version 9.2.

Results: All patients were mechanically ventilated and approximately 60% of patients in both measurement groups were diagnosed with infection during their ICU stay. There was a slight non-significant difference in mortality levels between the Mesoscale group (25%) and the IL-8 ELISA group (33%). IL-10 levels on ICU day 1 were significantly elevated in patients who ultimately developed infection versus patients not diagnosed with infection (p=0.0055 via t test). Neither age nor sex were significant in multivariate models relating IL-10 to infection. IL-8 levels were elevated in patients who ultimately died, this relationship remained significant in a multivariate model that included both IL-8 (p=0.0024, dichotomized, and p=0.0484 for the continuous variable), and age (p=0.0013). Cut-off values for both cytokines (20 pg/ml for IL-10 and 220 pg/ml for IL-8) yielded high specificity (>90%) and low sensitivity (<20%).


DEPLETING ABUNDANT PROTEIN ALSO DRAMMATICALLY DEPLETES CYTOKINES.B. Japp,* E. Schuller, and D. Remick. Boston University School of Medicine, Boston, MA 02118

It has been reported that the presence in plasma of high abundance proteins like albumin and IgG can mask the detection of lower abundance proteins such as the cytokines. This masking can have negative ramifications if the low abundance proteins are to be used for downstream proteomic or clinical applications. The present study tests the ability of the Qiagen Qproteome albumin/IgG depletion kit, a commercially available spin column, to specifically remove albumin and IgG in LPS stimulated whole human blood. Blood was drawn from normal volunteers into heparinized syringes and stimulated with 50 nG/mL of LPS for 6 hours @ 37°C and 5% CO2. 25ul of plasma was passed through the column to deplete albumin and IgG which removed 93% of the total protein content. Previous research has shown IgG and albumin account for only ∼76% of total protein content. Cytokine concentrations were measured using a multiplex ELISA platform. IL-4, IL-10, IL-12, GRO and IFN-y were below detectable limits. All of the detectable cytokines were reduced significantly by protein depletion.



THE ROLE OF INTERLEUKIN (IL)-18 IN LYMPHOCYTE APOPTOSIS DURING ENDOTOXIN-INDUCED SYSTEMIC INFLAMMATION.M. Watanabe,1* M. Aoyama,1* N. Iizuka,1* M. Miyoshi,1* T. Ueda,2* A. Miyawaki,2* M. Usami,1* and J. Kotani2. 1Department of Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japan, 2Department of Emergency and Critical Care Medicine, Hyogo College of Medicine, Nishinomiya, Japan

Introduction: Increased lymphocyte apoptosis is an important cause of immunosuppression and mortality in critical illness. Signaling through the Fas/Fas ligand (FasL) pathway plays a central role involved in the induction of apoptotic cell death, including lymphocytes. Serum IL-18 levels correlate with the severity of systemic inflammatory disease. However, it is unclear the difference of IL-18 effect between mature and immature T-lymphocyte under systemic inflammation.

Objective: The purpose of this study was to elucidate the role of endogenous IL-18 in T-lymphocyte apoptosis and maturing in thymus and spleen during endotoxin-induced systemic inflammation.

Methods: Male wild-type (WT) mice and IL-18 knock-out (KO) mice were injected intraperitoneally with PBS or LPS (40 mg/kg), followed by sacrifice and isolation of thymocytes and splenocytes at 18 h. Apoptosis (annexin V), phenotyping (CD3) and expression of Fas or FasL were assessed using three-color flow cytometry.

Results: Compared with WT mice, the IL-18 KO mice in endotoxemia 1) attenuated apoptosis of both immature (p<0.05) and mature T-lymphocyte; 2) increased the population of immature T-lymphocyte (p<0.05) but decreased the population of mature T-lymphocyte (p<0.05); 3) showed low levels of Fas and Fas-mediated apoptosis in T-lymphocyte (p<0.05).

Conclusion: Endogenous IL-18 induces mature and immature T-lymphocyte apoptosis and Fas expression during endotoxemia and plays a role of accelerating the recruitment of lymphocyte during endotoxin-induced systemic inflammation. Thus, IL-18 may be a new target for immunosuppression therapy.


GENDER DIFFERENCE OF INTERLEUKIN (IL)-18-RELATED SIGNAL TRANSDUCTION IN JEJUNUM DURING ENDOTOXIN-INDUCED SYSTEMIC INFLAMMATION.M. Aoyama,1,2* M. Watanabe,1* M. Miyoshi,1* N. Iizuka,1* N. Maeshige,1* T. Ueda,2* A. Miyawaki,2* M. Usami,1* and J. Kotani2. 1Department of Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japan, 2Department of Emergency and Critical Care Medicine, Hyogo College of Medicine, Nishinomiya, Japan

Introduction: Serum IL-18 levels correlate with the severity of systemic inflammatory disease. We reported that the endogenous IL-18 production was increased and survival rate was decreased only in male but not female mice after lethal LPS injection.

Objective: The purpose of this study was to elucidate the gender difference of endogenous IL-18 in jejunum during lethal endotoxemia.

Methods: Male and female C57BL/6J mice were injected intraperitoneally with LPS (40 mg/kg) or PBS, followed by sacrifice and collection of jejunum at 24 h. IL-18 mRNA was determined by quantitative real time PCR using GAPDH for internal standard. Apoptosis (cleaved caspase-3); IL-18 receptors (alpha: collate with signal transduction, beta: collate with affinity) were assessed by immunostaining (positive cell ratio in three field were counted by microscope).

Results: IL-18 mRNA was significantly higher in female mice than male mice. Cleaved caspase-3 did not significantly change in all groups. IL-18 receptor alpha was significantly decreased in LPS-injected mice with PBS in both male and female.

Conclusion: The expression of jejunum IL-18 receptor alpha was decreased during endotoxemia in both male and female. Although female mice produced higher levels of IL-18 in jejunum than male mice, the signal transduction in jejunum might be similar in both genders during endotoxemia.



ISOFLURANE PROTECTS AGAINST ACUTE LUNG INJURY FOLLOWING TRAUMA/HEMORRHAGIC SHOCK.M. Wohlauer,* E. Moore, M. Fragoso,* J. Eun,* J. Haenel,* and A. Banerjee. University of Colorado at Denver, Aurora, CO 80045

Background: Recent clinical work has shown that the inhaled anesthetic, isoflurane, reduced acute lung injury following elective surgery via modulation of pro-inflammatory cytokines. We hypothesized that isoflurane reduces acute lung injury following trauma/hemorrhagic shock.

Methods: Rats were either anesthetized with pentobarbital or isoflurane before hemorrhagic shock (MAP 30 mmHg × 45 min to achieve a base deficit greater than 20). Rats were sacrificed 3 hours postshock, and a bronchoalveolar lavage was performed to measure lung vascular permeability using Evans blue dye. CINC-1 levels were measured in the bronchoalveolar lavage fluid (BALF).

Results: Acute lung injury as measured by BALF protein was attenuated in the isoflurane group (0.14 mg/ml +/− 0.03 vs. 1.492 mg/ml +/− 0.53). Isoflurane virtually eliminated CINC-1 concentration in the BALF (210 pg/ml +/− 271 vs. 5930 pg/ml +/− 2934).

Conclusion: The substantial reduction of CINC-1 (murine analogue of human IL-8) in the BALF suggests a potential lung protective mechanism of inhaled isoflurane following trauma/hemorrhagic shock.



TYPE 1 DIABETES ENHANCES TLR-2-MEDIATED INFLAMMATORY RESPONSES IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS.J. Li,* C. Jin,* L. Ao,* D. Fullerton,* and X. Meng. Department of Surgery, University of Colorado Denver, Aurora, CO 80045

Toll-like receptor (TLR)-mediated endothelial inflammatory responses play an important role in tissue injury. While type 1 diabetes may alter the innate immune response in antigen-presenting cells, it remains unknown whether TLR2 responses are altered in endothelial cells of patients with type 1 diabetes. In this study, we examined the production of pro-inflammatory mediators in human coronary artery endothelial cells (HECs) from healthy and type 1 diabetic donors following stimulation of TLR2.

Methods and Results: Healthy and diabetic HECs, as well as mouse coronary vascular endothelial cells (MECs) from wild-type (WT) and TLR2 knockout (KO) mice, were treated with Staphylococcus aureus peptidoglycan (PGN, 10 μg/ml). NF-κB activation (phosphorylation and intranuclear translocation), and the production of ICAM-1, IL-6, IL-8 and TNF-α were analyzed by immunoblotting, immunostaining and ELISA. PGN induced NF-κB activation, and the production of ICAM-1, IL-6 and IL-8 in HECs and WT MECs, but had no effect on TNF-α production. The pro-inflammatory effects of PGN were absent in TLR2 KO MECs. Interestingly, the production of ICAM-1, IL-6 and IL-8 was significantly enhanced in diabetic HECs in comparison to that in healthy HECs. The enhanced inflammatory responses in diabetic HECs correlated with exacerbated NF-κB activation in the absence of a change in TLR2 or MyD88 protein level.

Conclusion: This study demonstrates that coronary artery endothelial cells from patients with type 1 diabetes exhibit enhanced inflammatory responses to TLR2 stimulation. Type 1 diabetes enhances TLR2 signaling, rather than the expression of TLR2 or the adaptor protein MyD88.



The inflammatory response of an injured or infected patient can cause significant morbidity and mortality. This stress response involves many processes including the glucocorticoid pathway. Although the stress response is expected to be similar in those who suffer similar insults, it is commonly not the case. Glucocorticoids bind to the glucocorticoid receptor (GR) and affect the transcription of genes involved in inflammation. Genetic variations in the GR could explain the differential responses to stress. In the survey of 97 human subjects, we identified a novel GR splice variant (81u-2) generated by an alternative splicing event that retained the intron between exons 8 and 9 (intron H). The predicted protein of 81u-2 has 727 amino acids (aa) in common with the human GR reference sequence (777aa) in conjunction with early termination at 745aa. This variant is expressed as a minor population in 3 individuals in the ongoing survey. Functional assay revealed an 80% reduction in transactivation potential of this isoform compared to the GR reference. In comparison with another early termination GR isoform of 743aa (HS22-22), the transactivation potential was very similar. This novel GR splice variant 81u-2 may differentially exist as a minor population in some individuals, and its interaction with other GR isoforms could explain, at least in part, the variations in stress response.



Abstract withdrawn.


PPARγ MEDIATES THE REGULATION OF INOS EXPRESSION BY C-JUN/AP-1.K. Ban,* Z. Peng,* and R.A. Kozar. University of Texas, Houston, TX 77030

We and others have demonstrated that iNOS, PPARγ, and c-Jun/AP-1 are implicated in intestinal injury and inflammation induced by ischemia/reperfusion (IR). However, the relationship of these genes after intestinal IR remains unclear and were therefore investigated in the current study.

Methods: IEC-6 cells were transfected with the PPARγ PPRE-x3-TK-Luc reporter vector and /or treated with the JNK inhibitor, SP600125 (20 μM); the PPARγ antagonist, GW9662 (10μM); or the iNOS inhibitor, 1400W (20μM) for 24 h, followed by H2O2 (250uM) for 6 h as an in vitro model of IR.

Results: After oxidant stress, SP600125 inhibited c-Jun phosphorylation (Fig. A), iNOS expression (Fig. A), and AP-1 activity (Fig. C), but increased activity of PPARγ (Fig. B). Inhibition of PPARγ by GW9662 had no effect on AP-1(Fig. C) but increased iNOS (Fig. A). Inhibition of iNOS by 1400W did not affect activity of PPARγ (Fig. B), or AP-1(Fig. C).

Conclusion: SP600125 inhibited phosphorylation of c-Jun/AP-1 which increased PPARγ activity and decreased iNOS expression. As GW9662 failed to affect AP-1 but increased iNOS, and 1400W changed neither AP-1 nor PPARγ activity, we speculate that inhibition of c-Jun/AP-1 by SP600125 resulted in increased of activity of PPARγ, and consequently decreased iNOS expression. Therefore, these results suggest that PPARγ mediates the regulation of iNOS by c-Jun/AP-1 after oxidative stress.



RAPID DEATH FROM SEVERE GUT ISCHEMIA IS PREVENTED BY GLYCOPYRROLATE, SUGGESTING A NEUROGENIC MECHANISM.A. Penn,* and G. Schmid-Schönbein. Department of Bioengineering, Univ. Calif. San Diego, La Jolla, CA 92093-0412

Splanchnic arterial occlusion (SAO) raises blood pressure (BP) in rats initially but within hours, even without reperfusion, BP falls irreversibly. These fatal blood pressure drops occur either over hours (slow) or in just minutes (fast). The fast BP drops occur without significant reduction of pulse pressure or pulse rate suggesting death is from neurogenic shock (i.e. dilation of peripheral blood vessels resulting in BP loss despite maintenance of cardiac contractility). Fast fatal pressure drops (FFPDs) occur more frequently and earlier if the rat is treated with intramuscular xylazine and glucose in the intestinal lumen, both stimulators of the parasympathetic nervous system (PNS). It is not know whether inhibition of the PNS can prevent FFPDs. Our objective was to determine if parasympathetic inhibition by glycopyrrolate, a muscarinic anticholinergic, in intestinal ischemia could reduce incidence of rapid death. We therefore added glycopyrrolate or saline to rats treated with glucose and xylazine before initiating SAO. The results show that glycopyrrolate (1 mg/kg) significantly delayed onset of the fatal pressure drop (115±31 vs. 71± 34 min post-occlusion) and completely prevented FFPDs (0 of 6 vs. 5 of 8 animals with a fast death without glycopyrrolate), but not the slow form of pressure reduction. These results suggest that in severe intestinal ischemia, parasympathetic blockade prevents rapid pressure reductions, an observation that is consistent with a similar protection seen after vagotomy. (Support: HL 67825, 76180, GM85072 and an unrestricted educational gift from Leading Ventures).


TRANSPORT OF PANCREATIC DIGESTIVE ENZYMES ACROSS THE INTESTINAL BARRIER IN EARLY STAGES OF SHOCK.M. Chang,* and G. W. Schmid-Schönbein. Department of Bioengineering, University of California San Diego, La Jolla, CA 92093

Transport of digestive enzymes across the mucosal epithelial barrier in the intestine sets the stage for shock, multi-organ failure and death due to autodigestion. We hypothesize that the intestinal mucin layer provides a physical barrier that prevents the entry of serine proteases normally contained within the lumen of the intestine and during ischemic states the mucin layer becomes diluted allowing access of these enzymes into the intestinal wall. In order to test these hypotheses we measured the location and levels of mucin and digestive enzymes in the intestinal wall. We occluded the superior mesenteric and celiac arteries up to 30 minutes as a model of splanchnic ischemia and used cyklokrapron to block luminal serine proteases. Jejunal sections were investigated by tissue in-situ zymography to quantify and visualize serine proteases entry and activity; alcian blue staining to visualize the carbohydrate part of mucin; immunohistochemistry to visualize goblet cell secreted mucin (mucin2) and membrane bound mucin (mucin13). Our results show intestinal damage and serine protease activity as early as 15 min of ischemia to a greater extent after 30 min of ischemia. Cyklokapron provided intestinal protection by reducing morphological damage and entry of chymotrypsin to the intestinal wall. We observe low levels of chymotrypsin entry into the mucin layer and intestinal wall of normal non-ischemic intestine. In contrast there is early swelling of the mucin layer and entry of chymotrypsin from the lumen of the intestine across the epithelium into the mucosa of ischemic intestine. These results suggest that dilution of the mucin layer allows entry of powerful pancreatic digestive proteases across the intestinal barrier, which is accompanied by the disruption of the epithelial barrier within minutes of intestinal ischemia. (Supported by HL76180 and GM85072).



Purpose: Glutamine (GLN) treatment can protect intestinal epithelial cells (IEC-18) in vitro from heat and oxidant stress. GLN's effect on heat shock protein (HSP) enhancement is implicated as the major mechanism of protection during heat stress. However, the effects of GLN treatment on specific HSPs during oxidant injury remain to be elucidated. Further, the effect of GLN on HSP32 (or HO-1) expression following oxidant injury in the intestinal epithelial cell (IEC) in vitro is unknown. The purpose of our study was to determine the effects of GLN on specific HSP expression in hydrogen peroxide (H2O2) injured IEC-18 cells.

Methods: Cells were treated for 15 min with either 0mM (CT) or 10mM GLN, and then subjected to either H2O2 (600uM) injury for 30 min, or a non-lethal heat stress (HS) of 43° for 45 min. Cells recovered for 2.5 hrs at 37°. Western blot analysis was used to determine total HSP25 and HO-1 expression. (N=3).

Results: GLN treatment increased HO-1 in both models of injury more than 3-fold (p=0.001 vs. H2O2 only, and p<0.001 vs. HS CT). HSP25 however, only increased with GLN treatment in the HS cells (p<0.01 vs. HS CT). H2O2 injured cells demonstrated a decrease in HSP25 with GLN treatment, (p<0.05 vs. H2O2 only).

Conclusions: This is the first data showing GLN induces a differential induction of HSP expression based on the type of cellular injury in the intestine. GLN treatment appears to consistently increase HO-1 expression during heat stress and oxidant injury, however HSP25 expression only increased in the HS groups. The mechanism of this differential expression may be due to the unique and diverse regulation of HO-1 expression by multiple transcription factors including heat-shock factor, NfκB, nuclear factor-erythroid 2, and AP-1.


ANALYZING THE MECHANISMS OF PROBIOTIC TREATMENT IN NECROTIZING ENTEROCOLITIS.J. Arciero,* G. Ermentrout,* J. Rubin,* and Y. Vodovotz. University of Pittsburgh, Pittsburgh, PA 15260

Objective: Necrotizing enterocolitis (NEC) is a severe disease of the gastrointestinal tract in preterm infants that may result in intestinal necrosis, systemic sepsis, and multisystem organ failure. In severe cases, surgical interventions are required, and mortality is nearly 30-50%. Studies have shown a reduced incidence and severity of NEC in neonates treated with probiotics, which are non-pathogenic species of bacteria that benefit the host. It is hypothesized that probiotic treatment improves intestinal barrier and immune function while limiting the presence of pathogenic bacteria and inhibiting Toll-like receptor 4 expression. The objective of this study is to analyze the protective potential of these mechanisms using a mathematical model.

Methods: Pathogenic and probiotic bacteria were simulated in two compartments representing the intestinal lumen and blood/tissue. The permeability of the intestinal wall was tracked by the model, and the inflammatory response was presumed to be activated once bacteria enter the blood/tissue.

Results: In most cases, the model predicted that the presence of probiotics would prevent bacterial efflux into the blood/tissue and the associated inflammatory response. However, in some instances, the model predicted that probiotics would contribute to a transient increase in total luminal bacteria, which can lead to an efflux of bacteria into the blood/tissue and a perpetuated inflammatory response. This paradox has been observed clinically, in which there were cases that probiotic treatment did not reduce the incidence of NEC and instead led to bacterial sepsis.

Conclusion: The model predicts some conditions under which probiotics may be beneficial, promoting the health of the host without exacerbating inflammation, and other cases in which this therapy could be detrimental.



Pancreatic digestive enzymes in the ischemic intestine play a central role in multisystem organ failure, which can be prevented by protease inhibition in the small intestine lumen. If allowed to circulate systemically, these enzymes may also result in shock. However, it is unclear whether enzyme liberation in an intestine with increased permeability alone leads to shock. To test this idea the nonischemic rat small intestinal lumen was perfused for two hours with either (A) digestive enzymes, (B) interventions designed to increase lumenal permeability (N-acetylcysteine (NAC) + atropine + increased perfusion flow) or (C) both, and animals were observed for shock and organ failure. Digestive enzymes perfused (Groups A and C) included trypsin, chymotrypsin, elastase, amylase and lipase at concentrations 2 log greater than baseline values. Group A (n=6) maintained stable hemodynamics as did other groups perfused with single enzymes alone. However all animals in Group C (n=6) developed hypotension, significant increases in gut permeability (p<0.001) and died (p<0.001). Group B (n=6) developed mild hypotension (NS) and increased gut permeability (p<0.05) compared to controls but there were no deaths. These experiments demonstrate that increased gut permeability such as occurs in ischemic conditions, in the presence of lumenal pancreatic digestive enzymes is sufficient to induce shock. Digestive enzymes, if allowed to penetrate the gut wall result in multiorgan failure and death. (Supported by NIH Grant GM085072).


PATTERN RECOGNITION USING 1H-NMR OF THE INTESTINAL EPITHELIAL CELL (IEC-6) UNDER OXIDATIVE STRESS.K. Nakata, N. Sato,* T. Asakura, K. Hirakawa, R. Zhu, T. Masuno, Y. Ohno, K. Koike,* and H. Yokota*. Dept of Emergency and Critical Care Medicine Nippon Medical School, Tokyo, 1138603

We have been studying mesenteric ischemia/reperfusion (IR)- induced gut mucosal injury and repair. Epithelial injury and restitution are central consequences of the gut mucosal damage. However, the metabolism of epithelial cell injury and restitution under the oxidative stress remain poorly. Pattern recognition using 1H-nuclear magnetic resonance (NMR) has been attracting attention as a new technology and comprehensive study of metabolites. To now test the utility of pattern recognition using 1H-NMR, we hypothesized that they could identify differences between metabolites of epithelial cells following oxidant stress.

Methods: IEC-6 cells were cultured to confluence then oxidant stress was induced by H2O2 (0.5mM). Cells were harvested after 6 hrs, 12 hrs and 24 hrs for pattern recognition of metabolites using 1H-nuclear magnetic resonance (NMR). Principle Component Analysis (PCA) and Cooman's plot analysis were performed from NMR spectra. Cell viability and cell migration were also assessed.

Results: The analysis showed the metabolite profiles of IEC-6 cell subjected to different time points of H2O2 were distributed to each different cluster area by PCA. The datasets were further analyzed by Cooman's plot, which showed clearly separated different time points. Cell viability was decreased and cell migration was gradually increased by time dependently.

Conclusion: Changes in metabolite profiles of IEC-6 cell following oxidant stress were identified pattern recognition using 1H-NMR. This technology provided insights into the pathogenesis of the epithelial cell restitution following oxidant stress.


PULMONARY AND SPLENIC IMMUNE CELL FUNCTION AFTER FEMORAL FRACTURE AND HEMORRHAGE IN WILD TYPE AND IL-6 KNOCKOUT MICE.F. Hildebrand,* P. Mommsen, C. Zeckey, T. Barkhausen, C. Krettek, and M. Frink. Trauma Department, Hannover Medical School, 30625 Hannover, Germany

The effect of femoral shaft fractures on immune cell function in multiple trauma patients remains unclear. The present study investigates the influence of femoral fracture and hemorrhage on the activation of splenocytes (Sp) and alveolar macrophages (AM). 36 male wild type (C57BL/6) and IL-6 knockout (B6;12952-IL6tmlKopf) mice underwent standardized femoral fracture and volume-controlled hemorrhage (orbital puncture). This was followed by fluid resuscitation (4 times the shed blood volume) and splint fixation of the fracture. Animals were sacrificed three hours after induction of hemorrhage and/or fracture. WT and IL-6−/− mice were randomly assigned to sham procedure (S) or experimental groups with either an isolated femoral fracture (Fx) or in combination with hemorrhagic shock (TH-Fx). Cytokine release of cultured Sp and AM were determined by FACS analysis. Interstitial edema and pulmonary PMN infiltration were determined by immuno-histochemical staining. In WT mice, a significant increase of cytokine and chemokine release of AM as well as a significant reduction of cytokine secretion of Sp was observed in groups Fx and TH-Fx. Compared to WT animals in IL-6−/− mice an increased TNF-α and decreased IL-10 secretion of Sp were detected. A decreased TNF-α and MCP-1 secretion of AM as well as a reduced interstitial edema and PMN infiltration in IL-6−/− animals was observed compared to WT. An isolated femoral fracture already has a significant impact on posttraumatic immune function. Our results suggest IL-6 as a potential therapeutic target to modulate the posttraumatic immune response.


INTRAVENOUS HYDROGEN SULFIDE DOES NOT INDUCE HYPOTHERMIA OR IMPROVE SURVIVAL FROM HEMORRHAGIC SHOCK IN A SWINE MODEL.T. Drabek, P. Kochanek,* J. Stezoski,* X. Wu,* H. Bayır,* R. Morhard,* W. Stezoski,* and S. Tisherman. Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA 15260

Several laboratory studies suggest that induced hypothermia during hemorrhagic shock (HS) improves survival. Inhaled hydrogen sulfide (H2S) was shown to induce hypothermia and decrease metabolism in mice and rats but not in piglets. We tested the hypothesis that intravenous H2S will induce hypothermia, reduce oxygen consumption (VO2) and improve outcome in a prolonged HS in pigs. We also assessed markers of organ injury (ALT, AST, CPK, creatinine and troponin) and levels of protein thiols. In a prospective randomized study, isoflurane anesthetized pigs were subjected to volume-controlled HS including spleen transection. Limited fluid resuscitation was provided after 40 min HS to maintain MAP ≥ 30 mmHg for 3 h. The study group received infusion of H2S (IK 1001) at 5 mg/kg/h based on the highest tolerable dose from pilot studies; the control group received vehicle. There was no difference in survival at 24 h (2/8 in H2S vs. 3/8 in control group). Heart rate increased similarly during shock over time in both groups. Cardiac output was better preserved in the delayed phase of HS in the vehicle group. Temperature was similar in both groups during shock (vehicle: BL 37.9±0.7°C, final, 38.0±0.4°C; H2S: BL 38.1±0.6°C, final 38.3±0.3°C) as well as VO2, lactate, glucose, and pH. Markers of organ injury markedly increased in both groups. Protein thiols increased in both groups during HS. However, there were no differences between groups. We were not able to demonstrate hypothermia-inducing effect or a reduction in VO2 from H2S infusion in our model of HS in pigs. Our data mirror those seen in studies with piglets and suggest inability to translate the hypothermia effects of H2S to large animals. (Support: Ikaria, Seattle, WA).


TLR4-IL-1RI CROSS-TALK MEDIATES HEMORRHAGIC SHOCK-PRIMED CASPASE-1 ACTIVATION IN LUNG ENDOTHELIAL CELLS.Y. Li,* M. Xiang,* M. Wilson, and J. Fan. Department of Surgery, University of Pittsburgh, and VA Pittsburgh Healthcare System, Pittsburgh, PA 15240

Lung endothelial cells (LEC) are critical in the mechanism of acute lung injury following hemorrhagic shock (HS) through release of inflammatory mediators, including IL-1β. Conversely, LEC are also targets of IL-1β. Our previous studies have shown that HS through HMGB1-TLR4 signaling activates LEC. We thus hypothesized that HS might upregulate IL-1RI expression through HMGB1-TLR4 and thus amplify the LEC response to IL-1β and prime for caspase-1 activation as well as subsequent inflammation.

Methods: WT and TLR4 mutant (C3H/HeJ) mice were bled to a MAP of 30 mmHg for 2h, and then resuscitated with Ringer's lactate. In some studies, the mice were injected (i.p.) with anti-HMGB1 neutralizing antibody (Ab) (600 μg/mouse) or control IgG 10 min before HS. IL-1RI in the lungs was detected 4 h after HS by Western blotting. In vitro experiments: Mouse LEC isolated from WT and TLR4-mutant mice were treated with HMGB1 (0.5 μg/ml) for 2 h, and IL-1RI in the cells was then detected. To address if the altered IL-1RI affects inflammasome activation, LEC were sequentially stimulated with HMGB1 at 0h and with IL-1β at 2h, followed by measurement of caspase-1 cleavage at 2 h after IL-1β.

Results: HS increased IL-1RI expression in the lungs of WT mice, but not of TLR4-mutant mice; Neutralizing Ab to HMGB1 prevented the HS-induced IL-1RI upregulation. In vitro, HMGB1 induced IL-1RI upregulation in WT LEC, but not in TLR4-mutant LEC. Furthermore, IL-1β caused a significant increase in caspase-1 cleavage in the WT LEC that were pretreated with HMGB1, but not in the TLR4-mutant LEC, in which IL-1RI upregulation was diminished. Thus, HS through HMGB1-TLR4 signaling upregulates IL-1RI expression in LEC, therefore amplifying the response of the LEC to IL-1β and priming for caspase-1 activation. The results suggest an important feedback mechanism of TLR4-IL-1RI cross-talk in enhancing post-HS inflammation.


C-PEPTIDE REDUCES LIVER NF-κB ACTIVATION FOLLOWING HEMORRHAGIC SHOCK.R. Chima, T. LaMontagne,* G. Piraino,* P. Hake,* and B. Zingarelli. Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229

Liver injury commonly occurs as a consequence of hemorrhagic shock. Mechanistically nuclear factor-κB is a crucial transcription factor that modulates the inflammatory response following hemorrhagic shock. Recent data suggest that C-peptide, a 31-amino acid peptide cleaved from proinsulin during insulin synthesis may modulate the inflammatory response. However, its mechanism of action remains undefined. Hence we investigated the effect of C-peptide on liver NF-κB activation following hemorrhagic shock. Hemorrhagic shock was induced in rats (age 3-4 months) by withdrawing blood from the femoral artery to a mean arterial pressure of 50mmHg. Animals were kept in shock for 3h at which time they were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, one group of animals received C-peptide (280 nm/kg IV) while another group received vehicle. In vehicle-treated rats hemorrhage and resuscitation significantly induced p65 activation in the liver which changed 5.5-fold at 1h and 1.5-fold over basal levels at 2h after resuscitation. Following hemorrhage and resuscitation, C-peptide-treated rats had a significant reduction in p65 activation in the liver when compared to vehicle-treated rats (p<0.05). Specifically, p65 activation in the liver changed 2.2-fold at 1h and 0.8-fold over basal levels at 2h after resuscitation. At 3h following resuscitation this difference in p65 activation with C-peptide treatment was associated with a significant reduction in liver neutrophil infiltration (93±7.4 vs. 153±12.8 U/100mg lung tissue, p<0.05) when compared to vehicle-treatment. Our data suggest that C-peptide may exert hepatoprotective effects by modulating neutrophil infiltration most probably through inhibition of the NF-κB pathway. (Supported by the Shock Society/Novo Nordisk, NIH K12 HD028827 & NIH AG-027990).


POSTSHOCK LUNG PMN SEQUESTRATION IS MEDIATED VIA THE 5-LIPOXYGENASE PATHWAY.J. Eun,* E. Moore, D. Mauchley,* X. Meng, J. Lee,* and A. Banerjee. University of Colorado Denver, Aurora CO 80045

Gut ischemia/reperfusion injury is the central event in the development of acute lung injury (ALI) following hemorrhagic shock (HS) and mesenteric lymph is the link between the two. Prior work has shown that metabolites of the 5-lipoxygenase (5-LO) pathway are critical to the development ALI following HS. Furthermore, a 6-fold increase in the delivery of arachidonic acid (AA) in post-shock mesenteric lymph has been shown. Leukotrienes are pro-inflammatory mediators derived from the metabolism of AA through the 5-LO pathway that recruit PMNs.

Hypothesis: The 5-LO pathway is integral to the development of PMN sequestration in the lung following HS.

Methods: 5-LO knock out (KO) mice and wild type (WT) controls (C57BL/6) underwent HS (30% of blood volume) via cardiac puncture and allowed to recover for 4 hours. Lungs underwent myeloperoxidase (MPO) analysis. An additional group of WT mice received the 5-LO inhibitor MK-886 (6mg/kg) IV, 30 minutes prior to the induction of HS.

Results: Shocked mice had a 5.5-fold increase in PMN sequestration vs. control animals (3.77 vs. 0.69 units/mg lung). Mice receiving a 5-LO inhibitor had a 39% decrease in MPO vs. vehicle control (2.35 vs. 3.87 units/mg lung). 5-LO KO mice had a 53% decrease in MPO vs. shock animals (1.77 vs. 3.77 units/mg lung).

Conclusion: The 5-LO pathway abrogates ALI following HS in part by attenuating pulmonary PMN sequestration.



CONTROLLED 60% HEMORRHAGE RESUSCITATION WITH SMALL VOLUME P188 IN MINIATURE SWINE.J. Burns,* L. Baer,* M. Dubick, and C. Wade. U.S. Army Institute of Surgical Research, Ft. Sam Houston, TX 78234

Objective: Evaluate small volume resuscitation using Poloxamer 188 (P188) following severe hemorrhage. P188 is a non-ionic block copolymer surfactant with rheologic, cytoprotective, anti-inflammatory and anti-thrombotic properties.

Methods: Seven mature male miniature swine were catheterized for hemorrhage and resuscitation and allowed to recover from anesthesia. The animals were sedated and bled 60% of EBV (∼ 39 ml/kg) exponentially over 1 hr, followed immediately by a 2 min infusion of 1.33 ml/kg P188 (150 mg/ml), with no additional resuscitation. Target survival time was 180 min. P188 data were compared against previous data from similarly hemorrhaged animals with no resuscitation (n = 16).

Results: Hemorrhage resulted in a characteristic hypotension and metabolic acidosis (Table). Survival time for the control swine was 64 ± 11.5 min with a 6% survival at 180 min. Survival time of 138 ± 19.2 min for the P188 swine was significantly greater than control (p = 0.009). However, a P188 survival of 44% at the target time of 180 min was not significantly different than the 6% control (p = 0.067). TEG data confirmed the in vivo and in vitro retardation of blood coagulation with a dramatic lengthening of R, decrease in α-angle and a decrease in MA with the use of P188.

Conclusion: In the presence of controlled bleeding P188 improved survival time, however, retardation of clotting raises serious concerns as to its use in the presence of uncontrolled bleeding.



POSTERIOR PITUITARY EXHAUSTION IN HEMORRHAGIC SHOCK AND RESUSCITATION.J. Rosenbloom,* Y. Guan,* E. Sawey,* L. Becker,* B. Sarani,* J. Pascual,* and C. Sims. University of Pennsylvania Division of Traumatology and Surgical Critical Care, Philadelphia PA 19102

Objective: To evaluate plasma and pituitary AVP levels during hemorrhagic shock and resuscitation.

Methods: Decompensated shock was simulated using a fixed-pressure hemorrhage model in Sprague-Dawley rats. Animals were bled to a MAP of 40 mmHg until the MAP could not be maintained without fluid infusion (Maximal Bleed Out Time, MBOT); 40% shed blood volume in Lactated Ringer's (LR) was then incrementally infused to maintain a MAP of 40mmHg (40% SVT, maximal shock); resuscitation with 4 times the total shed volume in LR subsequently occurred over 60 minutes (60R); and animals were followed post-resuscitation for 18 hrs (18H). Control animals (SHAM) were not hemorrhaged. Pituitaries (n=5) and blood samples (n=3) were analyzed for AVP using EIA. Statistical analysis was performed with Mann-Whitney test (significance=*p<0.05).

Results: MAP was significantly depressed throughout hemorrhagic shock and resuscitation (graph 1). Plasma AVP peaked at MBOT and then declined significantly (graph 1). Pituitary AVP concentration dropped from SHAM to MBOT and continued to fall by 18H (graph 2).

Conclusions: Plasma AVP peaked during decompensated shock (MBOT), then declined despite persistent hypotension. Pituitary AVP stores were depleted post-shock (18H) and were associated with decreased plasma AVP and reduced MAP. The use of exogenous vasopressin to supplement depleted pituitary stores following hemorrhagic shock may be beneficial and warrants further investigation.



MICROCIRCULATORY DISORDERS AFTER TRAUMA-HEMORRHAGIC SHOCK (THS): ASSESSMENT BY DIFFERENT TECHNIQUES.T. Berezina, A. Kirsanova,* I. Novoderzkina,* G. Kozinets,* and S. Zaets. UMDNJ-NJ Medical School, Newark, NJ 07103, Institute of General Resuscitation, Moscow, Russia 103031, and Novo Nordisk, Inc., Princeton, NJ 08540

Objective: THS results in microcirculatory disorders in various organs and tissues. This study is aimed to determine if there is a correlation between different methods of assessment of these abnormalities.

Methods: Rats were subjected to THS (laparotomy + blood withdrawal until mean arterial pressure of 30-40 mm Hg) or trauma sham-shock (TSS) (laparotomy, no bleeding) for 90 min. Microcirculation in liver and skeletal muscle as well as RBC shape was assessed before and up to 3 hours post-THS or TSS by means of laser flowmetry, intravital microscopy, and scanning electron microscopy. Correlation analysis of data received by different techniques was performed.

Results: THS shock caused a significant decrease in microvascular blood flow and RBC flow velocity (FV) both in liver and skeletal muscle as well as a significant increase in the percentage of abnormally shaped RBC. These alterations persisted up to 3 hours post-THS (Table). A correlation was revealed between microvascular blood flow in liver/muscle and RBC FV (r=0.68/0.65, p<0.01). There was an inverse correlation between the percentage of abnormal RBC and RBC FV or micro-vascular blood flow (r=−0.72, p<0.01 and r=−0.76, p<0.01, respectively).

Conclusion: Data describing microcirculatory alterations after THS, received via intravital microscopy and laser flowmetry, tightly correlate. RBC shape abnormalities contribute to microcirculatory disorders.



INFLUENCE OF AGING ON CARDIAC MITOCHONDRIAL GENE EXPRESSION FOLLOWING TRAUMA-HEMORRHAGE.B. Jian,* S. Yang,* D. Chen,* L. Zou, J. Chatham, I. Chaudry, and R. Raju*. University of Alabama at Birmingham, Birmingham, AL 35294

Cardiac dysfunction and mortality associated with trauma and sepsis increase with age. Mitochondria play a critical role in the energy demand of cardiac muscles, and thereby on the function of the heart. In order to obtain the information concerning the molecular changes occurring in mitochondria after trauma-hemorrhage (T-H), we performed a microarray analysis using our custom rodent mitochondrial gene chip (RoMitochip). Neonatal rat cardiomyocytes subjected to normoxia or hypoxia (1% oxygen for 24 hr) were used to validate the chip. To study gene expression changes following T-H, 6 and 22 month old rats were subjected to T-H or sham operation (n=4 in each age group) and sacrificed 2 hr following resuscitation. Left ventricular tissue was collected, RNA isolated, labeled and gene expression determined using the RoMitochip. RoMitochip has 419 probe sets from nuclear and mt DNA of the rat. The gene expression changes in rat cardiomyocytes following hypoxia were similar to that previously (Shock, in press) observed for the mice. Following T-H, while 151 probesets were significantly altered (45 up and 106 down) in 6 months old rats, only 69 were altered (31 up and 38 down) in 22 month old rats. 38 probesets (12 up and 26 down) showed the same trend in both groups. Among the genes upregulated in both age groups were HIF-1α, Sod2 and Hk2, when mitochondrial Ucp3 and Gpam were down regulated. Among the uniquely changed genes in the aged rats (22 months) were 11 tRNA transcripts on the mtDNA - all upregulated compared to sham. The results demonstrate a decrease in transcriptional alteration and an increased level of mtDNA tRNA transcripts following T-H in the aged.

Conclusion: There is a marked decrease in age dependent changes in the transcriptional profile following T-H, possibly indicating cellular senescence (Support:AG031440-RR).



The purpose of this study was to assess the effects of blood components vs. conventional fluid resuscitation on blood loss. Anesthetized, instrumented female pigs (8/group) underwent controlled hemorrhage of 24 ml/kg, 20-minute shock period, splenic injury with 15-minute initial uncontrolled hemorrhage, and administration of test fluids. The hypotensive (≤65 mmHg) resuscitation volumes were 15 ml/kg for Hextend® and all the blood component groups (fresh frozen plasma [FFP], 1:1 ratio of FFP: red blood cells [RBC], 1:4 FFP:RBC, and fresh whole blood) and 45 ml/kg for lactated Ringer's (LR) solution. Post-resuscitation blood loss (PRBL), hematocrit (hct), and coagulation status (INR and TEG-MA were measured post-spleen-injury up to 5h. The table shows the PRBL and the change from baseline (BL) at 60 minutes when all fluids were given, prior to any deaths. Blood products as initial resuscitation fluids can reduce PRBL from a non-compressible injury and preserve coagulation.



THE POTENTIAL FOR IMPROVED INTRAOSSEOUS FLOW AT LOW INFUSION PRESSURES.B. Rubal,* C. Neal,* C. Sartin,* M. Dubick, and R. DeLorenzo*. Brooke Army Medical Center, Fort Sam Houston, TX 78234

Objective: Intraosseous (IO) access is an effective route of fluid and drug administration during resuscitation efforts when attempts for intravenous access fail. Prior studies have suggested that a three parameter exponential decay model may be used to characterize IO flow dynamics in vivo under low IO flow conditions and that this model could be used to quantify improved IO flow following saline flush and heparin infusion. The present study examines low pressure IO flow dynamics in a model uncomplicated by the clotting cascade, vascular regulation or competitive flow from native circulation to assess the potential for improved flow under low infusion pressure conditions.

Methods: IO access was established in the proximal tibia (EZ-IO 9001 AD, Vidacare, San Antonio, TX) in six postmortem isolated swine hind limbs (Sus scrofa, ages: 3-5 months). Following a thorough saline flush of the IO cannula, stop-flow manometric methods were used to record manometer discharge pressures and IO flow. Discharge pressures were then fitted to an exponential decay model (Pman = Po e-bt + Pbone). Where Pman = the height of the manometer saline column during discharge into the IO compartment, Po = initial height of the discharging column (55 cm saline), b = decay constant, t = time in minutes, and Pbone = zero flow asymptote.

Results: An excellent fit (R20.996, P < 0.001) was noted with the model in all preparations. The mean ± 95% CI for the decay constants and the discharge pressure half-times were b = −0.083 ± 0.046 and t1/2 = 13.2 ± 10.6 seconds, respectively.

Conclusion: This study demonstrated increased IO discharge rates in an isolated cadaveric preparation using previously reported methods for assessing IO flow dynamics in vivo. Results suggest that low pressure IO infusion rates may be improved by modifying in vivo physiologic factors.



Hyperglycemia and insulin resistance induced by critical illness or injuries are associated with increased mortality and morbidity. The molecular mechanisms underlying the acute onset of insulin resistance following injuries remain poorly understood. Activation of the stress signaling pathways, including Inhibitory κB kinase (IKK) and c-Jun N-terminal kinase (JNK) pathways are thought to be involved in the pathogenesis of obesity- or Type 2 diabetes-related insulin resistance. In the present studies, the roles of acute activation of IKK and JNK kinases in the development of hepatic insulin resistance following injury were investigated. An animal model of injury, trauma and hemorrhage, resulted in increased phosphorylation/activation of IKK and JNK kinases in liver. The activation of hepatic IKK and JNK was observed as early as 15 minutes after trauma and hemorrhage, which was concomitant with rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate (IRS) 1. Inhibition of IKKα or β, or both, by adenovirus vector-mediated expression of dominant negative IKKα (Ad-DN-IKKα) or β (Ad-DN-IKKβ) in liver partially restored insulin signaling following trauma and hemorrhage. Similarly, inhibition of JNK1 kinase by expression of dominant negative JNK1 (Ad-DN-JNK1) also resulted in improved hepatic insulin signaling, indicating both IKK and JNK1 kinases contribute to the hemorrhage-induced insulin resistance in liver. In conclusion, IKK and JNK kinases are important mediators of injury/hemorrhage-induced insulin resistance and are potential targets for therapies to treat insulin resistance in the Intensive Care Unit.


LONG-TERM SURVIVAL AND RESUSCITATION IN HEMORRHAGIC SHOCK RATS BY PANCREATIC DIGESTIVE ENZYME BLOCKADE.F. A. DeLano,* and G. W. Schmid-Schönbein. Microcirculation Laboratory, Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093-0412

The sequence of events that lead to multi-organ failure after hemorrhagic shock involve compromises to key intestinal barrier function and tissue structure. Our evidence suggests that the inflammation in shock may involve pancreatic digestive enzymes in the intestine. Blockade of pancreatic proteases in the intestinal lumen serves to attenuate the production of inflammatory mediators and generation of a microvascular inflammatory cascade with reduced peripheral inflammation, tissue swelling and multi-organ dysfunction. The evidence has so far only been obtained in acute forms of shock without studies of survival after blockade of digestive enzymes in a chronic model of shock. Therefore we examined the impact of digestive enzyme blockade in the intestinal lumen on long-term survival in a Wiggers shock model in adult rats. Animals were exposed to acute hemorrhagic shock (mean arterial blood pressure of 30 mmHg) for 2 hours by hemorrhage from the femoral artery at which time all shed blood volume was returned. One hour after initiation of hypotension, the small intestine from duodenum to the cecum was subjected to intraluminal loading with and without an inhibitor of pancreatic proteases and lipases. Blockade of digestive enzymes in the lumen of the intestine was confirmed by zymography. Without blockade of digestive enzymes, more than 80% of the animals died (all within the first 12 hours), while with blockade more than 80% survived for a period of 1 month at which time animals had returned to initial body weight and normal weight gain (p<0.001, Chi-Square test). The treated animals when compared to animals without treatment showed both low levels of tissue damage in the heart and also reduced cell dysfunctions, e.g. insulin resistance. These results suggest a significant protection against multi-organ failure and insulin resistance by intraluminal blockade of digestive enzymes in the small intestine after severe hypotension.


PLASMA RESUSCITATION SUPPRESSES REACTIVE OXYGEN SPECIES PRODUCTION IN HEMORRHAGED RATS.M. Doursout,* Y. Liang,* X. Deng,* Y. Cao,* J. Salsbury,* T. Ko, J. Conyers,* and J. Holcomb. UTHSC Medical School at Houston, Houston, TX 77030

Hemorrhagic shock (HS) causes oxidative stress that leads to tissue injuries in various organs including the lung, liver, kidney and intestine. Specifically, reactive oxygen and nitrogen species, (ROS, RNS) specificially peroxynitrite, are potent inducers of tissue damage during systemic inflammatory response and circulatory shock. ROS caused injuries to the vascular endothelium resulting in endothelial dysfunction leading to multiple organ failure in HS. Our study assessed the production of nitrate, xanthine oxidase and peroxynitrite in hemorrhaged rats in the presence and in the absence of resuscitation fluids. Sprague Dawley rats were hemorrhaged at a rate of 2 ml/100g over 10 min until mean arterial blood pressure dropped to 30 mmHg. One hour following HS, rats were resuscitated with either Fresh Frozen Plasma (FFP) at Day 0 and Day 5 or Lactated Ringer (LR). Additional rats included HS alone and sham control. Blood samples were collected at baseline, end of HS/resuscitation and every 2 hrs for 6 hrs for nitrite/nitrate production. Animals were sacrificed at 6 hrs following HS/resuscitation. Lung tissues were harvested for xanthine oxidase and peroxynitrite determination. Our data demonstrated that Day 0 FFP increased nitrate production, supporting a vasodilatation in vascular beds as compared to Day 5 FFP; LR and HS alone. Our data also showed that xanthine oxidase, an enzyme that generates ROS, was predominant in HS alone and in LR-resuscitated rats whereas ROS production was decreased in rats resuscitated with Day 0 FFP. Finally, resuscitation with Day 0 FFP as compared to Day 5 FFP and LR decreased nitrotyrosine production. Our results indicate that Day 0 FFP improved resuscitation as compared to Day 5 FFP and/or LR in hemorrhaged rats.


EFFECTS OF DISUSE AND CONTROLLED HEMORRHAGE ON LIVER FUNCTION IN RATS.L.A. Baer, R. McCue, O. Hsu, and C.E. Wade. US Army Institute of Surgical Research, San Antonio, TX 78234

Hemorrhagic shock in the trauma patients can often lead to multiple organ failure. Extended bed rest may also be an underlying factor with recovery. The objective of this study was to examine liver function after a controlled hemorrhage in rats subjected to disuse for 14 days. Rats were randomly selected to the disuse (HU; n=9) or control (CONT; n=9) for 14 days prior to a 60% estimated blood volume controlled hemorrhage over 1 hr. Blood samples were taken at each time point during the hemorrhage period to determine hematocrit (HCT), lactate (Lac) and selected liver enzymes. HCT was not different between groups at the end of hemorrhage, but it did decrease over time. An overall significant increase in Lac was observed in HU. Livers/g/100g body mass were significantly smaller and liver enzymes specifically associated with trauma were significantly reduced in HU. Liver metabolism may not elicit a response to hemorrhage if extended bed rest is introduced prior to the severe blood loss.



SALUTARY EFFECT OF CALCIUM CHANNEL BLOCKER VERAPAMIL ON IGA TRANSCYTOSIS.T. Kamash,* L. Diebel, and D. Liberati*. Wayne State University Detroit, MI 48201

Objective: Calcium is a regulator of many cellular functions and it is known that calcium ion (Ca2+) homeostasis is disturbed following shock conditions. Intestinal epithelial cells (IEC) express L-type calcium channels and actively transport secretory IgA (SIgA), the principle Ig in mucosal defense, into mucosal secretions. Transcellular transport (transcytosis) of IgA is Ca+2 dependent and thus may be impaired following shock insults.

Methods: Confluent HT-29 IEC monolayers grown in a two-chamber cell culture system were held under hypoxic (5% CO2) conditions for 90 minutes followed by normoxia (21% O2) (H/R). Verapamil (8μM) was added to HT-29 cell subsets after hypoxic treatment. Dimeric IgA was added to the basal compartment and apical media was obtained at intervals to quantitate IgA transcytosis by ELISA. HT-29 cells held under normoxic conditions served as controls. HT-29 cell monolayer integrity was monitored by serial measurement of transepithelial electrical resistance (TEER).

Results: Apical chamber sIgA (mean ± SD, ng/ml, N = 4) TEER measurements remained stable throughout the experiment.

Conclusions: H/R led to increase IgA transcytosis across HT-29 cells. Addition of the calcium channel blocker (CCB) verapamil led to increased transcytosis in both control cells and cells subjected to H/R. CCB treatment may preserve gut barrier function by its effect on IgA transcytosis. Additional studies including in vivo confirmation in animal shock models are warranted.



OXYGEN DEBT REPAYMENT PREDICTS SURVIVAL IN A SWINE MODEL OF TRAUMATIC SHOCK.R. Barbee, P. Reynolds, N. White, M. Tiba,* and K. Ward. VCU Medical Center, Richmond, VA 23298

Objective: We hypothesized that oxygen debt repayment (O2DR) would be a reliable predictor of resuscitation efficacy and 3-hr mortality following severe hemorrhagic shock in a swine model. We predicted that survival time St would be negatively associated with absolute oxygen debt (O2 D) at any given resuscitation time, and positively associated with O2DR.

Methods: Forty male swine were hemorrhaged to a target O2D (80 ml/kg) and then resuscitated over 20 min with colloid, hypertonic saline, hemoglobin-based oxygen carrier (HBOC), or whole blood. O2D was calculated every 30 s during resuscitation. St was measured from the end of resuscitation to death or 3 hr. Animals were categorized as: failure to repay O2D; partial O2D repayment (0 to <75% in 2 hr); repayment of > 75% O2D by 2 hr. Association of O2DR with St was assessed by failure time models. Response to resuscitation was assessed by maintenance of MAP > 60 mm Hg. Both absolute debt and percent debt repaid (from the end of resuscitation) were predictive of survival as early as 30 min through one hour following resuscitation (p < 0.02). O2DR was a better predictor of St and MAP support than other common clinical vital signs. Hemorrhage models are notoriously variable; however O2DR is a promising metric for standardization of hemorrhage severity and resuscitation efficacy.

Relation of MAP support and O2D at 60 min post-resuscitation.

Survivors to 180 min •; Nonsurvivors ○



SEQUENTIAL LACTATE PREDICTS 3-HR SURVIVAL IN A SWINE MODEL OF TRAUMATIC SHOCK.P. Reynolds, R. Barbee, N. White, M. Tiba,* and K. Ward. VCU Medical Center, Richmond,VA 23298

Objective: We hypothesized that sequential lactate [Lac] will be a useful surrogate marker for predicting resuscitation response and 3-hr mortality following severe trauma and hemorrhage in a swine model. We predicted that rates of [Lac] reduction would be positively associated with both St and the rate of O2 debt (O2D) repayment.

Methods: Forty male swine were hemorrhaged to a target O2D (80 ml/kg) and then resuscitated over 20 min with colloid, hypertonic saline, hemoglobin-based oxygen carrier (HBOC), or whole blood. O2D and [Lac] were measured every 30 min. St was measured from the end of resuscitation to death or 3 hr. Association of variables with St was assessed by Weibull failure time models; estimates of [Lac] clearance between O2D repayment categories were obtained by random coefficient mixed models.

Results: Sequential lactate change tracked changes in O2D. Although absolute [Lac] post-resuscitation could not distinguish non-survivors from survivors, rates of repayment were highly predictive (p< 0.0001). St to 3 hrs was associated with the most rapid clearance rates (-0.02 mmol/L/min); animals surviving < 60 min showed increased [Lac] rate gain (+0.024 mmol/L/min). Animals that accumulated debt increased [Lac]; clearance rates were 2× higher for animals with complete debt repayment compared to those with partial repayment (p = 0.02). Rapid sequential [Lac] measurements rather than isolated measures are of more clinical utility in assessing response to resuscitation.



LONG-TERM EFFECTS OF HEMORRHAGIC SHOCK ON COAGULATION IN PIGS.W. Martini, B. Jordan,* K. Chung,* S. Colvin,* I. Terrazas,* D. Cortez,* N. Miranda,* and M. Dubick. US Army Institute of Surgical Research, Fort Sam Houston, TX 78234

Hemorrhagic coagulopathy increases trauma-related mortality. Mechanisms related to coagulation complications remain unclear. We investigated long-term (5 days) effects of hemorrhage on coagulation in pigs. Sixteen pigs were randomized into the control (C) and the hemorrhage and Lactated Ringers resuscitation (H-LR) groups. On day 1, hemorrhage was induced in the H-LR group by bleeding 35% of the total blood volume, followed by LR resuscitation at 3 times the bled volume. Pigs in C were not hemorrhaged or resuscitated. Measurements of hemodynamics and coagulation function were made at baseline, 15 min after H-LR, and 6h after H-LR. On day 2, 3, 4, and 5, all pigs were observed along with the same measurements. There were no significant changes of hemodynamics and coagulation in C during the study. Hemorrhage caused a decrease in mean arterial pressure and an increase in heart rate. LR resuscitation corrected these changes within 3h. Compared to baseline values on day 1 (BL), fibrinogen levels were decreased to 76 ± 6% by H-LR on day 1, increased to 217±16% on day 2 (both p<0.05), and remained at elevated levels afterwards; Platelet counts were decreased to 63±5% (p<0.05) by H-LR on day 1, remained at depleted levels until returning to BL on day 4. The initial clotting time did not change after H-LR on day 1(3.1±0.2 min), but was prolonged to 4.6±0.2 min on day 2 (p<0.05) and remained prolonged afterwards. Clot rapidity did not change on day1, but decreased on days 2 and 3, returned to BL on day 4. Clot strength was decreased from BL of 70±2 mm to 65±1 mm (p<0.05) by H-LR on day 1, but returned to BL by day 2. We conclude that hemorrhage caused different changing profiles in coagulation function and substrate availability. These data may help to facilitate the search for effective treatments of hemorrhagic coagulopathy.


EFFECTS OF SELECTIVE AUTONOMIC BLOCKADE ON HEART RATE COMPLEXITY AND SURVIVAL TIME DURING LETHAL HEMORRHAGIC SHOCK.A. Batchinsky, C. Necsoiu,* B. Jordan,* J. Burns,* L. Baer,* C. Wade, and L. Cancio. U.S. Army Institute of Surgical Research, Fort Sam Houston TX 78234

Background: We investigated the effect of selective autonomic blockade on survival time during lethal hemorrhagic shock; we explored the mechanism of this effect using Sample Entropy (SAMPEN, a measure of heart-rate complexity or HRC) and pNN50 (a measure of vagal cardiac input).

Methods: Midazolam-sedated, spontaneously breathing swine received atropine 0.005 mg/kg then 0.5 mg/min in Group A (n=13) or propranolol 0.5 mg/kg then 1 mg/min in Group P (n=13); Group C served as time Controls (n=12). Next 60% of blood volume was withdrawn in 1 hour using a controlled hemorrhage protocol. EKG was recorded at 5 time points: baseline (BL), after bolus (Bolus), at mid bleed (MB), end bleed (EB), and 30 min before death (BD). SAMPEN and pNN50 were calculated from 200-beat EKG sections. Survival was assessed at 1 and 3 hours.

Results: See Table (*p<.05; **p<.001; p<.0001 for between-group changes). Atropinization was evident from low pNN50 after Bolus. pNN50 and SAMPEN were not linearly related. Survival time in Group P was significantly lower (p<.0001) than other groups.

Conclusions: Sympathetic but not vagal blockade decreased survival time after HS. Atropine and HS independently decreased both vagal input and HRC. Vagal input is a major but non-exclusive contributor to HRC.



17β-ESTRADIOL SUPPRESSES THE STRESS RESPONSE FOLLOWING TRAUMA-HEMORRHAGE.H. Akabori,* F. Moeinpour,* K.I. Bland,* and I.H. Chaudry. Center for Surgical Research, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294

Although 17β-estradiol (E2) has been reported to alter the corticotropin-releasing hormone (CRH) neuronal activity in the hypothalamus after trauma-hemorrhage (T-H), it remains unknown whether E2 has any salutary effects in the hypothalamic-pituitary-adrenal (HPA) axis under those conditions. Prolactin-releasing peptide (PrRP), an important stress mediator in the central nervous system (CNS), is thought to stimulate CRH neurons. We hypothesized that T-H upregulates PrRP neuron-mediated stress response in the CNS, and E2 modulates the HPA axis via negative regulation of PrRP. Male Sprague-Dawley rats underwent sham operation (cannulation plus laparotomy) or T-H (midline laparotomy, mean BP of 35±5 mmHg for 90 min followed by resuscitation). Rats received vehicle or E2 (1 mg/kg BW i.v.) at the onset of resuscitation. Immediately after and 2 hr after resuscitation, rats were sacrificed. In T-H rats at the end of resuscitation, the hypothalamic PrRP and CRH contents were increased compared to shams (Fig. 1). In addition, plasma CRH and corticosterone levels were also significantly increased. E2 administration following T-H prevented these stress responses. Thus, E2 may regulate the stress response to T-H via PrRP neurons in the hypothalamus. (NIH RO1 GM39519)



VEGF AND ENDOTHELIAL FUNCTION IN LUNG FOLLOWING TRAUMA-HEMORRHAGIC SHOCK AND E2 TREATMENT.Z.F. Ba,* K.I. Bland,* and I.H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294

Vascular endothelial growth factor (VEGF) plays an important role in capillary tube formation and endothelial cell proliferation. However, the effects of trauma-hemorrhagic shock (T-H) with or without 17β-estradiol (E2) administration on VEGF and endothelial function in the lung remained unclear. To study this, pulmonary VEGF was determined by immunohistologic stain technique and pulmonary vascular endothelial cell function was also determined following T-H and E2 treatment. Fig. 1 shows VEGF (brown color) which was seen in epithelium of vessel smooth muscle, alveolar and endothelial cells. VEGF staining was significantly decreased at 2 h following T-H and resuscitation compared to sham; however, VEGF staining was markedly improved by E2 administration. Results also show that the central venous pressure and perfusion resistance were increased following T-H, but these were not observed in E2-treated T-H group. Fig. 2 shows that the pulmonary vascular endothelial cell function was significantly decreased following T-H; endothelial function was maintained in E2-treated group. Moreover, results showed that IL-6 was significantly increased after T-H, and was modulated by E2 treatment. These results indicate that E2 administration protects endothelial function following T-H and may partially prevent VEGF decrease under those conditions. (Supported by NIH 2 R01 GM 39519).

FIG. 1
FIG. 1:
VEGF (Immunohistologic stain with rabbit polyclonal antibody, Santa Cruz) in pulmonary tissue in sham, T-H and T-H+E2 groups. VEGF stain in brown color.
FIG. 2
FIG. 2:
Pulmonary Vascular Endothelial Function. Data are presented as mean ± SE and compared by one way ANOVA and Turkey's test: *p<0.05 vs Sham group.



Mast cells have been recognized as the key cells in the response of the intestine to infection and inflammation as well as other immune responses. Furthermore mast cell activation and the release of mast cell mediators, such as mast cell protease have been implicated to contribute to gut injury. The aim of this study was to test the hypothesis that trauma-hemorrhagic shock (T/HS)-induced gut injury is associated with mast cell activation.

Methods: Male SD rats were subjected to a laparotomy (trauma) and 90 min of sham shock (T/SS) or actual shock (35 mmHg × 90 min) (T/HS). After 3 hr of resuscitation, the rats were sacrificed and samples of the terminal ileum were processed for histology for gut injury and immunohistochemistry for rat mast cell protease II (RMCP II) expression. Serum samples were taken for the determination of RMCP II activity using ELISA.

Results: As shown in the figure below, T/HS-induced villous injury (T/SS=3.1 ± 1.1% vs T/HS=11.9 ± 3.0, p<0.01, N=6) as well as increased RMCP II immunopositive cells showing mast cell activation. In addition, the RMCP II level in serum of T/HS group was significantly higher than that of T/SS group (271.3 ± 8.4 ng/ml vs 171.0 ± 6.7, p<0.01).

Conclusion: Our novel findings suggest that T/HS-induced gut injury is associated with the activation of mast cells. This may be another potential mechanism by which mast cell proteases contribute to gut injury, inflammation and the production of biologically-active mesenteric lymph.



NON-INVASIVE HEMOGLOBIN MONITORING (SPHB) AND VOLUME EXPANSION IN HUMANS.M. Kinsky, M. Salter,* G. Kramer, H. Li,* D. Solanki,* and S. Henkel*. University of Texas Medical Branch, Galveston, TX 77555

Most often, surrogate endpoints of circulating volume are used to guide fluid therapy. However, over- and under-resuscitation remain a common complication.

Objective: We sought to determine if non-invasive hemoglobin monitoring could provide better predictors of vascular volume expansion compared to traditional endpoints after fluid loading.

Methods: Hemoglobin and hemodynamic data were obtained before and after a fluid bolus (25 mL/kg lactated Ringer's over 20 min (T0-T20)) in five normovolemic and hemorrhaged volunteers (bled 10 mL/kg). Blood hemoglobin, measured by co-oximeter (Co-ox), was compared to non-invasive Masimo Rainbow SET SpHb. Plasma volume (PV) was determined from indocyanine green (ICG) and blood hemoglobin over time. Measurements were recorded for 120 min (T120).

Results: Heart rate and arterial blood pressure did not change (p > 0.05) after fluid bolus. A small increase in cardiac output and decrease in systemic vascular resistance (p<0.05) following fluid bolus were observed. Hemoglobin decreased after fluid loading (p<0.001). A larger decrease was observed during hemorrhage. Comparative hemoglobin measurements by Co-ox and SpHb yielded a strong correlation (r2=0.7) and low bias (−0.9 g/dL). Hemoglobin by both SpHb and Co-ox was inversely proportional to PV changes.

Conclusion: SpHb provides an effective, novel, non-invasive indicator of vascular volume expansion. SpHb monitoring could aid in distinguishing acute blood loss from hemodilution. (Support: UTMB's CRC. Grants from ONR and Masimo).




Objective: We tested the hypothesis that intraluminal administration of high-molecular weight polyethylene glycol (PEG 15-20) attenuates trauma-hemorrhagic shock (T/HS)-induced gut injury.

Methods: Male SD rats underwent a laparotomy (trauma) and 30ml/kg ileal intraluminal injection of 5% PEG 15-20 or medium (sterile water) followed by shock (35mmHg × 90min). Trauma-sham shock (T/SS) animals underwent similar procedures but were not subjected to shock. At 3 hour post reperfusion, in vivo ileal permeability to fluorescein dextran (FD4), bacterial translocation to mesenteric lymph node (MLN) and histological ileal villus injury score were measured as markers of gut injury.

Results: Intraluminal administration of PEG 15-20 attenuates T/HS-induced gut injury as seen by a reduction in the percentage of ileal villi injured, permeability of ileum to FD4 and the incidence of bacterial translocation to MLN. There was no difference observed with either medium or PEG administration in T/SS animals.

Conclusion: Intraluminal administration of a mucin surrogate, high-molecular weight polyethylene glycol 15-20 attenuates T/HS-induced gut injury.



EXTENT OF LUNG INJURY IS PROPORTIONAL TO THE BASE DEFICIT DURING HEMORRHAGIC SHOCK.M. Wohlauer, E. Moore,* J. Eun, F. Wright, M. Fragoso, A. Morton, and A. Banerjee*. University of Colorado Denver, Aurora, CO 80045

Background: Fifteen years ago, our lab published the observation that base deficit is an early predictor of acute lung injury in the Surgical Intensive Care Unit. In a rat hemorrhagic shock model, the current method of controlled exsanguination to a predetermined MAP and length of time often results in varied and unpredictable degrees of lung injury.

Hypothesis: Measuring the base deficit will serve as a predictor of acute lung injury in a rat model.

Methods: Blood is removed from anesthetized rats to induce hemorrhagic shock (MAP 30 mmHg × 45 min). An ABG is taken at the end of shock to measure the base deficit. The rats are resuscitated with NS and one half of the shed blood over two hours, observed for one hour, and then sacrificed. Evans blue dye is injected and used as an index of lung permeability.

Results: Lung injury increases in proportion to the base deficit during hemorrhagic shock.

Conclusion: The base deficit at the end of shock is a predictor of acute lung injury, and can be a useful adjunct to standardize the depth of hemorrhagic shock in a rat model.



C-PEPTIDE EXERTS BENEFICIAL EFFECTS IN KIDNEY INJURY FOLLOWING HEMORRHAGIC SHOCK.G. Piraino,* G. Maltese,* T. LaMontagne,* P.W. Hake,* A. Denenberg,* M. O'Connor,* B. Zingarelli, and R. Chima. Geriatrics, Univ. Messina; Critical Care Med., Cincinnati Children's Hosp. Med. Center, Cincinnati, OH 45229

Multiple organ failure, including kidney failure, is a serious outcome of hemorrhagic shock. Recently, C-peptide has been shown to exert potent anti-inflammatory properties in sepsis and ischemia/reperfusion injury. Here, we investigated the effect of C-peptide on renal dysfunction following severe hemorrhage. Hemorrhagic shock was induced in male rats (3-4 months old) by withdrawing blood from the femoral artery to a mean arterial pressure of 50 mmHg. Animals were kept in shock for 3h at which time they were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, one group of animals received C-peptide (280 nm/kg intravenously) while another group received vehicle. Hemorrhagic shock resulted in significant rise in plasma levels of creatinine (1.37±0.31 mg/dl) in comparison with sham rats (0.34±0.06 mg/dl, p<0.05), suggesting kidney dysfunction. Hemorrhagic shock also induced kidney neutrophil infiltration as evaluated by myeloperoxidase (MPO) assay in vehicle-treated rats (3.06±0.17 versus sham value of 0.52±0.13 U/100 mg tissue, p<0.05). Treatment with C-peptide significantly (p<0.05) attenuated the rise in creatinine (0.70±0.09 mg/dl) and kidney MPO (1.13±0.25 U/100 mg tissue) when compared to vehicle group. At molecular analysis, kidney nuclear expression of c-Fos and DNA binding of the transcription activator protein-1 (AP-1) were increased in vehicle-treated hemorrhaged rats (2.71±0.23 versus sham 1.40±0.14 relative intensity, p<0.05). C-peptide treatment significantly attenuated AP-1 activity (2.06±0.19 relative intensity, p<0.05). Thus, our data suggest that C-peptide may exert renal protective effects through transcription modulation. (Supported by the Shock Society/Novo Nordisk Fellowship Grant and NIH R01 AG-0227990).


NITRIC OXIDE REGULATES AUTO-RESUSCITATION IN HEMORRHAGIC SHOCK.P. Van,* W. Holden,* N. Spoerke,* C. Sambasivan,* J. Differding,* J. Watters,* C. Phillips,* and M. Schreiber. Oregon Health & Science University, Portland, OR 97239

An increase in nitric oxide (NO) synthesis occurs in response to hemorrhagic shock (HS). A blockade of NO results in reversal of hypotension and an increased mean arterial pressure (MAP). Uncontrolled hemorrhage is associated with a spontaneous increase in MAP in animals and humans. We hypothesize that this "auto-resuscitation" is associated with changes in NO. Swine (n=16) were anesthetized, instrumented, and randomized to sham or hemorrhage (HEM) groups. Sham animals received a standard incision only. HEM animals underwent right groin incision and slash injury to the femoral vessels with ∼30% blood volume loss. MAP was monitored continuously and blood was drawn pre-injury and at 5, 10, 20, 30, 60, and 120 min post injury. Whole blood NO adducts were measured by chemiluminescence. MAP was similar in both groups at the end of the study. During hemorrhage (Table 1) a decrease in MAP corresponded with an increase in NO. During auto-resuscitation, an increasing MAP corresponded with a decreasing NO. Sham animals had a decreased MAP at 120 min (58.9 ± 1.62 to 54.6 ± 1.31, p < 0.01), but no significant change in NO (1.50 ± 0.17 to 1.65 ± 0.17, p = 0.06). In a swine model of HS, the auto-resuscitation phase corresponded with a significant decrease in concentrations of NO. Further study with NO blockade should be performed. (Supported by USAMRMC W81XWH-04-1-0104).

HEM group. MAP and whole blood arterial NO (WB-NOa). All values mean ± SEM


LOW LEVELS OF PLATELET MICROPARTICLES CORRELATE WITH POOR OUTCOMES IN SHOCK PATIENTS.N. Matijevic,* W. Y-W. Wang,* R. Kozar, E. Gonzales, and J.B. Holcomb. Center for Translational Injury Research, UTHSC-H, Houston, TX 77030

Background: Microparticles (MP) are small vesicles shed from damaged or activated cells. Circulating MPs are predominantly of platelet origin and have important role in hemostasis, inflammation and vascular function. They are also derived from other blood and vascular cells. Increased circulating MPs have been associated with better survival rates in septic shock patients.

Hypothesis: We hypothesized that elevated levels of circulating endothelial MPs (EMP) and low platelet MPs (PMP) are associated with poor outcomes, reflecting increased endothelial injury and impaired hemostatic response.

Methods: Study cohort consisted of 24 severely injured trauma patients (16 males, age 37 ± 4, ISS 32 ± 2) and 20 normal donors. Plasma samples were collected at admission and 7h after resuscitation. MPs were measured by flow cytometry, using a cell-lineage specific antibodies to detect PMP (CD61), EMP (CD146), leukocyte (LMP; CD45), and Annexin V-positive (AnnV+) MPs. Results were correlated with clinical outcomes.

Results: Both the MP levels and their relative phenotypic distribution were significantly different between normal (78.2% PMPs, 4.3% LMPs, 0.6% EMPs) and shock plasmas (56.2% PMPs, 16.4% LMPs and 21% EMPs). The PMP concentration was 4.5-fold lower (1417/μL vs. 6296/μL, p=0.02), and the EMP was 10-fold higher (529/μL vs. 48/μL, p=0.002) in shock patients compared to controls, and remained unchanged after the resuscitation. Non-survivors (n=7) had 45% lower total AnnV+ MP levels, and 62% lower PMP levels in comparison to survivors (n=17).

Conclusions: Low PMPs in the presence of elevated EMPs were associated with increased mortality after shock. A modified resuscitation protocol including more PLT transfusions might be beneficial.


PLATELETS ARE SUPERIOR TO PLASMA FOR HEMOSTATIC RESUSCITATION FROM DILUTIONAL COAGULOPATHY.E. Gonzalez,* E. Moore, C. Silliman, A. Banerjee, T. Berry,* and K. Land*. University of Colorado Health Science Center, Aurora, CO 80262

Objective: The acute coagulopathy of trauma is further exacerbated by hemodilution via crystalloid resuscitation. To offset hemodilution, replacement with plasma-rich components is utilized. By using rapid thrombelastography (rTEG) we assessed the capability of blood components to restore the hemostatic integrity of diluted whole blood (WB).

Methods: WB obtained from 10 donors was diluted with normal saline to a 20% hematocrit (Hct). Fresh frozen plasma (FFP), cryoprecipitate (CRY) and apheresis platelets (PLTs) alone or in combination, were then added in concentrations physiologically equivalent to transfusing 1, 2, or 4 units (U) of each. rTEG was initiated with tissue factor. The differences in TEG parameters in response to the addition of FFP, CRY, and PLTs were detected by paired ANOVA.

Results: Dilution to a 20% Hct prolonged ACT by 12%, and decreased angle, MA and G by 19%, 24% (p=0.02) and 48% (p=0.01), respectively. PLTs and CRY were superior to FFP in restoring angle when added as 2 or 4U. PLTs were superior to CRY and FFP in restoring MA and G when added as 1, 2 or 4U. Combinations of FFP:CRY: PLTs (1:1:1) and CRY:PLTs (1:1) were superior to individual products. There was no difference between FFP:CRY:PLTs and CRY:PLTs (Table). Classic kaolin-TEG was run in duplicate and produced analogous data.

Conclusions: PLTs are more effective and require fewer units than FFP to restore hemostasis.



PHYSIOLOGIC PARAMETERS CORRELATE TO METABOLOMIC MARKERS IN A PORCINE MODEL OF HEMORRHAGIC SHOCK.D. Lexcen,* J. Greenberg,* E. Lusczek,* N. Witowski,* K. Mulier,* and G. Beilman. University of Minnesota, Minneapolis, MN 55454

Introduction: Metabolomics is a new field of study that allows simultaneous evaluation of multiple metabolites in a given sample. In our porcine model of hemorrhagic shock and resuscitation, nuclear magnetic resonance (NMR) is used to identify and analyze the flux of metabolites in a biofluid. Currently, clinical decisions are made using available measures of a physiologic and biochemical variables. Our hypothesis is that we can correlate known prognostic indicators with identified metabolic biomarkers.

Methods: Animals (n=18) were hemorrhaged via IVC cannula, then resuscitated after a 45 min period of shock. Serum samples and physiologic/biochemical measurements were taken at specific time points. 1H-NMR spectroscopy was used to identify and quantify the concentrations of metabolites using Chenomx software. The resulting metabolite profiles were analyzed using R software. Spearman correlation coefficients were calculated between serum metabolites and physiologic variables.

Results: Metabolomic variables serine and lactate were highly correlated to lactate, heart rate, oxygen consumption, base excess, oxygen saturation and urine output in either a positive or negative manner. The amino acids (glutamine, arginine, histidine, phenylalanine,) all shared strong positive correlations with oxygen saturation (StO2) and urine output. Additionally, those same amino acids had a negative correlation with heart rate, oxygen consumption, and temperature.

Conclusion: In this study, we observed a correlation between known physiologic markers of severity of injury and metabolic variables in serum in animals undergoing shock and resuscitation. These findings suggest that metabolomics may be a useful tool to elucidate underlying biochemical pathways and to identify functional biomarkers in this process.


HYPERTONIC RESUSCITATION DIFFERENTIALlY MODULATES SOLUBLE ADHESION MOLECULES IN SHOCK PATIENTS.S.G. Rhind, S.B. Rizoli,* W.G. Junger, J. Cuschieri, D.B. Hoyt, A.J. Baker,* P.N. Shek,* and E.M. Bulger. Defence Research & Development Canada, and University of Toronto, Toronto, ON M3M 3B9, Canada

Background: Elevated cellular adhesion molecules (CAMs) play a central role in trauma-induced tissue injury and organ damage.

Objective: Evaluate the effects of initial resuscitation with hypertonic saline alone or with dextran on soluble vascular and intercellular adhesion molecule-1 (sVCAM-1, sICAM-1) and E-selectin.

Population: Thirty-two trauma patients in hypovolemic shock (systolic BP≤70 mmHg or 70-90mmHg plus heart rate>108) and 10 healthy controls (CT).

Interventions: Prehospital treatment with 250mL of 7.5% hypertonic saline (HS; n=9), 7.5% HS 6% dextran-70 (HSD; n=7) or 0.9% normal saline (NS; n=12).

Methods: Plasma concentrations (mean±SEM, ng/mL) of sVCAM-1, sICAM-1 and sE-selectin were measured by ELISA, in samples drawn at admission (ED Adm), 12 and 24-h post-treatment.

Results: Admission concentrations of sCAMs were not different between intervention groups and controls. Levels rose over time in both HSD and NS arms, with significant (P <.05) increases in sICAM-1 (272±35; 239±23), sVCAM-1 (1312±154; 1060±142) and E-selectin (43±6; 47±5) observed at 24-h. However, in HS-treated patients sCAM values remained similar to controls.

Conclusions: This study demonstrates that relative to NS or HSD, HS blunts the posttraumatic elevation in sCAM markers of endothelial activation/damage in hemorrhagic shock patients, suggesting differential immunomodulatory capacities of HS vs. HSD fluids. (ROC substudy supported by NIH R01-2007-000-20819-0; Defence R&D Canada).




Acute alcohol intoxication (AAI) aggravates hypotension and blunts neuroendocrine activation during hemorrhagic shock (HS) and impairs the pressor response to fluid resuscitation (FR) with lactated Ringer's (LR). Sympathetic outflow activation via acetylcholinesterase (ACEse) inhibitor administration improves the pressor response to FR with LR and decreases morbidity and mortality from AAI+HS. However, delayed FR with LR+ACEse resulted in greater end organ failure (EOF). We hypothesized that alternative FR with hypertonic saline (HTS) would improve blood pressure recovery and decrease EOF in AAI. Male Sprague Dawley rats received a primed intragastric alcohol infusion (2.5g/kg +.3 g/kg/h × 15h) or isovolumic dextrose (DEX) followed by fixed pressure HS (40mmHg for 60 min). Animals were then resuscitated with HTS (7.5%; 4ml/kg over 10 min) or LR (2.4× total blood removed over 60 min). Early MABP recovery was enhanced by HTS resuscitation in AAI (109±6 vs 80±5 mmHg) and DEX (114±6 vs 83±5 mmHg) compared to LR at 12 min post-HS. However, values were similar between all groups at the end of FR (60 min post-HS). FR with HTS attenuated the decrease in hematocrit associated with LR in AAI (33±2 vs 25±0.8%) and DEX (34±0.8 vs 26±0.9%). ALT was unaltered in DEX+HS regardless of FR utilized. AAI+HS produced a ∼4-fold increase in ALT following LR which was prevented by HTS. In contrast, the initial increase in BUN in AAI- and DEX+HS was not altered by HTS. BUN levels 3h post-HS were elevated by HTS in both AAI (237%) and DEX (245%) compared to LR. These results show improved hemodynamic recovery during FR with HTS in AAI+HS and improved hepatic outcome, but suggest decreased renal perfusion. The mechanisms underlying this differential organ response are currently under investigation. (DOD-PR-054196, BoR-110350057A, NIAAA-AA7577).


BIOFLUID ANALYSIS OF HEMORRHAGIC SHOCK USING METABOLOMICS.E. Lusczek,* D. Lexcen,* N. Witowski,* J. Greenberg,* K. Mulier,* J. Chipman, and G. Beilman. University of Minnesota, Minneapolis MN

Introduction: Metabolomics is a new field of study that profiles the entire repertoire of small molecules (metabolites) in a biological sample. Our laboratory has an established porcine model of hemorrhagic shock and resuscitation. Our goal was to correlate metabolomic findings in urine and serum during hemorrhagic shock and resuscitation.

Methods: After sedation and intubation, 13 pigs were prepared for protocol with splenectomy, placement of arterial line, pulmonary artery catheter, cystostomy, and inferior vena cava (IVC) cannulation. After stabilization, hemorrhagic shock was induced by withdrawing blood thru the IVC cannula to a goal systolic blood pressure of the mid 50s. After 1 hour of shock, 8 pigs received standardized resuscitation by algorithm. Five pigs were not resuscitated. Serum and urine were collected at fixed timepoints throughout. Samples were flash frozen and stored at −80°C until prepared for analysis by nuclear magnetic resonance spectroscopy (NMR). The NMR spectra were obtained with a Bruker Avance 700 MHz spectrometer and were analyzed with Chenomx software to identify and quantify the concentrations of metabolites present in both biofluids. The resulting metabolite profiles were analyzed using R software. Spearman correlation coefficients were calculated between metabolites in urine and serum.

Results: Heatmaps of correlations between urine and serum highlight metabolic shifts due to hemorrhagic shock. Positive correlations between the urinary and serum metabolites suggest that the urea cycle and ammonia recycling pathways are activated, and also highlight metabolites that are known to be associated with kidney injury.

Conclusions: Correlations between metabolites tracked in the serum and urine of pigs in a hemorrhagic shock model highlight metabolites that are known to be indicative of renal injury.


THERAPEUTIC HYPOTHERMIA CARDIOPROTECTION IN MURINE HEMORRHAGIC SHOCK/RESUSCITATION DIFFERENTIALLY AFFECTS P38α/P38γ, AKT AND HSP25.J. Li,* D. Beiser,* H. Wang,* A. Das,* E. Berdyshev,* S. Stern,* and T. Vanden Hoek. University of Chicago, Chicago, IL 60637

Objective: To characterize cardiac Akt, p38 and Hsp25 signaling regulated by therapeutic hypothermia (TH) and their association with improved cardiac performance and short term survival in a Wigger's prep mouse model of hemorrhagic shock (HS).

Methods: TH (33 ± 0.5 °C) was induced at 30 min HS and maintained until 60 min resuscitation via surface cooling. Heart tissue collected during both HS and after resuscitation was analyzed for p38, Akt and Hsp25 phosphorylation (p-p38, p-Akt and p-Hsp25) and expression. Akt-Hsp25 interaction, plasma cytochrome c, myocardial myeloperoxidase activity and TUNEL staining were analyzed.

Results: During HS onset, myocardial p-p38 and p-Hsp25 increased within 5 min, with p-p38 peaking over 10-fold by 30 min HS. By contrast, myocardial p-Akt demonstrated a reciprocal decrease and increased significantly in the late HS. TH decreased p-p38α and upregulated p-p38γ, inducing a p38 isoform switch. After resuscitation, TH increased p-Akt and total Hsp25 expression, and enhanced Akt-Hsp25 interaction as measured by co-immunoprecipitation. TH attenuated markers of mitochondrial injury, heart apoptosis and inflammation by 180 min resuscitation (R180), with decreased plasma cytochrome c concentrations (3.11 ± 0.56 and vs. 1.48 ± 0.02 μg/ml, p < 0.05), and myocardial MPO and TUNEL staining. Further, TH significantly increased R180 survival (88.5% vs. 50%, p = 0.001).

Conclusion: Cardiac p-Akt changes inversely to p-p38α during hemorrhagic shock and resuscitation. TH increases p-Akt, p-p38γ, Hsp25, cardiac function, and decreases indices of cardiac apoptosis and inflammation. Targeted Akt and p38 isoform specific treatments that enhance Akt-Hsp25 interaction may play a future role as adjuncts for TH treatment during HS.



Acute alcohol intoxication (AAI) impairs the neuroendocrine response to hemorrhagic shock (HS) and the restoration of MABP following fluid resuscitation (FR) with lactated Ringer's (LR). Central cholinergic stimulation restores the arginine vasopressin (AVP) response and improves MABP recovery in AAI+HS; suggesting that AAI-mediated disruption of central mechanisms controlling AVP release play an essential role in AAI-induced hemodynamic dysregulation in HS. In contrast, AAI does not impair the AVP response to a hyperosmotic challenge. We hypothesize that AAI-induced dysregulation of stimulatory angiotensin II (ANG II) and inhibitory nitric oxide (NO) mechanisms contribute to the blunted AVP response to HS; thus, FR with hypertonic saline (HTS) should restore AVP response and improve MABP recovery in AAI+HS. Male Sprague Dawley rats received a primed-constant 15h intra-gastric infusion of alcohol (2.5g/kg +.3g/kg/h) or isovolumic dextrose (DEX) followed by a fixed-pressure (40mmHg) HS followed by FR with HTS (7.5%; 4ml/kg over 10 min) or LR (2.4× total blood volume removed over 60 min). FR with HTS enhanced MABP recovery in AAI (109±6 vs 80±5 mmHg in LR) and DEX (114±6 vs 83±5 mmHg in LR) immediately post-FR (12 min). MABP was similar in HTS and LR groups at completion of 60 min FR. AVP levels were higher (∼66%) in HTS than in LR in both AAI and DEX animals at 3h post-HS. PVN NO levels in both AAI- and DEX-HS were ∼60% lower in FR with HTS than time-matched LR controls. AAI blunted the HS-induced ANG II response and this was partially and transiently restored by FR with HTS. These results suggest that restoration of AVP response following AAI+HS with HTS is predominantly the result of removing central NO inhibitory tone. (DOD-PR-054196, BoR-110350057A, NIAAA-AA7577).


HEMORRHAGIC SHOCK IN RATS MEDIATES EARLY AND PERSISTENT ER STRESS IN LIVER, BUT NOT IN KIDNEY.J. C. Duvigneau,1 I. Chaudry,3 A. Müllebner,1* C. Zifko,2* T. C. Hyatt,3 H. Redl,2 S. Bahrami,2 and A. V. Kozlov2. 1Univ. of Vet. Med., 1210 Vienna, 2Ludwig Boltzmann Inst. of Exp. & Clin. Traumatol., 1200 Vienna, Austria, 3Univ. of Alabama at Birmingham, Birmingham, AL 35294-0019

Objective: In rodent models we and others have previously shown that trauma-hemorrhagic shock (T-H) followed by resuscitation leads to endoplasmic reticulum (ER) stress in the liver - a possible mechanism underlying delayed organ dysfunction. To elucidate the role of ER stress in another organ susceptible to T-H, we followed ER stress markers in the kidney in comparison to the liver.

Methods: ER stress markers (alternative splicing of X-box protein (XBP1) mRNA, upregulation of C/EBP homologous protein (CHOP) mRNA and 78kDa glucose regulated protein (GPR78) mRNA levels) were investigated by real-time PCR in liver and kidney samples from rats subjected to different T-H models. Model 1 consisted of hemorrhage (40 mm Hg) until 60% of blood was removed (45′), followed by prolonged hypotension (2h). Model 2 consisted of hemorrhage (MAP of 35-40 mm Hg), a hypotensive phase until reaching decompensation (40-45′), followed by a low volume (45′), and a subsequent adequate fluid resuscitation (1h). Animals were sacrificed directly after T-H, during resuscitation, or after resuscitation (at 3h, or 18h after T-H).

Results: Splicing of XBP1 mRNA and upregulation of CHOP mRNA were significantly increased after the hypotensive phase in the liver, but not in the kidney. Both markers remained increased during reperfusion, and additionally GPR78 mRNA rose following T-H in the liver. All markers stayed elevated (until 18h after T-H) only in the liver.

Conclusion: T-H mediates early and persistent ER stress predominantly in the liver. Although the significance of ER stress mediating organ failure has to be elucidated, our data suggest that ER dysfunction may be an underlying mechanism for hepatic, but not for renal failure following T-H.



In several HS models, small volume resuscitation with HBOC reduces mortality as compared to standard crystalloids. HBOC infusion however, is associated with marked increases in systemic (SVR) and pulmonary vascular resistances limiting their clinical utility. The proposed mechanism for this effect is nitric oxide (NO) scavenging.

Hypothesis: In a model of combined TBI/HS, concomitant NTG infusion, as an NO donor, will attenuate the vasoactive effects of HBOC infusion.

Methods: 29 swine (27.5[2.4]kg) were subjected to 3.4(0.4) atm fluid-percussion TBI and bled to and maintained at a MAP of 30mmHg for 15 min (T=S15/R0) in the presence of an aortic tear. At T=S15/R0, animals were randomized to one of 4 groups: lactated Ringers (N=7; LR); HBOC (N=8); HBOC+NTG 2.5μg/kg/min (N=8; NTG-2.5); HBOC+NTG 5μg/kg/min (N=6; NTG-5). Fluids were infused to maintain MAP=60mmHg (T=R0-R105; pre-hospital). At 105 min (T=R105; in-hospital), the aortic tear was repaired and all animals were resuscitated with 0.9% NaCl and shed blood to normalize physiologic parameters. Animals were observed for 360 min (T=R360).

Results: % 6-hr survival were 71, 0, 38, and 33 (p=0.007; Fisher exact), while survival times were 318(90), 98(110), 179(152), and 165(135) min (p=0.0205; ANOVA) for LR, HBOC, NTG-2.5, and NTG-5 respectively. Hemorrhage volumes were higher in HBOC groups and were 23(7), 42(12), 41(11), and 30(10) mL/kg (p=0.003; ANOVA) for LR, HBOC, NTG-2.5, and NTG-5. PAP and SVR were greater and CO lower in all HBOC groups vs LR (p<0.0001; rmANOVA).

Conclusion: In this HS/TBI model, initially limited resuscitation with HBOC resulted in decreased survival and greater hemorrhage volume as compared to LR. NTG failed to blunt the HBOC associated vasoactive response.


PLASMA PROTEIN CONCENTRATIONS CHANGE DURING STORAGE AFTER THAWING OF FRESH FROZEN PLASMA.B. Gill,* W. Dubinsky,* E. Golunski,* J. Conyers,* and J. Holcomb. University of Texas Health Science Center, Houston, TX 77030

Background: Fresh frozen plasma (FFP) after thawing is commonly stored for up to five days before transfusion. The changes that occur in the plasma milieu during storage are poorly characterized. We investigated the changes that occur in plasma protein concentrations in five-day-old FFP when compared to FFP that has recently been thawed.

Methods: Pooled human plasma (5 donors) was sampled immediately after thawing (Day 0) and again after storage for 120 hours at 4°C (Day 5). Proteins were identified and changes in relative concentration quantified by tandem mass spectrometry (MS). Signal-to-noise ratio was improved by depletion of abundant plasma proteins via filtration on immunoaffinity HPLC columns.

Results: More than 250 proteins were identified by MS, the vast majority of which showed no significant change from Day 0 to Day 5. However, significant changes were seen in several proteins. In particular, fibrinogen beta chain (−27% in Day 5 vs. Day 0, p<0.02), serotransferrin (−10%, p=0.05), hemopexin (−22%, p<0.01), fibronectin (−22%, p<0.03), ApoE (−27%, p<0.01), laminin (−33%, p<0.01) and succinyl co-A ligase (−44%, p< 0.01) are all reduced in concentration at Day 5. Significant changes are also seen in several complement protein, glycoprotein, and immunoglobulin levels.

Conclusion: Significant changes occur in the protein constituents of thawed plasma during storage over 120 hours. These changes may result in differing biological activity of older plasma versus recently thawed plasma.


HEMOPERITONEUM ELEVATES CARBON MONOXIDE EXCRETION.S. Nicholson,* R. Johnson, T. Craig,* F. Johnson, J. Myers,* and R. Stewart*. U. Texas Health Science Center at San Antonio, San Antonio, TX 78229

Hemorrhage into body cavities is a common complication in blunt trauma. Since hemoglobin degradation liberates free heme which is degraded by heme oxygenase to form carbon monoxide (CO), we wanted to determine if such a change can be detected by measuring total CO excretion using solid phase gas chromatography.

Objective: Determine if experimentally generated hemoperitoneum can be detected and accurately quantified via non-invasive measurements of whole animal CO excretion.

Methods: Male inbred Lewis rats (250-275g) were divided into two experimental sets. Rats in the first experimental set received femoral catheters and then were allowed to stabilize for 3 days. Next, animals were hemorrhaged 30% of their calculated blood volume followed by intraperitoneal (IP) injections of fresh whole blood, or the fresh red blood cells or plasma fractions alone. In a second phase, additional non-hemorrhaged animals received IP injections of the fresh whole blood, red blood cells or plasma. Whole animal CO excretion was measured using solid-phase gas chromatography starting 3 days prior to the IP injections until 7 days after the treatments.

Results: In hemorrhaged rats receiving IP injections, whole blood and RBCs increased CO excretion whereas plasma did not (whole blood: 0.73 μmol/kg per hr vs. RBC: 0.72 μmol/kg per hr vs. plasma: 0.55 μmol/kg per hr). Elevated CO levels persisted for 4 days following hemorrhage and IP injection. Similarly, CO production was increased in rats without hemorrhage receiving IP whole blood (0.63 μmol/kg per hr) and RBCs (0.63 μmol/kg per hr) compared to IP plasma (0.51 μmol/kg per hr).

Conclusions: Hemoperitoneum increases endogenous CO excretion in inbred Lewis rats. The total CO excretion is equimolar to the heme in the blood that is injected. As such, the findings validate the use of total CO excretion as a means to quantify internal bleeding.


REGULATION OF PROTEIN PHOSPHATASE 2B ACTIVITY AND THE EXOCYTOSIS OF VON WILLEBRAND FACTOR IN HEMORRHAGIC SHOCK.N. Alrehani,* N. Matijevic, M.F. Doursout, J.F. Dong,* M.A. Cruz,* J.B. Holcomb, and K.V. Vijayan*. Baylor College of Medicine, Houston, TX 77030

Trauma induced hemorrhagic shock (HS) can generate oxidants like superoxide via increased activity of xanthine oxidase (XO). Superoxide can trigger rapid exocytosis of endothelial Weibel Palade body contents like P-selectin and von Willebrand factor (VWF), and also inhibit protein phosphatase 2B activity in an in vitro endothelial cell culture. Although, cellular protein phosphorylation and dephosphorylation is altered in HS, a role for phosphatases in VWF exocytosis during HS is unknown. VWF is secreted from the WPB as ultra long VWF (ULVWF) multimers and subsequently cleaved by plasma metalloprotease ADAMTS-13 to generate smaller hemostatically active VWF multimers. We have previously shown that the inhibition of protein phosphatase 2B (PP2B) activity triggers ULVWF exocytosis in endothelial cells by modulating the phosphorylation of vesicular trafficking proteins. Here, we investigated the correlation between XO activity, VWF secretion and PP2B activity in a HS rodent model. HS was induced in Sprague-Dawley rats by withdrawing blood and maintaining the mean arterial blood pressure to 35mm Hg for 1 hour. VWF antigen levels were assayed from HS plasma. PP2B and XO activity was analyzed from lung since this organ is frequently associated with post-injury multi-organ failure after HS. Compared to the sham rats, HS induced rats displayed ∼75% increased XO activity (p=0.05) in lung tissues. More importantly, HS rats showed inhibition (p=0.02) of PP2B activity along with an increase in VWF antigen level. These studies imply that the oxidant stress induced signaling generated in HS could potentially regulate PP2B activity and thereby in part control the systemic exocytosis of VWF.


STANDARD STORAGE OF FRESH FROZEN PLASMA (FFP) REDUCES ITS ANTI-APOPTOTIC EFFECTS DURING HEMORRHAGIC SHOCK (HS) RESUSCITATION.X. Deng,* Y. Cao,* M.-F. Doursout,* G. He,* R.A. Kozar, J.B. Holcomb, and T.C. Ko*. University of Texas Health Science Center, Houston, TX 77030

Objective: Recent clinical studies have shown that resuscitation with FFP, platelets and red blood cells is associated with improved outcome after severe HS. Under current clinical guidelines, FFP is either used immediately after thawing (Day 0) or given after storage at 4°C for up to five days (Day 5). Previously, we have reported that standard storage of FFP alters its growth factor composition (Shock 31(S1):56, 2009). The purpose of this study was to investigate the effects of FFP storage on reducing HS-induced endothelial cell (EC) apoptosis.

Methods: A rat HS model was used to study vascular apoptosis in vivo using a VasoTACS in situ Apoptosis Detection kit (R&D Systems). In vitro, human pulmonary microvascular ECs (HPMECs) were treated with FFP under hypoxia (72 h) or with FFP for 30 min followed by Staurosporine (2 h) treatment and analyzed using Caspase-Glo 3/7 Assay kit (Promega) and immunoblotting.

Results: Apoptosis was detected in the vasculature of the kidney, gut and liver of unresuscitated HS rats, and was significantly reduced in Day 0 FFP-resuscitated HS rats, but not in Day 5 FFP- or lactated Ringer's-resuscitated rats. Day 0 FFP was then used in vitro to examine FFP's effects on survival signaling. FFP inhibited hypoxia- and Staurosporine-induced Caspase 3/7 activation in HPMECs. FFP induced activation-phosphorylation of Akt1, a major survival signal, at Ser473. Akt1 is known to induce BAD phosphorylation and its binding to 14-3-3 leading to cell survival. We found that FFP dose-dependently increased BAD phosphorylation at Ser136 and BAD binding to 14-3-3 proteins.

Conclusions: Standard storage of FFP reduces its inhibition of HS-induced EC apoptosis. FFP activates the survival signaling of Akt1 and BAD suggesting that this pathway mediates the anti-apoptotic effects of FFP.


PLASMA ADIPONECTIN LEVELS DECREASE AFTER HEMORRHAGIC SHOCK (HS).C. Duan,* Y. Cao,* X. Deng,* W. Yang,* R.A. Kozar, E.A. Gonzalez, J.B. Holcomb, and T.C. Ko*. University of Texas Health Science Center, Houston, TX 77030

Objective: Recent clinical studies have shown that resuscitation with FFP is associated with improved survival after HS. Conceptually, the beneficial effects of FFP in HS have been attributed to its volume-expansion and hemostatic properties by replacing lost coagulation factors. However, we postulated that FFP also has direct beneficial effects on endothelial cells (ECs). We have shown that FFP induces activation of AMPKα1 in ECs (Shock 31(S1):34, 2009). AMPKα1 plays an important role in maintaining vascular structure and function. Plasma contains a plethora of proteins including adiponectin, a key activator of AMPK. However, the effects of HS and FFP-based resuscitation on plasma adiponectin levels are unknown. The purpose of this study is to examine plasma adiponectin levels in HS patients before and after FFP-based resuscitation.

Methods: 19 HS patient plasma samples before and after initial ED resuscitation were analyzed for adiponectin by ELISA compared to 9 plasma samples from healthy donors.

Results: Plasma adiponectin levels were significantly lower in HS patients before resuscitation compared to healthy donors (3.78±0.50 versus 7.82±1.64 μg/ml, p<0.05). Plasma adiponectin levels increased after FFP-based resuscitation (4.55±0.46 versus 3.78±0.50 μg/ml, p=0.19) although this did not reach statistical significance.

Conclusions: This is the first demonstration of a decrease in plasma adiponectin levels after HS. Furthermore, our data suggest that FFP-based resuscitation partially restores plasma adiponectin levels which may restore AMPKα1 signaling, maintain vascular structure and function, and improve survival.


HYPERTONIC RESUSCITATION OF SHOCK PATIENTS DOWNREGULATES NEUTROPHIL ACTIVATION.W.G. Junger, S.G. Rhind, S.B. Rizoli,* J. Cuschieri, D.B. Hoyt, A.J. Baker,* P.N. Shek,* and E.M. Bulger. Dept. of Surgery, BIDMC, Harvard Medical School, Boston, MA 02215

Background: Hypertonic saline may attenuate neutrophil (PMN) activation that is correlated with systemic inflammation and posttraumatic complications.

Objective: Evaluate if initial resuscitation of shock patients with hypertonic saline alone or with dextran affects PMN activation.

Population: 28 trauma patients in hypovolemic shock (systolic BP≤70 mmHg or 70-90 mmHg plus heart rate>108) and 10 healthy controls (CT).

Interventions: Prehospital treatment with 250mL of 7.5% hypertonic saline (HS; n=9), 7.5% HS plus 6% dextran-70 (HSD; n=7) or 0.9% normal saline (NS; n=12).

Methods: Multi-color flow cytometry was used to quantify PMN expression [mean fluorescence intensity (MFI) ± SEM] of cell-surface adhesion (CD11b, CD62L) and degranulation (CD63, CD66b, CD35) markers, assessed at admission (ED Adm), 12 and 24 h later.

Results: HS and HSD, but not NS significantly (p <.05) blunted initial increases in the intensity of CD11b and CD35 expression. HS reduced CD66b expression at ED Adm. In the HSD group, CD62L dropped below CT values after admission; CD63 expression did not differ among groups.

Conclusions: Hypertonic resuscitation diminishes PMN activation markers and may reduce initial PMN activation after trauma, ameliorating host tissue injury. (ROC substudy NIH R01-2007-000-20819-0; Defence R&D Canada).



GENDER DIFFERENCE IN CARDIAC TRANSCRIPTION FACTORS: NF-κB AND NRF2 FOLLOWING TRAUMA-HEMORHAGE (T).S. Hu,* S. Yang,* R. Raju,* K.I. Bland,* and I.H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294

Although T-induced cardiac depression is mediated via upregulation of cardiac NF-κB in male rats, and estrogen (E2) administration after T restored cardiac function and prevented the increase in cardiac NF-κB, it remains unknown whether gender has any effect on cardiac transcription factors NF-κB and Nrf2. T was induced in male: sham, sham-E2 (SE), T-cyclodextrin (CD. TC), T-E2 (TE), and T-ICI 182,780 (ICI)-E2 (TIE); and female rats: proestrus sham (PS), proestrus T (PT), ovariectomized (OVX)-sham (OS), OVX-T (OT), and OVX-E2-T (OET). E2, 1 mg/kg, iv was administered at the onset of resuscitation in males or 30 min before inducing T in OVX; ICI 3 mg/kg, IP, 30 min before E2 administration. Two hr after resuscitation, T induced a decrease in LV performance, cardiac Nrf2, estrogen receptor (ER) -α and -β in males and OVX females. In contrast, T induced a significant increase in cardiac NF-κB in males and OVX. However, LV performance, cardiac NF-κB, Nrf2, and ER were normalized/enhanced in E2-treated males, PE, and E2-pretreated OVX. ICI abolished salutary effects of E2 on above parameters in males. Moreover, plasma E2 levels were significantly higher in PE, E2-treated OVX and males. Thus, gender difference exists in cardiac function and transcription factors, i.e., T not only induced upregulation in cardiac NF-κB, but also downregulated Nfr2, which were prevented in PE and E2-pretreated OVX females, and E2-treated males (NIH R01 GM 39519).



AUTOPHAGY FOLLOWING TRAUMA-HEMORRHAGE (T-H).F. Moeinpour,* M. Athar,* K.I. Bland,* and I.H. Chaudry. Center for Surgical Research, Dept. of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294

Autophagy is an evolutionarily conserved catabolic pathway for turnover of damaged proteins and organelles. While physiological level of autophagy might be protective, high levels of autophagy caused by stress conditions may cause organ self digestion and cell death. Our aim was to determine whether T-H produces any alterations in autophagy. Male mice underwent T-H (removal of 60% circulating blood volume; mean blood pressure decreased from 100 to 35 mmHg for 90 min) followed by fluid resuscitation. Two hrs thereafter, liver samples were fixed and embedded in paraffin, sectioned and stained for immunoflouresence detection of Beclin-1 and Atg4. In another group of mice, liver tissues were prepared for electron microscopy examination of vacuole formation. Our preliminary results show that Beclin-1 and Atg4, the critical genes of mammalian autophagy, were upregulated in T-H mice compared to controls. Furthermore, electron microscopy shows that vacuolization in liver was significantly increased after T-H compared to shams. Although the exact role of autophagy after T-H remains unknown, our results show that T-H induces vacuolization and upregulation of autophagy genes in the liver (Supported by NIH R01 GM37127).



ESTROGEN (E2)-DERIVED INCREASE OF CARDIACO-GlcNAc FOLLOWING TRAUMA-HEMORRHAGE (T-H) IS VIA ENHANCEMENT OF CARDIAC NUCLEARO-GlcNAc TRANSFERASE (OGT) PROTEIN LEVELS.S. Yang, S. Hu,* R. Raju,* K.I. Bland,* and I.H. Chaudry. Center for Surgical Research & Department of Surgery, Univ. of Alabama at Birmingham, Birmingham, AL 35294

We have shown that one of the mechanisms of 17β-estradiol (E2)-mediated cardioprotection after T-H is through E2-derived increase of cardiac O-GlcNAc. To further explore the mechanism of E2-derived upregulation of cardiac O-GlcNAc, T-H was induced in male adult rats and the rats received E2 (1 mg/kg, IV) or vehicle (cyclodextrin, CD, 20.7 mg/kg, IV) during resuscitation. In an additional E2-treated T-H group, ER antagonist ICI 182,780 (ICI, 3 mg/kg, IP) or OGT inhibitor alloxan (ALX, 25 mg/kg, IV) was administered 30 min before E2. Two hr after resuscitation, LV performance was determined, heart tissue and plasma harvested, followed by cardiac nuclei extraction. Our results show that T-H induced decrease in LV performance, cardiac O-GlcNAc, and cardiac nuclear but not cytosol OGT protein; however, E2 restored all those parameters. Moreover, T-H-induced decrease of cardiac ER expression was normalized by E2. Both ICI and ALX abrogated the salutary effects of E2 on the above parameters. Thus, E2 cardioprotection following T-H is due to upregulation of cardiac O-GlcNAc, which is via enhanced cardiac nuclear OGT (NIH R01 GM 39519).



TRANSFUSION-INDUCED IMMUNOSUPPRESSION VARIES WITH BLOOD AGE.P. Efron, D. Nacionales,* A. Cuenca,* K. Kelly-Scumpia,* M. Delano,* D. Ang, and L. Moldawer. Department of Surgery, University of Florida College of Medicine, Gainesville, FL 32610

Introduction: Packed red blood cell (PRBC) transfusion is associated with increased infections and mortality in the critically ill. Although still debated, some studies indicate longer PRBC storage is associated with these outcomes. We wished to determine how PRBC age affected murine immunosuppressive phenotypes following transfusion.

Methods: C57Bl/6 mice (3-6/group) underwent a 100 μL hemorrhage (15% TBV) followed by a 200 μL retro-orbital infusion of PBS or allogeneic Balb/c mouse PRBC stored for 1, 7 or 21 days. PRBC were created by centrifuging and refrigerating murine whole blood in preservative solutions (Adsol™ and CPD). Spleens and blood were harvested 3 days post transfusion and phenotypes determined by flow cytometry.

Results: Similar to our previous reports, 1 day old (DO) blood induced a loss of CD8+ T-effector cells in the spleen. However, only 7 DO blood increased the splenic CD4+CD25+ T regulatory population (PBS 1.1 ± 0.2% vs. PRBC 1.5 ± 0.2%, p=0.03). Interestingly, 21 DO blood significantly decreased the percentage of circulating dendritic cells (PBS 0.8 ± 0.1% vs. PRBC 0.4 ± 0.1%, p=0.02).

Conclusions: Transfusion of allogeneic PRBC induces leukocyte alterations associated with immune suppression in the recipient's circulation and lymphoid tissue. These specific immunosuppressive phenotypes, such as the loss of effector cells (CD8+ T cells and dendritic cells) or the generation of immune suppressive cells (T regulatory cells), appear to depend on the age of the transfused PRBC. These varying WBC alterations may explain transfusion-associated morbidity as well the complexity that will be encountered in resolving PRBC age-related affects on the immune system.


Genetic Determinants of Resting Serum C3a and C5a Levels in Humans.C. Nypaver, and J. Younger*. U Michigan, Ann Arbor MI 48109

Complement C3a and C5a are potent inflammatory mediators that contribute to injury in sepsis, trauma, and reperfusion, and C3a and C5a levels have been used as markers of illness severity in animals and humans. We examined the link between 50 known single nucleotide polymorphisms (SNPs) and resting C3a and C5a levels in healthy humans. Subjects were genotyped using a mass spectroscopy based system (Sequenom) and C3a/C5a levels were measured using ELISA. 80 patients were studied. Relationships between genotype and C3a/C5a level were modeled as linear allele dose responses (e.g., aa = 0, aA = 1, AA = 2 doses). Median response, and 2.5% and 97.5 percentile ranges for those estimates, were generated with bootstrapping. Explanatory strengths of relationships (as R2) were handled in a similar fashion. Several SNPs were found to be tied to resting serum levels, and one was shared by both C3a and C5a (Factor H, rs 1061147) This SNP is a known risk for macular degeneration, but has not been described in systemic disease. These are the first descriptions of genetic influence on C3a and C5a in serum, and lay groundwork for studies in septic patients.



EARLY ESTRADIOL-DHEA ADMINISTRATION PREVENTS NEURONAL DAMAGE IN MALE RATS AFTER CARDIAC ARREST.I.V. Ostrova, M.Sh. Avrushchenko, and A.V. Volkov. Institute of General Reanimatology n.a. V.A. Negovsky, Russian Academy of Medical Sciences, Moscow, 107031, Russia

It is known that cerebral ischemia outcome is influenced by biological sex and endogenous sex steroids. Estradiol and its precursor, dehydroepiandrosterone (DHEA), exert marked neuroprotective activity in the central nervous system. The aim of this study was to appreciate effect of estradiol with DHEA (Gynodian-Depot, Schering, Germany) on hypoxia-sensitive neuronal populations of the brain in male and female rats after ischemia/reperfusion. Cardiac arrest of 12 min duration in ether-anaesthetized white rats was evoked by intrathoracic clamping of the cardiac vascular bundle. Animals (n=12) were treated with intramuscular injections of estradiol (1mg/kg) with DHEA (50mg/kg) on the 30th minute after resuscitation. Untreated rats (n=12) received equivalent volumes of normal saline. Control group included intact rats (n=12). On the 14th day of post-resuscitation period the rats were sacrificed, the brains were removed and fixed. The total density and composition of pyramidal neurons in fields CA1 and CA4 of the hippocampus, in layer V of the sensorimotor cortex and Purkinje cells from the lateral region of the cerebellum were determined by morphometric analysis. In resuscitated males loss of neurons, viz., a decrease in total density of neuronal population, was observed in the cerebellum and field CA4 of the hippocampus, and dystrophic changes of neurons were found in the cortex and field CA1 of the hippocampus. In females neuronal loss was revealed only in field CA1 of the hippocampus. Estradiol with DHEA prevented neuronal loss in the cerebellum and hippocampus of male rats but had no effect in females. Thus, our data indicate that sex-dependent differences exist in neuronal damage caused by cardiac arrest. Brain lesions are more prominent in males than in females. Estradiol-DHEA protect neurons from ischemia/reperfusion injury only in males.


PERIPHERAL ADMINISTRATION OF HUMAN ADRENOMEDULLIN AND ITS BINDING PROTEIN ATTENUATES STROKE-INDUCED BRAIN INJURY IN RATS.W.W. Chaung,* R. Wu, Y. Ji,* Z. Wang,* W. Dong,* X. Qiang,* H. Wang, and P. Wang. Department of Surgery, North Shore-LIJ Medical Center, Manhasset, NY 11030

Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM) increases under various inflammatory conditions, and counter-balances inflammatory responses. However, the regulation of AM activity after the onset of ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (i.e., AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally at 10 min after MCAO, exogenous human AM in combination with human AMBP-1 reduced the brain infarct volume at 24 h post-MCAO in rats, an effect not observed after the treatment of human AM alone (Figure below). Moreover, administration of human AM in combination with human AMBP-1 significantly reduced neuron apoptosis and injury, downregulated neuronal nitric oxide synthase (nNOS) expression, inhibited neutrophil infiltration in the brain, and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 reduces stroke-induced brain injury in rats. AM/AMBP-1 may be developed as a novel therapeutic agent for patients with ischemic stroke.



HUMAN GHRELIN PROTECTS RATS FROM RENAL ISCHEMIA/REPERFUSION INJURY.D. Rajan,* R. Wu, K. Shah,* A. Varghese,* G. Coppa,* and P. Wang. Department of Surgery, North Shore-LIJ Medical Center, Manhasset, NY 11030

Acute kidney injury (AKI) occurs in various clinical settings with varied manifestations ranging from a minimal but sustained elevation in serum creatinine to anuric renal failure. Ghrelin, a stomach-derived peptide, has been shown to reduce the systemic inflammatory response in a rat model of sepsis. The purpose of this study was to explore possible beneficial effects of human ghrelin after renal ischemia/reperfusion (I/R) in rats. To study this, male adult rats were subjected to AKI by bilateral renal pedicle clamping for 60 min followed by reperfusion. Human ghrelin (4 nmol/rat) or saline (vehicle) was given intravenously over 30 min immediately after reperfusion and 24 h later, blood and organ samples were collected for various measurements. As shown in the table below, human ghrelin attenuated kidney injury and decreased inflammatory responses in the kidney after renal I/R.


In addition, elevated serum levels of liver enzymes (i.e., ALT & AST) and hepatic, pulmonary and intestinal edema after renal I/R were also reduced by human ghrelin treatment. Thus, human ghrelin protects rats from renal I/R injury. Ghrelin may be developed as a novel treatment for patients with acute kidney injury induced due to I/R.


THE RENOPROTECTIVE EFFECT OF MILK FAT GLOBULE EGF-FACTOR VIII AFTER ISCHEMIA AND REPERFUSION INJURY IN MICE.A. Matsuda,* R. Wu, H. Komura,* M. Zhou, Z. Wang,* D. Rajan,* and P. Wang. Department of Surgery, North Shore-LIJ Medical Center, Manhasset, NY 11030

Milk fat globule epidermal growth factor (EGF) factor VIII (MFG-E8) mediates the clearance of apoptotic cells by phagocytes. MFG-E8 attenuates acute inflammation in sepsis. However, it remains unknown whether MFG-E8 has beneficial effects on ischemia/reperfusion (I/R) injury. To investigate this, male C57BL/6J mice (8-12 weeks old) were subjected to renal I/R (45 min ischemia followed by 20 h reperfusion) or sham operation. The I/R mice were treated with saline (vehicle) or recombinant murine MFG-E8 (rmMFG-E8) at 0.4 μg/20 g BW i.p. at the beginning of reperfusion. Biochemical markers (plasma BUN, creatinine, and ALT), tissue (kidneys and liver) water content, MFG-E8 gene and protein expression, and cytokine gene expression (IL-6, IL-1ß, and MIP-2) in the kidneys were assessed. Renal I/R significantly decreased MFG-E8 gene and protein expressions in kidneys (by 45% and 44%, respectively; P <.05). As shown in the table below, renal I/R induced organ injury and inflammatory cytokine expression were attenuated by rmMFG-E8 treatment. Thus, MFG-E8 treatment prevented organ damage in renal I/R at least in part through its anti-inflammatory property.




Hepatic ischemia and reperfusion (I/R) injury occurs in a variety of clinical settings and activates many of innate immune systems including toll-like receptors (TLRs) pathway. It has been shown that a TLR signaling cascade is required for ROS production and the release of high-mobility group box (HMGB) 1. Recently, TLR signaling is identified to be interconnected to local induction of heme oxygenase (HO)-1, a potent redox enzyme. Chlorogenic acid (CGA) is one of the most abundant polyphenols in human diet and its antioxidant and anti-inflammatory properties have been reported. In this study, we investigated the protective mechanisms of CGA against I/R injury. Rats were subjected to 60 min of partial hepatic ischemia followed by 5 hr of reperfusion. CGA was administered intraperitoneally 15 min before ischemia and 5 min before reperfusion. CGA treatment markedly improved hepatic function and histology in a dose-dependent manner, and suppressed oxidative stress as indicated by hepatic lipid peroxidation and GSH/GSSG ratio, as compared with vehicle-treated group. To determine the protective mechanisms of CGA against IR injury, liver TLR2, TLR4, HO-1, and serum-released HMGB1 expression were measured by Western blot. I/R increased TLR4 (not TLR2), HO-1, and serum-released HMGB1 levels after reperfusion. CGA significantly attenuated TLR4 and HMGB1 expressions, whereas it augmented HO-1 expression. The mRNA levels of proinflammatory mediators such as tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, inducible nitric oxide were increased after reperfusion, which were significantly attenuated by CGA. Our data suggests that CGA inhibits hepatocellular damage resulting from I/R by inducing HO-1, suppressing TLR4 overexpression and HMGB1 release.


EFFECTS OF AORTAL CLAMPING AND DECLAMPING ON VALUES OF BRAIN NATRIURETIC PEPTIDE AND INTERLEUKINE-6.S. Asmussen,* V. Forchheimer,* M. Pook,* W. König,* M. Schneider,* A. Gauss,* and R. Meierhenrich*. Departments of Anesthesiology and Cardiology, University Hospital of Ulm, Germany. (D.L.Traber, Dept. of Anesthesiology, UTMB, Galveston, TX)

Objective: Aortic clamping and declamping maneuvers induces cardiovascular complications during vascular surgery. We hypothesized that the clamping and declamping maneuvers during major vascular operative procedures increase the plasma levels of brain natriuretic peptide (BNP) and Interleukine-6 (IL-6).

Method: Thirty patients were included in this pilot study, 15 had abdominal aortic aneurysms and underwent transabdominal aortic graft surgery (aortal clamping time 1-3 hrs), and 15 patients had peripheral cardiovascular disease (CVD) grade 4 that underwent small vessel surgery. Both groups were matched for cardiovascular risk factors. Blood samples for BNP, IL-6 and creatine kinase (CK), CK-MB, Troponin I and C-reactive proteine (CRP) were taken before anesthesia at baseline (BL), before clamping, just after declamping and 2, 6, 24 and 48 hrs post declamping.

Results: Interleukine-6 increased from 6 to 24 hrs in the aortal aneurysm patients (833±13 ng/l vs BL 63±13 ng/l, p<0.05) in comparison to CVD (49±11 ng/l, p<0,05). BNP increased slightly in comparison to BL (0 pg/ml) in CVD patients (71±475 pg/ml) and aortal aneurysm group (105±141 pg/ml, p=0.25). Although CK tended to rise above baseline in the aortic graft patients 24 hrs after declamping the change could not be shown to be statistically different (p=0.18). The levels of CK-MB, Troponin I and CRP also remained at baseline levels.

Conclusion: Interleukine-6 but not BNP might be used as a potential marker for possible cardiovascular complication in abdominal aortic surgery. On the other hand the influence of the reperfusion injury after declamping on these markers has to be discussed. Further studies with more patients included are needed to explore those important problems.


MITOCHONDRIA CATALYSE THE REDUCTION OF NITRITE IN CARDIOMYOCYTES AND HEPATOCYTES.P. Dungel, A. Banerjee, D. Dopler, O. Andrukhov, H. Redl, and A.V. Kozlov. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria

Objective: Nitrite can protect heart and liver from ischemia/hypoxia injury. The protection is probably mediated by the reduction of nitrite to nitric oxide (NO) and activation of cGMP synthesis. However, the mechanism of nitrite reduction is still unclear. The objective of this study was to clarify the predominant mechanism(s) of nitrite bioactivation in an in vitro model of hypoxia in cardiomyocytes and hepatocytes.

Methods: Cell lines of cardiomyocytes (HL-1) and hepatocytes (BRL-3A) were incubated with nitrite at various oxygen tensions. Targeted inhibitors were used to investigate the relevance of specific enzymes. NO was determined via nitrosyl complexes with hemoglobin by EPR, cGMP levels was determined by ELISA.

Results: HL-1 and BRL-3A are both able to reduce nitrite to NO in an oxygen-dependent manner, with highest yields under anaerobic conditions. Nitrite reduction was accompanied by significant increase of intra- and extracellular cGMP levels in both cell lines. However, extracellular cGMP was 5-fold and 10- fold increased in BRL-3A and HL-1 respectively, compared to intracellular levels. In both cell lines myxothiazol, a specific mitochondrial inhibitor, significantly reduced cGMP levels induced by nitrite. Allopurinol, an inhibitor of xanthine oxidase, and L-NAME, a NOS inhibitor, had no effect on cGMP levels. NO gas, however, had the same effect as nitrite which was also abolished by myxothiazol.

Conclusion: Our data suggest that under hypoxic conditions nitrite is reduced to NO resulting in cGMP generation and that in both cardiomyocytes and hepatocytes this reaction is catalyzed predominantly by mitochondria. (Funded by FWF grant P 21121-B11).



Objective: Secretory IgA (SIgA) is the principle antibody in mucosal defense and is therefore a critical component of the mucosal barrier to luminal pathogens. In a previous study we demonstrated that SIgA protected against intestinal epithelial cell (IEC) apoptosis and cell monolayer permeability following exposure to bacteria and hypoxia-reoxygenation (H/R). The transcription factor NFκB plays an important role in triggering local and systemic effects of intestinal reperfusion injury (I/R). We postulated that SIgA would modulate NFκB nuclear translocation following H/R and studied this in vitro.

Methods: Established Caco-2 IEC monolayers were placed in hypoxic conditions (5% O2) followed by normoxia (21% O2). In some experiments a commensal strain of Escherichia coli (EC) was added with or without SIgA. After 90 minutes of hypoxia followed by 3 hours of normoxic conditions nuclear extracts were obtained from Caco-2 cells and NFκB (p65) was measured using an ELISA. Data were normalized to total nuclear protein. Caco-2 cells held under normoxic conditions served as controls.

Results: NFκB (mean ± SD, ng/mg protein, N=4).


Conclusion: EC and H/R and the combined insults increased nuclear translocation of NFκB. This effect was blocked by SIgA. The protective effects of SIgA on barrier function are likely due to downregulation of downstream events following NFκB activation.


MICROCIRCULATORY EFFECTS OF LOCAL AND REMOTE ISCHEMIC PRECONDITIONING AFTER SUPRACELIAC AORTIC CLAMPING.N. Erling Jr,* J.W. Cruz,* P. Sannomiya,* J.C. Baptista-Silva,* P. Sannomiya,* and L.F. Poli de Figueiredo. Dept Surgery, University of São Paulo School of Medicine, Brazil

Objective: We hypothesized that both local (LIPC) and remote ischemic preconditioning (RIPC) promote microcirculatory benefits after supra-celiac aortic cross-clamping (SCAC).

Methods: Anesthetized male Wistar rats (N=56) were randomly assigned for 4 groups of 7 rats each: CT, controls, with no aortic clamping; IR, 20 min of SCAC, followed by 120 min of reperfusion; LIPC, 2 cycles of SAC (5 min of SCAC and 5 min reperfusion), followed by 20 min of SCAC and 120 min of reperfusion; RIPC, 2 cycles of infrarenal aortic clamping (10 min ischemia and 10 min reperfusion), followed by 20 min of SCAC clamping and 120 min of reperfusion. Microcirculatory blood flow, as well as PMN-endothelial cell interactions in the mesentery were evaluated by intravital microscopy at the end of experiment. In another set of similar experiments (N=28), the intestine was removed at the end of the experimental protocol for ICAM-1, P- and E-selectins.

Results: IR increased the expression of both P and E-selectins, while LIPC and RIPC lead to a down expression of these molecules, with P-selectin even lower than controls. IR also increased rolling leukocytes and decreased rolling velocity, while LIPC and RIPC promoted marginally significant protection. IR increased ICAM-1 expression, but not in preconditioned animals. Stickers and migrated leukocytes showed 5- to 6-fold increases, while LIPC and RIPC significantly lessened this process.

Conclusion: We conclude that visceral and muscular ischemia induced by supraceliac aortic clamping promote intense systemic and tissue inflammatory reactions, modifying leukocyte behavior and adhesion molecules expression. Local and remote IPC render tissue more resistant to the deleterious microcirculatory effects of the prolonged and extensive lower torso IR injury. (FAPESP grant 04/15964-6).


HUMAN MYOCARDIAL INFLAMMATORY RESPONSE TO SURGICAL ISCHEMIA: ROLE OF EXTRACELLULAR HSP27.C. Jin,* L. Ao,* J. Cleveland,* J. Li,* D. Fullerton,* and X. Meng. Department of Surgery, University of Colorado Denver, Denver, CO 80045

Heat shock proteins (HSPs) could have pro-inflammatory functions when released into extracellular spaces. We found that mouse heart releases constitutive HSP70 (HSC70) following global ischemia and that extracellular HSC70 induces myocardial cytokine production via a TLR4-dependent mechanism. In this study, we examined human myocardial inflammatory responses to global ischemia and investigated the potential role of extracellular HSP27 in the inflammatory responses. In patients who underwent cardiac surgery with cardiopulmonary bypass, levels of HSC70 and HSP27, particularly the latter, were elevated in coronary sinus blood after global ischemia. The release of HSPs correlated with increased production of MCP-1 and ICAM-1. In vitro studies show that recombinant HSP27 induced the phosphorylation of NF-κB and p38 MAPK, and up-regulated MCP-1 and ICAM-1 production in both human and mouse coronary endothelial cells. TLR2 KO or TLR4 mutation markedly attenuated HSP27-induced NF-κB phosphorylation in mouse coronary endothelial cells, but neither had an effect on p38 MAKP phosphorylation. However, HSP27-induced production of MCP-1 and ICAM-1 was significantly reduced in coronary endothelial cells from TLR2 KO mice or TLR4 mutant mice. These results demonstrate: 1) human myocardium releases HSP27 following global ischemia, which is associated with myocardial inflammatory responses, 2) extracellular HSP27 induces the production of pro-inflammatory mediators in human and mouse coronary endothelial cells, 3) both TLR2 and TLR4 are involved in mediating the effect of extracellular HSP27 and 4) NF-κB, rather than p38 MAKP, appears to play a major role in post-receptor pro-inflammatory signaling.



Background: While regional intraischemic hypothermia (IH) prevents intestinal injury and inflammation during mesenteric ischemia-reperfusion injury (IRI), the distant organ effects remain unknown. We investigated lung structural, cellular, and transcriptional changes during mesenteric IRI and hypothesized that IH would suppress neutrophil trafficking and the associated lung inflammatory transcriptome.

Methods: Male Sprague-Dawley rats (∼300gm) underwent laparotomy (sham) or SMA clamp for 60 min (IRI) ± IH. Gut temperature was maintained at 15-20°C during IH, and systemic normothermia (37°C) was maintained throughout the study period. At 6 or 24 hours, lung tissues (n=3/group) were collected for 1) histology, 2) myelo-peroxidase (MPO) levels, and 3) total RNA isolation and hybridization to Illumina's Sentrix BeadChip (>22,000 probes) which were simultaneously analyzed by BeadStudio_1.5 and SAM_2.0. Genes with fold change (FC)>1.5 and false discovery rate (FDR) <5% were considered significantly affected by IRI.

Results: At 24 hrs, histology revealed increased lung neutrophils and alveolar hemorrhage in IRI rats compared to sham, sham+IH, and IRI+IH. Lung MPO was increased in IRI (7.1±0.4, p<.05) compared to sham (5.9±0.3), sham+IH (6.3±0.4), and IRI+IH (6.5±0.4). At 6 hrs, we identified 287 significant IRI-specific lung candidate genes that were suppressed by IH, and analysis revealed the top differentially expressed genes between IRI and IRI+IH (ie. CXCL-1, Lipocalin-2, and CXCL-2) are prominent pro-inflammatory genes involved in leukocyte activation and trafficking. Quantitative RT-PCR confirmed concordant FC expression patterns between array and RT-PCR.

Conclusion: IH preferentially regulates lung neutrophil trafficking and activation and modulates the mesenteric IRI-activated lung transcriptome.


EFFECTS OF TEMPERATURE ON COAGULATION, IMMUNE RESPONSES AND APOPTOSIS DURING HEMORRHAGIC SHOCK.S. Burruss,* A. Andakyan,* S. Romanov,* N. Semiletova,* J. Hiatt,* and H. Cryer. University of California, Los Angeles, Los Angeles, CA 90095

Background: Hemorrhagic shock (HS) leads to a systemic ischemic injury characterized by dyshomeostasis of coagulation cascades and immune responses.

Methods: 7-wk-old SD rats underwent a progressive hemorrhage protocol until death. The animals were divided into three groups: HS-cold, hemorrhage without a heating pad; HS-warm, body temperature maintained at 37°C; Control, body temperature maintained at 37°C without hemorrhage (n=5 in each group). 1mL of blood was withdrawn Q5 mins until 4mL, then Q10 mins until death (total 13mL). Liver and lung tissues were collected for histology and gene expression tests.

Results: Hemorrhage resulted in a significant increase in IL-6 and MMP-9 in liver mRNA in both cold and warm HS while Egr-1 significantly increased by 25% in cold HS (p=.01) but decreased 12% in warm HS (p=.03). Coagulation factors were altered similarly in cold and warm HS with fibrinogen decreasing by 42% in warm HS (p=.0009) and 55% in cold HS (p=.0001) while prothrombin liver mRNA increased similarly in both groups. Of note, protein C levels were not significantly different from control in either cold or warm groups. Pro-apoptotic Bax significantly increased by 75% in cold-HS (p=.01) and 53% in warm-HS (p=0.04) livers and were not different between warm and cold groups.

Conclusions: Upregulation of inflammatory, coagulation and apoptotic markers occurs early in shock, prior to resuscitation. Only Egr-1 activation was minimized by maintaining normothermia during hemorrhagic shock in our model to suggest that temperature may not be an important factor prior to resuscitation in severe hemorrhage.



Introduction: Acute renal failure in patients with shock is generally ascribed to ischemia of the tubular epithelium leading to epithelial dysfunction and acute tubular necrosis. The renal tubular system is a complex interplay of cellular activity and differential permeability; understanding the shift from disease to health requires a dynamic representation of the cellular and molecular mechanisms involved. Agent-based modeling provides a means of integrating this knowledge via an in-silico framework for evaluation of treatment effects and identification of potentially paradoxical behavior. Here we present a cell-level agent-based model (ABM) of renal tubular function and the dysfunction associated with ATN.

Methods: We constructed an ABM of the renal tubular complex using NetLogo 4.0.5. A literature review of cellular and molecular mechanisms involved with renal tubular function formed the basis of the rules for the ABM. Validation of the model consisted of confirmation of baseline renal tubular behavior with experiments of the effects of diuretics and the implementation of putative mechanisms associated with ATN.

Findings: The ABM incorporated the essential cellular types of the glomerular complex and activities for normal renal function. Interventions with simulated loop and osmotic diuretics produced expected alterations in fluid and electrolytes. Simulating ATN reproduced sodium wasting, acidosis and the progression from non-oliguric to oliguric renal failure.

Discussion: The ABM of renal function captured the complexity of cellular interactions and the evolution of ATN in an abstract but qualitatively valid fashion. This type of computational model has the potential for investigating preventive/protective strategies and the treatment effect from novel mechanism-based interventions.


CHOLINERGIC AGONISTS ATTENUATE LPS-INDUCED ACUTE KIDNEY INJURY.C.N. Metz, X. Xue,* O. Dowling,* and P.K. Chatterjee*. The Feinstein Institute for Medical Research, Manhasset, NY 11030

Objective: The kidney is a major target for sepsis-related injury and acute kidney injury (AKI) significantly enhances sepsis mortality. Sepsis-associated AKI is accompanied by marked upregulation of inflammatory mediators within the kidney followed by leukocyte infiltration. Based on our previous studies demonstrating the regulation of leukocyte trafficking by cholinergic agonists, we examined their effect on LPS-induced AKI.

Methods: Swiss Webster mice were treated with saline or cholinergic agonists, nicotine (1 mg/kg, i.p.) or GTS-21 (5mg/kg, i.p.), followed by LPS (2.5 mg/kg, i.p.). Mice were euthanized 3.5hrs and 24hrs post LPS. Kidney damage was assessed and both kidney and circulating inflammatory mediators were measured by ELISA. IκB degradation and NFκB activation within the kidney was determined. Using renal proximal tubule cells (HK-2), primary mesangial cells, and renal glomerular endothelial cells, we investigated the effect of cholinergic agonists on LPS-induced cytokine and chemokine production ex vivo.

Results and Conclusions: Cholinergic agonists significantly attenuated LPS-induced AKI, as determined by decreased: kidney injury (BUN levels), kidney leukocyte infiltration (MPO levels), endothelial cell activation (soluble ICAM-1 levels), systemic inflammation (serum TNF levels), and local kidney inflammation (MCP-1, IP-10, TNF, NFκB activation). We found that cholinergic agonists significantly suppressed cytokine and chemokine production by LPS-treated kidney cells (HK-2 epithelial, primary human renal glomerular endothelial, and mesangial cells) supporting the hypothesis that cholinergic agonists regulate local cellular inflammatory responses within the kidney following LPS. These data support the development of cholinergic agonists for the prevention and treatment of AKI during sepsis. (Grant support: NIGMS R01 GM070727 (CNM)).



Objective: To investigate if combined inhibition of the neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) attenuates pulmonary morbidity in sheep subjected to burn and smoke inhalation injury.

Methods: Sixteen chronically instrumented sheep were randomized to either an uninjured, untreated group (n=5), an injured control group (48 breaths of cotton smoke and 3rd degree burn of 40% TBSA; n=6) or an injured group treated with the nNOS inhibitor 7-NI and the iNOS inhibitor BBS-2 (n=5) for the entire 48-hr study period.

Results: Combined burn and smoke inhalation trauma induced a significant increase in stable metabolites of NO in the plasma (p<0.05) which was inhibited by the treatment. The injury-related decline in oxygenation index (P/F ratio; Fig. 1) and increases in pulmonary shunt fraction, lung lymph flow, and lung water content were also attenuated in the treatment group (p<0.05 each). The insult was further associated with marked elevations in airway pressures and histologically determined airway obstruction score. In treated animals, these parameters were not significantly altered towards the sham group. Additionally, the development of systemic vascular leakage was significantly inhibited by the treatment.

Conclusions: The combination of nNOS and iNOS blockade attenuated the severity of pulmonary morbidity following ovine burn and smoke inhalation injury. Because no endothelial NOS (eNOS) inhibitors are currently available, the role of eNOS-derived NO should be investigated in future studies using gene knock out mice.




Objective: To investigate the effects of epinephrine nebulization on airway hyperemia and pulmonary oxygenation following severe smoke inhalation injury in sheep.

Methods: Twenty chronically instrumented sheep were randomized to either an sham-injured, untreated group (n=5), an injured control group (48 breaths of cotton smoke; n=6), and injured groups treated with 2mg (n=3) or 4mg (n=6) nebulized epinephrine every 4 hrs, respectively.

Results: The injury led to significant increases in tracheal, main bronchial, and distal bronchial blood flow, resulting in elevated whole lung water content, increased ventilatory pressures and severely disturbed oxygenation index (PaO2/FiO2 ratio) and pulmonary shunt fraction. In the treatment groups, the increases in tracheal and right main bronchial blood flow were attenuated (Fig. 1), but elevated distal bronchial blood flow and whole lung water content were not affected. Additionally, the injury-related increases in ventilatory pressures as well as deteriorations in P/F ratio (Fig. 2) and pulmonary shunt fraction were dose-dependently attenuated by the treatment.

Conclusions: Nebulization of epinephrine may represent a beneficial treatment option for patients with inhalation injury, probably due to its vasoconstrictive effects and the attenuation of injury-induced airway hyperemia.

FIG. 1
FIG. 1:
Impact of epinephrine nebulization on left main bronchial blood flow.
FIG. 2
FIG. 2:
Impact of epinephrine nebulization on oxygenation index.


ROLE OF PEROXYNITRITE IN OVINE BURN AND SMOKE INHALATION INJURY.M. Lange,* C. Szabo, P. Enkhbaatar, E. Horvath, L. Traber, and D. Traber. University of Texas Medical Branch, Galveston, TX 77550

Objective: In response to acute lung injury (ALI), large amounts of nitric oxide exert cytotoxic effects by reacting with superoxide radicals, yielding reactive nitrogen species such as peroxynitrite (ONOO-). ONOO- may exert a deleterious influence by nitrating/nitrosating proteins and lipids and inducing the nuclear repair enzyme poly(adenosine diphosphate ribose) (PAR) polymerase and/or vascular endothelial growth factor (VEGF). We hypothesized that administration of the ONOO- decomposition catalyst INO-4885 attenuates ALI in chronically instrumented sheep.

Methods: Animals were randomized to a sham-injured group (n=7), an injured control group (48 breaths of cotton smoke, 3rd degree burn of 40% TBSA; n=7) or an injured group treated with INO-4885 (n=6).

Results: The double-hit injury contributed to a significant drop in PaO2/FiO2 ratio and significant increases in pulmonary shunt fraction, lung lymph flow, lung vascular permeability index, lung wet/dry weight ratio, and ventilatory pressures. All these changes were significantly attenuated by the treatment. In addition, the injury-related increases in protein expression of 3-nitrotyrosine, a marker of ONOO- (Fig. 1), VEGF, and PAR in lung tissue as well as the immuno-histochemical PAR score were significantly attenuated in treated animals.

Conclusions: ONOO- plays a crucial role in the pathogenesis of ALI following burn and smoke inhalation injury in sheep. Administration of an ONOO- decomposition catalyst may represent a potential treatment option for this injury. (Support: NIH-P012 GM066312, NIH R01 GM060688, SBI#8541, SBI #8450. INO-4885 was kindly provided by Inotek Pharmaceuticals, Beverly, MA).

FIG. 1
FIG. 1:
Impact of INO-4885 on protein expression of 3-nitrotyrosine.


INFLAMMATORY RESPONSE TO HIGH FREQUENCY PERCUSSIVE VENTILATION.K.K. Chung,* M.C. Shelhamer,* J.K. Aden,* G.A. Merrill,* M.G. Schwacha, L.C. Cancio,* E.M. Renz,* C.E. Wade, L.H. Blackbourne,* and (Spon: S.E. Wolf). US Army Institute of Surgical Research, Ft Sam Houston, TX 78234

Introduction: High frequency percussive ventilation (HFPV) is a commonly utilized mode of mechanical ventilation in select burn intensive care units (BICU), especially to support those with smoke inhalation injury. We recently reported the clinical results of a single center prospective randomized trial comparing HFPV (n=31) to conventional lung-protective mechanical ventilation (CV) (n=31) in BICU patients which demonstrated no difference in our primary outcome of ventilator-free days at 28 days. Given the potential for large background tidal volumes, our hypothesis was that a HFPV-based strategy would be associated with a greater systemic inflammatory response than a CV-based strategy as seen with the ARDSnet trial.

Methods: We conducted an analysis of selected markers at 1, 3 and 7 days after randomization in each arm. IL-1β, IL-6, IL-8, GM-CSF, and TNF-α were measured. Greater than 90% of values for IL-1β, GM-CSF, and TNF-α fell below the lower limit and thus were not further analyzed.

Results: When comparing the HFPV group to the CV group via ANOVA after log transformation we found no difference over time (1, 3, and 7 days) or between groups. Crude mean±SE (HFPV vs CV) for plasma IL-6 concentrations (pg/ml) at days 1, 3, and 7 were 138±50 vs 285±156 (p=0.39), 118±32 vs 158±56 (p=0.55), and 128±41 vs 107±26 (p=0.69) respectively. Crude mean±SE (HFPV vs CV) for plasma IL-8 concentrations (pg/ml) at days 1, 3, and 7 were 78±16 vs 234±123 (p=0.23), 78±16 vs 143±51 (p=0.23), and 230±85 vs 163±46 (p=0.51) respectively.

Conclusion: The systemic inflammatory response for HFPV is no different from a low-tidal volume based ventilator strategy. This suggests that HFPV may confer the same level of 'lung protection' against inflammation as low-tidal volume ventilation.


EFFECTS OF MUSCARINIC ANTAGONIST THERAPY ON BRONCHIAL SUBMUCOSAL INFLAMMATION AND EDEMA IN SHEEP AFTER INHALATION AND BURN INJURY.S. Jacob,* Y. Zhu,* C. Jonkam, E. Kraft, S. Rehberg, Y. Yamamoto, L. Traber, D. Traber, P. Enkhbaatar, H. Hawkins,* and R. Cox. Shriners Hospital for Children and the University of Texas Medical Branch, Galveston, TX 77550

Nebulization of a muscarinic antagonist reduces airway obstruction and significantly improves pulmonary function in sheep with smoke inhalation and burn (SB) injury This study test the hypothesis that nebulized tiotropium bromide (TB), an M1 and M3 specific muscarinic receptor antagonist, would reduce airway submucosal inflammation and edema compared to injured non-treated animals. Uninjured sham sheep (n=4), and injured sheep received a standardized 40% TBSA, 48 breaths of cooled cotton smoke. Injured sheep were treated with 18 μg (n=6) 1 hr after injury and 18 μg TB every 4 hrs for the 24 hr study period. Control sheep (n=6) received equal volumes of saline nebulization as the treated animals. Following sacrifice, main bronchial tissue was systematically sampled for microscopic quantitation of bronchial submucosal neutrophils (PMNs) and for determination of tissue wet to dry weight ratios (W/D). Submucosal inflammation was significantly increased in the control and TB-treated animals compared to sham, p=0.02. A strong trend of increased inflammation was seen in the TB-treated animals compared to controls, although the difference was not significant, p = 0.33. W/D ratios were significantly increased in control and TB-treated animals compared to sham. TB-treated animals showed a significant decrease in edema compared to the control group, p=0.01. TB-treated animals showed an increase in acute inflammation, although the W/D ratio was statistically decreased in animals treated with TB. These results suggest that TB may inhibit the trafficking of PMNs to the airway lumen and reduce the activity of PMN within the submucosa.


COMBINED RHAPC AND CEFTAZIDIME PREVENT ONSET OF ARDS IN OVINE ACUTE LUNG INJURY.M.O. Maybauer, D.M. Maybauer, J.F. Fraser, L.D. Traber, and D.L. Traber. Dept. of Anesthesiology, UTMB, Galveston, TX

Objective: Aim of this study was to investigate the effects of combined recombinant human activated protein C (rhAPC) and Ceftazidime (CEF) in our established model of acute lung injury (ALI) and sepsis.

Methods: Thirty sheep (35-40 kg) were operatively prepared for chronic study, and were randomly allocated either to sham, control, CEF, rhAPC, or CEF + rhAPC groups (n=6 each). After a tracheostomy, ALI and sepsis was produced in all groups, following an established protocol (1,2), except the sham group that received the vehicle. The sheep were studied for 24h in awake state and were ventilated with 100% oxygen. PaO2/FiO2 ratio was determined at baseline (BL) and every 3 h. CEF (3 g) was administered intravenously 1 and 13 h post injury. RhAPC was given as a continuous infusion (24mcg/kg/h), starting 1h post injury. The animals were resuscitated with Ringer's Lactate Solution to maintain filling pressures and hematocrit. Statistical analysis: two-way ANOVA and Student-Newman-Keuls post hoc comparison. Data are expressed as mean ± SEM. Significance P<0.05.

Results: PaO2/FiO2 ratio remained stable in sham (BL: 518±13 vs. 24h: 500±19) animals. The control group showed a significant decrease in PaO2/FiO2 ratio (BL: 497±21 vs. 24h: 76±5). The CEF group also showed a fall in PaO2/FiO2 ratio (BL: 524±11 vs. 24h: 165±47), as well as the rhAPC (BL: 541±12 vs 24h: 118±17) group. Both groups showed a significant improvement of the PaO2/FiO2 ratio compared to the control group. The PaO2/FiO2 ratio of the CEF + rhAPC group (525±10 vs. 24h: 280±20) was significantly higher than in all other injured groups.

Conclusion: Administration of CEF + rhAPC after ALI associated with sepsis improved oxygenation more than CEF or rhAPC alone. References: (1) Maybauer MO et al., Crit Care Med 2006;34(9):2432-2438; (2) Maybauer MO et al., Intensive Care Med 2007;33(7):1219-1227.


BURN-INDUCED ACUTE LUNG INJURY REQUIRES A FUNCTIONAL TOLL-LIKE RECEPTOR 4.M. Krzyzaniak,* C. Peterson,* L. Reys,* W. Loomis,* J. Putnam,* J. Cheadle,* B. Eliceiri, A. Baird,* V. Bansal, and R. Coimbra. Division of Trauma, University of California-San Diego, San Diego, CA 92103

Background: The role of the Toll-like receptor 4 (TLR4), as a component of the innate immune system, on the development of burn-induced ALI has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation following trauma and hemorrhagic shock. We hypothesized that burn-induced ALI is a TLR4 dependent process.

Methods: Male C57BL/6J (TLR4 Wild Type, WT) and C57BL/10ScN (TLR-4 Knock Out, KO) mice were subjected to a 30% total body surface area, full thickness steam burn. Animals were then sacrificed at 6 and 24 hours after the initial insult. Lung specimens were harvested for histological examination after staining with hematoxylin/eosin. In addition, lung MPO and ICAM-1 immunostaining was performed. Lung myeloperoxidase (MPO) was measured by an enzymatic assay. Total lung IL-8 content was measured using a commercially available ELISA kit.

Results: ALI, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intra-alveolar hemorrhage, and neutrophil infiltration, was seen in TLR4 WT animals at 24 hours after burn injury. In contrast, lung histology of TLR4 KO animals was similar to both KO and WT sham. MPO and ICAM-1 immunostaining of TLR4 KO animals at 24 hours was also similar to KO and WT sham. In addition, a 46% reduction in MPO enzymatic activity was observed in TLR4 KO mice at 24 hours (p<0.006) and a 65% reduction in IL-8 levels at 6 hours (p<0.002), compared to their WT counterparts.

Conclusion: Burn-induced acute lung injury develops within 24 hours following the initial thermal insult in our model. TLR4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR-4 dependent process.


THE RELATIONSHIP BETWEEN ARGINASE, NOS, AND ADMA TO ACUTE LUNG INJURY.L. Sousse,* Y. Yamomoto,* P. Enkhbaatar, L. Traber, D. Herndon, and D. Traber. University of Texas Medical Branch, Galveston TX 77555

Inhalation injury causes restrictive lung disease in burn victims. The objective is to instigate long-term lung dysfunction in an ovine model of burn and smoke inhalation injury. We hypothesize that lung arginase activity is increased by burn and inhalation injury, which contributes to increased collagen deposition and pulmonary dysfunction.

Methods: Ewes were randomly divided into sham/uninjured (n=6) and injured (n=7) groups. The injured sheep were subjected to 20% TBSA burn and 36 breaths of cotton smoke under deep anesthesia. The sheep were monitored for 96 hrs, 2 wks, or 3 wks.

Results: Asymmetrical dimethyl-arginine (ADMA), an endogenous NOS inhibitor, increased in plasma from 2 wks to 3 wks in injured sheep (32.7 uM ± 9.1 vs 48.0 ± 6.5). Lung myeloperoxidase, an index of neutrophils, significantly increased after 96 hrs vs sham sheep (0.6 uM ± 0.1 vs 0.4 ± 0.1,p<0.05). Expression of inducible NOS in the lung significantly decreased 2 wks after injury vs sham sheep (4505 ± 1128 vs 14040 ± 3520,p<0.05), and plasma nitrites and nitrates increased over time after injury vs sham sheep. Lung arginase activity (0.2 uM urea/uG protein ± 0.1 vs 0.1 ± 0.1,p<0.05) and hydroxyproline, which is a marker of collagen deposition (0.1 ± 0.17 vs 0.7 ± 0.1,p<0.05), were significantly increased 3 wks post-injury vs sham sheep. The increases in arginase and collagen are associated with significantly deteriorated final pulmonary gas exchange (PaO2/FiO2: 293 ± 64 vs 479 ± 21,p<0.05) and worst gas exchange (252 ± 41 vs 487 ± 6.3,p<0.05) after 3 wks vs sham sheep.

Conclusions: Metabolites of NOS inhibit arginase, and arginase increases because ADMA inhibits NOS. The increase in arginase contributes to the increase in collagen deposition, which leads to pulmonary dysfunction. Arginase inhibitors may prevent detrimental sequelae of the burn and inhalation injury.


ROLE OF DEATH RECEPTORS IN THE PATHOGENESIS OF CHEST TRAUMA-INDUCED SEPTIC ACUTE LUNG INJURY (ali).M. Perl, B. Baumann,* M. Huber-Lang, M.G. Bachem,* and F. Gebhard. Dept. of Orthopedic Trauma, Univ. of Ulm, 89075 Ulm, Germany

The pathomechanisms of septic ALI still remain largely unclear. Importantly, it is unknown whether programmed cell death is involved in the pathogenesis of septic ALI. To study this, male mice with a mutation in the Fas-receptor (lpr) or TNF-receptor-I (TNF-RI) deficient or wild type mice (C57) underwent blunt chest trauma (TxT), induced by a single blast wave. 24hrs later, sepsis (CLP) was induced via cecal ligation and puncture; lung tissue (n=8/group) or bronchoalveolar lavage fluid (BALF) (n=6/group) were harvested 12 or 24hrs thereafter. Lung cytokines were quantified via cytometric bead array or ELISA, lung active caspase-3 (C-3) by Western blotting. Lung myeloperoxidase activity (MPO) was also assessed. The degree of lung injury was determined via total protein in BALF and lung histology (H and E). TUNEL staining was performed in lung sections. Two-way ANOVA and SNK. In all animals lung KC, MCP-1, G-CSF were increased at 12 and 24hrs after TxT+CLP. Lung IL-6 and IL-1β were enhanced at 12 and, in C57, also at 24hrs after TxT+CLP compared to sham. KC, MCP-1, IL-6, IL-1β were decreased at 24 and G-CSF at 12hrs after TxT+CLP in lpr and TNF-RI compared to C57. IL-10 and MIP-1α were increased at 12 and 24hrs after TxT+CLP in C57 and at 12hrs also in TNF-RI compared to sham. At 24hrs IL-10 and MIP-1α were decreased in lpr and TNF-RI compared to C57. At 12hrs after the insult lung MPO was higher in C57 compared to sham, lpr or TNF-RI. BALF-protein, TUNEL and C-3 were increased at 12hrs in all animals after TxT+CLP, but were markedly lower in lpr, and for TUNEL and C-3, also in TNF-RI compared to C57. Thus, activation of Fas and TNF-Receptor-I is involved in the pathogenesis of trauma induced septic ALI by regulating both, lung apoptosis and inflammation. These pathways might represent potential therapeutic targets in ALI in the future. (Supported by DFG-PE 908/2).


THE ROLE OF CAMKI IN ANIN VIVOLPS MODEL OF ACUTE LUNG INJURY.J. Stripay, R. Collage, L. Guo, X. Zhang, and M. Rosengart. University of Pittsburgh, PA 15213

Our prior studies suggest that the multifunctional calcium/calmodulin-dependent protein kinase (CaMK) cascade mediates the monocyte/ macrophage inflammatory response during sepsis. Here, we hypothesize that the family member CaMKI mediates the systemic and pulmonary cytokine response and pulmonary dysfunction in an in vivo LPS model of acute lung injury.

Methods: C57BL/6 mice were administered CaMKI or scrambled, non-target (NT) siRNA (6mg/kg) by hydrodynamic tail vein injection, and after 72 hours underwent intratracheal instillation of LPS (1.5mg/kg). Mice were euthanized after 6 hours, the lungs were broncholavaged (BAL), and blood and organs were harvested. Serum and BAL cytokine concentrations were assayed by ELISA. Autophagy and CaMKI expression were assessed by immunoblot and immunofluorescence.

Results: CaMKI, not NT, siRNA effectively reduced the expression of CaMKI in the lung. CaMKIRNAi mice displayed reduced pulmonary concentrations of TNF-α (1361 vs. 944 pg/mL; p=0.08) and IL-6 (619 vs. 367 pg/mL; p=o.001) by comparison to NTRNAi mice. CaMKIRNAi mice also exhibited reduced neutrophil infiltration (84% vs. 92%, p=0.06) and protein concentration (0.17 vs. 0.24 mg/ml, p=0.32) by contrast to NTRNAi mice, but these differences did not attain statistical significance. Immunofluorescence revealed a decrease in autophagy as evidenced by reduced induction of LC3b, a key autophagic protein, in BAL cells. There was no significant difference in systemic cytokine concentrations.

Conclusion: These data support that LPS induces pulmonary inflammatory cytokine production and neutrophil recruitment through a CaMKI-dependent pathway. This correlates with an inhibition of autophagy, though subsequent studies are need to causally link these processes.


NEBULIZED HEPARIN DOSE-DEPENDENTLY IMPAIRS THE BENEFITS OF INTRAVENOUS ANTITHROMBIN FOLLOWING BURN AND SMOKE INHALATION INJURY.S. Rehberg,* Y. Yamamoto,* L. Sousse,* L. Traber, D.L. Traber, and P. Enkhbaatar. Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555

Objective: To compare the effects of two different doses of nebulized heparin on the efficiency of the combined therapy with intravenous (iv.) recombinant human antithrombin (rhAT) and nebulized tissue plasminogen activator (TPA) in an ovine model of burn and smoke inhalation injury.

Methods: Chronically instrumented sheep were subjected to a 40% total body surface area 3rd° burn and 48 breaths of cotton smoke under deep anesthesia. Sheep were randomly assigned to receive an iv. infusion of 6 U·kg-1·h-1 rhAT (started 1h post injury) combined with nebulized TPA (2 mg every 4h, started 4h post injury) and heparin (5,000 [low-dose] or 10,000 IU [high-dose], respectively, every 4h, started 2h post injury) or 0.9% NaCl iv. and aerosolized (control; n=6 each). All sheep were awake, mechanically ventilated and fluid resuscitated for 48h. Data are expressed as mean±SEM at 48h.

Results: Both strategies attenuated lung injury, as suggested by higher PaO2/FiO2 ratios (low-dose: 276±44 mmHg, high-dose: 352±25 mmHg, control: 134±30mmHg) and lower airway peak pressures (27±2cmH2O, 27±1 cmH2O, 36±2 cmH2O). Notably, the combination with low-dose heparin reduced pulmonary transvascular fluid flux (16±2mL/h, 40±5mL/h, 51±4m/h) and permeability index (9±1mL/h, 19±2mL/h, 25mL/h) and increased plasma protein (4.6±0.1 g/dL, 3.9±0.2g/dL, 4.0±0.3g/dL) vs. both other groups (p<0.05 each). Cumulative net fluid balance was lower in the low-dose heparin group (2.1±0.2L) vs. control animals (3.5±0.4L; p<0.05).

Conclusions: With the lower dose of heparin the systemic anti-inflammatory effects of iv. rhAT on vascular leakage are more pronounced, while the local, beneficial effects of nebulized heparin on gas exchange are preserved. A reduction of systemic interaction between heparin and rhAT represents a possible explanation.


MYCOPLASMA PNEUMONIAETOXIN IN THE ICU.M. Muir,* S. Cohn, and J. Baseman*. University of Texas Health Science Center, San Antonio, TX 78229

We used novel assays targeting the Community Acquired Respiratory Distress Syndrome (CARDS) toxin, a unique virulence factor of Mycoplasma pneumoniae, to determine the incidence of this organism among ICU patients. Our hypothesis was that M. pneumoniae would be frequently detected in ventilator-dependent ICU patients using CARDS toxin assays. We conducted a prospective observational study enrolling mechanically ventilated subjects undergoing bronchoscopy with bronchoalveolar lavage (BAL) in the Surgical Trauma ICU at a Level 1 Trauma Center. BAL fluid and serum were collected from each subject and tested for the presence of M. pneumoniae using the CARDS toxin assays. We analyzed the BAL fluid using PCR and antigen capture to detect CARDS toxin DNA and protein, respectively. We also tested for anti-CARDS toxin antibodies in serum. BAL and serum samples from 37 subjects were analyzed, with 15 of 37 (41%) testing positive for M. pneumoniae using CARDS toxin assays. These results are summarized in Table 1. M. pneumoniae is detectable in a significant proportion of ICU subjects. While PCR is the most sensitive solitary assay, a panel of tests targeting CARDS toxin is more sensitive for the detection of this pathogen than any single assay. Further investigation is needed to identify the clinical implications of M. pneumoniae in this population.

Summary of results, by assay type, for the 15 subjects testing positive for CARDS toxin, classified by PCR-positive and PCR-negative status.


INTRATRACHEAL TAT-HSP INCREASES HSP-70 ABUNDANCE IN THE LUNGS FOLLOWING CECAL LIGATION AND DOUBLE PUNCTURE (2CLP).M.M. Lyons,* A. Ruggieri,* N.R. Raj,* and C.S. Deutschman. University of Pennsylvania, Philadelphia, PA 19104

Introduction: The Heat Shock Response (HSR) is a highly conserved endogenous mechanism that protects cells from injury. The 70 kD HSP70 in particular is increased in response to many different types of noxious stimuli. 2CLP in rats causes lung injury, with neutrophil accumulation and histologic changes consistent with ARDS. Previous work has shown that 2CLP reduces or eliminates HSP70 from the lungs. In the past we have used an adenoviral vector to increase HSP70 in lung tissue after 2CLP. This protected rats from lung injury. The HIV TAT peptide may be an alternative method to increase HSP70 abundance after 2CLP.

Hypothesis: Intra-tracheal administration of TAT-HSP will increase HSP70 abundance in lung tissue following 2CLP in rats.

Methods: 50 to 100μg of TAT-HSP70 was administered into the trachea of male Sprague-Dawley rats at the time of 2CLP. Tissue was harvested 24 and 48 hours post-2CLP. Controls consisted of unoperated animals (TO), animals subjected to sham operation (SO) and given either intra-tracheal PBS (SO-P) or TAT-HSP and 2CLP rats receiving PBS. Immunoblotting was performed on lung homogenate and fixed tissue was examined for histologic evidence of lung injury and neutrophil accumulation.

Results: Relative to T0, SO and PBS-treated 2CLP rats, 2CLP treated rats exhibited increased HSP70 abundance in lung tissue. Further, 2CLP rats given TAT-HSP had less neutrophil accumulation than PBS-treated 2CLP rats.

Conclusion: Intratracheal administered of TAT-HSP is a novel delivery mechanism. TAT-HSP may be a useful treatment for lung injury secondary to 2CLP.


ROLE OF CASPASE-3 IN REGULATING MICROVASCULAR ENDOTHELIAL CELL HYPERPERMEABILITY.B. Tharakan, D. Sawant,* F. Hunter,* and E. Childs. Department of Surgery, Texas A&M Health Science Centre College of Medicine and Scott and White Memorial Hospital, Temple, TX 76508

Objective: Microvascular hyperpermeability occurs due to the disruption of the endothelial adherens junction complex. Studies from our laboratory have demonstrated the role of apoptotic signaling in microvascular hyperpermeability. β-Catenin which is an integral component of the adherens junction complex is cleaved by caspase-3 during apoptotic signaling. The dynamics of β-catenin following caspase-3 activation and how cell-cell contacts are re-established are not known. Our objective was to determine how β-catenin recovers and re-establish the barrier integrity following cspase-3 activation.

Methods: Rat lung microvascular endothelial cell (RLMEC) monolayers were transfected with pro-apoptotic BAK peptide (5μg/ml) for 0-4 hrs. BAK is an inducer of caspase-3 activation. FITC-albumin flux across the monolayer was measured as an indicator of hyperpermeability. The recovery of β-catenin following caspase-3 activation was studied by transfecting RLMEC with active caspase-3 (5μg/ml) for 0-4 hrs followed by immunofluorescence of β-catenin. Role of protein synthesis in β-catenin recovery was tested by transfecting RLMEC with active caspase-3 (0-4 hrs) after actinomycin D (Act D; transcription blocker) or cycloheximide (CHX; translation blocker) treatment.

Results: Transfection of RLMEC with BAK induced hyperpermeability followed by its recovery at 3 hrs (p<0.05). Active caspase-3 induced significant loss of β-catenin at 1 hr and 2 hrs followed by its recovery at 3 hrs. Act D or CHX did not affect the recovery of β-catenin.

Conclusion: Caspase-3 induces disruption of β-catenin leading to microvascular hyperpermeability. β-Catenin time dependently recovers following caspase-3 activation and the recovery is not due to newly synthesized β-catenin.


HYDROGEN SULFIDE INCREASES ENDOTHELIAL NITRIC OXIDE SYNTHASE ACTIVITY IN RAT SINUSOIDAL ENDOTHELIAL CELLS.A. Blade,* S. Larion,* E. Norris,* C. Culberson,* and M.G. Clemens. Department of Biology, The University of North Carolina at Charlotte, Charlotte, NC 28223-0001

Hydrogen sulfide (H2S) is a gaseous mediator that is synthesized endogenously from L-cysteine via cystathionine gamma-lyase (CSE) which is induced in inflammation. H2S may have vasorelaxant effects on vascular smooth muscle by acting on potassium channels (KATP) but has also been suggested to be a vasoconstrictor. While the mechanism of H2S is not as well known as other endogenous gases it has been suggested that H2S may interact with nitric oxide (NO). Since NO production by sinusoidal endothelial cells is an important determinant of liver blood flow regulation in shock, the aim of our study was to test whether H2S affects endothelial nitric oxide synthase (eNOS) activity in primary rat liver sinusoidal endothelial cells (rLSECs). Treatment with hydrogen sulfide (50 μmol/l) resulted in a significant increase of eNOS activity as measured by production of L-citrulline. This effect was similar in magnitude to the effect of endothelin-1, a known activator of eNOS. Combined stimulation with ET-1 and H2S increase eNOS activity only slightly compared to either alone. Since H2S has been shown to hyperpolarize cells by activation of KATP channels, we tested whether this might increase eNOS activity by enhancing L-arginine transport. Accumulation of 3H-L-arginine was inhibited by the L-arginine analog L-NAME, but was not different between control and H2S treatment. These data indicate that H2S up-regulates the eNOS activity in sinusoidal endothelial cells. The mechanism appears to be independent of enhanced -arginine transport. This effect may be important in regulating liver microcirculation during shock. (Supported by DK38201 and ARRA supplement).


EXOGENOUS HYDROGEN SULFIDE INFUSION IS ASSOCIATED WITH HEPATIC SINUSOIDAL VASOCONSTRICTIONIN VIVO.E. Norris,* G. Aoyagi,* C. Culberson,* and M. Clemens. Department of Biology, University of North Carolina at Charlotte. Charlotte, NC 28223

An expanding body of evidence suggests that hydrogen sulfide (H2S) is a critical mediator in maintaining vascular tone and may be upregulated in inflammation. H2S activates KATP channels causing vasodilation, but also is a putative scavenger of nitric oxide. Our lab has shown that H2S augments the activity of Nitric Oxide Synthase (NOS) while others reported that intravenous infusion of the H2S donor, Sodium Hydrosulfide (NaSH) increased mean arterial blood pressure suggesting vasoconstriction. This suggests complex responses in different vascular beds. However, the effect of H2S on hepatic sinusoids is not known. Therefore, we investigated the vasoactive properties of exogenous H2S on the liver sinusoids in vivo using intravital microscopy in a mouse model. We observed a significant increase in sinusoidal diameter fluctuations (p<.01, N=4) after 5 minute infusion of NaSH (10μmoles kg-1 min-1) as compared to saline controls. Additionally, our analysis of FITC-labeled red blood cells revealed the development of a significant amount of heterogeneity as evidenced by a doubling in the standard deviation of red cell velocities in individual sinusoids between the experimental group and the control group. Our findings demonstrate a net direct vasoconstrictive effect of H2S on the hepatic sinusoids. However, this was associated with dilation of some sinusoids leading to an increased heterogeneity of microvascular flow such has been shown to occur in various shock-related conditions. These findings demonstrate that the role of hydrogen sulfide in hepatic microvasculature is complex, likely involving multiple pathways in different vascular cells. We propose that this complex response contributes to the heterogeneous microvascular perfusion observed in shock. (Supported by DK38201 and ARRA supplement).


RESTITUTION OF SYNDECAN-1 IS MODULATED BY AGE OF FRESH FROZEN PLASMA (FFP).Z. Peng,* R. Haywood-Watson, S. Pati, J. Dong,* V. Vijayan,* J.B. Holcomb, T. Ko, and R.A. Kozar. University of Texas, Houston, TX 77030

We have shown that fresh (day 0) plasma (FFP) preserves endothelial cell (EC) permeability better than aged (day 5) FFP. Here, we evaluated the effect of age in restitution of syndecan-1 (sdc-1), a cell surface heparan sulfate proteoglycan, important to membrane permeability.

Methods: HUVEC's were treated for 2 hrs with day 0 or day 5 FFP or media alone under control conditions or after heparinase (a sdc-1 shedding enhancer, 10 U) then stained with anti sdc-1 Ab.

Results: Cells incubated in day 0 or day 5 FFP demonstrated enhanced expression of sdc-1 on the surface of HUVECs compared to media alone, but expression was greater after day 0 than day 5 under both control and heparinase treatment conditions.

Conclusion: Fresh plasma reconstitutes EC sdc-1 expression and may explain the protective properties of plasma in maintenance of EC permeability.




Heme oxygenase-1 (HO-1) is a shock protein that degrades heme to form iron, biliverdin and carbon monoxide (CO). We recently have shown that hemorrhagic shock can drive heme oxygenase activity to increase vascular tone preferentially at low pressures, and that this effect arises in an endothelium independent manner. While we have shown that HO activity can limit vasodepression and delay circulatory collapse, the specific product(s) responsible for this effect were unknown.

Objective: Determine which HO product(s) promote increased vascular myogenic tone preferentially at lower pressures.

Methods: Male Sprague-Dawley rats were used to harvest 1st order gracilis muscle arterioles that were mounted in a microvessel chamber. Vessel luminal diameters were measured in response to decreased intraluminal pressures (80-20mmHg, no flow); subsets of vessels were treated with iron (2μM), biliverdin (15μM) or CO (10μM, luminal). To identify effects arising in the vascular smooth muscle, the series was repeated using vessels denuded of endothelium.

Results: In response to decreased intraluminal pressures, intact vessels (N=34) treated with iron or CO displayed enhanced constriction (alpha= 0.05) at pressures below 40 mmHg (min diameter 133±5, 113±1 and 114±4μm; Veh, CO and iron); biliverdin had no effect. In vessels denuded of endothelium (N=32), CO still promoted enhanced tone with respect to vehicle controls (117±1 vs 141±5μm, respectively), but neither iron nor biliverdin had an effect.

Conclusions: When hemorrhagic loss drives HO activity to selectively heighten vascular tone at reduced pressures, it is largely an effect of endogenously formed carbon monoxide acting directly on vascular smooth muscle.


HOST RESPONSE TO INJURY: THE ROLE OF MITOCHONDRIAL DAMPS IN ACTIVATION OF PBMC.Q. Zhang, K. Itagaki,* and C. J. Hauser. Harvard Medical School and Beth Israel Deaconess Medical Center, Boston MA 02215

Background: In our recent studies, we have demonstrated that trauma and injury cause tissue damage and subsequent release of endogenous damage-associated molecular patterns (DAMPs), such as mitochondrial formyl peptides (mtFP) and mitochondrial DNA (mtDNA). Both mtFP and mtDNA activate neutrophils and contribute to post-trauma Systemic Inflammatory Response Syndrome (SIRS). However, it is unknown whether mitochondrial DAMPS activate monocytes. The monocytes are the main source of cytokines, which play key roles in the network of immune reactions. We hypothesized that mitochondrial DAMPs (mtFP and mtDNA, which have the similarity with bacteria), might activate monocytes and induce cytokines release, potentially contributing to SIRS.

Methods: Human peripheral blood mononuclear cell (PBMC) were isolated and incubated for 4 hours, then subjected to wash with medium. The adherent cells (mainly monocytes) were further incubated with human liver-derived mtFP and mtDNA overnight. PBMC activation was assessed as IL-6 release by ELISA.

Results: Both mtFP and mtDNA induced IL-6 release in PBMC in a dose-dependent manner.

Conclusions: mtFP and mtDNA activate PBMC, subsequently lead to release of IL-6. Mitochondrial DAMPs stimulate innate immunity and play important roles in the genesis of SIRS after tissue trauma.



NEUROLOGICAL OUTCOME FOR UNCONSCIOUS POST-CARDIAC ARREST SYNDROME PATIENTS CAN BE PREDICTED BY BISPECTRAL INDEX.M. Aibiki, S. Kikuchi, K. Umakoshi, M. Ohshita, H. Matsumoto, S. Ohtsubo, and T. Nishiyama. Ehime Univ Hosp, Dept of Emerg Med, Shitsukawa 454, Tohon, Ehime, 791-0295, Japan

Introduction: The prediction of neurological outcome is crucial but challenging in unconscious post-cardiac arrest syndrome (PCAS) patients. Therefore, we require a reliable predictor for neurological prognosis in PCAS patients in such situations.

Methods: In consecutive unconscious twenty-five PCAS patients, using a BIS XP (a portable bed-side monitor), we examined the time-course changes in bispectral index value (BIS: an index combining EEG with EMG) and suppression ratio (SR: a ratio of cortical silence in the EEG) within 72 hours after admission. Statistical analysis was done by Mann-Whitney U-test. Specificity and sensitivity for BIS values and SR ratios as outcome predictors were also examined.

Results: PCAS patients regaining consciousness had much higher BIS values and lower SRs as compared to those with poor prognosis when no EMG signals were obtained after administration of muscle relaxant. BIS value and SR at certain levels had high sensitivity and specificity enough to predict not only poor, but also good neurological outcome of PCAS patients (Table).

Conclusions: BIS value and SR are reliable parameters predicting neurological outcome in unconscious PCAS patients. However, we should be careful on determining the prognosis of PCAS patients at the early phase, especially within 24 hrs after admission.



REFRACTORY POSTINJURY THROMBOCYTOPENIA IS ASSOCIATED WITH MULTIPLE ORGAN FAILURE AND ADVERSE OUTCOMES.T. Nydam,* J. Kashuk, E. Moore, J. Johnson, C. Burlew,* C. Barnett,* and A. Sauaia. Denver Health Medical Center and University of Colorado, Aurora Denver, CO 80204

Background: Postinjury multiple organ failure (MOF) remains the leading cause of morbidity and late mortality after severe trauma. Our previous work consistently identified an association between thrombocytopenia and progression to MOF. Additionally, recent studies suggest that platelets play a critical role in postinjury hyperinflammation. Therefore, we hypothesized that postinjury thrombocytopenia is a marker for progression to MOF.

Methods: 1,415 critically injured SICU patients surviving >48 hrs were prospectively collected over 12 years. Variables studied included age, ISS, RBC/12 hrs, MOF (Denver MOF score), death, infectious complications, and non-infectious complications. Thrombocytopenia was defined as platelets <80K. Logistic regression was applied to identify independent predictors of MOF and death.

Results: Mean ±SEM ISS, age, and RBC were: 29.3 ±11.3; 37.4±16.6 years; and 4.4±5 units. MOF developed in 346 (24%) patients and 118 (8%) died. Thrombocytopenia occurred in 35% of patients within 48 hours postinjury and was associated with a significant increase in ISS, RBC transfused, and age. Logistic regression confirmed that thrombocytopenia was a major independent risk factor for all adverse outcomes with an odds ratio of 2.4 for developing MOF and 3.4 for death. After adjustment for these factors, a relative increase in platelet count from day 3 to day 10 was associated with a significantly lower likelihood of MOF and death.

Conclusion: Early postinjury thrombocytopenia is an independent risk factor for MOF, death and other complications. Following platelet count dynamics over the first several days postinjury can help predict which high risk patient will develop these adverse outcomes.


LIPOPOLYSACCHARIDE DIRECTLY INHIBITS CALCIUM OSCILLATIONS AND THE HYPERPOLARIZATION-ACTIVATED, NON-SELECTIVE CATION CURRENTIf IN HL-1 CARDIOMYOCYTES.R. Wondergem,* B. Graves,* T. Ozment-Skelton, C. Li, and D. Williams. Departments of Physiology and Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

Lipopolysaccharide (LPS) has been implicated in endotoxin-mediated heart failure and chronic cardiac myopathy. We determined whether LPS directly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration, [Ca2+]i, in HL-1 cardiomyocytes. In HL-1 [Ca2+]i oscillated (<0.4 Hz) with slow and transient components. LPS (1 μg/ml), derived from E. coli or S. enterica, reversibly abolished Ca2+ oscillations and decreased basal [Ca2+]i by 30-40 nM. HL-1 expresses TLR2 and TLR4. We differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of ultra-pure LPS, a TLR4 ligand. Ultrapure LPS had no effect on basal [Ca2+]i, but it reduced the rate of Ca2+ oscillations. In contrast, Pam3Csk4, did not affect either Ca2+ parameter. When LPS and Pam3Csk4 were co-administered the effects were similar to that of ultra-pure LPS alone. Since others have shown that LPS impairs pacemaker current, If, which is expressed in HL-1 cells, we utilized whole-cell voltage clamp techniques to demonstrate that LPS (1 μg/ml) reduced the hyperpolarization-activated, non-selective cationic current, If, in HL-1 cells. However, this inhibition was significant only at very negative potentials (p < 0.05 at −150 & −160 mV). Thus, we evaluated effects of LPS on fully-activated If. Here, LPS reduced the non-selective cationic current over a range of voltages ranging from −60 to 40 mV. We conclude that LPS directly impairs regulation of [Ca2+]i in cardiomyocytes. The primary action of LPS appears to be on If although effects on other pacemaker currents cannot be ruled out.


STORED PRBCS CAUSE ALI AS THE FIRST EVENT IN A TWO-EVENTIN VIVOMODEL.C. Silliman, M. Kelher,* E. Moore, and S. Khan*. Bonfils Blood Ctr., Denver, CO 80230

Objective: Transfusion of stored PRBCs has been linked to increased morbidity for injured patients. Transfusion of stored (>21 days) PRBCs is also an independent risk factor for the development of multiple organ failure (MOF) in patients with 35>ISS>15 who have been transfused >6 units in the first 12 hours. We hypothesize that transfusion of older stored PRBCs activates human pulmonary microvascular endothelial cells (HMVECs) and serves as the first event in a two-event model of ALI.

Methods: Leukoreduced PRBCs were drawn and stored per AABB criteria for 42 days at 4°C. Serial samples were drawn throughout storage, and the plasma was isolated by centrifugation. HMVECs were incubated with 10% (v:vFINAL) of fresh (day 1) vs. stored (day 42) PRBC plasma and HMVEC activation was quantified by the surface expression of intercellular adhesion molecule-1 (ICAM-1) by flow cytometry, chemokines via ELISA, and adherent neutrophils (PMNs) by MPO assays. Rats were injected with 10% PRBC plasma (1st event) followed by Evans Blue dye (EBD), incubated for 6 hours, and IV LPS (100 μg/kg) or saline (NS) were given as the 2nd event. ALI was quantified by EBD leak.

Results: Day 42 plasma induced increases in ICAM-1 (MFI, Day 42: 34±5* vs. D1: 10±2, n=6) and the release of chemokines: IL-8, growth related oncogene-α (GROα, and epithelial neutrophil activating factor-78 (ENA-78) vs. day 1 (Table). Day 42 plasma increased MPO 12.1±1.0% vs. day 1: 4±1% (p<.05). Day 42 plasma also elicited ALI as the first event in the two-event model while day 0 plasma did not (Table).

Conclusions: We conclude that plasma from stored, but not fresh, PRBCs induced HMVEC activation and served as the first event in a two-event in vivo model of ALI.



EFFECT OF AEROSOLIZED HYPERTONIC SALINE ON LUNG INJURY FOLLOWING HEMORRHAGIC SHOCK.M. Wohlauer,* E. Moore, J. Eun,* F. Wright,* E. Peltz,* M. Fragoso,* F. Gamboni-Robertson,* and A. Banerjee. Univ. of Colorado at Denver, Aurora, CO 80045

Objective: Intestinal ischemia and reperfusion play a central role in acute lung injury (ALI) and subsequent multiple organ failure (MOF) following hemorrhagic shock. Intravenous hypertonic saline (HTS) modulates inflammation and can attenuate ALI/MOF, however, lung-directed HTS therapy may be less prone to systemic complications and has not yet been investigated. We hypothesized that inhaled, aerosolized hypertonic saline therapy given at the onset of resuscitation will decrease acute lung injury following hemorrhagic shock.

Methods: Rats are placed on a T-piece (FI02 0.4, 2 LPM) at the start of hemorrhagic shock (MAP 30 mmHg × 45 min to achieve a base deficit greater than 20). Hypertonic saline (7.5% NS) is delivered via jet nebulizer (MiniHeart Hi-Flo Nebulizer. Westmed, Tucson, AZ) at a flow rate of 1-2 LPM for 15 min at the start of resuscitation, and at 1 hr and 2 hr of resuscitation, respectively. Upon infusing a lethal dose of pentobarbital, a bronchoalveolar lavage was performed to measure lung vascular permeability using Evans blue dye (3 hours postshock).

Results: Hemorrhagic shock caused marked lung permeability (5.41 ± 0.72, N=5), which was attenuated with administration of aerosolized 7.5% hypertonic saline (2.93 ± 0.37, N=5).

Conclusion: Hypertonic saline nebulizer administration during resuscitation attenuates acute lung injury following experimental trauma/hemorrhagic shock. A clinical investigation to validate organ-directed hypertonic therapy for acute lung injury in the trauma population is needed.



HMGB1 CONTRIBUTES TO THE DEVELOPMENT OF LIVER INJURY AFTER HEAT STROKE.T. Kawasaki, C. Kawasaki,* K. Okamoto,* and T. Sata*. University of Occupational and Environmental Health, Kitakyushu, JAPAN 8078555, and Sugioka Memorial Hospital, Fukuoka, JAPAN 8130017

Objective: High mobility group box-1 protein (HMGB1), recently identified as a lethal late-phase mediator, closely correlated with the development of sepsis. Although heat stroke resembles sepsis in many aspects, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. The purpose of this study was to investigate the contribution of HMGB1 to development of liver injury in experimental heat stroke.

Methods: Male C3H/HeN (8-10 weeks) mice were randomly assigned to heat stroke group (HS) or normothermic control group (NC). Heat stroke was induced by exposure to ambient temperature of 42°C for 60 mins. The heat-stressed mice were returned to room temperature (25°C) after the end of the heat exposure. After heat stress, the levels of cytokines (TNF-alpha and IL-6), plasma HMGB1 concentrations, liver dysfunction; plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations, and immunohistochemical and histopathological characteristics of liver were determined.

Results: In HS, plasma TNF-alpha and IL-6 increased significantly, peaking at 2 hr after heat stress (p<0.05, ANOVA). Plasma HMGB1 concentration also increased in HC, peaking at 12 hr after heat stress (p<0.05, ANOVA). In NC, plasma cytokines and HMGB1 did not increase at any time point assayed. Plasma AST and ALT concentrations increased at 12 hr after heat stress (p<0.05, ANOVA). In HS, strong and extensive immunostaining for HMGB1 was observed. In addition, there was extensive hepatocellular necrosis and collapse of nuclei observed in HS.

Conclusion: This study demonstrated that HMGB1 contributes to the development of liver injury after heat stroke. To regulate the expression of HMGB1 may be a beneficial treatment for heat stroke patients.


LEUKOCYTE ESTROGEN RECEPTOR EXPRESSION AND PLASMA ESTROGEN LEVELS ARE ASSOCIATED WITH TREATMENT OUTCOME IN PATIENTS WITH SEVERE ACUTE PANCREATITIS.C.H. Hsieh,* C.W. Lu,* Y.C. Hsieh,* and I.H. Chaudry. Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294

Severe acute pancreatitis (SAP) is a complex pathologic state with a high mortality rate. Continuous regional arterial infusion (CRAI) of protease inhibitors and antibiotics has been shown to be therapeutically useful for SAP. While plasma estrogen (E2) levels were shown to be associated with mortality in critically injured patients, it remains unknown whether they are also associated with patient outcome in SAP. We hypothesized that plasma E2 levels can be used as a predictor of treatment outcome in SAP, and are related to estrogen receptor (ER) expression. In 2009, 43 consecutive patients diagnosed as SAP received standard treatment while additional CRAI therapy was given to the last 8 patients. Disease grading, complications and patient outcome, and plasma levels of sex hormones were recorded. We found that plasma levels of testosterone, progesterone and follicular stimulating hormone did not correlate with disease severity; however, elevated E2 levels at admission correlated with increased risk of infection and higher mortality. Also, patients with successful CRAI therapy had gradually decreasing E2 levels. In contrast, leukocyte ER mRNA expression in these patients was significantly decreased at admission, but was restored to levels of healthy control group if CRAI therapies were effective. Our results suggest that E2 levels can be used as a prognostic predictor in SAP patients; the underlying mechanism could be a feedback response due to the suppression of ER expression by SAP (NIH R01 GM37127).



TROPISM, CYTOTOXICITY, AND INFLAMMATORY PROPERTIES OF TWO ENVELOPE GENES OF MURINE ENDOGENOUS RETROVIRUSES OF C57BL/6J MICE.Y. Lee,* A. Chew,* D.G. Greenhalgh, and K. Cho. Shriners Hospitals for Children Northern California and Department of Surgery, University of California, Davis, Sacramento, CA 95817

Endogenous retroviruses (ERVs) constitute approximately 8% and 10% of the human and mouse genomes, respectively. Certain ERVs envelope (env) proteins are reported to participate in a range of pathophysiological processes, such as placental morphogenesis and neuronal degeneration. In this study, we characterized pathophysiologic properties of two murine ERV (MuERV) env genes, which were cloned from the normal ovary during a survey of MuERV expression profile in C57BL/6J mice. The two env subgenomic genes (named ENVOV1 and ENVOV2) with 1,926 bp coding region originated from two MuERV loci on chromosomes 8 and 18, respectively. They share 98% identity at amino acid level and ENVOV1 has not been reported previously. Overexpression study revealed that ENVOV1 and ENVOV2 were ∼75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions using a retroviral packaging system. Tropism trait and infectivity of ENVOV2 were similar to the amphotropic env controls; however, ENVOV1 had very low level of infectivity for both cells of mouse and non-mouse origins. In addition, it was evident that overexpression of ENVOV2, but not ENVOV1, exerted cytotoxic effects to HeLa cells. Similarly, ENVOV2, but not ENVOV1, substantially induced expression of COX-2, IL-1β, IL-6, and iNOS in RAW264.7 alveolar macrophage cells while no significant changes in ICAM-1 and TNF-α levels were detected by the ENVOV2. The findings from this study suggest that the MuERV ENVOV1 and ENVOV2 are capable of serving as an env protein for assembly of retroviral particles, and they exert differential cytotoxicity and modulation of inflammatory mediators.


PREVALENTDE NOVOSOMATIC MUTATIONS IN SUPERANTIGEN GENES OF MOUSE MAMMARY TUMOR VIRUSES IN THE GENOME OF C57BL/6J MICE AND ITS POTENTIAL IMPLICATION IN IMMUNE SYSTEM.S. Chiu,* Y. Lee,* A. Chew,* D.G. Greenhalgh, and K. Cho. Shriners Hospitals for Children Northern California and Department of Surgery, University of California, Davis, Sacramento, CA 95817

Superantigens (SAgs) of mouse mammary tumor viruses (MMTVs) play a crucial role in T cell selection in the thymus in a T cell receptor (TCR) Vβ-specific manner and SAgs presented by B cells activate T cells in the periphery. The peripheral T cell repertoire is dynamically shaped by the steady induction of T cell tolerance against self antigens throughout the lifespan. We hypothesize that de novo somatic mutation of endogenous MMTV SAgs contributes to the modulation of the peripheral T cell repertoire. The SAg coding sequences were cloned from the genomic DNAs and/or cDNAs of various tissues of C57BL/6J mice (normal as well as stressed). A total of 68 unique SAg sequences (54 translated sequences) were identified from the genomic DNAs of normal mice, which harbor only three endogenous MMTV loci (Mtv-8, Mtv-9, and Mtv-17). Similarly, 69 unique SAg sequences (58 translated sequences) were cloned from the cDNAs of normal mice. cDNAs from the bone marrows of no burn and burn mice yielded 128 unique SAg sequences (100 translated sequences); however, no significant difference in SAg profiles between no burn and burn groups was observed. Examination of TCR Vβ specificity revealed that a substantial percentage of the putative SAg isoforms may have Vβ specificities different from SAgs of Mtv-8, Mtv-9, or Mtv-17. The pool of diverse SAg isoforms, generated by de novo mutation, with altered TCR Vβ specificity of affinity may contribute to the shaping of the peripheral T cell repertoire, including the autoimmune T cell population.



Background: As an assay for endotoxin (etox), the Limulus amebocyte lysate assay (LAL) has several desirable properties: sensitivity, specificity, and potential for adaptation to a quantitative format. The chromogenicmodifications allow quantitative measurement of etox and also improve its application to blood samples for detecting etox translocation.

Material and Method: Plasma was collected during and after major abdominal- (n=50), thyroid- (n=20), heart surgery (n=60) and colonoscopy n=20). Plasma was assessed for etox and the endotoxin neutralizing capacity (ENC), 10 healthy volunteers, were sampled in the same manner as a control group.

Results: A significant increase in plasma levels of etox and a decrease of ENC were found in the major abdominal and cardiac surgery groups following induction of anesthesia and in thyroid surgery after skin incision. No chance was found in the control group. During endoscopy increased endotoxemia was only detected when comparing the plasma levels before with the highest levels during endoscopy. The timed etox plasma levels did not change significantly by use of the conventional LAL test. However, ENC was found to diminish significantly 5 min after the onset of endoscopy with maximal values at the end which recovered completely 24 h later. These results, (patients did not receive any infusions) demonstrate that the half life of etox in the circulation seems to be short and therefore etox cannot itself be detected. But small amounts of etox reaching the blood stream are capable to decrease ENC which can be analyzed by a modified LAL test.

Conclusion: With the use of ENC and plasma endotoxin determinations, we are able to show significant endotoxemia even during a minimal invasive procedure such as colonoscopy.


EFFECT OF CHANGES IN DIETARY NITRITE ON VENTILATION.D. Rayburn,* S. Byzek,* I. Gist,* and J. Atkins. Walter Reed Army Institute of Research, Silver Spring, MD 20910

Recent studies have identified several proteins that display nitrite reductase activity at low oxygen tensions, converting nitrite to nitric oxide (NO) (Lundberg et al, Nat Chem Biol 5(12):865, 2009). Dietary nitrate is converted to nitrite by the commensal bacteria of the mouth. NO production in the brain influences ventilation and increased ventilation is an important initial adaptive response to hypoxia. We sought to determine if changes in dietary nitrite could influence the hypoxic ventilatory response. Sprague-Dawley rats were placed on a low nitrite/nitrate diet for seven days and some of the animals had nitrite or nitrate added to their drinking water. Conscious animals were placed in a plethysmograph breathing room air. After a 30 minute equilibration time, the gas flowing into the box was changed to 10% oxygen balance nitrogen. Minute ventilation was calculated from changes in pressure, humidity and temperature in the box (Buxco). Initial studies measuring hypoxic response every day demonstrated a gradual decline. We therefore measured ventilatory response after 7 days on diet without intervening measurements. Following this protocol, minute ventilation increased in all animals, but contrary to our expectations the ventilatory response tended to be greatest in the low nitrite/nitrate animals (372±73 SEM, n=5) as compared to those receiving nitrate supplementation (147±13, n=5) or nitrite supplementation (220±56, n=5) (Anova ≤0.03; Holm-Sidak p≤ 0.02 diet vs nitrate). These results suggest that diets low in leafy vegetables (the major dietary source of nitrate) would increase the hypoxic ventilatory response. (Supported by a Grant from the Office of Naval Research #42237).


CD11C+ DENDRITIC CELLS MODULATE THE LOCAL INFLAMMATORY RESPONSE AND HOST DEFENSE TO CUTANEOUSSTAPHYLOCOCCUS AUREUSINFECTION.K. Kelly-Scumpia,1* P. Scumpia,1* A. Cuenca,1* L. Miller,2* and L. Moldawer1. University of Florida Gainesville, FL 326101, UCLA Los Angeles, CA 900952

Staphylococcal wound infections, including cellulitis and abscesses are a major source of morbidity in critically ill patients. Many host factors contribute to cutaneous host defense against bacterial pathogens including antimicrobial peptides, TLR activation, local keratinocyte or mast cell activation, and recruited neutrophils. Although dendritic cells (DCs) are vital for allergic skin reactions, their role in S. aureus skin infection is currently unknown. Here, using CD11c-Diphtheria toxin receptor transgenic mice (CD11c-DTR) to deplete DCs, we show that CD11c+ DCs play an integral part in local host defense to S. aureus. We find that at 24 and 72 hrs following subcutaneous injection of S. aureus, bacterial colonization of mice depleted of DCs is increased 3-fold and 1.5-fold, respectively. Depletion of CD11c+ DCs results in decreased concentrations of IL-1β and and IL-6, but not TNF-α in the skin wound at 24 and 72 hours. Interestingly, depletion of CD11c+ cells did not affect recruitment of neutrophils histologically or neutrophil activity, as measured by myeloperoxidase activity. These data suggest that skin CD11c-expressing DCs are essential for local skin immunity to S. aureus by regulating the local microenvironment, and not by regulating neutrophil chemotaxis or activity. Augmenting cutaneous DC function following cutaneous injury may prove effective as an adjunct to prevent staphylococcal infections.


IDENTIFICATION OF HUMAN ENDOGENOUS RETROVIRUS VIRION RNAS IN TRANSFUSION BLOOD.H. Rah,* K. Cho, D. Greenhalgh, and T. Palmieri. Department of Surgery, University of California, Davis and Shriners Hospitals for Children Northern California, Sacramento, CA 95817

Background: Allogeneic blood transfusion, which can be life-saving, has been linked to immunosuppresion. Human endogenous retroviruses (HERVs) constitute approximately 8 % of the human genome and have been associated with a range of pathogenic processes. One potential causative agent responsible for transfusion-associated immunosuppression is HERVs. The purpose of this study was to determine if HERVs exist in allogeneic packed red blood cell (PRBC) transfusion blood.

Methods: Allogeneic PRBC transfusion blood was separated into cells and plasma, and viral particles were isolated from the plasma after ultracentrifugation followed by viral RNA extraction. The genomic RNAs for 21 different HERV families were surveyed by RT-PCR amplification of the respective LTR regions.

Results: The viral genomic RNAs, which were presumed to be derived from the HERV-E and HERV-R virions were detected in the transfusion blood examined. Ten unique LTR sequences were isolated from the viral genomic RNAs. A survey of the NCBI human genome database using the LTR sequences as a probe identified five putative HERVs (four from HERV-E family and one from HERV-R family). Evaluation of coding potential for retroviral polypeptides for each putative HERV revealed that the provirus isolated using the HERV-R probe has an intact env coding sequence.

Conclusions: The preliminary findings from this study provide evidence suggesting that virus particles from certain HERVs exist in the PRBC transfusion blood. These HERV particles may be transmitted to the recipient during blood transfusion. It may be important to confirm the HERV transmission to the transfusion recipient by further investigations.



Sepsis-induced muscle atrophy is produced in part by a decrease in protein synthesis mediated by via inhibition of mTOR kinase activity. The purpose of the present study was to ascertain alterations in protein-protein interactions within the mTORC1 which might contribute to such a defect. Cecal ligation and puncture in male rats decreased in vivo translational efficiency in gastrocnemius after 24 h, and reduced the phosphorylation of 4EBP-1, S6K-1 and mTOR, compared to time-matched pair-fed control values. Sepsis coordinately decreased T308- and S473-phosphorylated Akt and T246-phosphorylated PRAS-40, and reciprocally increased S792-phosphorylated raptor in muscle. Despite the change in PRAS40 phosphorylation, sepsis did not alter its binding to raptor. Furthermore, sepsis did not alter the amount of the mTOR-raptor complex. In contrast, the binding and retention of both 4E-BP1 and S6K1 to raptor was increase in sepsis. Finally, there was a sepsis-induced decrease in the binding of raptor with eIF3. These changes in mTORC1 protein-protein interaction were associated with enhanced AMPK activity (e.g., increased AMPK and ACC phosphorylation) which appeared TSC-independent. Overall, our data suggest that the sepsis-induced decrease in muscle protein synthesis is mediated, at least in part, by the inability of 4E-BP1 and S6K1 to be phosphorylated and released from mTORC1 as well as decreased recruitment of eIF3 which is necessary to form a functioning 48S complex. (Supported by GM38032).


PCR-BASED DISCRIMINATION OF SEPSIS FROM SIRS.K. Itagaki,* D. Mu,* Q. Zhang, T. Sursal,* and C.J. Hauser. Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215

Optimal clinical care of sepsis due to bacterial infection and of SIRS due to trauma is markedly different. Telling SIRS from sepsis may be difficult since bacterial DNA (bDNA) released by infection and mitochondrial DNA (mtDNA) released by shock or injury can each activate innate immune cells via TLR9. We hypothesized that assays of the relative abundance of bDNA and mtDNA in plasma would reflect the relative contributions of sterile (endogenous) mediators and septic (exogenous) mediators to systemic inflammation. Primers were thus synthesized that recognized consensus bacterial 16S-RNA sequences shared by Gram (+), Gram (-) and anaerobic bacteria but that did not recognize mtDNA. Similarly, we synthesized primers that recognized mtDNA (CytB) but that did not recognize bDNA. We then used these primers to perform qPCR on genomic bDNA from Staph. Aureus, E. coli and B. Fragilis as well as on mtDNA from human liver. The 16S primers recognized all three types of bDNA equally with a threshold concentration of ∼1 fg/mL (Figure1). mtDNA was not recognized even at 1 μg/mL. Conversely, CytB primers recognized mtDNA at <1 fg/mL but fail to recognize bDNA at up to 1 ng/mL (Figure 2). These methods will now be studied prospectively to determine whether patients can be assigned to early antibiotics vs. early SIRS modification therapies on the basis of bDNA / mtDNA abundance.

FIG. 1
FIG. 1:
16S primers.
FIG. 2
FIG. 2:
Cyt-B primers.


SYNERGISTIC EFFECTS OF POLYETHYLENE GLYCOL AND HEPARIN ON ENTEROCYTE INTERACTIONS WITHSTAPHYLOCOCCUS AUREUS.M. Henry-Stanley, D. Hess, M. Shepherd,* M. Sivertson,* and C. Wells. University of Minnesota, Minneapolis, MN 55455

S. aureus remains a frequent cause of morbidity and mortality in the USA, and intestinal colonization likely represents an important portal of entry for systemic infection. We have shown that either high molecular weight polyethylene glycol (HMW PEG) or intact heparin (HP), inhibit bacterial interactions with the intestinal epithelium both in vitro (cultured enterocytes) and in vivo (orally inoculated mice), and that low MW PEG (LMW PEG, an active ingredient in over-the counter laxatives) is less effective. The gentamicin protection assay was used to determine if intact HP or HP disaccharides (I-S, II-H, IV-H) added to LMW PEG decreased S. aureus interactions with enterocytes to a greater extent than obtained with LMW PEG alone. Enterocytes were incubated with 108S. aureus in the presence of 25μg/ml of a heparin moiety plus 5% LMW PEG. Compared to LMW PEG alone, LMW PEG plus HP or selected HP disaccharide alone decreased enterocyte internalization (see figure, *P<0.05, **synergistic effect). Thus, combining LMW PEG with HP disaccharides may represent a novel approach to inhibit S. aureus interactions with epithelial cells, including enterocytes.



HEPARIN PROTECTS AGAINST SEPTIC MORTALITY VIA APOE-ANTAGONISM.B. Leung,* K. Chuang,* N. Hsu,* and H. Harris. Department of Surgery, University of California, San Francisco, San Francisco, CA 94143

Apolipoprotein (apoE) E, a component of plasma lipoproteins, plays an important, but poorly defined role in sepsis. Injecting apoE increases septic mortality in a rodent model of sepsis, presumably by enhancing lipid antigen presentation to antigen-presenting cells via the LDL receptor (LDLR). Thus, we sought to determine whether apoE antagonism would protect against septic mortality in mice. To antagonize apoE-LDLR binding we used heparin and its octasaccharide derivative, which have been shown to inhibit the LDLR binding site of apoE. Lepirudin served as our control. Wild type mice (C57BL6) were subjected to cecal ligation and puncture (CLP) and given an infusion of heparin, octasaccharide, or lepirudin via intraperitoneal osmotic pump. Serum partial thromboplastin time (PTT) was measured at 24 hours and survival was monitored for 7 days after CLP. In addition, mouse embryonic fibroblasts (MEF) expressing and lacking the LDLR (MEF+ and -) were incubated with apoE and heparin at multiple concentrations for 12 hours. We then quantified apoE internalization via immunoblot. Heparin and its octasaccharide each decreased CLP-induced mortality by approximately 50% versus saline-treated controls. Lepirudin increased septic mortality. Anticoagulation did not correspond to survival for any of the three treatments. MEF+ cells displayed decreased uptake of apoE when treated concurrently with heparin for 12 hours (p<0.05). MEF- cells did not demonstrate any significant uptake of apoE compared to controls. Our study demonstrates that heparin is protective against septic mortality independent of its anticoagulant effect. The protective effect of heparin against septic mortality is associated with the inhibition of apoE-LDLR binding. These findings indicate a potential clinical role for apoE-antagonism in the treatment of sepsis.


EFFECTS OF THERAPEUTIC HYPOTHERMIA AND SPEED OF REWARMING ON SEPSIS IN RATS.J.H. Lee, K.P. Lim, I.S. Cho, Y.W. Nam, Y.H. Jo, K. Kim, J.H. Lee, J.E. Rhee, T.Y. Kim, W.Y. Kwon, and G.J. Suh. Department of Emergency Medicine, Seoul National University Bundang Hospital, Seongnam-si Gyeonggi-do, 463-707, Korea

Objective: Therapeutic hypothermia (HT) has been evaluated in a variety of clinical situations including cardiac arrest, traumatic brain injury, stroke and experimental sepsis model. After the therapeutic HT, rewarming is necessary, but the duration of rewarming has rarely been investigated. This study was performed to evaluate the effect of HT and speed of rewarming on mortality in sepsis model of rats.

Methods: To induce the very severe sepsis, adult male Sprague-Dawley rats anesthetized and distal 1/3 of cecum was ligated with 3-0 silk and 0.5cm sized incision to cecum was done. Then the cecum was squeezed to spill the stool into the abdominal cavity. Rats were randomly assigned to normothermia (NT, 36-38°C), 4hr HT (30-32°C) with rewarming for 1hr (HT+RW1) and 4hr HT with rewarming for 2hr group (HT+RW2, N=10 per group). The survival time was observed for 24 hours.

Results: All animals died within 12 hours after the induction of sepsis. The mean survival times in NT, HT+RW1 and HT+RW2 groups were 6.53±0.94, 7.27±0.80 and 8.34±0.67 hrs, respectively. The HT+RW2 group survived longer than the NT and HT+RW1 groups.

Conclusion: Gradual rewarming after TH improved survival in sepsis models in rats.



INCREASED SUSCEPTIBILITY TO CANDIDA INFECTION FOLLOWING CECAL LIGATION AND PUNCTURE.J. Muenzer,* C. Davis, T. Ferguson,* and R. Hotchkiss. Washington University School of Medicine, St. Louis, MO 63122

Background: Immunosuppression following septic insult is a major contributor to secondary infections. C. albicans is the most common fungal species seen in superficial and systemic infections. Candida species are also ranked as the 4th most common nosocomial infection.

Objective: The purpose of this study was to evaluate the susceptibility of septic mice to secondary fungal infection. Additionally, we evaluated the effect of "priming" on the host's ability to eradicate secondary infection in the face of ongoing immunosuppression.

Methods: Survival studies: B6 mice (6-8 wks) were separated into 4 groups (n=12/group). The first two groups received CLP or sham followed by IV C. albicans (40ul of 0.3McF) two days later. For priming purposes the next two groups were given 2 SC injections (100ul of 0.3 McF Candida) 2 weeks apart. Three weeks after the priming period they underwent CLP or Sham surgery followed by a secondary injury of C. albicans. Acute studies: Delayed type hypersensitivity (DTH) tests were performed on all groups to assess the effect of priming on their immune response. Finally, blood cultures were obtained from all groups 24 hours after C. albicans infection.

Results: In survival studies, C. albicans infection following CLP had significantly higher mortality compared to C. albicans alone (92% vs. 50%, p<0.02). Priming animals prior to CLP improved survival to secondary challenge with C. albicans (58% vs. 8.3%, p<0.02). Primed CLP animals showed significant increases in their DTH response compared to non primed CLP animals (249um vs. 10um, p<0.01). Consistent with survival and DTH results, primed animals had decreased fungemia (2.6 vs. 0.9 log CFU, p<0.01).

Conclusion: Immunosuppression following CLP leads to increased susceptibility to C. albicans infection. Priming of animals prior to double injury increases survival.


CECAL LIGATION AND DOUBLE PUNCTURE (2CLP) DECREASES ACTIVITY IN THE OREXINERGIC NERVOUS SYSTEM.E. McGuire,* J. Anson,* N. Raj,* and C. Deutschman. University of Pennsylvania, Philadelphia, PA 19104

Introduction: Sepsis and the multiple organ dysfunction syndrome (MODS) are the leading causes of mortality in critically ill patients. These syndromes impair the normal physiologic feedback mechanisms that produce an orchestrated, tightly regulated homeostatic response to internal and external changes. Feedback involves hormones, white cell products such as cytokines and output from the central nervous system. While the first two have been studied extensively in sepsis, little is known about the effects of sepsis on neural control of organ function. The orexinergic nervous system modulates a number of basic physiologic functions (sympathetic outflow, temperature, respiration, arousal, control of pituitary hormones) that are altered in sepsis/MODS.

Hypothesis: We propose that septic pathophysiology reflects a dysregulation of central neuronal control of basic physiologic parameters. Therefore, we hypothesize that experimental sepsis will decrease activity in the orexinergic nervous system.

Methods: We performed 2CLP on C57Bl6 mice. Animals were sacrificed by cervical dislocation 24 and 48 hours after surgery. Findings were compared to similarly euthanized un-operated controls. Brains were removed, fixed and sectioned. The hypothalamic sections known to contain orexinergic neurons were stained for orexin and for c-fos, a marker for neuronal activity. Co-localization indicated an active cell in the orexinergic system.

Results: Using fluorescence staining, we found that, relative to unoperated controls, 2CLP reduced the number of cells positive for both orexin and c-fos.

Conclusion: 2CLP reduces activity in the orexinergic nervous system. Therefore, loss of activity in this group of neurons may play an important role in the sepsis-induced loss of homeostatic integration.



Background: Mitochondria replicate themselves via a process called biogenesis. This requires expression of proteins encoded by both nuclear and mitochondrial (mt) DNA. Impaired biogenesis has been implicated in the pathogenesis of a number of disorders. Therefore, we examined the intra-hepatic abundance of the mitochondrial transcription factor A (Tfam) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), two key regulators of biogenesis, following CLP.

Hypothesis: CLP decreased Tfam and PGC-1α levels in the liver.

Methods: Three sets of C57Bl/6 mice were subjected to CLP or sham operation (SO). Mice were sacrificed at 3, 6, 16, 24, 48, and 72 hours post surgery. Hepatic tissue was harvested and mitochondria isolated. Protein abundance was determined using immunoblotting.

Results: Relative to SO and T0 controls, Tfam and PGC-1α abundance both decreased significantly 24 and 48 hours following CLP.

Conclusions: Tfam and PGC-1α expression decreased following CLP, indicating impaired biogenesis. These data suggest that 1) sepsis impairs biogenesis in hepatocytes via a mechanism that involves nuclear-encoded proteins and 2) failed biogenesis is important in septic pathophysiology.


DIFFERENTIAL CYTOKINE RESPONSES IN BABOONS FOLLOWING SHIGA TOXIN TYPE-1 AND -2 CHALLENGE.S-Y. Oh, D. Weiner,* D.J. Stearns-Kurosawa, and S. Kurosawa. Boston University School of Medicine, Boston, MA 02118

Enterohemorrhagic E.coli that release Shiga toxins (Stx1, Stx2) cause bloody diarrhea, hemolytic uremic syndrome and severe renal injury with significant morbidity. The toxins are very similar structurally with a shared glycolipid receptor, but Stx2 is associated with renal damage in children after a 3-6 day delay. Unlike other species, the baboon (Papio) Stx models recapitulate many human responses. The current study characterized inflammatory responses induced by Stx1 or Stx2.

Methods: Anesthetized baboons were challenged with i.v. bolus of Stx1 (10-100ng/kg) or Stx2 (10-50ng/kg) and monitored for at least 7 days. Cytokine levels in timed plasma and urine samples were quantified by xMAP™ multiplex assays (Luminex® 200 IS, Millipore).

Results: Results revealed similarities, but also unexpected differences in some cytokines for timing and magnitude. In plasma and urine, there was a 1-3 day delay in Stx2-induced cytokine changes. In plasma, both toxins raised MCP-1 and IL-6 levels, but only Stx1 increased IL1-Ra and GCSF. In urine, both toxins elevated MCP-1, IL-6 levels and IL-8, but only Stx1 increased IL-1Ra and IL-1b. TNFα was either not detectable or very low in all samples. Finally, a number of inflammatory mediators in plasma (GM-CSF, IL-1β) did not show changes following toxin challenge.

Conclusions: We demonstrate that Stx1 and Stx2 elicit systemic and distinct cytokine response patterns in nonhuman primates. The differences were prominent among the mediator induced, time course and the magnitude of the responses induced by Stx1 or Stx2. It is possible that the toxins have different effects on cytokine mRNA stability and/or gene transcription events. The current disease paradigm views the toxins essentially interchangeably, but these results demonstrate that the in vivo consequences are very different.


DISTINCT PATHOPHYSIOLOGIC RESPONSES IN BABOONS AFTER CHALLENGE WITH SHIGA TOXIN TYPE-1 OR -2.D.J. Stearns-Kurosawa, V. Collins,* S. Freeman,* S-Y. Oh,* and S. Kurosawa. Boston University School of Medicine, Boston, MA 02118

Shiga-toxin producing enterohemorrhagic E. coli (O157:H7) are a principal source of regional and sporadic outbreaks of bloody diarrhea and hemolytic uremic syndrome, and the leading cause of acute renal failure in US pediatric patients. Primary bacterial virulence factors are the Shiga toxins type-1 and -2 (Stx1, Stx2). We performed parallel analyses of the pathophysiology elicited by the toxins in nonhuman primates to identify shared and unique consequences of the toxemias. Baboons (Papio) were anesthetized and challenged with i.v. bolus of Stx1 (10-100ng/kg) or Stx2 (10-50 ng/kg) and monitored for at least 7 days. Both toxins altered hematologic parameters and targeted the kidneys, resulting in thrombocytopenia, anemia, red blood cell fragmentation, progressive anuria, proteinuria, and reduced glomerular filtration function in a dose-dependent manner. There were unexpected timing and magnitude differences in physiologic responses between the toxins. For example, while the absolute loss of platelet counts was similar, a high dose of Stx1 resulted in fairly abrupt thrombocytopenia by 24-48 hours, whereas loss of platelets after Stx2 was a gradual daily decrease until either euthanasia at day 4-5 or resolution and recovery. The animals were more sensitive to Stx2 with mortality at lower doses, but Stx2-induced renal injury and mortality was delayed by 2-3 days compared to Stx1 challenge. Renal corticomedullary congestion in the kidney was more severe after Stx2 challenge, whereas Stx1 targeted the small intestine with resultant hemorrhage. This study is the first parallel comparison of the in vivo consequences of the toxins in nonhuman primates. The Stx1 and Stx2 toxemia baboon models provide a platform for reproducible testing of immunotherapeutics and anti-toxin compounds in a near human setting with definable outcomes that have clinical meaning.


CARBENOXOLONE RESCUES MICE FROM LETHAL SEPSIS BY INHIBITING HMGB1 RELEASE.W. Li,* J. Li,* M. Ashok,* S. Zhu,* A.E. Sama,* and H. Wang. The Feinstein Institute for Medical Research, North Shore - LIJ Health System, Manhasset, NY 11030

Our seminal discovery of HMGB1 as a late mediator of lethal endotoxemia and sepsis has prompted further investigation for developing new experimental therapeutics. Carbenoxolone is a derivative of glycyrrhizinic acid (a major component of Chinese herb, liquorice), and has been traditionally used as a drug for oesophageal ulceration.

Objectives: To further explore its therapeutic potential in the treatment of sepsis, we examined its effects on endotoxin-induced HMGB1 release, and animal lethality in experimental sepsis (induced by cecal ligation and puncture, CLP).

Methods: Murine macrophage-like RAW 264.7 cells were stimulated with bacterial endotoxin (lipopolysaccharide, LPS, 1000 ng/ml) in the absence or presence of carbenoxolone disodium, and HMGB1 levels in the culture medium were determined 16 hours later. Male Balb/C mice (20-25 g, 6-7 weeks old) were subjected to experimental sepsis by CLP, and the effects of carbenoxolone on animal survival rates were monitored for more than 2 weeks.

Results:In vitro, carbenoxolone dose-dependently inhibited LPS-induced HMGB1 release, with a maximal suppression (> 90 ± 10%) when given at a concentration as low as 10 μM. In vivo, intraperitoneal administration of carbenoxolone (6.0 mg/kg, at 24 h, 48 h, and 72 h post CLP) significantly reduced circulating HMGB1 levels (by 40 ± 5.0%) in septic mice (at 52 h post CLP), and increased animal survival rate from 42% (in control group receiving saline, N = 26 mice/group) to 85% (in experimental group receiving carbenoxolone; N = 27 mice/group).

Conclusions: Carbenoxolone significantly rescued mice from lethal sepsis, and thus hold potential for treating human sepsis. (Supported by the National Institute of General Medical Sciences Grant R01 GM063075 to H.W.).


THE AQUEOUS EXTRACT OF MUNG BEAN SKIN (VIGNA RADIATA) PROTECTS MICE AGAINST LETHAL SEPSIS.S. Zhu,* J. Li,* W. Li,* M. Ashok,* A.E. Sama,* and H. Wang. The Feinstein Institute for Medical Research, North Shore - LIJ Health System, Manhasset, NY 11030

The pathogenesis of sepsis is in part mediated by bacterial endotoxin, which stimulates macrophages to sequentially release early (e.g., TNF, IL-1, and IFN-gamma) and late [e.g., high mobility group box 1 protein (HMGB1)] proinflammatory mediators. Our discovery of HMGB1 as a late mediator of lethal systemic inflammation has initiated a new field of investigation for the development of experimental therapeutics. Mung bean has been traditionally used in the treatment of inflammatory conditions such as heatstroke and summer heat syndrome (restlessness, irritability, thirst, and etc).

Objectives: To explore the therapeutic potential of Mung bean skin extract in animal models of sepsis (induced by cecal ligation and puncture).

Methods: Murine macrophage cultures were stimulated with endotoxin (LPS, 1000 ng/ml) in the absence or presence of Mung bean extract, and HMGB1 release was assessed 16 hours later. Male Balb/C mice (20-25 g, 6-7 weeks old) were subjected to CLP, and aqueous extract of Mung bean was administered orally at 24, 48, and 72 h post CLP.

Results: Aqueous extract of Mung bean dose-dependently attenuated endotoxin-induced HMGB1 release by stimulating cytoplasmic HMGB1 degradation in macrophage cultures. Oral administration of Mung bean extract significantly increased animal survival rate from 29.4% (in control saline group, N = 17 mice) to 70% (in experimental Mung bean extract group, P < 0.05).

Conclusions: These data suggest that Mung bean skin extract exerts protective effects against lethal sepsis in part by attenuating endotoxin-induced release of late proinflammatory mediators. (Supported by the National Institute of General Medical Sciences Grant R01 GM063075 to H.W.).


THE IMPACT OF SEPSIS ON FIBROCYTE: T-CELL INTERACTIONS.C. Fry,* and J. A. Nemzek. University of Michigan, Ann Arbor, MI 48109

Bone marrow-derived fibrocytes are recruited to sites of inflammation. Previous work in our lab demonstrated significantly increased fibrocyte recruitment to the peritoneum 24hrs post-CLP. In addition, we have shown a 50% increase in T-cell proliferation and 20% increase in activation when T-cells were exposed to fibrocytes grown in culture. However, fibrocytes raised in culture may behave differently from those freshly harvested or from septic animals. Therefore, transgenic mice that express GFP on the collagen promoter had 21 gauge CLP or Sham surgeries. After 24hrs, peritoneal lavage was performed and viable cells counted on a hemacytometer. Recruitment of fibrocytes was assessed by flow cytometry; GFP expressing fibrocytes were 0.5% 0.75% and 3.5% of the total peritoneal cells in the normal, sham, and CLP animals, respectively. In addition, GFP+ CD45+ fibrocytes were sorted using a FACSVantage SE Cell sorter. Sorted septic or sham fibrocytes were co-cultured at a 1:10 ratio with isolated, splenic, CFSE loaded T-cells from normal, sham, and CLP mice for 1wk. Co-cultures were assessed for total T-cell proliferation, as well as CD4 and CD8 T-cell proliferation. The results demonstrated a 1.5 to 2.5-fold increase in proliferation of T-cells from normal and Sham animals when co-cultured with either Sham or CLP fibrocytes, compared to T-cells alone. However, there was a significant increase when septic T-cells were co-cultured with CLP or Sham fibrocytes, compared too CLP T-cells cultured alone. This was a result of increases in both CD4 and CD8 T-cell proliferation. Collectively, these data suggest fibrocytes derived from a septic environment may retain the ability to enhance T-cell proliferation. Due to the association of increased mortality and T-cell apoptosis in sepsis, a better understanding of fibrocyte:T cell interactions is important to the study of sepsis.


LOW DOSE DEXAMETHASONE-INDUCED MODERATE IMMUNE SUPPRESSION ENHANCES SURVIVAL FOLLOWING CLP IN MICE.J.W. van den Berg, M. van der Zee, J.C.P.A. van Holten, J.N.M. IJzermans, R. Benner, R.W.F. de Bruin, and W.A. Dik. Erasmus Medical Center, Rotterdam, The Netherlands

Anti-inflammatory therapies so far did not reduce sepsis related mortality in humans. Studies using IL-6 KO mice and IL-6 neutralizing antibodies in CLP-induced sepsis, and low dose dexamethasone (DEX) administration in L. monocytogenes infection suggest that moderate downregulation of the inflammatory response may be beneficial in an early septic event while strong immune suppression is detrimental. To test this hypothesis we examined the effect of DEX (varying doses from high to low) on CLP-induced mortality and inflammation. C57BL/6 mice were subjected to CLP, DEX was administered i.v. (2.5, 0.25, or 0.05 mg/kg.) 20 min. postoperatively. Mice receiving PBS served as controls. Survival (up to 28 days) and cytokine plasma levels at 6 h. following CLP were determined. In the control group survival was 50%. DEX 0.05 mg/kg treatment improved survival to 73% whereas treatment with higher concentrations of DEX (0.25 mg/kg and 2.5 mg/kg) was associated with reduced survival (both 33%). Treatment with either 2.5 or 0.25 mg/kg DEX was associated with significantly (p<0.05) reduced IL-6, CCL-2, KC, eotaxin, MIP-2, TNF-sr1, TNF-sr2, and IL-1ra plasma levels as compared to control mice at 6 hours post-CLP. In contrast, treatment with 0.05 mg/kg DEX was associated with a moderate, but non significant, downregulation of plasma IL-6, CCL-2, KC, eotaxin, MIP-2, TNF-sr1, TNF-sr2, and IL-1ra levels. These data demonstrate that only a moderate downregulation of the early sepsis associated inflammatory response improves survival, probably because this level of immune suppression still allows the immune system to battle the invading pathogen(s) sufficiently. We propose that success of anti-inflammatory therapies in a septic setting fundamentally depends on finding the maximum level of immune suppression that still allows adequate defense against pathogens.


CB2 CANNABINOID RECEPTORS CONTRIBUTE TO BACTERIAL INVASION AND MORTALITY IN POLYMICROBIAL SEPSIS.G. Haskó, Z. Németh,* Z. Spolarics, S. Federici,* P. Pacher,* E. Deitch, and B. Csóka. University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ

Objectives: Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB2 receptors on immune cells. Here we examined the role of CB2 receptors in regulating the host's response to sepsis.

Methods: The role of CB2 receptors was studied by subjecting CB2 receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture.

Results: We found that CB2 receptor inactivation by knockout decreased sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB2 receptor inactivation decreased kidney and muscle injury, suppressed splenic nuclear factor-B activation, and diminished the production of IL-10, IL-6 and MIP-2. Finally, CB2 receptor deficiency prevented apoptosis in lymphoid organs and augmented the number of CD11b+ and CD19+ cells during CLP.

Conclusions: Taken together, our results establish for the first time that CB2 receptors are important contributors to septic immune dysfunction and mortality, indicating that CB2 receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.


INCREASED ADIPOSE TISSUE EXPRESSION OF ADIPONECTIN: POTENTIAL THERAPEUTIC TARGET IN INFECTION?H. Linge, K. Cheng, K. Takahashi, Y. Al-Abed, and E. Miller. The Feinstein Institute for Medical Research, Manhasset, NY 11030

Background: We here investigate the effects of staphylococcal stimulation of the lung on abdominal adipose tissue and whether its effect on adipokines could influence the progression of infection. Adiponectin is produced by mature adipocytes is found in circulation at around 5μg/ml. Besides insulin-sensitizing, adiponectin acts anti-inflammatory by neutralizing LPS and altering monocyte pro-inflammatory pathways. Its role in G+ infection remains unstudied. The aim of this study was to determine if adiponectin expression during infection could be enhanced pharmacologically.

Methods: C57/Bl6 mice were instilled i.t. with LTA (10μg/kg) and PGN (33 μg/kg) and sacrificed after either 3h or 20h. Controls received saline. Post mortem the omental fat pad was examined for adiponectin expression by qPCR. Expression was also determined after i.p. injection of a selective α7 nicotinic Ach receptor agonist alone or in combination with i.t. LTAPGN. In cell culture experiments, 3T3-L1 adipocytes were monitored for altered expression following agonist treatment.

Results: Compared to controls, LTAPGN instillation induced a 7.8 and 13.5-fold elevation of adiponectin expression in omental adipose tissue after 3h and 20h, respectively. Treatment of mice with the α7 agonist alone resulted in a 38-fold increase compared to controls. In LTAPGN instilled mice, the agonist increased adiponectin expression to 48.4 times above normal. Cultured adipocytes responded to the agonist by increasing the expression of adiponectin.

Conclusion: Response to pulmonary stimulation can increase adiponectin expression. α7 nicotinic Ach receptor agonism can elevate adiponectin expression in adipose tissue, aiding the innate host defense in combating infections.



Circulating fibrocytes are recruited to sites of injury and inflammation where they secrete pro-inflammatory cytokines. Our previous work showed that MHC Class II positive fibrocytes significantly increase in the peritoneum 24 hrs post-CLP as compared to Sham. In addition, we have shown that fibrocytes increase T-cell proliferation and activation in co-cultures. Although fibrocytes express several chemokine receptors, there have been no studies concerning fibrocyte recruitment toward sites of infection. Therefore, CLP was performed on CCR2 KO and WT mice. After 24 hours, peritoneal lavage fluid and mesenteric lymph nodes were harvested. Fibrocytes were identified by staining for CD45 and collagen I, followed by flow cytometry analysis. Total peritoneal lavage fluid counts were significantly higher in WT mice as compared to KO mice. However, CCR2 KO mice recruited significantly fewer fibrocytes (0.2 +/- 0.1 × 106) to the peritoneum as compared to WT mice (4.0 +/- 1.5 × 106). The number of fibrocytes in the peritoneum of CCR2 KO mice was no different than constitutive numbers found in normal mice. In addition, CCR2 KO mice had 50% fewer fibrocytes in mesenteric lymph nodes (1.8 +/- 0.8 × 105) as did WT mice (3.6 +/- 1.6 × 105) at 24 hours post-CLP. It is unknown at this time whether the recruitment of fibrocytes to the lymph node is due to active migration or a function of passive peritoneal drainage. However, the results strongly show that CCR2 has a major role in the recruitment of fibrocytes to the peritoneum in response to CLP. This marks the first report on the mechanisms behind fibrocyte recruitment in bacterial infection.


MILK FAT GLOBULE EGF FACTOR 8 ATTENUATES SEPSIS-INDUCED APOPTOSIS AND ORGAN INJURY IN ALCOHOL-INTOXICATED RATS.R. Wu, W.W. Chaung,* M. Zhou, Y. Ji,* W. Dong,* Z. Wang,* X. Qiang,* and P. Wang. North Shore-LIJ Medical Center, Manhasset, NY 11030

Controlling high morbidity and mortality due to infections in alcohol abusers remains a prominent challenge. Our previous studies have shown that milk fat globule epidermal growth factor-factor VIII (MFG-E8), a protein required to opsonize apoptotic cells for phagocytosis, is protective in inflammation. However, it remains unknown whether MFG-E8 ameliorates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats. The aim of this study was to determine whether recombinant murine MFG-E8 (rmMFG-E8) attenuates organ injury after acute alcohol exposure and subsequent sepsis. To study this, male adult rats were subjected to acute alcohol intoxication by a bolus injection of intravenous (iv) alcohol at 1.75 g/kg BW, followed by an iv infusion of 300 mg/kg BW/h of alcohol for 10h. Sepsis was induced at the end of alcohol infusion by cecal ligation and puncture (CLP). rmMFG-E8 was iv administered three times (i.e., at the beginning of alcohol injection, the time of CLP, and 10h post-CLP) at a dose of 20 μg/kg BW each. Blood and tissue samples were collected 20h after CLP. As shown in the table below, administration of rmMFG-E8 reduces alcohol/sepsis-induced apoptosis in various organs.


In addition, administration of rmMFG-E8 decreases serum levels of TNF-α and IL-6, and attenuates organ injury after alcohol exposure and subsequent sepsis. Thus, rmMFG-E8 attenuates sepsis-induced apoptosis and organ injury in alcohol-intoxicated rats and MFG-E8 may be a novel approach to treat alcoholic patients with sepsis.


EARLY T CELL ACTIONS CONTROL THE INNATE IMMUNE RESPONSE TO SEPSIS.K. Kasten,* and C. Caldwell. Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH 45267

The early interplay between T cells and the innate immune response during sepsis remains poorly elucidated. Previously we have shown decreased neutrophil oxidative burst in CD4-deficient mice and decreased neutrophil recruitment in γδ T cell-deficient mice. We hypothesized that maintenance of TCR activation can control the innate immune response in sepsis. Here, mice were subjected to a moderately severe cecal ligation and puncture (CLP). Using IL-10 GFP mice we demonstrate that macrophages modestly produce IL-10 during sepsis. Macrophages insensitive to IFN-γ have early decreased active Stat1 and decreased IL-10 production, while direct administration of IL-10 reduces neutrophil oxidative burst. We observed that increased production of IFN-γ following IL-7 treatment augmented CXCL10/IP-10 release, recruitment of γδ T cells with elevated IL-17 production, and ultimately, accelerated neutrophil recruitment to the peritoneum with decreased bacterial load. Using transgenic OT-II mice, we demonstrate that too little CD4 T cell activation leads to decreased bacterial clearance and survival, while too much CD4 T cell activation leads to effective bacterial clearance but with increased tissue damage and mortality. Altogether, early T cell production of IFN-γ mediates macrophage function, as well as neutrophil recruitment and function. (Supported by NIH R01GM072760).

FIG. 1
FIG. 1:
Proposed schematic of CD4+ T cell role in control of the innate immune response to sepsis.


PULMONARY CONSEQUENCES OF MURINE SEPSIS: INJURY OR JUST INFLAMMATION.F. Craciun,* L. Vaickus,* E. Schuller, S. Natarajan,* B. Belikoff,* D. Horton,* J. Kim,* and D. Remick. Boston University School of Medicine, Boston, MA 02118

Objective: Although 40% of septic patients develop lung injury, respiratory failure is usually not the cause of death. The murine sepsis model of cecal ligation and puncture (CLP) was used to study the association between lung injury development and sepsis mortality.

Methods and Results: Following CLP in female ICR mice, plasma IL-6 measured at 24 hours was used to predict outcome and separate mice in 2 groups: predicted to survive the first 5 days post-CLP (Live-P) or die within 5 days post-CLP (Die-P); a group with no surgical intervention (Normal) was also included. We measured a comprehensive set of parameters starting with a broad view of lung function. By unrestrained whole body plethysmography Die-P mice had reduced respiratory rate, peak inspiratory and expiratory flow compared to Normal and Live-P. But Die-P still had normal tidal volumes and pulse oximetry showed normal arterial oxygen saturation, indicating preserved capacity for gas exchange. Lung edema was assessed and the wet/dry lung weight ratio was similar for the 3 groups. Bronchoalveolar lavage (BAL) total protein and albumin were decreased compared to Normal, but Die-P and Live-P values were similar. Neutrophils were not recovered in the BAL from either Live-P or Die-P. Lung homogenate myeloperoxidase levels were similar in Die-P and Normal and lower then the two in Live-P. Histology confirmed these results as lung injury score was the same in Live-P and Die-P. Die-P had higher plasma levels of numerous pro and anti-inflammatory cytokines and also higher BAL levels of IL-1β, IL-6, MIP-2, MCP-1, IL-1ra, IL-10.

Conclusion: Our data shows lung inflammation but not injury in septic mice 24 hours post-CLP and consequently no association between lung injury and outcome.


POST-CLP IMMUNOSUPPRESSION IN MICE IS DEPENDENT UPON CASPASE-1.E.D. Murphey, and E.R. Sherwood. Dept of Anesthesiology, UTMB, Galveston, TX 77555

Bacterial clearance is impaired in mice that have been subjected to cecal ligation and puncture (CLP). We hypothesized that that outcome is dependent upon a caspase-1 mechanism and tested that by measuring caspase-1 after CLP and by measuring clearance of a bacterial challenge in caspase-1-deficient mice after CLP. Wild-type mice subjected to CLP had increased caspase-1 activity in liver homogenates, had increased expression of caspase-1 and IL-1β production in splenocytes, and had impaired clearance of a Pseudomonas aeruginosa challenge when compared to sham controls (Table 1). Caspase-1-/- mice subjected to CLP had no splenocyte production of IL-1β and no impairment of bacterial clearance compared to sham or WT CLP (Table 2).

WT mice subjected to CLP or sham CLP and challenged with Ps. aeruginosa (1 × 108 cfu i.v.)
Casp-1 -/- mice subjected to CLP or sham CLP and challenged with Ps. aeruginosa (1 × 108 cfu)

Increased caspase-1 expression and activity (also evidenced by increased IL-1β) after CLP injury appears to contribute to diminished innate immune function.


ECTO-5'-NUCLEOTIDASE (CD73) GENERATED ADENOSINE ATTENUATES IMMUNE DYSFUNCTION IN SEPSIS.Z. Németh,* B. Csóka, P. Pacher,* B. Koscsó,* R. Rolandelli,* Z. Spolarics, E. Deitch, and G. Haskó. Morristown Memorial Hospital, Morristown, NJ 07962

Objective: Adenosine is produced in response to cellular stress during sepsis and organ injury. CD73 is a cell surface protein with ecto-5′-nucleotidase enzyme activity that catalyzes the dephosphorylation of 5′-AMP to adenosine. Therefore, this molecule plays a key role in the generation of extracellular adenosine.

Methods: The immunological status of animals was assessed by measuring cytokine levels and bacterial counts from blood and peritoneal lavage fluid. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatine kinase (CK) levels were measured 16 h after cecal ligation and puncture (CLP). Markers of immune cell apoptosis were examined in the protein extract from thymus and spleen tissue using Western blotting.

Results: CD73 gene knock-out (KO) mice exhibited a significantly higher bacterial burden and higher interleukin- (IL)-10, IL-6, and inflammatory protein 2 (MIP-2) levels than their wild-type (WT) counterparts. Pharmacological inhibition of CD73 with AMPCP similarly increased the levels of serum and lavage IL-10 and IL-6 concentrations. In addition, CD73 deficiency caused immune cell apoptosis, measured as increased levels of cleaved caspase-3, and PARP in the thymi of CD73 KO mice relative to those of CD73 WT animals. Furthermore, septic CD73 KO mice showed signs of kidney injury as evidenced by higher levels of BUN in those mice.

Conclusion: CD73-derived adenosine decreases bacterial load, cytokine and chemokine levels, and immunocyte apoptosis, as well as organ injury during sepsis. Taken together our findings indicate that adenosine receptor modulation might represent a new therapeutic method in the clinical treatment of patients with sepsis or serious systemic inflammation. (This work was supported by the National Institute of Health (NIH) Grant R01 GM66189).


HEAT SHOCK PROTEIN 70 PREDICTS MORTALITY, INFECTION, AND HOSPITAL LENGTH OF STAY IN ADULT INTENSIVE CARE PATIENTS.K. Queensland,* A. Edwards,* L.B. Weitzel,* D. Heyland,* and P.E. Wischmeyer. University of Colorado, Denver (Aurora, CO 80045)

Objectives: Heat Shock Protein 70 has been shown to be a vital intracellular protective protein following injury. However, the role of plasma HSP70 (pHSP70) in critical illness is unclear as extracellular HSP70 can function as a signaling molecule or "chaperkine". We investigated the relationship of pHSP70 to mortality, infection, and hospital length of stay (LOS) in adult ICU patients.

Methods: 103 ICU patients (> 18 y.o.) had blood collected serially for HSP70 analysis (ICU Day 0-7). Patients from non-selected subgroup obtained from patients enrolled in a prospective observational ICU trial. pHSP70 analysis via Mesoscale analysis.

Results: pHSP70 levels on ICU day 5 are significantly related to 28-day mortality when controlling for age (p<0.02). Day 5 pHSP70 levels ≥ 50 ng/mL was predictive of mortality at Day 28. (Sensitivity = 43.48%, Specificity = 83.54%, OR = 4.17 (95% CI 1.40-12.36)). Increased pHSP70 levels over time post-ICU admit are correlated with improved 28 day survival (p=0.05), but longer hospital LOS (p=0.001). pHSP70 levels increase over ICU stay in patients who develop infection (p<0.0004), however, patients who ultimately do not survive post-infection have decreasing levels of pHSP70 (p<0.04).

Conclusion: Our data reveals increasing levels of pHSP70 over time are beneficial to survival and survival post-infection. We also show pHSP70 may be a very early marker of increased risk of infection and death, as pHSP70 levels at ICU admission are higher in patients who ultimately expire. pHSP70 may be a useful tool for early prediction of ICU survival. (Supported: R01 GM078312).


MODELING OF SEVERE SEPSIS PATIENTS WITH COMMUNITY ACQUIRED PNEUMONIA (CAP).D.D. Marathe,* J. Sarkar,* K.W. Hurst,* A.M. Inglis,* J.A. Kellum, D.C. Angus, Y. Vodovotz, and S. Chang. Immunetrics Inc., Pittsburgh, PA 15203

The progression of sepsis involves dysfunction of vital organs and is governed by dynamic interaction among cytokines, immune cells, tissues and pathogens. We used our proprietary modeling platform to build a multi-scale, equation-based mechanistic simulation that encompasses these biological components, along with the simulated administration of standard-of-care interventions. This "virtual clinician" alters the dynamics of the disease state in a clinically relevant manner. This model was fit to time course data consisting of various biomarkers and clinical-markers, including MAP, PaO2, creatinine, TNF-α, IL-6, and PAI-1, from published human endotoxemia studies, and CAP severe sepsis patients in the GenIMS study. The model accounts for differences in physiological/immune-function of patient demographics. It was fit to median time course data for survivors/non-survivors. Five co-morbidity related parameters were changed to fit the model to 9 co-morbidity related sub-groups. The model reproduced the entire spectrum of patients in the GenIMS study by altering only a handful of parameters related to pathogen, antibiotic efficacy and baseline patient status. The model simulations provided evidence of changes in disease progression and likelihood of survival as a function of various treatments, patient stratification on the basis of outcome and time of death, and predictive ability for patient outcome beyond hospital discharge. Model training and optimization led to the identification of a minimum set of temporal analyte data required to predict future trajectories and outcomes of patients. Overall, the reproduction of the GenIMS study with a handful of parameter changes demonstrates the model's robustness, along with its capability to assess the efficacy/outcome of new sepsis-related drugs and treatment strategies in an anticipated patient cohort. (Funded by NIH grants- GM61992 and HL080926).


COMPUTERIZED PROTOCOL FOR SEPSIS MANAGEMENT.B. McKinley, M. Sailors,* A. Valdivia,* L. Moore,* J. Sucher,* S. Todd,* K. Turner,* and F. Moore. The Methodist Hospital, Dept of Surgery, Houston TX 77030

Objective: We compare paper and computer protocol management of surgical sepsis.

Methods: A guideline based comprehensive 24 hr protocol was implemented in 2008 (paper) and 2009 (computer) in a 30 bed surgical ICU. Process and outcome are compared. (t, Χ2 tests).

Results: Cohorts tended to less sepsis severity and physiologic derangement ([lactate], [platelet], INR) from 2008 to 2009. Shock cohorts differed in presenting coagulopathy and early LR volume. Sepsis management using a computer (2009) compared to a paper protocol (2008) comprising the same clinical logic was associated with decreased mean time from protocol start to antibiotic intervention and tendency for improved survival rates for severe sepsis and septic shock.

Conclusion: Computerization of complex protocol logic improves process of care.



IMPACT OF FLUID RESUSCITATION ON HEART RATE AND BLOOD PRESSURE VARIABILITY IN SEPSIS.T. Ben-Jacob,* S. Zanotti-Cavazzoni, M. Guglielmi,* J. Skaf,* and S. Hollenberg. Cooper University Hospital, Camden, NJ 08103

Objective: The goal of this study was to evaluate the impact of aggressive fluid resuscitation on HR volatility (HRV) and BP volatility (BPV) in a murine model of sepsis.

Methods: In C57BI/6 mice (8-12 weeks, 30g, n=25) radiotelemeters for continuous measurement of BP and HR were implanted into the carotid artery. After recovery, baseline data were acquired. Cecal ligation and puncture (n=21) or sham surgery (n=4) was then performed. Septic mice received one of two resuscitation regimens: Low resuscitation (n=7): NaCl 0.9% 35 ml/kg subcutaneously (SQ) intraop only or High resuscitation (n=14): 35-100 ml/kg fluid SQ after surgery and then q6hr. Both groups received antibiotics intramuscular (IM) q6hrs. Continuous HR and BP were collected (96 hrs). MATLAB was used to calculate standard deviations (SD) for every 5 minute interval. The lowest 5% of SDs for the baseline period (24hrs) was utilized to define low volatility in the experimental period (72 hrs). The % of low volatility intervals during the experimental period was calculated.

Results: Compared to baseline, there was a non significant increase in the % of low HRV and BPV intervals in the sham-operated mice [HRV: 5.1±0.2 vs. 12.3±12.7 (p =0.3)], [BPV: 5.2 vs. 12.4± 6.5, (p=0.1)]. Low HRV intervals increased significantly in septic mice compared to sham-operated controls in the L group [67.3±1.9 vs. 12.3±12.7 (p= 0.001)] and to a less extent in the H group [50.7±32.4 vs. 12.3±12.7 (p< 0.01)]. Similarly, BPV was significantly increased in the L group [38.5±14.5 vs. 12.4±6.5, (p<0.01)], with a smaller increase in the H group [32.5± 19.8 vs. 12.4±6.5, (p<0.01)].

Conclusions: HRV and BPV are abnormal in sepsis. A trend toward improved HRV and BPV with aggressive resuscitation was identified. Further investigations are required to determine if these parameters could be used as resuscitation endpoints.


LIPOPOLYSACCHARIDE INDUCES PRODUCTION OF ENDOGENOUS RETROVIRAL VIRIONS IN MURINE LYMPHOID CELLS, PRIMARILY IN B LYMPHOCYTES.D. Kwon,* Y. Lee,* D.G. Greenhalgh, and K. Cho. Department of Surgery, University of California, Davis and Shriners Hospitals for Children, Sacramento, CA 95817

Endogenous retroviruses (ERVs), which constitute a substantial portion of the human and mouse genomes, are activated in mice during the course of experimental sepsis. Lipopolysaccharide (LPS) plays a critical role in sepsis pathogenesis. We investigated whether LPS-elicited stressors induce the production of murine ERV (MuERV) virions in cells derived from lymphoid organs. The primary cell suspensions and sorted B and T cells from seven lymphoid organs (spleen, thymus, bone marrow, and four different lymph nodes [cervical, inguinal, mesenteric, and axillary]) were treated with LPS. There were significant increases in production of putative MuERV virions in response to LPS-elicited stressors in certain lymphoid organs. These changes were specific to the B cell population. A total of 49 unique MuERV U3 sequences clustered into eight size groups were cloned from the virion genomic RNAs. A survey of the U3 sequences for transcription regulatory elements determined that the nuclear respiratory factor 1 binding site is present only in the U3 sequences of the 406 bp size, which were induced after LPS treatment. Using the U3 sequences as a probe, 58 putative MuERV loci were mapped on the C57BL/6J genome and 15 of them were presumed to be full-length proviruses with intact coding potential. Furthermore, transfection of recombinant MuERVs, which originated from three of the 58 loci, into RAW264.7 macrophage cells followed by LPS treatment identified one LPS-responsive putative MuERV with full coding potential. The findings from this study suggest that LPS-elicited stressors differentially activate MuERV virion production in lymphoid organs in a B cell-specific manner. Further investigation is needed to define the ERVs' roles in LPS signaling and in sepsis pathogenesis involving Gram-negative bacteria.


A ROLE OF PPARγ AGONIST IN SALUTARY EFFECTS ON OMENTAL ADIPOCYTE FUNCTIONS FOLLOWING POLYMCROBIAL SEPSIS.T. Matsutani,1 A. Matsuda,1 H. Yoshida,1 E. Uchida,2 M. Miyashita,2 Y. Tsujimura,3 M. Kutsukake,3 K. Tamura,3 and K. Sasajima1. Dept. Surgery, 1Nippon Medical School Tama-Nagayama Hospital, Tokyo 206-8512, Japan; 2Nippon Medical School, Tokyo; 3Dept. Endocrine Pharmacology, Tokyo Univ. Pharmacy and Life Sciences, Tokyo

Objective: To examine the mechanism by which peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, pioglitazone, improves omental adipocyte function following polymicrobial sepsis.

Methods: Male mice received pioglitazone (10 mg/kg/day) for 7 days before cecal ligation and puncture (CLP). The survival rate was monitored for 7 days. The blood sample and omental tissues were collected before, on 24, and 48 h after CLP.

Results: The survival rate in the pioglitazone group for 7 days was significantly higher than that in control group after CLP. The serum levels of adiponectin in the pioglitazone group were higher compared with the control group before CLP. The serum levels of adiponectin after CLP in the both groups were decreased compared to those before CLP. However, administration of pioglitazone significantly prevented the reduction of adiponectin levels on 48 h after CLP. The serum levels of interleukin-6 and monocyte chemoattractant protein-1 were elevated following CLP. Administration of pioglitazone attenuated these serum levels and omental mRNA expressions on 24 h after CLP. Furthermore, mRNA expressions of omental TNF-α and interleukin-1β following CLP were also inhibited by pioglitazone.

Conclusions: These data suggest that pioglitazone administration on polymicrobial sepsis improves survival rate and might attenuate the inflammatory response through the activation of adipocyte function. (Supported by Grant for Japanese Scientific Research (C) 20591537).


GENIPIN PROTECTS MICE FROM CECAL LIGATION AND PUNCTURE INDUCED POLYMICROBIAL SEPSIS.T.H. Kim,* S.J. Yoon,* and S.M. Lee. School of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea

Sepsis is an acute life-threatening clinical condition and remains the major cause of death in intensive care units. The high rate of mortality in patients with sepsis results from an inappropriately amplified systemic inflammatory response to infection. Gardenia fructus, a traditional Chinese medicine, has long been used for the treatment of inflammatory diseases. Genipin functions as the main bioactive compound to exhibit the pharmacological activities of gardenia. The aim of this work was to evaluate the anti-inflammatory activity of genipin in vitro and the protective effect of genipin during polymicrobial sepsis induced by cecal ligation and puncture (CLP). RAW264.7 cells were exposed to lipopolysaccharide (1 μg/ml) for 24 hr in the absence of genipin (1-100 μM) or not. The pretreatment of genipin markedly inhibited the release of nitrites, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, -6 and monocyte chemoattractant protein (MCP-1) and high mobility group box 1 (HMGB1) into the culture media. In polymicrobial sepsis, intravenous administration of genipin immediately after CLP significantly elevated the survival rate at doses of 2.5 and 5 mg/kg. In septic mice, the serum levels of TNF-α, IL-1β, IL-6, Interferon-γ and MCP-1 were significantly increased 6 hr after CLP. Furthermore, the serum level of HMGB1 protein, the late mediator of lethal systemic inflammation in sepsis, was increased 24 hr after CLP. These increases were attenuated by genipin. Our data suggest that genipin has a protective effect on polymicrobial sepsis by modulating inflammatory cytokines. Thus, genipin may serve as an effective therapeutic agent for septic patients.


THE SELECTIVE V1a AGONIST POV ATTENUATES VASCULAR LEAKAGE BY REDUCING ANGIOPOIETIN-2 CONCENTRATIONS IN MRSA-INDUCED SEPSIS.S. Rehberg,* P. Enkhbaatar, L. Sousse,* Y. Yamamoto,* L. Traber, and D.L. Traber. Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555

Objective: To compare the effects of the selective V1a-receptor agonist [Phe2Orn8]vasotocin (POV) and the mixed V1a/V2-receptor agonist [Arg8]vasopressin (AVP) on vascular leakage associated with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced septic shock using an established ovine model.

Methods: Chronically instrumented sheep were subjected to 48 breaths of cotton smoke and 4×1011 CFU MRSA were placed into both lungs under deep anesthesia. Sheep were randomly assigned to receive POV, AVP or vehicle (0.9% NaCl) intravenously (n=6 each). POV and AVP were titrated to keep mean arterial pressure above baseline-10mmHg. All sheep were awake, mechanically ventilated and fluid resuscitated. Data are expressed as mean±SEM and normalized for individual survival times.

Results: Compared to vehicle, AVP and POV significantly reduced fluid input (vehicle: 10±0mL/kg/h; AVP: 8±1mL/kg/h; POV: 6±1mL/kg/h), positive net fluid balance (8±0mL/kg/h; 5±1mL/kg/h; 0±1mL/kg/h), thoracic (2±0mL/kg/h; 1±0mL/kg/h; 0±0mL/kg/h) and abdominal fluid (0.29±0.13mL/kg/h; 0.10±0.04mL/kg/h; 0.01±0.01mL/kg/h) as well as extravascular lung water (23±3mL/kg; 23±1mL/kg; 16±2mL/kg) and increased plasma oncotic pressures (9±0mmHg; 13±1mmHg; 16±1mmHg). POV was superior to AVP in all cases (p<0.05 each). Angiopoietin-2 levels were lower in the POV group than the AVP or vehicle group (lung: 10±1pg/mg; 11±0pg/mg; 12±1pg/mg; heart: 11±1pg/mg; 15±1pg/mg; 15±1pg/mg, respectively, p<0.05 each).

Conclusions: In MRSA-induced ovine septic shock, the selective V1a-receptor agonist POV reduces tissue levels of angiopoietin-2 and vascular leakage more effectively than the mixed V1a/V2-receptor agonist AVP.


ADIPONECTIN DEFICIENCY PROMOTES THE PRODUCTION OF INFLAMMATORY MEDIATORS WHILE SEVERELY EXACERBATING HEPATIC INJURY IN MICE WITH POLYMICROBIAL SEPSIS.Y. Uji,1 H. Yamamoto,2 H. Tsuchihashi,2 H. Akabori,2 T. Shimizu,2 Y. Endo,2 and T. Tani2. 1Department of Surgery, Shin-Koga Hospital, 2Department of Surgery, Shiga University of Medical Science

Objective: Adiponectin (APN), which is an adipose tissue-derived hormone, is known as an anti-inflammatory cytokine. In this study, the possible involvement of APN in polymicrobial sepsis was investigated using adiponectin-knockout (APN-KO) mice that had cecal ligation and puncture (CLP).

Methods: Wild type (WT) and APN-KO mice underwent CLP. The plasma and hepatic levels of inflammatory mediators, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were measured after CLP. A histological analysis of the liver was examined to evaluate hepatic injury.

Results: APN deficiency resulted in significant increases in the plasma levels of TNF-α, IL-6, and MCP-1 at 24 hours after CLP. Hepatic MCP-1 and plasma AST levels in APN-KO mice were significantly higher than those in WT mice at 48 hours after CLP. A steatosis change and MCP-1 expressions in hepatocytes were induced in APN-KO mice during sepsis.

Conclusion: These results suggest that an APN deficiency induces an excessive systemic inflammatory status and exacerbates hepatic injury during polymicrobial sepsis.



Vascular catheters are the most common cause of blood stream infections in critically ill patients. There is increasing awareness that bacteria colonize the catheter lumen within vegetations termed biofilms. Little is known about factors that influence the initial stages of bacterial adherence to indwelling catheters. Vascular catheters (indwelling for 2 days to almost 3 yr) were removed from 25 patients with no microbiological evidence of systemic infection, placed immediately in glutaraldehyde-based fixative, and processed for scanning electron microscopy. All catheters contained noticeable vegetations that varied in ultrastructural appearance but contained one or more of the following characteristics: amorphous material, fibrillar elements, platelets, red blood cells, and an occasional leukocyte. The extent of these vegetations varied, but often occluded at least one-half of the catheter lumen. Catheters from four patients were additionally rinsed with saline, cut into three to six 1-cm segments, and injected with ∼108/ml Staphylococcus aureus (washed cells from overnight broth). Bacteria were allowed to dwell within the catheter at room temperature for 15 min, rinsed, sonicated, and processed for quantitative microbiological culture, with data compared to control (unused) catheter segments. Results were variable. Compared to control segments, catheters from two patients had significantly decreased (P<0.01) numbers of adherent S. aureus. The catheter from a third patient had increased numbers of S. aureus (P<0.05) compared to control segments, and the catheter from the fourth patient did not appear to affect bacterial adherence. Thus, vascular catheters from patients with no evidence of systemic infection have luminal vegetations of varying sizes, and these vegetations can modulate bacterial adherence to the catheter surface.


ZINC SUPPLEMENTATION IMPROVES SURVIVAL IN POLYMICROBIAL MURINE SEPSIS.J. Nowak,* K. Harmon,* and H. Wong. Cincinnati Children's Hospital Medical Center Research Foundation, Cincinnati, OH

Objective: Genomic analysis has shown that human sepsis causes widespread repression of genes involved in zinc (Zn) homeostasis (Physiol Genomics 30:146; 2007) and Zn deficiency increases mortality in murine sepsis (Crit Care Med 37:1380; 2009). Our objective was to determine if Zn supplementation could improve survival in murine sepsis, and if so, through which mechanisms.

Methods: 4-5 wk old C57BL/6 mice were given 10 mg/kg of Zn gluconate (n=30) or placebo (n=30) i.p. daily. On the 4th day polymicrobial sepsis was induced through i.p. injection of a slurry of fecal contents (1.5 mg of fecal content/gm). The mice continued to receive either Zn gluconate or placebo daily and were observed for 72 hrs to determine mortality. Sixteen additional mice (8/arm) were treated in the same manner, except they were sacrificed 24 hrs after induction of sepsis and organs were harvested to evaluate either bacterial clearance via culture (n=10) or NF-κB activity in the lung via electrophoretic mobility shift assay (EMSA) (n=6).

Results: Survival was greater in the mice who had received Zn supplementation (p=0.026, Kaplan-Meier analysis). The survival differential was seen at 24 hrs, and persisted at each 24 hr interval (Table). There was no difference in bacterial colony counts in blood, spleen, or lung. Preliminary EMSA data indicate that NF-κB activity was greater in Zn supplemented animals.

Conclusions: Supplementation with Zn improves survival, leads to increased NF-κB:DNA binding in the lung, and does not appear to affect bacterial clearance in polymicrobial murine sepsis. (Supported by T32GM008478 and R01 GM064619).



ROLE OF BIOMARKERS ON MORTALITY PREDICTION IN SEVERE SEPSIS AND SEPTIC SHOCK.J.H. Lee, K.P. Lim, I.S. Cho, Y.W. Nam, Y.H. Jo, K. Kim, J.H. Lee, J.E. Rhee, T.Y. Kim, W.Y. Kwon, and G.J. Suh. Department of Emergency Medicine, Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, 463-707, Korea

Objective: Several prognostic scales have been used to predict mortality in severe sepsis and septic shock. We performed this study to investigate whether biomarkers could increase the accuracy of prognostic scale to predict 28-day mortality.

Methods: This study was a prospective observational study. When patient was diagnosed as severe sepsis or septic shock, early goal-directed therapy was provided. Cholesterol, albumin, C-reactive protein (CRP), lactate, N-terminal pro-B-type natriuretic peptide (NT-proBNP), D-dimer, fatty acid-binding protein (FABP) were measured when patients were enrolled. In each case, the Acute Physiology And Chronic Health Evaluation (APACHE) II score was calculated.

Results: Of a total 116 patients, 68 (59%) were male and mean age was 69.9±13.4 years. Forty five (39%) patients died. The APACHE II score, lactate, FABP and NT-proBNP in non-survival group were significantly higher than those of survival group (p<0.001). Serum cholesterol and albumin level were significantly lower in non-survival group (p<0.001). The area under the curve (AUC) of APACHE II, lactate, FABP, NT-proBNP, cholesterol and albumin were 0.784, 0.712, 0.726, 0.692, 0.663 and 0.759, respectively. Adding each biomarker to APACHE II did not significantly increase of AUC. However combination of two biomarkers such as lactate and albumin to APACHE II, albumin and NT-proBNP to APACHE II, and lactate and FABP to APACHE II significantly increased AUC from 0.784 to 0.874, 0.869 and 0.865, respectively.

Conclusion: Adding lactate and albumin, albumin and NT-proBNP, and lactate and FABP to APACHE II score improve the 28-day mortality prediction in severe sepsis and septic shock.


THE ROLE OF IP-10/CXCR3 IN THE NEONATAL IMMUNE RESPONSE TO A SEPTIC INSULT.A. Cuenca, J. Wynn, K. Kelly-Scumpia, P. Scumpia, D. Nacionales, S. Kunkel, P. Efron, and L. Moldawer. Department of Surgery, University of Florida Gainesville, FL 32610

Despite significant advances in critical care, the mortality rate to neonatal sepsis has remained unchanged over the past two decades. Though several studies have identified functional deficiencies in neonatal innate immunity (ie poor neutrophil extracellular trap formation or reactive oxygen species (ROS) production), the mechanisms underlying recruitment and/or modulation of neonatal innate immunity during infection remain poorly defined. In adults, blockade of CXCL10 (IP-10) has been shown to increase mortality to experimental sepsis, although the mechanism is not well known. Therefore, we sought to define the role of IP-10/CXCR3 signaling in neonatal sepsis. Here, we provide evidence that increased recruitment of peritoneal neutrophils and macrophages is closely associated with increases in peritoneal IP-10 concentrations and CXCR3 expression on innate immune effector cells recruited to the peritoneum following septic challenge. In addition, blockade of IP10 is shown to not only decrease peritoneal macrophage and neutrophil numbers, but is also to worsen survival following sepsis compared to control antibody treated mice (25% vs 65% p<0.05). To better understand the effect of IP-10 on neutrophil and macrophage function, neonatal splenocytes were incubated in vitro with recombinant IP-10 (rIP-10, 100 ng/mL) and subsequently assayed to determine phagocytic function (FITC-labeled latex beads) or ROS production (phorbol stimulation). Though rIP-10 had no effect on ROS production, rIP-10 significantly enhanced both macrophage and neutrophil phagocytic ability. This data suggests that IP-10/CXCR3 signaling is required for the recruitment and phagocytic function of innate immune effector cells in vivo and may represent a novel therapeutic to modulate neonatal innate immunity and improve outcome to neonatal sepsis.


THE ANTIMICROBIAL PEPTIDE LIPOCALIN 2 IS A VERY EARLY MARKER IN THE RESPONSE TO SEPSIS.D.E. Vazquez,* V.T. Le,* A. De Maio, and D.M. Cauvi*. University of California San Diego, La Jolla, CA 92093

Introduction: Lipocalin 2 (Lcn2) is an antimicrobial peptide that binds to iron-laden siderophores, limiting bacterial growth. Lcn2 is a key component of the innate immune system and its production is mediated by activation of Toll-like receptors (TLR). The role of this novel antimicrobial peptide in sepsis has not been fully elucidated.

Methods: Eight-week-old, male A/J and C57BL/6J (B6) mice, which display a difference in the inflammatory response after a variety of stimuli, were used. Sepsis was induced by cecal ligation and puncture (CLP). Lungs were harvested 1, 2, 3, 6 and 20h after CLP. Lcn2 was measured by quantitative RT-PCR. Statistical analysis was performed by student t-test.

Results: Lcn2 expression in lungs was detected as early as 1h after CLP in both A/J and B6 mice compared to non-treated animals (p<0.05). Lcn2 peaked at 2h in B6 mice and 3h in AJ mice. Expression significantly dropped in both strains by 6 h. A second Lcn2 expression peak was observed at 20h in B6 mice, but not in A/J mice. Throughout all time points B6 mice show a greater amount of Lcn2 in comparison with A/J mice.

Conclusions: Lcn2 appears as a very early marker in the response to sepsis. The expression of this peptide may play a key role in the control of bacteremia during sepsis. In addition, Lcn2 expression is modulated by the genetic background of the subject.



Cardiac dysfunction is associated with both aberrant inflammation and remodeling of the extracellular matrix through elevated activity of inflammatory cytokines and matrix metalloproteinase's. Our study demonstrates that TNF triggers cardiac expression and activation of MMP's, particularly MMP9, during septic challenge. Neonatal cardiomyocytes were isolated from 1-2 day old mouse pups (WT and TNF-/-) and were treated with recombinant TNF (20ng/ml). Samples were removed at 30 m, 1, 2, 4, 8, 12, and 24 hr. Real-time PCR was used to measure mRNA levels for MMP2, 8, 9 and 12, TIMP2, IL-6, IL-1β, and MIF. Samples were also isolated for gelatin zymography to measure MMP protein levels and activity. Examining the mRNA expression of the MMP's, MMP9 specifically was enhanced in WT cardiomyocytes as early as 1 hr, but showed a 35-fold increase by 12 hr. In both strains, rTNF significantly induced expression of MMP8 (6-fold) and MMP12 (2-fold), in a TNF independent manner. Both WT and TNF-/- mice were subjected to S. pneumonia type 3 sepsis. At 24, 48 and 72 hr post inoculation, cardiac function was measured by echocardiography along with sham controls for both strains. Fractional shortening was significantly decreased in the WT at 48 hr post inoculation (51.7±1.2 vs 16.2±1.9). The differences in TNF-/- mice were greatly attenuated (52.7±1.3 vs 48±2.2). Cardiac samples from both groups showed a consistent decrease in MMP9 in the TNF-/- mice as compared to WT at all times. The in vitro and in vivo results shown in this study provide a direct link between TNF and MMP's, with TNF as an upstream regulator of MMP induction and cardiac dysfunction during septic insult.


EXPRESSION OF ENDOPLASMIC RETICULUM STRESS-RELATED PROTEINS INCREASE IN ADRENAL GLANDS IN SEPSIS.T. Suzuki,* N. Matsuda,* H. Teramae,* S. Beppu,* M. Futatsugi,* and K. Koike. Department of Primary Care and Emergency Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

Introduction: Although recent studies have shown that endoplasmic reticulum (ER) stress is involved in the pathophysiology of chronic inflammatory diseases, it remains unclear whether ER stress has a critical role under acute inflammatory conditions.

Hypothesis: ER stress is implicated in the pathophysiology of sepsis in adrenal glands.

Methods: Male mice underwent cecal ligation and puncture (CLP). The adrenal gland samples were collected at 24 hours after CLP and immunohistochemical staining was performed. Normal mice were used as a control group.

Results: Three ER stress sensor proteins, IRE1α, PERK and ATF6, as well as ER chaperon-protein, BiP, expressed stronger in the adrenal cortex than in the medulla. In addition, CHOP, apoptosis associated protein induced by ER stress, also expressed more in the cortex. However, the expression of all these proteins in the adrenal medulla was greatly increased following CLP. Moreover, phosphorylation of PERK was also augmented in the zona glomerulosa of the cortex, in which mineral corticoids were produced, by polymicrobial sepsis.

Conclusions: The present results suggest the possibility of the involvement of ER stress in the pathophysiology of sepsis in the adrenal medulla. On the other hand, the adrenal cortex, which has higher constitutive expression of ER stress-related proteins, appears to have more innate ability to manage ER stress compared to the medulla.

CHOP in the adrenal cortex. Bar: 30μm.


MAL MUTATIONS RESULT IN DECREASED NF-kB UPREGULATION IN RESPONSE TO LPS.K. Banton, K. McCulloch, K. Wasiluk, and Spon: G. Beilman. University of Minnesota, Minneapolis, MN 55455

Objective: To investigate the effect of amino acid (aa) substitutions within Mal, an adapter protein of Toll-like receptor 4 (TLR4), on NF-kB expression in lipopolysaccharide(LPS)-responsive cells.

Methods: We modified 6 charged aa in Mal by site-directed mutagenesis of full-length Mal cloned into the expression vector pcDNA4/HisMax™ and cotransfected into RAW 264.7 cells with an NF-kB luciferase reporter vector. Transfected cells were then stimulated with LPS. NF-kB upregulation was measured by luciferase assay. Double mutants contained P145H, comparable to the TLR4 P712H mutation known to abrogate TLR4 signaling.

Results: Without LPS, all mutants with P125H as well as D142A, R141A, and D105N had reduced NF-kB upregulation as compared with baseline (Figure). With LPS, all constructs had increased NF-kB upregulation, as compared with no LPS, but only D142A, D107A, and D105R/P145H had NF-kB levels higher than baseline. Intriguingly, P145A resulted in slightly diminished NF-kB upregulation as compared with native Mal. R141A had the least NF-kB upregulation.

Conclusions: Mutations within Mal may decrease the susceptibility of individuals to infections, limiting the inflammatory response to gram-negative bacteria. Mutations in R141, P145, and D142 have the greatest effect on NF-kB upregulation, while mutations of D107, and D223 have minimal effects. Knowledge of structure/function relationships at the level of single aa will lead to further understanding of the molecular mechanisms regulating the inflammatory response.




Timely identification and initiation of care for septic patients remains a clinical challenge. We conducted a prospective before-and-after study to evaluate electronic prompting of ED staff on sepsis care. Adult ED patients were continuously screened for the presence of >= 2 SIRS criteria and 2 systolic blood pressures <= 90 mmHg using information system software. In the 'Before' phase (3 months), detected patients were registered only. In the 'After' phase (3 subsequent months) caregivers were notified by pager of patients' condition and prompted regarding sepsis bundle recommendations. Outcomes were frequency of 3 studies (blood culture, blood lactate, and chest x-ray) and 1 intervention (antibiotics). Analysis included chi-squared testing and Cox proportional hazard modeling of time-to-event data. 451 patients were studied. The system was 100% sensitive and 47% specific in identifying severe sepsis among patients. Of the 4 sepsis bundle outcomes studied, only the obtaining of blood cultures was statistically significantly improved by the use of the system (p = 0.02). This appeared to be due to extensive delay between presentation and patients manifesting ≥ 2 SIRS criteria and hypotension; the mean time to these findings was 138 minutes after presentation. In ∼ half of these patients, caregivers initiated sepsis bundle steps based on other patient features. Monte Carlo simulations based on our data indicate that for every one minute of hastened delivery of blood culture, lactate, chest X-ray, or antibiotics, future search algorithms would need to identify patients 4 minutes sooner. In conclusion, patients ultimately diagnosed with severe sepsis in the ED do not typically present meeting diagnostic criteria, but do so only after hours of evaluation. Software for hastening care of septic patients in an emergency department should pursue patient features other than SIRS and hypotension, as these are likely to manifest themselves relatively late.


SEPSIS INDUCES CHANGES IN INTESTINAL EXPRESSION OF PD-1:PD-L1.S. Patel,* N. Shubin,* A. Ayala, and C.S. Chung. RI Hospital, Providence, RI 02903

Costimulatory signals, whether stimulatory and inhibitory, are critical to an effective/balanced immune response. Recent studies have demonstrated that programmed death-1 (PD-1), an inhibitory receptor, and its ligands (PD-L1 /PD-L2) are involved in the pathogenesis of autoimmune diseases and intestinal mucosal inflammation. Previous studies from our laboratory have also shown that the PD-1 may play a role in mediating the inflammatory response seen in sepsis. PD-1 deficient (PD-1 -/-) mice are protected from septic lethality and this is associated with enhanced bactericidal activity, reduced inflammation and preserved tissue integrity including small intestine in these mice. However, little is known about the role of PD-1:PD-L1 in local intestinal pathology in response to sepsis. Therefore, the aim of this study was to examine the expression of PD-1/PD-L1 and assess their role in local inflammatory response of the small intestine after sepsis. To do this, male C57BL/6 mice were subjected to sham or cecal ligation and puncture (CLP). At different times post-surgery, the small intestine was harvested and intestinal cells were isolated and assessed for phenotypic expression of PD-1 and PD-L1 in combination with various cell surface markers by FACS. Our results indicate that PD-1 expression was mainly evident on EpCam+ and less on CD45+ intestinal cells. Although the percentage of PD-1 expressing cells was no change after sepsis compared to shams, the actual numbers of PD-1+EpCam+ cells were significantly increased in septic mice (n=2-6/group, Mann-Whittney U test). Similarly, PD-L1 expression was observed on EpCam+ and CD45+ intestinal cells and sepsis appeared to induce an increase in the numbers of PD-L1 expressing cells. These findings suggest that PD-1:PD-L1 pathway may play an important role in sepsis-induced intestinal dysfunction.


HYPERTONIC FLUIDS IN SICU SHOCK PATIENTS REDUCES FLUID AND OXYGEN REQUIREMENTS.J. Pascual,* P. Rosen,* A. Horan,* B. Sarani,* C. Sims, and C. Schwab*. U. of Pennsylvania, Philadelphia, PA 19104

Multiple animal studies have reported benefits of hypertonic saline (HTS) resuscitation but the few human studies have shown conflicting results in prehospital trauma patients. No study to date has evaluated HTS in surgical intensive care unit (SICU) patients days after admission. We hypothesized that HTS in SICU patients would reduce fluid needs and improve organ function.

Methods: Charts of SICU patients receiving HTS for shock or volume depletion from 7/07-2/08 were reviewed for demographics, hemodynamics, fluid requirements, serum electrolytes, urine output and oxygen requirements (FiO2) 24hrs before (pre) and 24hrs after (post) a bolus of 500mls of 3% HTS. Paired t-test and chi square analysis established significance at p<0.05.

Results: 42 patients received HTS in a long protracted SICU stay. Mean age was 53, 70% were male, 52% were trauma patients, 48% postoperative patients. 7.1L of crystalloid boluses were required 24 hrs before HTS as compared to 4.8L after (*p<0.05) and colloid decreased by 0.5L (Fig1). [Na+] (140 to 143mEq/L) & [Cl-] (109 to 114mEq/L) rose after HTS (p<0.001). FiO2 use dropped by 14% after HTS (*p<0.01) (Fig2). Urine output and arterial & central venous pressure increased. Hemoglobin dropped (11.1 to 10.2 g/dL, p<0.001). Pressor use was unchanged. 8 patients died (27%), 1 required renal replacement therapy and none suffered myocardial ischemia.

Conclusion: The use of HTS resuscitation in SICU patients reduces the need for fluid boluses, may reduce oxygen requirements and appears safe with minimal hypernatremic risks. A prospective trial is needed to further delineate advantages.




Introduction: Pre-injury use of statins, a class of drugs that lower blood cholesterol levels, have been shown to modify trauma outcomes. Some studies suggest that patients who were using statins prior to injury had improved outcomes. However, if the medication was abruptly discontinued, there was an increased risk of post-injury complications including organ failure. In this study, we investigated the effects of simvastatin withdrawal, on the endotoxin induced pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC).

Methods: Whole blood was obtained by venipuncture from healthy human volunteers and PBMC were isolated by Ficoll-Paque density gradient centrifugation. Cells were plated and stimulated with 100ng/ml of LPS after the following conditions: 1) no treatment, 2) 0.2uM simvastatin treatment for 48hrs, 3) 0.2uM simvastatin treatment for 24hrs and simvastatin withdrawal for 24hrs, 4) 0.2uM simvastatin treatment for 24hrs, withdrawal for 24hrs and simvastatin reinstitution for 8hrs. Cell culture supernatants were harvested and analyzed for IL-6 and TNF-alpha by ELISA.

Results: When simvastatin was withdrawn after 24 hrs of pretreatment, LPS stimulation augmented both pro-inflammatory cytokines, IL-6 and TNF. However, when simvastatin was reinstituted during LPS stimulation, IL-6 and TNF levels were attenuated.

Conclusion: These results suggest that critically ill patients with pre-injury statin use withdrawn should have the drug restarted after injury to minimize an exaggerated inflammatory response.



ROS-DEPENDENT MODULATION OF TLR4 SIGNALING AND SEPSIS.X. Kong,* R. Thimmulappa,* and S. Biswal. Johns Hopkins University, Baltimore, MD 21205

Rationale: Reactive oxygen species (ROS) play crucial roles in TLR4 activation and hence in the pathobiology of sepsis by regulating immune cell activation and end organ injury. Excess ROS have the ability to prime the phagocytes (macrophages and neutrophils) against an acute hyper-inflammatory response. Host factors that regulate cellular ROS levels may act as important modifiers in pathogenesis of sepsis. NADPH oxidase is the primary generator of ROS in phagocytes. Nrf2 is a primary transcription factor that regulates cellular antioxidant defenses. The present study was designed to investigate if ablations of NADPH oxidase, Nrf2 and or both modulate ROS generation, TLR4 downstream signaling, lung inflammation and mortality.

Method: Wild type (Nrf2+/+), Nrf2-deficient (Nrf2-/-), Gp91phox-deficent (gp91phox-/-) and Nrf2 and gp91phox double knockout mice (Nrf2-/-//gp91phox-/-) mice were used. ROS, PKC activity, phosphorylation of P47phox, TLR4 signaling and cytokine expression was measured in phagocytes. LPS-induced lung inflammation and CLP-induced mortality was investigated.

Results: Nrf2-directed GSH regulation suppressed PKC mediated NADPH oxidase activity and ROS generation in macrophages. Relative to Nrf2+/+, LPS induced greater activation of TLR4 signaling as evident by TLR4 surface trafficking, downstream recruitment of MYD88 and TRIF, and phosphorylation of IKB and IRF3 in Nrf2-/- macrophages that was diminished by ablation of gp91phox. Ablation of NADPH oxidase component, gp91phox in Nrf2-/- mice significantly suppressed LPS-induced lung inflammation and CLP-induced mortality.

Conclusion: Redox homeostasis in innate immune cells is critical in sepsis pathogenesis. Nrf2 modulates immunopathogenesis of sepsis by redox regulation of innate immune response. (Supported by NIGMS R01GM079239).



Rationale: Recent studies have shown critical role of CD4 cells in suppressing gut epithelial apoptosis and mortality after sepsis. Nrf2 is a bZIP transcription factor that regulates a robust cytoprotective defenses and protects from apoptosis. Whole-body disruption of Nrf2 augments inflammation and mortality after cecal ligation and puncture (CLP). The present study was designed to investigate if ablations of Nrf2 specifically in CD4 cells modulate sepsis.

Methods: Mice with conditional deletion of Nrf2 in CD4 cells (CD4-Nrf2-/-) was generated by crossing Nrf2 flox/flox (Nrf2f/f) with CD4-cre mice. CD4-Nrf2-/- and Nrf2f/f was subjected to CLP.

Results: Antioxidant defenses (NADPH- quinone reductase, GSH biosynthesizing enzymes, heme oxygenase-1) in purified CD4 cells isolated from CD4-Nrf2-/- mice were significantly lower compared to Nrf2f/f. Ex vivo studies indicated greater propensity of Nrf2-deficient CD4 cells and splenocytes for apoptosis after H2O2 treatment. CLP induced greater mortality in CD4-Nrf2-/- mice compared to Nrf2f/f mice.

Conclusion: Nrf2-dependent cytoprotective response is critical in modulating sepsis pathogenesis. (Supported by NIGMS R01GM079239).


ULTRASTRUCTURAL CHANGES AND UNDERLYING MOLECULAR MECHANISMS IN RAT LIVERS INDUCED BY ENDOTOXIC SHOCK.S. Nuernberger,*1,2 S. Gupta,*3 E.T. Kavanagh,*3 J.C. Duvigneau,4 I. Miller,*4 O. Hori,*5 A. Samali,*3 H. Redl,1 and A.V. Kozlov1. 1L. Boltzmann Inst. Exp. Clin. Traumatol. in AUVA Res. Center Vienna, 2Med. Univ. Vienna, Dept. Traumatol, Austria; 3Dept. Biochem., National Univ. of Ireland, Galway, Ireland; 4Dept. Nat. Sci., Univ. Vet. Med. Vienna, Austria; 5Dept. Neuroanat., Kanazawa University, Japan

Objective: In the present study we investigated livers of rats subjected to endotoxic shock.

Methods: Rats treated with LPS were subdivided into four groups in accordance to their ALT blood levels. Morphological analyses of livers were done by light and electron microscopy (EM); RT-PCR was used for gene expression analyses.

Results: EM-examination did not reveal changes in mitochondrial structure of hepatocytes. However, dramatic changes were observed in endoplasmic reticulum (ER) in close vicinity to mitochondria, appearing as dilated ER (Fig.). Apoptosis was visible only in blood cells infiltrating liver tissue, not in hepatocytes. While the mRNA levels of some ER stress markers were slightly upregulated (SCOTIN, GRP78), the protein levels of ER stress markers (GRP78, CHOP, Herp) remained unchanged. These findings suggest the development of unresolved ER stress in liver cells. Some transcripts of Bcl-2 family members associated with ER stress and apoptosis were upregulated suggesting the activation of apoptotic pathways. Other markers of apoptosis (BIM, NOXA, BNIP3, BMF, Puma, Bax) were unchanged suggesting that apoptosis remained incomplete.

Conclusion: Ultrastructural changes in hepatocytes in response to severe endotoxic shock are associated with ER. The specific location of dilated ER suggests that mitochondria are involved in pathways leading to ER stress and ER dilation.



SUBCELLULAR FRACTIONS OF LIVERS OBTAINED FROM RATS IN ENDOTOXIC SHOCK - A PROTEOMIC STUDY.I. Miller,1* B. Gesslbauer,1* H. Redl,2 and A.V. Kozlov2. 1University of Veterinary Medicine Vienna, 1210, Austria; 2L. Boltzmann Inst. Exp. Clin. Traumatol. in AUVA Res. Center, 1200 Vienna, Austria

Objective: Sepsis and endotoxic shock lead to the development of multiple organ failure, which often is not accompanied by histological changes in relevant organs, suggesting that organ failure is the result of cellular dysfunction. Cellular dysfunction may often be reflected in the protein patterns of the affected organs. This study investigates changes in the proteome in the relevant subcellular fractions (mitochondrial, endoplasmic reticulum (ER), and cytosolic) of rat liver in response to LPS challenge.

Methods: Livers from Sprague-Dawley rats were obtained after 16 hours of LPS challenge (8 mg/kg, i.p.). Subcellular fractions were prepared by differential centrifugation, proteins labelled with fluorophores and separated by two-dimensional electrophoresis (2D-DIGE). Spots differentially regulated between treated and control animals were identified by mass spectrometry methods.

Results: Upon LPS challenge, SOD-2, catalase and the alpha-chain of ATP synthase were upregulated in the mitochondrial fraction. The cytosol contained increased levels of intact carbamoylphosphate synthase (CPS), 60 kDa heat shock protein, and one upregulated peroxiredoxin-1 spot. The most remarkable changes were observed in ER: many functional proteins were down-regulated (e.g. GRP78, protein disulfide isomerase A3, argininosuccinate synthase, transitional ER ATPase). At the same time, the expression levels of proteins responsible for antioxidant capacity were increased in mitochondria, slightly increased in cytosol, and markedly decreased in ER. Fragmentation of CPS, an important member of the urea cycle, was increased in mitochondria of treated animals.

Conclusion: Our data suggest that protein patterns of ER are more sensitive to endotoxic shock than protein patterns of cytoplasm and mitochondria.


MITOCHONDRIA IN SYSTEMIC IMMUNE RESPONSE: A VICTIM OR AN OFFENDER?.A.V. Kozlov,1 I. Kehrer,1* J.C. Duvigneau,2 S. Nürnberger,1* J. Paier-Pourani,1 I. Miller,2* and H. Redl1. 1L. Boltzmann Inst. Exp. Clin. Traumatol. in AUVA Res. Center, Vienna, Austria; 2University of Veterinary Medicine Vienna, Austria

Objective: To clarify the contribution of mitochondria to liver dysfunction induced by systemic inflammatory response (SIR).

Methods: SIR was induced in rats by LPS injection in vivo; liver tissue and mitochondria were investigated. In vitro rat hepatocytes were incubated with medium (conditioned medium = CM) obtained after ex vivo incubation of rat white blood cells with LPS.

Results: Injection of LPS in rats was accompanied by a reversible inhibition of mitochondrial function, observable only in vivo (ATP-levels), but not in isolated mitochondria. An irreversible increase of mitochondrial ROS (mROS) production was detected both in vivo and in isolated mitochondria. Electron microscopy revealed unchanged mitochondrial morphology, impaired structure of ER and the absence of apoptosis in hepatocytes. Histological examination revealed focal necroses affecting less than 10% of liver tissue. In vitro experiments were performed to better understand the impact of mROS. The incubation of hepatocytes with CM resulted in the elevation of mROS levels, ER-stress markers (GRP78, XBP-1), and IL-6, a marker of acute phase reaction, but did not remarkably induce oxidative stress (lipid peroxidation). A mitochondria-targeted ROS scavenger, Mito-TEMPO significantly attenuated the levels of mROS in liver cells. Simultaneously it attenuated increased IL-6 levels, but further up-regulated ER-stress markers. The latter was observed both in control cells and in cells treated with CM.

Conclusion: Our data suggest that SIR neither induces apoptosis nor impairs mitochondria, but causes reversible inhibition of mitochondria in vivo. Increased mROS production does not contribute to the development of oxidative stress, but it interferes with the expression of genes, relevant for liver dysfunction.



Objective: To evaluate the effect of Monophosphoryl Lipid A (MPLA), a clinically useful immunomodulator, on the innate immune response during clinically relevant models of systemic infection in mice.

Methods: Mice received treatment with MPLA or vehicle on two consecutive days prior to CLP. In further experiments, mice with 20% TBSA burns were treated with MPLA on days 3 and 4 post-burn followed by burn wound inoculation with Pseudomonas on day 5. Endpoints included survival, rectal temperature, cytokine production and leukocyte recruitment.

Results: Mice treated with MPLA showed significantly improved survival and less hypothermia during sepsis caused by CLP or Pseudomonas burn wound infection. MPLA treatment improved local bacterial clearance and decreased the systemic dissemination of bacteria. The production of MyD88-regulated pro-inflammatory cytokines such as TNFα, IL-1β and IL-6 were attenuated in MPLA-treated mice at the site of infection and systemically but production of TRIF-associated factors such as IFNβ, G-CSF, MCP-1 and RANTES remained intact. The recruitment of granulocytes, inflammatory monocytes and immature myeloid cells to sites of infection was increased in MPLA-treated mice compared to control mice and the percentage and total numbers of myeloid cells mediating bacterial phagocytosis was increased.

Conclusions: These studies show that MPLA treatment improves the innate host response to infection by selectively attenuating pro-inflammatory cytokine secretion but enhancing bacterial clearance. The enhanced bacterial clearance is mediated, in part, by increased recruitment of myeloid cells with effective phagocytic functions. (Supported by NIH R01GM66885 and F31).


AGE-RELATED RESPONSES TO THE TWO-HIT MOUSE MODEL OF POST-TRAUMATIC SEPSIS.S. Drechsler,* K. Weixelbaumer, H. Redl, M. van Griensven, S. Bahrami, and M. Osuchowski. Ludwig Boltzmann Institute for Clinical and Experimental Traumatology and UAVA Research Center, Vienna, Austria

In critical illnesses, age modulates immuno-inflammatory response (IFR) and outcome. To evaluate the impact of age upon pathophysiology of the acute sepsis, we studied the natural evolution of selected IFR and organ function parameters in young and mature mice developing sepsis after trauma-hemorrhage. 3 (n=58) and 15 (n=37) month old female CD-1 mice were subjected to a unilateral femur fracture immediately followed by a sublethal haemorrhage (TH). 48h later, mice were subjected to cecal ligation and puncture (CLP). Blood (20μl) was sampled daily until day 7 post-TH. For all comparisons, mice were retrospectively divided into surviving (SUR) or dead (DIE) by day 5 post-CLP. Post-CLP survival was similar regardless of age (approx. 40% by day 5). In SUR animals, circulating NEU were two-fold higher in young compared to mature mice starting 48h post-CLP (0.5 vs. 1.5 K/μl). Between days 1-5 post-CLP, LYM counts in young mice were consistently higher (by approx. 25%) than in mature mice regardless of outcome. The post-CLP trajectories for Hb and platelets were similar in both age groups. In both outcome groups, young mice had consistently higher RBC count and glucose level (by approx. 20%) compared to mature animals (days 1-5 post-CLP). In SUR animals, ALT was 20-30% higher in young vs. mature mice at 24 and 48h post-CLP. The same was true for the DIE group, but the difference wasn't apparent until 48h post-CLP. Regardless of outcome, LDH was always higher in young mice: up to six-fold (e.g. 500 vs. 3000U/l at 48h post-CLP) between DIE mice and up to two-fold (e.g. 500 vs. 1000U/I at 24h) between SUR animals. Differences in studied parameters accompanied by identical outcome indicate diverse survival and/or pre-lethal mechanisms in young versus mature subjects. (Supported by the WWTF grant).


TH17 TYPE CYTOKINES ARE INCREASED LOCALLY AND SYSTEMICALLY IN A MOUSE SEPSIS MODEL.A. Elkhal,* Y. Sumi,* M. Bhate,* Y. Chen, Y. Yao,* T. Woehrle,* and W.G. Junger. Department of Surgery, BIDMC, Harvard Medical School, Boston, MA 02215

IL-17 type cytokines are crucial mediators of systemic inflammation, neutrophil recruitment, and tissue damage in allergic diseases. IL-17 is a robust pro-inflammatory cytokine that induces lung inflammation, asthma, and COPD. Trauma patients are characterized by tissue damage that is mainly caused by excessive neutrophil recruitment to host tissues, leading to organ failure and ARDS. Here we investigated in a mouse model of sepsis whether Th17 type cytokines are increased. Balb/c mice were subjected to cecal ligation and puncture (CLP) and mRNA levels of Th1, Th2, and Th17 cytokines were measured by real-time RT-PCR in cecum and lung samples collected at different times after sepsis. Th17 cytokines (IL-17A, IL-17F) were dramatically increased in both tissues while the upregulation of Th1 (INFγ, IL-2) and Th2 (IL-4, IL-5) cytokines was less dramatic and more transient compared to Th17 cytokines. Because they are involved in neutrophil recruitment, Th17 type cytokines seem to play an especially important role in local and systemic inflammatory responses in sepsis and ARDS.



CONTINUOUS HEART RATE PREDICTS WORSENING ORGAN FAILURE AND MORTALITY IN SEPSIS.R.C. Arnold, G. Greene, L. Glaspey, S.M. Hollenberg, and A.J. Seely. Cooper University Hospital, Camden, NJ 08103

Objective: To evaluate the feasibility of continuous heart rate variability (HRV) monitoring in emergency department (ED) with sepsis, and assess if progressive change of a multi-parameter HRV panel occurs in association with the development of worsening organ failure or mortality.

Methods: Patients with systemic infection were identified during their initial ED evaluation. Heart rate was continuously monitored for 2 hours in each subject. HRV was characterized continuously using time, frequency, complexity, and fractal domain techniques using a web-enabled continuous individualized multi-organ variability analysis ( tool. Subjects were then followed for the composite outcome of worsening organ failure (SOFA increase ≥ 1) at 24 hours or all-cause in-hospital mortality. The absolute value and the change in each parameter of HRV were calculated (percent change) every 5 minutes.

Results: 31 subjects were enrolled. The composite outcome occurred in 12/31 (38%) with an in-hospital mortality rate of 25% (8/31). Continuous HRV analysis was performed on 584,581 R-R intervals. The dynamic change of complexity and fractal parameters demonstrated progressive loss in those with worsening organ failure or in-hospital mortality. The interval change of each parameter better differentiated the outcome incidence than did the absolute value.

Discussion: Continuous HRV monitoring enables the assessment of absolute as well as interval changes in variability parameters. The progressive loss of variability parameters may assist in identifying subsequent deterioration or risk of death, and suggest a potentially novel target for therapy.



IL-6 AND TGF-α COSTIMULATE MESENCHYMAL STEM CELL VEGF PRODUCTION BY ERK, JNK, AND PI3K-MEDIATED MECHANISMS.J. Herrmann,* B. Weil,* A. Abarbanell,* Y. Wang,* J. Poynter,* M. Manukyan,* and D. Meldrum. Indiana University School of Medicine, Indianapolis, IN 46202

Background: Mesenchymal stem cells (MSCs) protect ischemic tissues in part through paracrine growth factor production. Interleukin (IL)-6, which is upregulated in the heart during ischemia, has been shown to enhance stem cell proliferation and migration. The effect of IL-6 on MSC paracrine function, however, remains unknown. In addition, transforming growth factor (TGF)-α increases MSC VEGF production and may share downstream signaling pathways with IL-6 involving ERK, JNK, and PI3K. We hypothesize that cotreatment with IL-6 and TGF-α will result in greater MSC VEGF production than by either treatment alone via these signaling pathways.

Methods: Murine MSCs were treated with IL-6 (0.05 ng/ml) with or without TGF-α (250 ng/ml) and in combination with inhibitors of ERKI/II, JNK, and PI3K. VEGF concentrations in the supernatants were measured using ELISA. Kinase phosphorylation was measured using Western blot analysis.

Results: IL-6 increased MSC VEGF production at a dose 0.05 ng/ml. The combination of IL-6 and TGF-α (250 ng/ml) increased VEGF production to a greater extent than IL-6 or TGF-α alone. IL-6 induced phosphorylation of ERK, JNK, and PI3K alone and in combination with TGF-α, however, there was no significant increase in phosphorylated levels following cotreatment with TGF-α compared to IL-6 treatment alone. IL-6-stimulated VEGF production was suppressed following inhibition of ERKI/II, JNK, and PI3K, and suppression by each inhibitor was overcome at least in part by cotreatment with TGF-α and IL-6.

Conclusions: IL-6 induces MSC VEGF production via shared intracellular signaling pathways as TGF-α involving ERK, JNK, and PI3K. Pretreating MSCs with IL-6 and TGF-α may be a useful strategy for optimizing stem cell paracrine function prior to therapeutic use.


NEUTROPHIL RECRUITMENT MEDIATES LUNG INJURY AFTER PULMONARY CONTUSION.J. Hoth, J. Wells,* B. Yoza,* W. Meredith, and C. McCall. Wake Forest University Health Sciences, Winston-Salem, NC 27157

Pulmonary contusion is a common and potentially lethal injury. Previous studies have demonstrated that acute lung injury following contusion is inflammatory in nature and associated with pulmonary neutrophilia, adhesion receptor expression, and the elaboration of various inflammatory mediators including CXC chemokines. This study used a mouse model to test the hypothesis that acute lung injury from pulmonary contusion is mediated by neutrophil activation and chemotaxis to the injured lung. Mice were made neutropenic using anti-Ly6g prior to blunt chest injury. Other mice were pretreated with anti-leukinate (CXCR2 inhibition), or with neutralizing antibodies for CXCL1, CXCL2/3, or ICAM-1 before injury. At 24H after injury, tissue, blood, bronchoalveolar lavage (BAL) were collected and analyzed. We found that neutropenia and inhibition of CXCR2, ICAM-1, CXCL1, or CXCL2 resulted in a significant decrease in neutrophil recruitment to the injured lung. Reduced neutrophilia correlated with significant improvement in lung PaO2:FiO2. We conclude that acute injury after pulmonary contusion is dependent on neutrophil activation and recruitment. Inhibition of neutrophil responses will improve lung function after injury.



ALTERNATIVE MODES OF TRANSPORTATION AND THEIR CRASH OUCOMES.M. Miggins,* P. Efron, L. Moldawer, H. Liu,* and D. Ang. University of Florida, Gainesville, FL 32610

Objective: Alternative sources of transportation have become more popular in recent years secondary to a volatile economy and rising gas prices. With this change in the transportation profile, we sought to examine the risk of severe or fatal injury among moped and scooter drivers, identify unique characteristics that put this population of drivers at risk and provide potential variables for injury prevention.

Methods: Moped crashes occurring in Florida between 2002 and 2008 were identified using the Florida Traffic Crash Records Database. The crashes were separated into two cohorts, severe and non severe, according to injury status. Outcomes were compared between the two cohorts using multiple variable logistic regression and adjusted for age, gender and insurance status.

Results: There were no statistical differences among the socio-demographic variables between the two cohorts. Independent variables associated with severely injured drivers were driving speed > 20mph OR 2.02(1.73,2.36), posted speed > 30mph OR1.4(1.22, 1.62), alcohol and drugs OR 2.09(1.64,2.66), being in rural areas OR 1.55 (1.33,1.8), dark lighting conditions OR 1.69(1.23,2.32), cloudy weather OR 1.25(1.02, 1.52), unpaved roads OR 1.57 (1.3,1.88), 4 lanes OR1.83 (1.04, 3.21) and divided highways OR 1.83(1.04,1.4).

Conclusions: Moped and scooter drivers exhibit many of the same risk factors as motor vehicle drivers however some variables are unique to this group and amenable for intervention. Reducing the posted speed to ≤ 30mph in areas with a high density of mopeds as well as not allowing them to drive on roadways with ≥ 4 lanes may decrease their risk for severe injury. Focused driver education should be implemented in areas where the density of moped drivers is small since rural areas tend to be a significant risk factor for severe injury. Also the use of protective equipment did not alter crash outcomes.


HYPERTONIC SALINE DELAYS NF-κB NUCLEAR TRANSLOCATION BUT NOT BY ALTERING IMPORTIN CARRIER AVAILABILITY.L. Santiago,* E. Moore, F. Gamboni-Robertson,* and A. Banerjee. University of Colorado Health Sciences Center, Aurora, CO 80262

Objective: TNF-α stimulation induces nuclear translocation of NF-κB. Hypertonic saline delays translocation in pulmonary epithelial cells (PEC) after TNF-α stimulation, a process correlated to reduced expression of inflammatory cytokines. A transport system involving importins α3, α4 and β1 facilitates passage of NF-κB through the nuclear pore complex (NPC). Our objective was to evaluate the effect of HTS on the net nucleo-cytoplasmic traffic of importins. We hypothesized that HTS would deplete cytoplasmic carriers, thus delaying NF-κB translocation in TNF-α-stimulated cells.

Methods: A549 PEC were cultured in chamber slides, and 6-hour time courses were conducted for the following groups: isotonic control, hypertonic control (180mM NaCl); TNF-α (10μg/mL) stimulation in isotonic medium; and HTS pre-treatment for 30 minutes, followed by TNF-α stimulation in hypertonic medium. Immunofluorescence microscopy was used to analyze the effect of HTS on the subcellular localization of NF-κB and importins α3, α4 and β1.

Results: HTS delays peak nuclear translocation of NF-κB from 60 minutes to over 120 minutes (p < 0.001). TNF-α affected importin α4 distribution in isotonic and hypertonic media (p < 0.01). However, HTS did not affect it in control or TNF-α-stimulated cells. Similar results were observed for importins α3 and β1 (data not shown).

Conclusion: HTS delays NF-κB translocation but not due to a change in the net nucleo-cytoplasmic traffic of importins α3, α4 and β1 under basal or stimulated conditions.



THE IMPACT OF GENDER ON MORBIDITY AND MORTALITY AFTER PELVIC FRACTURE.J. Adams,* M. Cockburn,* J. Luján,* J. Bilaniuk,* B. Siegel,* L. DiFazio,* B. Csóka,* and Z. Németh*. Morristown Memorial Hospital, Morristown, NJ 07962

Objective: We examined the effects of gender on clinical outcome and acute complications after pelvic fracture.

Methods: The trauma registry of the Morristown Memorial Hospital, an ACS Level I regional trauma center, was used to conduct this cohort study. Data on age, mechanism of injury, Injury Severity Score, type of pelvic fracture, ICU length of stay, hospital length of stay, ventilator days, pneumonia, other infections, admission prothrombin time, transfusion of blood products, development of deep venous thrombosis, development of pulmonary embolism, solid organ injuries, thoracic injuries, other long bone fractures, operations and death were collected. Patients were stratified by gender and all the above parameters were compared between the two groups.

Results: A significantly higher percentage of males (67.6%, or 50 out of 74) were operated on, compared to females (35.1%, or 13 out of 37). This difference reached statistical significance with p-value of 0.014 (Chi-Square test). While 19 male patients out of 74 (25.7%) developed pneumonia after pelvic fracture compared to 3 out of 37 (8.1%) female patients (p<0.05, Chi-Square test). We also found that male patients with pelvic fractures spent a significantly longer time (16.3 days vs. 9.1 days) in the hospital and in the ICU (18.5 days vs. 7.9 days) than female patients. Other infections also occurred more often among men than women. Nevertheless, we were not able to detect significant differences in posttraumatic mortality between the two genders.

Conclusion: While several parameters point toward the markedly beneficial effects of female gender on morbidity (e.g. pneumonia, other infections, hospitalization, operation, ventilation, and ICU care) after pelvic trauma, the overall mortality rates were not different between the two genders.


NOVEL METHOD OF EPINEPHRINE DELIVERY DURING CARDIAC ARREST IN SWINE.R.M. Lima, E.R. Kraft, L.H.C. Navarro, W.G. Dominguez, P. Enkhbaatar, and G.C. Kramer. University of Texas Medical Branch, Galveston, TX 77555

Background: Intravenous access (IV) can be delayed during rescue of cardiac arrest. We tested a novel dry powder endotracheal drug delivery system for delivery of ephinephrine (DPET) that it is an effective alternative during cardiopulmonary resuscitation (CPR) to IV and to conventional endotracheal (ET) access for the delivery of epinephrine into the systemic circulation.

Methods: Propofol anesthetized swine were subjected to ventricular fibrillation (VF) cardiac arrest with an electric shock, after which they were randomized into groups dependent on mean of epinephrine delivery: IV, ET, or DP. After 6 minutes of VF alone, following by 2 minutes of chest compressions (100 compressions/minute), we delivered epinephrine: IV = 0.01 mg/kg, ET = 0.4 mg/kg, or DP = 0.4 mg/kg and continued chest compressions. Ventilation was provided by passive oxygen insufflations. Hemodynamic data and arterial blood samples were collected.

Results: The mean arterial pressure was significantly higher after IV epinephrine compared with ET and DP (Fig. 1). Larger increase in carotid blood flow (Fig. 2) and PaO2 were observed after DP epinephrine delivery when compared with IV and ET.

Conclusions: DEPT may offer an effective alternative to conventional ET delivery of epinephrine during CPR.



COMPETITIVE INHIBITION OF SDF-1/CXCR4 ALTERS HOMING OF BONE MARROW CELLS FOLLOWING INJURY.B. Keck,* K. Beiermeister,* Z. Sifri, A. Mohr, W. Alzate,* D. Barlos,* and D.H. Livingston. UMDNJ-New Jersey Medical School Newark, NJ 07103

Background: Stromal cell-derived factor-1 (SDF-1) is a chemokine known to play an important role in the mobilization and homing of HPCs from bone marrow (BM) to the periphery. AMD3100 is a competitive inhibitor of SDF-1 at the CXCR4 receptor. This study delineates the role of the SDF-1/CXCR4 interaction on HPC growth and homing to injured tissue.

Methods: Male Sprague-Dawley rats were either given daily injections of AMD3100 or saline for 5 days then subjected to unilateral lung contusion (LC). Lung tissue was then harvested for growth of HPCs (CFU-GEMM, BFU-E, and CFU-E). HPC homing was assessed by tagging BM with a fluorescent dye +/- AMD3100. Following LC, lung tissue was examined for immunofluorescence.

Results: LC results in increased HPC growth in injured lung and AMD3100 prevented growth of all HPC progenitors (CFU-GEMM 25±7 vs. 0, BFU-E 23±6 vs. 1, CFU-E 33±1 vs. 1). The table below demonstrates that when BM is tagged and re-injected peripherally the homing of HPCs to injured tissue does occur and is partially inhibited with AMD3100.

Conclusion: Following lung contusion, rats pretreated with AMD3100 have complete inhibition of HPC growth in injured lung. In addition, AMD3100 inhibited homing of HPCs to the injured lung. By blocking the SDF-1/CXCR4 interaction, homing of BM cells to sites of injury is impaired.



ALTERED AUTOPHAGIC ACTIVITY OF SPLENIC DENDRITIC CELLS FOLLOWING TRAUMA IN MICE AND THEEX VIVOREVERSAL EFFECT OF RAPAMYCIN.X. Fan,* H. Liang,* X. Yang,* X. Wang,* Q. Wei,* and Spon: L. Zhou. Research Institute of Surgery & Daping Hospital, Third Military Medical University, Chongqing 400042, PR China

Objectives: The autophagic activity of dendritic cells (DC) in traumatized mice was investigated and the ex vivo effect of rapamycin, an autophagy inducer, on DC function was studied.

Methods: A murine model of trauma (two hind femurs fracture plus hemorrhage of 35% blood volume) in BALB/c mice was performed, splenic DC (CD11C+) were isolated and their autophagic activity was detected, expression of MHC class II, CD40, CD80, and CD86 on DC surface, IL-12 and IL-10 levels in lipopolysaccharide (LPS)-stimulated DC supernatants and DC-induced mixed lymphocyte reaction (MLR) were measured, ex vivo effect of rapamycin on these parameters were observed.

Results: The basal autophagy levels of splenic DC from trauma mice were reduced from 12 hours to 7 days post-trauma as evidenced by decreased percentage of punctuated monodansylcadaverine (MDC) staining cells and lack of autophagosomes. Moreover, induced autophagy by lipopolysaccharides treatment in DC from control group was enhanced while that from 1 day post-trauma group had resistance to this autophagy induction factor. However, ex vivo administration of rapamycin could significantly reverse the suppression of autophagic activity of DC and DC-induced MLR after trauma, elevate the expression of MHC class II and IL-12 secretion, decrease IL-10 secretion, while unaltering the inhibited expression of CD40.

Conclusions: These findings suggest that autophagy in DC, a protective mechanism in response to stress and inflammation was suppressed following traumatic injury. Ex vivo administration of rapamycin can partially ameliorate this change and further enhance the capacity of DC in inducing T cell responses.


SUPPRESSED CLEARANCE OF APOPTOTIC CELLS AND ACCELERATED PRO-INFLAMMATORY RESPONSE FOLLOWING TRAUMA IN MICE.H. Liang,* X. Fan,* X. Yang,* X. Wang, Q. Wei,* and Spon: L. Zhou. Research Institute of Surgery & Daping Hospital, Third Military Medical University, Chongqing 400042, PR China

Objective: The clearance of apoptotic cells is critical in maintaining normal immunity. It remains unclear whether or not serious trauma can affect this ability performed by antigen presenting cells. In this study the clearance of apoptotic cells by peritoneal macrophages in traumatized mice was investigated.

Methods: A murine model of trauma (two hind femurs fracture plus hemorrhage of 35% blood volume) in BALB/c mice was performed. Apoptosis was induced in mouse thymocytes by dexamethasone incubation. At multiple time points after traumatic injury, peritoneal macrophages were isolated and the in vitro phagocytic index (PI) for apoptotic thymocytes were determined. In addition, apoptotic thymocytes, stained with the red fluorescent dye PKH26, were injected intraperitoneally, then unengulfed PKH26 positive cells from lavage liquid were calculated, proliferative response of splenocytes and serum levels of interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected at multiple time points.

Results: The PI of peritoneal macrophages from traumatic mice was significantly lower than that of sham group from day 1 to day 4 post trauma. Moreover, unengulfed apoptotic cells from lavage liquid were increased and proliferative response of splenocytes, without ex vivo additional stimulation, was enhanced and serum IL-6 and TNF-alpha were significantly higher than that sham group and trauma group alone (without injection of apoptotic cells).

Conclusions: These findings suggest that capability of clearance for apoptotic cells was suppressed following traumatic injury, which may result in increased late apoptotic (necrotic) cells, and then stimulate lymphocytes proliferation and accelerate pro-inflammatory response evoked by serious trauma attack.


PREVENTION OF BONE MARROW DERIVED CELLS MOBLIZATION RESULTS IN IMPAIRED HEALING AFTER INJURY.E. Hannoush,* I. Elhassan,* Z. Sifri, A. Mohr, W. Alzate,* and D. Livingston. UMDNJ-New Jersey Medical School Newark, NJ 07103

Background: We have shown that mobilized bone marrow derived cells (BMDC) home to sites of injury of blunt lung contusion (LC) and differentiate into pneumatocytes. Mesenteric lymph duct ligation (LDL) prior to shock resulted in decreased BMDC mobilization and bone marrow suppression. We hypothesized homing of mobilized BMDC is necessary for healing of injured tissues.

Methods: Male Sprague-Dawley rats were subjected to unilateral right LC + laparotomy with LDL (LC+LDL). LC alone and LC + laparotomy w/o LDL (LC and LC+LAP) groups were used as controls. Lung tissue was harvested at days 1, 3, 5, 7 and 14 after LC and the degree of injury was assessed using a histologic lung injury score (LIS) based on inflammatory cells, interstitial and pulmonary edema and alveolar integrity. N=4/group. Data expressed as mean±SEM. *P<0.05 by Mann-Whitney test.

Results: Histologic healing after LC alone occurred at day 7 post-injury. The figure demonstrates that LDL prior to LC delayed healing at 5 and 7 days. At day 14 there was still evidence of continued inflammation in the LC+LDL vs. the LC and LC+LAP groups (LIS 1.5±0.6 vs. 1±0 and 0.75±0.5).

Conclusion: Prevention of BMDC mobilization and/or homing by mesenteric lymph diversion significantly delays healing of injured lung tissue. Understanding the regulation of BMDC mobilization following tissue injury is essential in developing potential therapeutic modalities to reduce the morbidity associated with lung contusion.



INHALED HYDROGEN SULFIDE INDUCES SUSPENDED ANIMATION, BUT DOES NOT ATTENUATE THE LOCAL INFLAMMATORY RESPONSE AFTER CHEST TRAUMA.D.H. Seitz,* U. Niesler,* A. Palmer,* J. Fröba,* F. Wagner,* P. Radermacher, F. Gebhard, and M.W. Knöferl. Dept. of Trauma Surgery, University of Ulm, Germany

The treatment of septic complications after blunt chest trauma is still a challenge. Inhaled hydrogen sulfide H2S caused a hibernation-like metabolic status, which protected against lethal hemorrhage due to an attenuated systemic inflammatory response. Therefore, we tested the hypothesis that inhaled H2S-induced suspended animation may attenuate lung inflammation after pulmonary contusion. Male CD rats were kept for 6h in an exposition chamber under a continuous flow of H2S (100 ppm) or control gas. Body temperature, heart rate and activity were measured by an implantable transmitter. In a second experiment animals were subjected to blunt chest trauma (single blast wave) or sham procedure prior to H2S (6h, 100 ppm) or control gas inhalation. At 6, 24 or 48h after trauma, alveolar macrophages (AM) and neutrophils (PMN) were counted in cytospins of bronchoalveolar lavage (BAL). Protein concentrations and levels of IL-1β, IL-6, CINC and MCP-1 were determined in BAL. The results indicate that temperature and activity but not heart rate were significantly reduced during inhalation of H2S, compared to rats inhaling control gas. The trauma induced rise in AM counts was abrogated at 48h after trauma and H2S inhalation, whereas PMN counts did not differ. Early after trauma, BAL protein, IL-1β and CINC levels and late, BAL IL-6 and MCP-1 were significantly increased in both, H2S and control animals. These findings indicate that inhaled H2S induced suspended animation and that this route of administration did not increase lung inflammation. Nevertheless, this treatment did not beneficially modify local inflammation after lung contusion except decreasing mononuclear cell immigration. Thus, further studies should focus on the effects of H2S on systemic inflammation after blunt chest trauma (KFO 200, DFG KN 475/5-1).



Introduction: Balance of parasympathetic (P) and sympathetic (S) tone is critical for maintaining cardiac function. Heart rate variability was used to study S and P modulation of sinoatrial node (SAN) function in trauma patients.

Methods: EKGs were recorded daily in 10 trauma patients. Root Mean Square of Successive Differences of RR intervals (rMSSD) (varies directly with P tone) was plotted against Standard Deviation of RR intervals ((SD) varies inversely with S tone). Coefficients for the equation y = mx + b and correlation (r) were determined. Differences between slopes were determined using ANOVA (mean (95% confidence interval)) and demographics compared using non-parametric t-test.

Results: Linkage between SD and rMSSD was preserved with two patterns seen. Group A (n=7) resuscitated with volume alone, Group B (n=3) also required vasopressors. All patients survived. No difference between the groups was seen for demographics or the duration or severity of shock. The regression line slopes differed significantly: Group A (m=1.561 (1.50-1.62)) and Group B (m=0.824 (0.685-0.963)).

Conclusions: Linkage between P and S modulation of SAN function is preserved in patients surviving major trauma but varies depending upon the patients need for vasopressors to successfully resuscitate from shock.



INDUCTION OF HBD-2 AND LL-37 IN BLOOD OF MULTIPLE TRAUMATIZED PATIENTS AND PATIENTS WITH AN ISOLATED FEMUR FRACTURE.S. Oestern,* D. Varoga,* T. Klueter,* S. Lippross,* T. Pufe,* M. Daniels-Wredenhagen,* O. Schroeder,* and A. Seekamp. Department of Trauma Surgery, UK-Schleswig-Holstein, Campus Kiel, Arnold Heller Strasse, 24105 Kiel, Germany

Antimicrobial peptides (AMP) such as defensins and cathelicidins belong to the innate immune response and hold broad-spectrum activity against bacteria, fungi and viruses. Trauma is still one of the main reasons for death among the population worldwide. Trauma implies a risk of bacterial infection and sepsis. In this study we determined the expression and production of AMPs in serum samples from patients after multiple trauma or an isolated femur fracture. Expression of AMPs was examined in serum samples of multiple traumatized patients using Elisa from day of trauma until 10 days after trauma and compared to a healthy control group. Serum and soft tissue samples of patients with an isolated femur fracture were analysed using Elisa and the expression was compared with AMP expression of a healthy control group. Statistical differences between the groups were evaluated using the t-test. Compared to the healthy control group, serum of multiple trauma patients showed significantly higher levels of AMPs expression. Tested serum samples of patients with an isolated femur fracture showed a low increase in antimicrobial peptide expression. Soft tissue samples additionally showed an upregulation of antimicrobial peptides. Serum samples of multiple traumatized patients showed high expression of AMPs, which may initially protect the severely injured patient from overwhelming bacterial infection. The AMP expression also appears to corrrelate with the degree of trauma suffered.


TRANSFUSION-RELATED ACUTE LUNG INJURY (TRALI) IN A RAT TRAUMA-HEMORRHAGE MODEL.F. Johnson, S. Nicholson,* R. Johnson, T. Craig,* J. Myers,* W. Durante,* and R. Stewart*. University of Texas Health Science Center at San Antonio, TX 78229

Major trauma often leads to transfusion-requiring hemorrhage and predisposes to TRALI (1st hit). TRALI is a cause of complications and death related to factors in stored blood products (2nd hit).

Objective: To develop a clinically-relevant two-hit model of trauma-hemorrhage and TRALI.

Methods: Male Lewis rats were used. Rat packed red blood cells (PRBC) were prepared from whole blood in a manner similar to human blood banking. Recipients were implanted with femoral arterial and venous catheters under isoflurane anesthesia, then after 16hr recovery subjected to 30% controlled arterial hemorrhage (1st hit). Following a 60min shock period rats were resuscitated with crystalloid solution and PRBC (0-35 days old, 3:1 ratio). Animals were followed for up to 6hrs. Lung edema was evaluated by Evans Blue Dye (EBD) and protein accumulation in bronchoalveolar lavage fluid (BALF) and iSTAT analyzer was used to measure arterial blood gases.

Results: We found that 30-min survival was reduced in rats transfused with 28 and 35-day old PRBC compared to the 0-day group (0-day: 100% vs. 28-day: 45.4% and 35-day: 0%). BALF EBD (0-day: 0.002±0.00 vs. 28-day: 0.174±0.06 and 35-day: 0.262±0.05 %EBD/min) and protein accumulation (0-day: 3.7±1.1 vs. 28-day: 281.0±111.3 and 35-day: 533.8±112.6 μg/min) was increased in rats transfused with 28 and 35-day old PRBC compared to the 0-day group. Arterial PO2 was decreased in rats transfused with 28 and 35-day old PRBC (0-day: 104±9 vs. 28-day: 59±28 and 35-day: 50±22 mmHg). However, PCO2 was not different between groups.

Conclusions: These results suggest that transfusion of 28 and 35 days old PRBC reliably promotes lung edema in a rat model of catheter surgery and hemorrhage. We propose that this model can be used as a clinically-relevant two-hit model of trauma-hemorrhage and TRALI.


META-ANALYSIS OF EFFECT OF FFP: RBC RATIOS ON SURVIVAL IN MASSIVE TRANSFUSION.M. Agapova,* B. Custer,* and P. Spinella. Blood Systems Research Institute, San Francisco CA 94118

Objective: The influence of increased FFP use on survival in massive transfusion is unclear. This analysis sought to understand general trends and sources of divergence in results.

Methods: Survival results from 25 peer-reviewed articles were eligible for a meta-analysis after a literature search of EMBASE and MEDLINE. Nine studies met the inclusion criteria: massive transfusion defined as ≥8 or ≥10 units of RBCs in 12 or 24 hours, respectively; at least 1 group of patients received FFP: RBC unit ratio ≥1:1.5 (HiR) and at least 1 group received FFP: RBC <1:1.5 (LoR). Overall or 30 day mortality was primary outcome. Using a random-effects analysis, a pooled odds ratio (OR) was obtained by binary data meta-analysis.

Results: The pooled OR from 9 studies (1654 LoR and 1154 HiR patients) was 0.32 (95%CI: 0.21-0.48) indicating mortality is less likely to occur in the HiR group. Although studies report almost uniformly the benefit of HiR there was significant heterogeneity (τ=0.32). Inter-study variance may be explained by two conflicting sources of survivor bias: 1) Grouping patients, post facto, by the sum FFP and RBC units received favors recipients who survive to receive higher levels of FFP, a bias toward HiR. 2) Treating the FFP: RBC, as a time-dependent covariate by dividing patients into groups by the ratio received at a specific time introduces a bias in favor of LoR as HiRs are often transfused after LoRs and to persons who do not survive.

Conclusion: To confirm the benefit of HiR, future study designs must account for sources of survivor bias and prospectively define and match recipients in HiR and LoR groups.



ASSOCIATION OF TRIAGE PARAMETERS AND INFLAMMATION BIOMARKERS IN TRAUMA SURVIVORS.R. Namas, A. Ghuma,* R. Namas,* R. Zamora, G. Bachuwa,* J. Ochoa, T. Billiar, and Y. Vodovotz. Department of Surgery, University of Pittsburgh, Pittsburgh, PA and Hurley Medical Center, Flint, MI

Introduction: Allocating treatment following trauma/hemorrhage (T/HS) requires triage. T/HS-induced inflammation may be linked to triage parameters.

Methods: Serum samples from 14 T/HS survivors (12 males, 2 females; age: 32±3, ISS: 31±3, height: 178±2 cm, weight: 82±3 kg, pulse: 105±7 bpm, maximal systolic blood pressure: 169±7 mmHg) were collected at 1-30 days post-admission and assayed for cytokines and chemokines using Luminex™. Primary inflammatory drivers were identified using Principal Component Analysis (PCA). Triage parameters were temp. (max. and min.), pulse, maximal systolic blood pressure (MBP), body mass index (BMI), basal metabolic rate (BMR), and Glasgow Coma Scale (GCS). Triage parameters and day 1 cytokine values was correlated using Spearman Rank Order Correlation.

Results: Based on PCA, inflammation in this cohort was driven by IL-10 and IL-1ra, which correlated positively with pulse (IL-10 [P<0.04], IL-1Ra [P<0.03]). Additionally, BMI correlated positively with IFN-γ (P<0.04), IL-6 (P<0.022), IL-2R (P<0.04), and Eotaxin (P<0.027). BMR correlated negatively with TNF-α (P<0.04), GM-CSF (P<0.04), IFN-α (P<0.01), IL-5 (P<0.03), IL-2p70/p40 (P<0.04), IL-13 (P<0.04), IL-17 (P<0.04), MIG (P<0.04), and MIP-1β (P<0.03). MBP correlated positively with IL-6 (P<0.01) and negatively with IL-17 (P<0.04). Minimum temp. correlated negatively with IL-8 (P<0.02). Maximum temp. correlated positively with IL-10 (P<0.04) and IP-10 (P<0.03). GCS correlated negatively with IL-6 (P<0.04), IL-8 (P<0.03) and MCP-1 (P<0.04).

Conclusion: Our findings suggest a link between a strong anti-inflammatory response and pulse, and highlight the power of PCA to discern novel patterns in highly-dimensional inflammation biomarker data.


INTEGER HEART RATE COMPLEXITY AND TROPONIN ARE ASSOCIATED WITH TRAUMA ICU MORTALITY.P. Norris, T. Barrett,* I. Jones,* A. Storrow,* and J. Morris Jr. Vanderbilt University Medical Center, Nashville, TN 37212

Objective: Reduced heart rate complexity (HRC), or "randomness" in heart rate variability, predicts trauma patient mortality. We sought to assess the contribution of myocardial injury, as reflected by serum troponin, to the relationship between HRC and mortality.

Methods: Over 5 years, 336 trauma ICU patients had serum troponin results and sufficient integer HR data (6 continuous hours within 6 hours of troponin). HRC was calculated using Costa's multiscale entropy (sum of scales, m=2, r=.15, Relationships between individual variables were evaluated using non-parametric statistical tests. Logistic regression (LR) characterized the simultaneous relationship of HRC and troponin to risk of death, controlling for age and injury severity score (ISS).

Results: Study population acuity was high (30% mortality, median ISS=27). Reduced HRC was weakly correlated with increased troponin (rho=-0.16, p=.004) and each was associated with mortality (p<.01, figure). The relationship of troponin to risk of death was eclipsed by HRC and other variables in LR (table).

Conclusions: 1) Integer heart rate complexity is associated with trauma mortality even in high-acuity subgroups; 2) Myocardial injury is weakly associated with reduced HRC, and contributes only slightly to this association; 3) Future studies of waveform-based metrics of HRC may yield more sensitive indicators of myocardial injury.



A SYSTEMS VIEW OF INFLAMMATION PRE- AND POST-MENOPAUSE IN FEMALE TRAUMA SURVIVORS.R. Namas, A. Ghuma,* R. Namas,* R. Zamora, J. Sperry, G. Bachuwa,* J. Ochoa, T. Billiar, and Y. Vodovotz. Department of Surgery, University of Pittsburgh, Pittsburgh, PA and Hurley Medical Center, Flint, MI

Objective: Outcomes following trauma/hemorrhagic shock (T/HS) have been reported to be better in women as compared to men. However, the role of menopausal status vis-à-vis inflammation has not been explored. In this study, we sought to compare the inflammatory response of pre- and postmenopausal women.

Methods: All studies were carried out following IRB approval and with informed consent. Based on prior studies, the age cutoff separating pre-menopausal (PrM) and post-menopausal (PoM) status was 48. Serum samples from female T/HS survivors (11 PrM [age: 29±11; ISS: 35±3] and 7 PoM [age: 61±6 {P = <0.001 vs. PrM}]; ISS: 29±1 [P = NS vs. PrM]) were collected at <6h, ≥6h, and ≤24h post-T/HS and assayed for cytokines, chemokines and NO2-/NO3- using Luminex™ and nitrate reductase, respectively, and compared using Student's t-test (P<0.05). Principal Component Analysis (PCA) was used to identify primary inflammatory drivers over the full follow-up time course (0-29 d for PrM; 0-22 d for PoM).

Results: PCA suggested that post-T/HS inflammation in PrM was driven by TNF-α, GM-CSF, IL-2, IL-8, and IL-10, while in PoM it was driven by TNF-α, MIP-1β, RANTES, IL-10, and IL-15. Of the post-T/HS inflammation biomarkers altered significantly, most (IFN-γ, IFN-α, IL-1β, IL-2, sIL-2R, IL-4, IL-15, and IL-17) were higher at <6h in PoM; only IL-10 was higher in PrM. At >6h < 24h, h post-T/HS, however, roughly the opposite was observed: IL-6, IL-7, IL-10, IL-15, GM-CSF, MIP-1α, and NO2-/NO3- were higher in PrM; only TNF-α was higher in PoM.

Conclusion: PCA may discern novel patterns in inflammation biomarkers in PrM vs. PoM. Further studies are needed in order to establish if the effects observed are a function of age, sex hormone status, or other factors.


AROMATASE LEVELS ARE INCREASED IN ASTROCYTES FOLLOWING INCREASED PRESSURE.J. Gatson, J. Simpkins,* and J. Wigginton. UT Southwestern Medical Center, Dallas, TX 75390

Introduction: In traumatic brain injury (TBI), the persistent demise of neuronal and glial populations leads to decreased brain function. Endogenous and exogenous estrogens may protect these vulnerable cells. In this study, we hypothesize that increased pressure leads to an increase in aromatase expression and estrogen production.

Methods: We subjected primary glia to increased pressure for (25mmHg) for 1, 3, 6, 12, 24, 48, and 72 hours. Total aromatase protein and RNA levels were measured using Western analysis and RT-PCR, respectively. In addition, we indirectly measured aromatase activity by measuring estrone levels after administration of androstenedione (aromatase converts androstenedione to estrone). Human levels of CSF estradiol were measured with the IMMULITE® immunoassay system.

Results: In TBI patients, individuals with a good outcome showed a rapid increase in the levels of estrogen in the CSF compared with the serum levels. Increased pressure applied to the astroglia cultures resulted in a significant increases in both aromatase RNA (2-fold) and protein (∼95% and 90%).To extend these findings, we also analyzed aromatase activity in glia during increased pressure. We found that increased pressure resulted in a significant increase in estrone levels.

Conclusion: In this study, we believe that following TBI, increased intracranial pressure leads to more aromatase production and subsequent estrogen production. This increase in estrogen presumably results in increased cell survival in the brain.

©2010The Shock Society