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doi: 10.1097/SHK.0b013e3181e2f17c
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CTLA-4 BLOCKADE BOOSTS T CELL ACTIVATION AND IMPROVES SURVIVAL IN SEPSISS. Inoue*, K. C. Chang*, D.F. Osborne, R. S. Hotchkiss. Departments of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110

Background: CTLA-4 is one of the critical inhibitory regulators of the early stages of T cell expansion, opposing the actions of CD28-mediated costimulation. Sepsis is a lethal condition where apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality.

Objective: Determine if anti-CTLA-4 antibody prevents sepsis-induced immunosuppression, modulates cytokines, and improves survival in a mouse peritonitis sepsis model.

Methods: CD1 mice underwent 1×27 gauge cecal ligation puncture (CLP) or sham surgery. After 6 and 24hrs, 50μg mouse ant-CTLA-4 was administered I.P.. Spleens were harvested 48 hrs later, and absolute cell counts, apoptosis, cytokine production, and survival were quantified.

Results: Compared to CLP, CLP mice treated with anti-CTLA-4 had an increase in absolute cell counts by 80% in CD4 T cells and 69% in CD8 T cells (p<0.05). Caspase 3 analysis demonstrated 21.9% CD4+ T cell apoptosis and 17.0% CD8+ T cell apoptosis in control vs 10.5% and 8.8% in anti-CTLA-4 treated mice (p<0.01). IL-10 in supernatant were 3020pg/ml in control vs 1993pg/ml in anti-CTLA-4 treated mice (p<0.05). The survival benefit of anti-CTLA-4 was dose-dependent. At 7days survival was 70% in anti-CTLA-4 treated mice vs 7.1% overall in control (p = 0.0028).

Conclusion: Anti-CTLA-4 antibody represents an exciting novel approach to sepsis because it not only blocks apoptosis but modulates the immunosuppression.


GHRELIN SUPPRESSES INFLAMMATION AND EXCITOTOXICITY IN FOCAL CEREBRAL ISCHEMIA VIA THE VAGUS NERVEC. Cheyuo*, R. Wu, M. Zhou, A. Jacob*, G. Coppa, and P. Wang. Department of Surgery, North Shore-LIJ Medical Center, Manhasset, NY 11030

Inflammation and excitotoxicity contribute to the pathogenesis of stroke. Ghrelin, an endogenous 28-amino acid peptide, is shown to exert anti-apoptotic and anti-inflammatory properties. However, the effect of ghrelin in permanent focal cerebral ischemia and the role of the vagus nerve in its actions remain unknown. To study this, male Sprague-Dawley rats (∼350g) underwent right-sided permanent middle cerebral artery occlusion (MCAO) with or without prior bilateral truncal vagotomy. This was followed by infusion of saline as vehicle or 4 nmol human ghrelin as treatment. Neurological scores were determined at 3h and 24h post-MCAO. Rats were sacrificed at 24h and brains rapidly removed and analyzed for infarct size, markers of inflammation (IL-6 by ELISA, MMP-9 by Q-PCR) and excitotoxicity (nNOS by Q-PCR). As shown in the table below, the increase in infarct size, IL-6, MMP-9 and nNOS observed in MCAO was markedly reduced by ghrelin treatment (p < 0.05). Moreover, ghrelin treatment resulted in 50% reduction in neurological scores in stroke animals. Prior vagotomy, however, completely eliminated the benefit of ghrelin in stroke rats (data not shown). Thus, ghrelin suppresses inflammation and excitotoxicity in focal cerebral ischemia through the vagal pathway.



INJURY RAPIDLY INDUCES TCR-MEDIATED SIGNALING RESPONSES IN CD4+ T-REGS BUT NOT IN NON-T-REGSM. Hanschen*, G. Tajima, F. O'Leary*, K. Ikeda*, J Lederer. Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115

Severe injury enhances the regulatory activity of CD4+ T regulatory cells (Tregs), but the mechanisms responsible for Treg activation following injury remain unclear. Objectives of this study were (i) to test whether injury differentially activates T cell receptor (TCR) signaling in CD4+ Treg and non-Treg cells and (ii) to investigate whether Treg activation following injury is TCR dependent. We employed phospho-flow cytometry rather than Western immunoblots to measure the phosphorylation of the TCR signaling molecules, ZAP-70, PKC-theta, NFATc1, and GSK-3beta in FoxP3+ Tregs and non-Tregs. In a first set of experiments, C57BL/6 mice were subjected to 25% total body surface burn injury or sham treated and lymph nodes (LN) and spleens (SPL) were harvested to measure signaling activation at early time points - 15, 30, 60, 120, and 240 minutes - after sham or burn injury. In a second set of experiments, the TCR-dependency of signaling activation following injury was investigated using mice with a highly restricted TCR repertoire (OT-II TCR transgenic mice). Following burn or sham injury, the expression and phosphorylation of TCR signaling molecules was analyzed in Tregs vs. non-Tregs derived from wild-type or OT-II mice. Burn injury induced a significant differential signaling response in Tregs vs. non-Tregs derived from the LN of WT mice as early as 15 min after injury. This effect could not be observed in Tregs derived from SPL. Non-Tregs did not show an early signaling response to burn injury. In contrast to WT Tregs, no rapid signaling activation was detectable in OT-II Tregs, indicating a potential role for the TCR in Treg activation following injury. In conclusion, we show that injury induces differential signaling by Tregs and non-Tregs and provide evidence to indicate that burn injury preferentially activates TCR signaling by Tregs in the LNs draining the injury site.


THE DUAL FUNCTIONS OF RIP1 IN FAS INDUCED HEPATOCYTE DEATH DURING SEPSISS. McNeal*, C-S. Chung,, A. Ayala. Department of Surgical Research, Division of Biology and Medicine, Brown University, Providence, RI 02903

In examining the liver's response to sepsis our lab has found that septic hepatocytes exhibit a higher degree of necrosis when compared to septic thymocytes, which typically die through the canonical apoptotic pathway. Recently an adaptor protein associated with the Fas/TNF death receptor pathway, Receptor Interacting Protein 1 (RIP1) has been shown to be critical for determining whether a cell's death is apoptotic or necrotic. We propose to test the central hypothesis that RIP1 activation by death receptor (Fas) during sepsis determines whether the hepatocytes fate is apoptotic vs. necrotic. We approached this problem by delivering RIP1 siRNA in vivo to C57Bl/6 mice and observing changes in mortality after septic challenge. Contrary to our hypothesis, RIP1 silenced mice did not survive as long as scrambled sequence injected controls (22.2% vs. 50.0% 14 days post CLP respectively). In light of this we carried out a series of comparative histological studies using a pharmacological/synthetic antagonist of RIP-1 kinase, Necrostatin 1 (Nec-1). Septic mice pre-treated with Nec-1 exhibit a preservation of liver architecture and function (represented by PAS stained glycogen stores). On the other hand siRNA treated mice exhibit lower glycogen stores and altered morphology. The data suggests the kinase domain of RIP1 is responsible for the alterations in the liver during sepsis. We speculate these confounding results are due to the dual signaling responsibilities of RIP1. RIP1 through it's kinase domain can complex with RIP3 to create the so-called complex II, inducing necrotic death through Fas ligation. However, RIP1 can also initiate pro-survival signaling through NFκB by way of it's intermediate domain. (Supported by NIH GM53209).


LXRα AGONIST T090137 REDUCES THE INFLAMMATORY RESPONSE AFTER SEVERE HEMORRHAGEP. Solan,*, P. Hake*, G. Piraino*, and B. Zingarelli. Dept of Surgery, Univ of Cincinnati; Div of Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229

LXRα is a nuclear transcription factor important in cholesterol metabolism that has also been shown to regulate inflammation. Here, we investigated the effects of an LXRα agonist on inflammation caused by hemorrhagic shock. Hemorrhagic shock was induced in male Wistar rats by withdrawing blood from the femoral artery to a mean arterial pressure of 50 mmHg, which was sustained for 3 hours. After 3 hours, resuscitation was performed by rapidly replacing the shed blood. Rats then received the LXRα agonist T0901317 (50 mg/kg i.p.) or vehicle at the time of resuscitation and every hour thereafter for 3 hours. Treatment with LXRα agonist significantly reduced hemorrhage-induced tachycardia at three hours post-resuscitation (p=.0001). Neutrophil infiltration in lung, as measured by myeloperoxidase assay, was significantly decreased in LXRα agonist treated rats as compared to vehicle (613 ± 23 U/100 mg tissue and 728 ± 23 U/100 mg tissue, respectively. p<0.05). Treatment with T090137 also increased the lung nuclear expression of LXRα as evaluated by western blot. Additionally, LXRα agonist decreased the plasma levels of the pro-inflammatory cytokines MIP1α and TNFα at 3 hours post-resuscitation (Figure). These results confirm that administration of an LXRα agonist does reduce the inflammatory response induced by severe hemorrhagic shock. (Supported by NIH Grants AG-027990 and 5 T32-GM8478-17).



INTENSIVE INSULIN THERAPY IN THERMALLY INJURED PEDIATRIC PATIENTS: RESULTS OF A PROSPECTIVE RANDOMIZED TRIALM. Jeschke1,2, G. Kulp*1,2,3, R. Kraft1,2, C. Finnerty1,2, R. Mlcak1,2, D. Herndon1,2. The University of Texas Medical Branch, Galveston, TX 77555-1220. This study was registered at #NCT00673309

Background: Hyperglycemia and insulin resistance have been shown to increase morbidity and mortality in severely burned patients, and glycemic control appears essential to improve clinical outcomes. However, to date no prospective randomized study exists that determines whether intensive insulin therapy is associated with improved post-burn morbidity and mortality.

Methods: Two-hundred thirty-nine severely burned pediatric patients with burns >30% of their total body surface area (TBSA) were randomized (block randomization 1:3) to intensive insulin treatment (n=60) or control (n=179). Demographics, clinical outcomes, sepsis, glucose metabolism, organ function, inflammatory and acute-phase and hypermetabolic responses were determined.

Results: Demographics were similar in both groups. Intensive insulin treatment significantly decreased the incidence of infections and sepsis compared to controls, p<0·05. Furthermore, intensive insulin therapy improved organ function as indicated by improved serum markers, DENVER2 scores, and ultrasound, p<0·05. Intensive insulin therapy alleviated post-burn insulin resistance and the vast catabolic response of the body, p<0·05. Intensive insulin treatment dampened inflammatory and acute-phase responses by deceasing IL-6 and acute-phase proteins compared to controls, p<0·05. Mortality was 4% in the intensive insulin therapy group, while it was 11% in the control group, p=0·1.

Conclusion: In this prospective randomized clinical trial, we showed that intensive insulin therapy improves post-burn morbidity.


REGULATORY T CELLS DEMONSTRATE AN INJURY-SPECIFIC RECALL RESPONSEG. Tajima, M. Hanschen*, F. O'Leary*, K. Ikeda*, A. Delisle*, M. Oukka*, V. Kuchroo*, and J. Lederer. Brigham and Women's Hospital/Harvard Medical School, Boston, MA 02115

In a previous study, we demonstrated that burn injury caused rapid activation of FoxP3+ regulatory T cell (Treg) in injury-site lymph nodes in mice. Given this observation, we wished to test whether Tregs might develop an injury-specific recall response. To accomplish this, we transferred CD4 T cells from sham or burn FoxP3-GFP knock-in mice into sham and burn congenic mice, and tracked the expansion and activation of injury-experienced and inexperienced Tregs. FoxP3-GFP mice underwent sham or burn injury. One or 4 weeks later, CD4 T cells were purified and then transferred into CD45.1-congenic mice. Recipient mice underwent sham or burn injury to represent a second injury response. After 7 days, transferred CD45.2 positive cells and FoxP3-GFP positive cells were detected by FACS to measure expansion and stained for cell-surface T cell activation and memory markers (CD62L, CD44, ICOS, and CTLA-4). We found that the combination of burn CD4 T cells into burn recipient mice caused significantly greater FoxP3+ Treg expansion and activation than other combinations. This recall response by Tregs was much higher in Tregs transferred 1 week after injury than Tregs from 4 weeks. Moreover, Tregs transferred at 1 week after the first injury showed a greater memory-like CD4 T cell phenotype (CD44high CD62Llow) than non-Tregs. The observation that injury-experienced Tregs respond more vigorously to the second injury suggests that Tregs develop an injury-specific recall response. Conventional CD4 T cells also showed a recall response to injury, but there were no significant difference between 1 week and 4 weeks after injury. These findings are novel and support the concept that Tregs are injury-responsive and can develop an early, injury-specific memory-like response.


COLD INDUCIBLE RNA-BINDING PROTEIN (CIRP) AS A NOVEL PROINFLAMMATORY CYTOKINE IN SHOCKX. Qiang*, R. Wu, M. Zhou, Y. Ji*, W. Dong*, H. Yang, H. Wang, G. Coppa*, K.J. Tracey, P. Wang. The Feinstein Institute for Medical Research, Manhasset, NY 11030

CIRP, an 18-KDa nuclear protein, was first characterized for its overexpression upon cold, and it can also be induced by other stresses such as hypoxia. Here we investigated its potential role in the pathogenesis of shock. Using a rat model of hemorrhagic shock, we found that plasma levels of CIRP as well as its gene expression in the liver and heart were markedly increased. To determine the effects of CIRP on tissue injury and inflammation, a dose of newly expressed recombinant murine CIRP (rmCIRP, 1 mg/kg BW) was given intravenously (iv) to healthy rats. The results indicate that rmCIRP significantly increased serum levels of liver enzymes and cytokines (i.e., TNF-α & HMGB-1) 4 h after its administration. In cultured macrophage-like RAW cells, rmCIRP dose-dependently upregulated TNF-α production. To determine the effect of blocking CIRP after hemorrhage, anti-CIRP antibodies (10 mg/kg BW) was given iv during fluid resuscitation after 90-min hemorrhage. We found that CIRP neutralization decreased TNF-α, IL-6 and HMGB-1, reduced organ injury, and improved survival after hemorrhage and resuscitation. In contrast, CIRP−/− mice had a much higher survival rate after hemorrhage as compared to the wild-type (WT) mice. To identify the receptor for CIRP, peritoneal macrophages isolated from various knockout mice (i.e., TLR4−/−, TLR2−/−, or RAGE−/− mice) were stimu-lated with rmCIRP. rmCIRP-induced TNF-α production in macrophages from RAGE−/− or TLR2−/− mice was similar to that in WT mice. However, rmCIRP-induced TNF-α pro-duction in macrophages from TLR4−/− mice was much lower than that in other groups. Moreover, TLR4 antibodies, but not the endotoxin-neutralizing agent polymyxin B, signify-cantly reduced rmCIRP-induced TNF-α release in RAW cells. In conclusion, CIRP is a new proinflammatory cytokine in shock and TLR4 may serve as CIRP receptors.


CYTOKINE STIMULATION BY HMGB1 REQUIRES TLR4 AND CYSTEINE IN POSITION 106H. Yang, H. Wang, M. Ochani, M. Rosas, U. Andersson*#, H. Hgreggvidsdottir*#, J. Li*, B. Lu*, S. Chavan*, K.J. Tracey. The Feinstein Institute for Medical Research, Manhasset, NY 11030. #Karolinska Institute, Stockholm, Sweden

High mobility group box 1 (HMGB1) has been implicated as a cytokine mediator in the pathogenesis of inflammatory diseases (Yang et al. BBA, 2009). To study mechanisms of HMGB1 action, we examined receptors involved in HMGB1 signaling and cytokine stimulation. Using primary macrophages from TLR2, TLR4 or RAGE knock out (KO) mice, we found that HMGB1 requires TLR4 to induce TNF release, as compared to wild type (WT), TLR2 or RAGE KO (Fig, n=4-5 experiments). Surface plasmon resonance (Biacore) analysis revealed that HMGB1 binds TLR4 in a concentration-dependent manner, giving direct evidence of HMGB1-TLR4 interaction. HMGB1, with a point mutation of cysteine 106 (C106) to alanine, loses the cytokine-stimulating activity and no longer binds to TLR4 (Biacore). Synthetic 20-mer peptide covering C106 on B box portion of HMGB1 is active in inducing TNF release from primary human macrophages; whereas the same sequence with C106 replaced by serine is not active, confirming the importance of C106 on HMGB1 cytokine activity. Hence, our results showed that TLR4 is essential for HMGB1-mediated cytokine release from macrophages; this effect requires C106 (Supported by grant from NIH, NIGMS, to KJT).

HMGB1-induced TNF release from macrophages.



Epidermal growth factor (EGF) treatment attenuates gut injury and decreases mortality in peritonitis-induced sepsis, but its role in extra-abdominal sepsis is unknown. Our aim was to determine if EGF preserves gut integrity and decreases mortality in P. aeruginosa pneumonia-induced sepsis. Mice were intratracheally injected with 2-4×108 CFU P. aeruginosa or saline and treated with or without EGF (150 μg/kg/d i.p.). At 24 hr, guts were evaluated for apoptosis by caspase-3 staining, proliferation by BrdU staining, and villus length. For survival studies, EGF was given immediately postoperatively or 24 hr later and mice were followed for 7 days. To determine if EGF protection is gut-specific, transgenic mice that overexpress EGF exclusively in enterocytes and WT mice were subjected to pneumonia and followed for survival. Compared to shams, septic mice had increased gut apoptosis (11±1 vs. 4±1; p<0.001), while EGF normalized apoptosis to shams (7±1; p=ns). Septic mice had decreased proliferation compared to shams (672±54 vs. 1170±33; p<0.001), while EGF increased proliferation (858±55 vs. 672±54; p<0.05). Septic mice exhibited villus atrophy compared to shams (244.5±7.4 vs. 422.2±8.3 μm; p<0.001), while EGF increased villus length (386.4±13.2 vs. 244.5±7.4 μm; p<0.001). Septic mice given EGF immediately after the onset of sepsis had improved survival compared to untreated septic mice (90% vs. 35%; p<0.001), which was maintained even when EGF treatment was delayed for 24 hr (73%; p<0.05). Septic mice that overexpress EGF exclusively in enterocytes had improved survival compared to WT septic mice (p<0.05). In conclusion, the protective effects of EGF are not specific to the anatomical site or type of infection. Further, EGF protection appears to be mediated in an intestine-specific fashion. Thus, EGF may be a novel therapeutic agent for the treatment of sepsis.



Multiple therapeutic applications of bone marrow derived mesenchymal stem cells (MSCs) have been reported. ARDS and SIRS frequently occur in patients after hemorrhagic shock and resuscitation. Little is known about interactions between MSCs and pulmonary endothelial cells (PECs), one of the first cell types that they interact with when administered intravenously for therapeutic applications. We hypothesized that MSCs have stabilizing effects on endothelium that prevent inflammation, endothelial permeability and vascular tight-junction disassembly in the lungs after HS.

Methods: To determine the effects of MSCs on ECs in vitro, MSCs were co-cultured with PECs. To determine the effects of MSCs in vivo we used a rat model of hemorrhagic shock which results in pulmonary vascular compromise. Rats were pretreated with MSC and Lung tissue and plasma were collected and analyzed.

Results:In vitro MSC-PEC interactions result in decreased PEC permeability (50%) and enhanced expression of VE-cadherin and β-catenin at the cell membrane. In vivo HS alone results in ablation of tight junction proteins VE-Cadherin, Occludin, and Claudin-1, all of which were preserved in MSC treated rats. Systemic levels of IL-6 and MCP-1 were decreased 5-fold in MSC treated rats whereas induction of TNFα and MIP-1α by HS were completely inhibited to baseline in MSC treated rats.

Conclusions: We show here that MSCs exert significant anti-inflammatory and stabilizing effects on the vasculature in the lungs and decrease systemic inflammation induced by HS. These results demonstrate a novel a mechanism of action of MSCs and bring attention to the potentially beneficial effects of MSCs as a resuscitative adjunct in HS.


AUGMENTER OF LIVER REGENERATION (ALR): A NOVEL INDUCER OF ACUTE INFLAMMATION IN SEPSIS, TRAUMA, AND HEMORRHAGEC. Gandhi*1,2, R. Zamora1, Y. Vodovotz1. 1Department of Surgery, University of Pittsburgh, Pittsburgh, PA and 1,2VAPHS, Pittsburgh, PA

The liver is a central organ involved in inflammatory processes, including the elaboration of acute-phase proteins. One such acute phase protein is Augmenter of Liver Regeneration (ALR), which also participates in liver regeneration and in maintaining the viability of hepatocytes. We examined the possibility that ALR may be involved in modulating inflammation induced by various stimuli. In vivo, ALR was elevated in rats subjected to intraperitoneal administration of LPS alone, surgical trauma followed by intraperitoneal administration of LPS, liver cirrhosis, and portacaval shunt. ALR was also elevated rapidly in the course of experimental Gram-negative sepsis (induced by implantation of a fibrin clot impregnated with live E. coli bacteria) in rats, and remained elevated for at least 24 hours. In this bacterial sepsis model, ALR was elevated sooner than other classical pro-inflammatory (TNF-α, IL-6) or anti-inflammatory cytokines (IL-10). Further, ALR was elevated in portacaval shunt in rats prior to alterations in liver expression of IL-1β, TNF-α, and IL-6. ALR was also altered in mice subjected to experimental hemorrhagic shock. Human trauma patients exhibited varying levels of ALR, associated with their clinical outcome. In vitro, ALR induced TNF-α and IL-6 production by Kupffer cells. Thus, ALR appears to be a novel pro-inflammatory agent, whose detection or modulation may have therapeutic applications. Since it mediates normal physiology in resting hepatocytes but can stimulate inflammatory responses, ALR also fits the definition of a Damage-associated Molecular Pattern (DAMP) molecule. ALR may thus serve as a potential diagnostic marker of, or therapeutic target for, acute inflammatory conditions.


AGE-RELATED CHANGES OF KLOTHO PATHWAY CORRELATE WITH KIDNEY INJURY IN HEMORRHAGIC SHOCKG. Maltese*, G. Piraino*, M. O'Connor*, A. Denenberg*, A. Lasco*, P. Hake*, B. Zingarelli. Geriatrics, Univ. of Messina, Italy, and Critical Care Med., Cincinnati Children's Hosp. Med. Center, Cincinnati, OH 45229

Age is an important factor for the development of multiple organ, including kidney, dysfunction in trauma patients. Klotho is a membrane-bound anti-aging PPARg-dependent protein, also found in a secreted form, which has been shown to counteract oxidative stress in kidney ischemia-reperfusion injury. Here, we investigated whether hemorrhage-induced kidney injury is age-dependent and whether it is associated with modification of klotho pathway. Hemorrhagic shock was induced in anesthetized young (2-3 months old) and mature male rats (11-12 months old) by withdrawing blood into a reservoir to a mean arterial pressure of 50 mmHg. After 3h, rats were rapidly resuscitated by infusing the shed blood. In young rats, kidney injury was characterized by mild edema and infiltration of neutrophils (myeloperoxidase activity 2.63±0.20 U/100 mg tissue) and elevated plasma creatinine levels (0.41±0.05 mg/dl). At Western blot analysis, plasma klotho levels increased significantly (4.19±0.54 relative intensity) at 3 h after resuscitation when compared to basal levels (1.50±0.50 relative intensity; P<0.05). In contrast, severe tubulo-interstitial injury was observed in mature rats. Tissue myeloperoxidase activity and plasma creatinine levels were significantly higher (4.73±0.84 U/100 mg tissue and 0.77±0.13 mg/dl, respectively) when compared to young rats (P<0.05). Interestingly, in mature rats plasma klotho levels did not increase after resuscitation (1.23±0.32 relative intensity) and were similar to basal levels (1.13±0.13 relative intensity). Our data suggest that kidney injury after severe hemorrhage is an age-dependent phenomenon and it may be associated with down-regulation of the renal protective klotho hormone. (Supported by NIH Grant R01 AG-0227990).



Introduction: NEFA are mobilized in critically ill patients. High levels are associated with dysregulated mitochondrial function and potential alteration in immunity. The relationship of NEFA and outcome in trauma has not been previously investigated. Thus, we set out to deter-mine if plasma NEFA levels are predictive in a population of severely injured trauma patients at risk of mitochondrial dysfunction and poor outcome.

Methods: Fifty eight severely injured patients at a Level I trauma center were evaluated. Clinical and outcome variables were prospectively collected. Blood samples were collected 24 hours-post injury, followed by enzymatic assay analysis to determine plasma NEFA level. A NEFA level was determined for the subsequent development of multiple organ dysfunction (MODS) by developing a ROC and defining the optimal NEFA level using the Youden index. Outcome based on NEFA was assessed by a step-wise regression model.

Results: A ROC generated for NEFA and MODS demonstrated an AUC of 0.876 and NEFA level of 0.38 mmol/L having the highest predictive value. A NEFA of 0.38 mmol/L or greater was associated with both the development of ARDS (OR 4.8, 95% CI: 1.1-21.8) and MODS (OR 8.98, 95% CI: 1.98-40.7) after adjusting for age, sex, injury severity, and blood transfusions. A NEFA level greater than 0.38 mmol/L was associated with increased ICU days (8.5 + 3.4 vs. 10.8 + 4.7, p=0.03), and reduction in ventilator free days (21.9 + 3.4 vs. 19.2 + 3.8, p=0.05). No difference was seen in the rate of infection or mortality.

Conclusions: NEFA are key mediators known to be involved in alteration mitochondrial function and immunity. Elevated NEFA following injury appears to correlate with MODS. This measurement may be useful as a biomarker for mitochondrial dysfunction and risk for MODS that may benefit from novel therapeutic interventions.


HYPERTONIC RESUSCITATION MODULATES MONOCYTE SUBSET ACTIVATION AND CYTOKINE PRODUCTIONJ. Cuschieri, S.G. Rhind, S.B. Rizoli*, W.G. Junger, P.N. Shek*, A.J. Baker*, D.B. Hoyt, E.M. Bulger. Dept. of Surgery, University of Washington, Harborview Medical Center, Seattle, WA 98104

Background: Blood monocytes are a phenotypically and functionally heterogeneous population of innate immune cells, whose capacity is altered in posttrauma patients.

Objective: Assess the impact of hypertonic saline ± dextran on distinct CD14hiCD16-'classical' and CD14loCD16+ 'proinflammatory' monocyte subsets.

Population: 25 adult trauma patients in hypovolemic shock (systolic BP≤70 mmHg) and 10 healthy controls (CT).

Interventions: Prehospital treatment with 250mL of 7.5% hypertonic saline (HS; n=7), 7.5% HS plus 6% dextran-70 (HSD; n=7) or 0.9% normal saline (NS; n=11).

Methods: Monocyte counts (×109/L), surface marker (CD14, CD16, HLA-DR) and intracellular cytokine (TNF-α, IL-10) expression [mean fluorescence intensity (MFI) ± SEM] were analyzed flow cytometrically in samples obtained at admission (Adm), 12 and 24-h post-treatment.

Results: Total CD14+ monocyte and CD14loCD16+ subset counts were significantly (p <.05) elevated in patients vs. CT at all time-points. NS-treated patients had the highest CD14loCD16+ counts compared to HS and HSD groups. HLA-DR was reduced by more than half in patients (60±7.0) vs. CT (122±10) throughout the 24-h period. Inflammatory CD14loCD16+ monocytes displayed twice the level of TNF-α as the more abundant CD14hiCD16- subset (28±4 vs.14±1); with peak levels (40±9) observed in the NS group on Adm. IL-10 expression was above CT levels in both CD14loCD16+ and CD14hi CD16- subsets, with higher values in HS and HSD groups vs. NS.

Conclusions: Our findings confirm shock patients resuscitated with HS or HSD have less proinflammatory CD14loCD16+ monocytes with a reduced capacity for TNF-α, but higher IL-10 production (ROC substudy supported by NIH R01-2007-000-20819-0; Defence R&D Canada).


TLR9 INHIBITION IMPROVES IMMUNE RESPONSE IN LATE POSTTRAUMATIC MICEX. Ruan*, S.S. Darwiche*, M. Scott, T.R. Billiar. Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213

Objective: To investigate the role of TLR9 in the immune response following severe trauma, we analysis the resistance of TLR9 mutant mice as well as inhibitory CpG injected C57BL/6 (WT) mice to immunosuppression in a novel pseudofracture (PFx) model.

Methods: Male TLR9 mutant, WT, inhibitory CpG sequence and control CpG injected WT mice were randomly assigned to sham operation or PFx. Blood were collected and splenocytes were isolated and stimulated with concanavalin A, antiCD3e, or lipopolysaccharide (LPS) for T and B lymphocytes proliferation.

Results: PFx increased serum il-6 and il-10 level at 6 h, and returned to sham level at 48 h posttraumatically in all groups. In WT mice, PFx decreased stimulated T cell proliferation to sham level by 45.9% (with concanavalin A), and 54.5% (with antiCD3e) respectively, and stimulated B cell proliferation by 45.0% (with LPS). In TLR9 mutant and inhibitory CpG injected WT mice, the inhibitory effects of PFx on stimulated splenocytes proliferation were abrogated, resulting in T and B cell proliferation similar to their sham group. Levels of il-6 were significantly increased by PFx in the supernatant of stimulated WT splenocytes, but not in that of TLR9 mutant and inhibitory CpG injected WT splenocytes. And fluctuations of il-10 in the supernatant induced by PFx were similar among groups.

Conclusions: Our results provide conclusive evidence that trauma contribute to a delay immunosuppression of T and B lymphocytes. TLR9 mutant and inhibitory CpG injected WT mice do not show the suppression indicating TLR9 signaling plays a key role in immunosuppression of T and B lymphocytes in late posttraumatic model. TLR9 blockade is a potential strategy for the treatment of posttraumatic immunosuppression in human severe trauma.


INSULIN IMPROVES HEPATIC STRUCTURE AND FUNCTION BY ATTENUATING HEPATIC ER STRESS/UPR, APOPTOSIS, AND AUTOPHAGYD. Boehning, R. Kraft, J. Song, G. Gauglitz, R. Cox, N. Brooks*, C. Finnerty, G. Kulp*, D. Herndon, M. Jeschke. University of Texas Medical Branch, Galveston, TX 77555

Background: Severe stress causes hepatic injury with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). ER stress/UPR due to calcium store depletion leads to cell apoptosis, autophagy, and activation of the JNK signaling pathway. Insulin attenuates hepatic damage and we therefore hypothesized that insulin administration improves liver structure and function by attenuating hepatic ER stress/UPR, apoptosis and autophagy.

Methods: Male Sprague Dawley rats received a 60% TBSA burn injury to induce a stress response. Animals were randomized to receive saline (controls) or insulin (2.5-5 IU/kg q. 24 hours) and sacrificed at 24 and 48 hours post-burn. ER stress

Results: Burn induced hepatocyte apoptosis via caspase-3 activation. Insulin significantly decreased hepatocyte apoptosis and caspase-3 activation at 24 and 48 hours post-burn, p<0.05. Insulin furthermore decreased hepatocyte autophagy compared to burn 48 hours post-burn, p<0.05. Insulin markedly decreased ER stress and unfolded protein response when compared to burn, which was associated with improve mitochondrial State-3 respiration and mitochondrial pore transition at 24 and 48 hours post-burn, p<0.05. Improved ER stress and mitochondrial function was associated with decreased ER cytochrome c expression, p<0.05. Transmission electron microscopy confirmed the biomedical markers and demonstrated markedly improved hepatic morphology with insulin administration.

Conclusion: Insulin significantly alleviates ER stress and UPR which is associated with decreased hepatocyte apoptosis and autophagy, leading to an improved liver structure post-burn.


CASPASE-1 BLOCKS REGULATES INDUCTION OF AUTOPHAGY AFTER HEMORRHAGIC SHOCK IN MICEQ. Sun*1, T. Billiar2, M. Scott2. 1Department of Pathology and 2Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213

Activation of the inflammasome by reactive oxygen species (ROS) produced during ischemia/reperfusion and hemorrhagic shock leads to maturation of caspase-1, which cleaves pro-inflammatory cytokines (IL-1β, IL-18) and causes their subsequent release. Our previous data showed that oxidative stress induces activation of the inflammasome and caspase-1, as well as upregulation of autophagy in mice hepatocytes. We hypothesized that tissue ischemia induced by hemorrhagic shock (HS) would also activate autophagy in a caspase-1 dependent way.

Methods: Caspase−1−/-and C57BL/6 (WT) mice underwent sham surgery (femoral artery cannulation only) or HS (1.5h hemorrhagic shock + 4.5 h resuscitation) (n=3-4/experimental group). Plasma cytokines were measured. Whole liver lysates were collected and immunoblotted for autophagic marker (LC3).

Results: In WT mice, IL-18 levels were significantly elevated after HS (125±40pg/ml HS vs nondetectable (ND) in Sham), suggesting inflammasome activation and maturation of caspase-1 (Fig. 1). As expected, IL-18 levels were nondetectable in all caspase-1-/-groups. LC3 levels (indicating autophagy induction) were significantly elevated after HS in the liver of WT mice, but the up-regulation of LC3 was blocked in caspase-1-/-mice (Fig. 2).

Conclusion: Induction of autophagy in the liver after hemorrhagic shock is dependent on caspase-1 activation, through a mechanism that has yet to be identified. Elucidating mechanisms of autophagy activation and inflammation may help understand the regulation of inflammation after trauma and hemorrhage.



FETUIN PROTECTS MICE AGAINST LETHAL SEPSIS BY MODULATING BACTERIAL ENDOTOXIN-INDUCED HMGB1 RELEASE AND AUTOPHAGYH. Wang, W. Li*, S. Zhu*, J. Li*, M.F. Ward*, H. Yan*, H. Yang, K.J. Tracey, P. Wang, and A.E. Sama*. The Feinstein Institute for Medical Research, North Shore - LIJ Health System, Manhasset, NY 11030

The pathogenesis of sepsis is complex, but in part mediated by bacterial endotoxin, which stimulates macrophages to release early (e.g., TNF, IL-1) and late (e.g., HMGB1) pro-inflammatory mediators. Various inflammatory stimuli (e.g., endotoxin, cytokines, and oxidative stress) similarly induce autophagy, a catabolic degradation process responsible for eliminating damaged cytoplasmic components during infection. A negative acute phase protein, fetuin-A (fetus protein in Greek), was recently characterized as a negative regulator of inflammation by opsonizing cationic anti-inflammatory molecules (e.g., spermine).

Objective: To further elucidate the role of fetuin-A in lethal experimental sepsis.

Methods: We examined its effects on endotoxin-induced HMGB1 release and autophagy in vitro, and determined whether administration of exogenous fetuin-A protects mice against lethal experimental sepsis (induced by cecal ligation and puncture, CLP) in vivo.

Results:In vitro, fetuin-A (25- 100 microgram/ml) effectively inhibited endotoxin-induced (100 ng/ml) HMGB1 release (by 60-90%), but enhanced endotoxin-induced autophagy in macrophage cultures. In vivo, intraperitoneal administration of fetuin-A (100 mg/kg, once daily, for three days) beginning at 24 hour post CLP, significantly increased animal survival rates from 40% (in saline vehicle group, N = 22 mice/ group) to 90% (in fetuin-A group, N = 22 mice/group, P< 0.05).

Conclusions: Fetuin-A occupies a protective role in experimental sepsis by attenuating a late mediator of lethal systemic inflammation. (Supported by the National Institute of General Medical Sciences Grant R01 GM063075 to H.W.)



Introduction: In a variety of diseases systemic inflammation may be associated with ATP release in organs and tissues. Concomitantly, increased numbers of immature phagocytic cells sharing properties of either dendritic cells or macrophages can be enriched from peripheral blood of patients with SIRS and sepsis by in vitro cultures. Clinically, the occurrence of this antigen presenting cell-(APC-) type was related to the reactivation of herpes virus infections and impaired natural killer-(NK-) cell function. This study aimed at the comparative analysis of immunological, functional and ultrastructural characteristics of such APC.

Methods: Caspase measurements using chemi-luminescence were applied to study ATP induced effects and to investigate the susceptibility for NK-lysis. Herpes virus reactivation was tested by a multiplex PCR identifying EBV genes characterizing latency states I, II and III. High pressure freeze substitution transmission electron microscopy (TEM) was used to study morphological criteria.

Results: Caspase 3/7 activation was significantly downregulated in the presence of exogeneous ATP but was increased when APC were coincubated with NK-92 killer effectors. However, a subgroup of APC with very low CD80/86 and HLA-class II was even resistant to NK-92. We found evidence for EBV latency state II infection in these immature APC by the detection of EBV-specific LMP-1 and EBNA 2 genes. TEM studies identified impressive macroautophagy, mitochondrial degradation and multivesicular body formation.

Conclusions: The present study illustrates novel aspects of APC maturation deficiency in patients suffering from SIRS and sepsis. These APC are often susceptible to NK-92 cytolysis but are resistant to ATP-induced apoptosis. Since ATP is an important DAMP in SIRS and sepsis, results provide an explanation how ATP contributes to severe immune deficiency. Persistence of these immature APC supports the survival of active virus infections and autophagy is a novel morphological criterion associated ATP specific anti-apoptosis.


CARDIOPROTECTION BY RAPAMYCIN IN SEPSIS: A NOVEL MECHANISM VIA COMPLETE INDUCTION OF AUTOPHAGYY.C. Hsieh*, H.W. Hsueh*, S.S. Yuan*, C.H. Hsieh*. (Spon: I.H. Chaudry) Department of Medical Research, E-Da Hospital/I-Shou University, Kaohsiung 824, Taiwan, ROC

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Autophagy functions predominantly as a pro-survival pathway in the heart during cellular stress by removing protein aggregates and damaged organelles, protecting the heart against famine and ischemia. We hypothesized that autophagy is involved in cardiac dysfunction in sepsis, and induction of autophagy can attenuate cardiac dysfunction and increase ATP levels as well as inhibit pro-inflammatory mediators during sepsis. Male mice were subjected to cecal ligation and puncture (CLP) or sham operation. At 1 h after CLP operation, mice received either rapamycin (autophagy inducer), bafilomycin A1 (autophagy inhibitor), or vehicle (10% DMSO). The results showed that autophagosomal protein LC3-II was increased in the heart at 24 h after CLP, whereas Atg5/12 complex and cathepsin D, required for initiation and degradation of the autophagosome, were decreased, indicating an incomplete autophagic process. This was consistent with the morphologic finding that septic heart exhibited more autophagic vacuoles but less digestion within the vacuole (Fig. 1). Administration of rapamycin, but not bafilomycin A1, after CLP resulted in the complete induction of the autophagic process, and increased cardiac output and stroke volume, as well as ATP levels. This is accompanied by the decreases of cardiac IL-6, MCP-1, and HIF-1α levels. This indicates that autophagy may serve as a cell survival mechanism to protect against cardiac dysfunction and inflammatory mediator releases during sepsis.

FIG. 1


HETEROTRIMERIC Gαi PROTEINS ARE MODULATED BY TOLL LIKE RECEPTOR (TLR) SIGNALING AND ARE BENEFICIAL IN SEPSISH. Fan, B. Zingarelli, K. Borg, P. Halushka, L. Birnbaumer, J. Cook. Medical Univ. of SC, Charleston, SC 29425

Previous studies implicated heterotrimeric Gαi proteins as negative regulators of lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Gαi proteins are directly modulated by TLR signaling and are beneficial in sepsis. Raw 264.7 cells were stimulated with E. coli 0111:B4 LPS (500ng/ml, List) and immunoprecipitated with a Gαi protein activation assay. In a second series of studies, Gαi2 protein knockout (KO) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP). Twenty-four hours after CLP, mice were sacrificed. Peritoneal fluid, blood, and lung samples were collected. CFU counts were used to determine bacterial load. A second group of mice was subjected to CLP and was monitored every 24 hours for 120 hours for survival analysis. We found that Gαi proteins are rapidly activated by LPS followed by rapid inactivation (fig, p<0.05, N=7). These studies provide the first evidence that Gαi proteins are directly modulated by TLR signaling. In following studies, Gαi2 KO mice exhibited impaired bacterial clearance as evidenced by increased bacterial load in peritoneal fluid, blood and lung tissue (41±18 fold, 10±5 fold, and 13±5 fold, respectively). The survival rate post-CLP was significantly decreased (33% decrease, p<0.05) in the Gαi2.

KO mice compared to WT mice. Understanding the role of Gαi2 protein in regulation of inflammatory response in sepsis may provide novel targets for treatment of sepsis. (Supported by NIH GM27673, GM67202, AI079248).



EFFECT OF HERITABLE AND ACQUIRED CONDITIONS ON MORTALITY OF 854 US PATIENTS WITH SEVERE SEPSISE. Watanabe, B.A. Zehnbauer*, S. Oda, T. Shimada*, H. Hirasawa, T.G. Buchman. Washington University School of Medicine, St. Louis, MO 63110

Objectives: Our aim is to evaluate the influence of genetic polymorphisms in selected genes and to assess relative effect on the heritable factors versus the physiologic derangement of severe sepsis.

Design: Collaborative case-control study.

Settings: Heterogeneous population of patients from 12 USA intensive care units (ICUs) represented by the Genetic Predisposition to Severe Sepsis (GenPSS) archive.

Measurements and Main Results: From the GenPSS archive, we selected 854 patients with severe sepsis. Fifty-three percent of the patients were male with 87.3 % of Caucasian racial type. Overall mortality was 35.1 %. Average age (SD) was 63.0 (16.05) years old. We initially analyzed 6 functional single nucleotide polymorphisms (SNPs) present before the first codon of TNF at-308, IL-1β at-511, IL-6 at-174, IL-10 at-819, and CD-14 at -159, and in the first intron of LT-α at +252 (LTA(+252)). We also compared the contributions of the genetic factors with preexisting conditions (cardiovascular diseases, diabetes, malignant neoplasm, etc.) as well as sequelae (acute organ dysfunctions, deep venous thrombosis, etc.) and treatments regarding the outcome of severe sepsis. A logistic regression analysis was performed. The presence of 3 acute organ dysfunctions and the necessity to use vasopressin infusion and renal replacement therapy were larger prognostic factors for the adverse outcome (OR = 11.8, 3.27, 2.57, P =.031,.004,.003, respectively). Variants of IL1B(-511), LTA(+252) and TNF(-308) had lesser influence on the outcome of severe sepsis than the preexisting conditions and sequelae.

Conclusions: Neither genetic factors nor chronic illnesses are by themselves sufficient to predict the outcome of severe sepsis. Acquired conditions contributed to the sepsis outcome more than heritable factors in the study population.


APOLIPOPROTEIN E DEFICIENCY PROTECTS AGAINST SEPTIC MORTALITYB. Leung*, O. Kattan*, N. Presser*, H. Harris. Department of Surgery, University of California, San Francisco, CA 94143

Although apolipoprotein E (apoE), a component of triglyceride-rich lipoproteins, has well-established roles in lipid metabolism, its role in sepsis is still poorly understood. ApoE has been suggested to bind and traffic lipid antigens for natural killer T (NKT) cell activation. We have previously demonstrated that serial injections of apoE increased septic mortality in rats in a dose-dependent manner, presumably by driving antigen presentation and the downstream secretion of cytokines by NKT cells. Thus, we sought to further characterize apoE's contribution to septic mortality by looking at subphysiological levels of apoE and its effect on serum cytokine levels and NKT cell activation. Hypomorphic apoE mice (Apoeh/h) expressing 2-5% of wild-type serum levels of apoE were subjected to cecal ligation and puncture (CLP). Survival was monitored for 28 days after CLP. At 24 hours, serum cytokines were measured by multiplex analysis, and the spleen, liver and thymus were harvested for NKT cell isolation and FACS analysis. Non-induced (low apoE) hypomorphic mice consistently had a greater survival rate following CLP than induced (wt apoE) mice, independent of the severity of septic insult. Higher apoE levels were associated with increased TH1 cytokine secretion, as the serum cytokine profile showed a trend towards greater TH1 cytokine levels in septic, induced mice compared to septic, non-induced mice (TNF-α 156.2 vs. 70.55 pg/ml, IL-1β 9.34 vs. 2.72 pg/ml, IFN-γ 3.67 vs. 0.47 pg/ml). NKT cell frequencies (as a percentage of total lymphocytes) was increased by 700% and 20% in the liver and thymus, respectively, in septic, induced mice vs. septic, non-induced mice although that of the spleen was unchanged. Our findings suggest that decreasing apoE lowers pro-inflammatory cytokine levels and the degree of NKT cell activation, and thus, promotes resistance to septic mortality. This further implicates apoE and lipoproteins as regulators of host microbial immunity.


SEPSIS-INDUCED IMMUNOSUPPRESSION IS MEDIATED BY TRAIL-EXPRESSING CD8+ REGULATORY T CELLSJ. Unsinger*, H. Kazama*, J. McDonough*, T. Griffith*, R. Hotchkiss, T. Ferguson*. Washington Univ. Sch. of Medicine, St. Louis, MO 63110

Septic patients often display severely compromised immune function, manifested by impaired delayed-type hypersensitivity (DTH) response (loss of skin testing to recall antigens). Since sepsis induces significant apoptosis in lymphoid and myeloid cells and apoptotic cells are themselves tolerogenic, we tested the hypothesis that suppression of DTH is mediated by tolerogenic properties of the apoptotic cells generated during sepsis.

Methods: Mice were subjected to cecal ligation and puncture (CLP) or sham surgery. The ability to mount an immune response toward a newly introduced antigen was measured at different time points post surgery. Results were compared to mice with disrupted apoptotic pathways due to overexpression of the anti-apoptotic protein Bcl-2 or knockout of the proapoptotic protein Bim.

Results: Wt mice subjected to CLP demonstrated a loss of DTH for 7d post surgery however, the immune response returned to normal by day 10. Blocking sepsis-induced apoptosis via Bcl-2 overexpression or Bim deficiency prevented loss of DTH. Injection of apoptotic cells into Bim−/−mice suppressed an effective DTH response, thereby suggesting a causal link between apoptotic cells and immune suppression. Surprisingly, TRAIL null mice had significant apoptosis but retained their DTH responses. Further studies revealed that apoptotic cells generated during sepsis induced a CD8+ regulatory T cell that suppressed DTH by TRAIL production.

Conclusions: These results establish a link between apoptotic cells and immune suppression during sepsis, and suggest TRAIL may be a viable therapeutic target for boosting the adaptive immune response following sepsis.


CALCIUM SUPPLEMENTATION DURING SEPSIS INCREASES MORTALITY: A CAMKK1 MECHANISMR. Collage, J. Stripay, X. Zhang, L. Guo, M. Rosengart. University of Pittsburgh, Pittsburgh, PA 15213

Introduction: Considerable resources are devoted to monitoring and treating the hypocalcemia of sepsis. However, altered immune cell calcium (Ca2+) regulation is postulated to fuel the aberrant inflammation underlying organ dysfunction. Here, we hypothesize that Ca2+ supplementation increases inflammation and mortality through a mechanism involving calcium/calmodulin-dependent protein kinase kinase 1 (CaMKK1).

Methods: C57Bl/6 and CaMKK1 knockout mice were subjected to cecal ligation puncture (CLP; 21-gauge, single puncture+imipenem/cilastatin). KN93 was utilized in vivo as a broad CaMK inhibitor. After 12h, a timepoint corresponding with hypocalcemia, mice were administered either CaCl2 (2.5mg/kg) or equivolume saline control. After 4h serum was isolated and analyzed for cytokines and HMGB1 by ELISA and immunoblot. A cohort population underwent a 7d survival study. Data were analyzed by ANOVA and log-rank. A p<0.05 was significant.

Results: Ca2+ supplementation after CLP markedly heightened systemic TNFα (35 vs. 167 pg/ml, p=0.05), IL-10 (173 vs. 3173 pg/ml, p=0.008) and HMGB1 concentrations. By comparison to its less active analogue KN92, KN93 significantly reduced TNFα (259 vs. 17.9, p=0.008), IL-10 (4702 vs. 125 pg/ml; p=0.008) and HMGB1 concentrations. Mice receiving Ca2+ had significantly worse survival by comparison to septic mice receiving saline control (33% vs. 88%, p=0.02), however, survival was significantly improved in CaMKK1 mice (33% vs. 67% p=0.02).

Conclusions: These data support that Ca2+ administration during sepsis worsens survival and highlights the need to reevaluate our perspective of managing hypocalcemia of critical illness. The observed survival benefit of inhibiting CaMKK1 suggests that a detailed understanding of the CaMK-dependent mechanisms regulating sepsis may provide therapeutic targets.



Background: Host injury involved in the pathogenesis of multi-organ system failure during sepsis results from massive infiltration of active neutrophils into tissue. A central player in this process is the cell surface adhesion molecules called integrins. The best characterized integrin-binding motif is the RGD sequence, which is present in a variety of extracellular matrix proteins. Clinical trials with RGD peptide mimetics show favorable anti-cancer efficacy, but relatively little information is available regarding their effects on inflammatory diseases.

Methods: RGD/Control peptide was administered in C57BL/6 male mice following cecal ligation & puncture (CLP) or LPS injection. Survival rate, cytokine production, bacterial clearance and leukocyte infiltration into peritoneum were compared. In vivo imaging of Gr1+ granulocytes with RGD administration was performed with intravital two-photon microscopy. Integrin expression on PMNs isolated from septic animals or septic patients was determined using flow cytometry.

Results: Administration of RGD peptide resulted in improved survival in both CLP (RGD=50% vs Control=20%) and endotoxemic (RGD=60% vs Control=20%) animals. Similarly, RGD group showed improved bacterial clearance in peritoneal lavage (PL) (RGD=4,746±4,172 vs Control=21,850±5,157 cfu/100ul), reduced leukocyte (Gr1+, F480+/CD11b+) infiltration, and lower levels of CD11b expression. CD18 and CD49c (RGD-binding integrin) were highly upregulated peaking at 6 h and 12 h, respectively, on Gr1hi granulocytes both in PL and peripheral blood following CLP or LPS injections. Neutrophils from septic patients demonstrated higher expression of RGD-binding integrins compared to control subjects.

Conclusions: Our results suggest that inhibition of interstitial migration of neutrophils with a RGD peptide enhances sepsis survival by promoting bacteria clearance and suppressing immature respiratory burst.


IMPLICATIONS OF UNPHOSPHORYLATED STAT3 IN SEPSISL. Ulloa*, G. Peña, B. Cai, E.A. Deitch. UMDNJ-New Jersey Medical School, Newark, NJ 07101

Objective: The role of STAT3 in infectious diseases has remained elusive due to irreconcilable results suggesting that both STAT3 depletion and activation are detrimental for sepsis. Here we report that STAT3 mediates the anti-inflammatory potential of cholinergic agonists and unphosphorylated STAT3 prevents systemic inflammation and improves survival in sepsis.

Results: Bacterial endotoxin induced STAT3 tyrosine phosphorylation in splenocytes and activated macrophages. Alpha7nAChR activation or JAK2 inhibition prevented endotoxin-induced STAT3 tyrosine phosphorylation. Pharmacological inhibition of STAT3 tyrosine phosphorylation mimicked the alpha7nAChR-signaling to control NF-kB and cytokine production in splenocytes and macrophages. Transfection of macrophages with a dominant negative form of STAT3 inhibited cytokine production and abrogated the anti-inflammatory signaling of alpha7nAChR. Transfection of macrophages with a mutant STAT3 to specifically prevent its tyrosine phosphorylation enhanced the alpha7nAChR signaling. However, inhibition of STAT3 expression enhanced cytokine production and abrogated the alpha7nAChR signaling. Alpha7nAChR can control TNF production in macrophages through a mechanism that requires STAT3 expression, but not its tyrosine phosphorylation. In vivo, pharmacological inhibition of STAT3 activation attenuated cytokine production in the spleen, prevented systemic inflammation, and improved survival in endotoxemia and polymicrobial peritonitis. STAT3 also prevented the production of late mediators of sepsis including HMGB1 extracellular secretion from macrophages. Pharmacological inhibition of STAT3 activation, started after the onset of sepsis, rescued animals from polymicrobial sepsis.

Conclusions: These results reveal the clinical implications of STAT3 in the cholinergic regulation of the immune system and pharmacological advantages of unphosphorylated STAT3 in infectious diseases.


IMMUNOABLATIVE THERAPY FOR SEPSISA. Shawo*, E. Uzosike*, H. Harris. University of California, San Francisco, San Francisco, CA 94143

Objectives: Immunoablation of circulating leukocytes with cyclophosphamide has been shown to effectively treat severe multiple sclerosis. Since sepsis and multiple sclerosis are both diseases characterized by a dysregulated host immune response, we sought to improve survival in a murine model of sepsis via removal of activated leukocytes using high-dose cyclophosphamide treatment.

Methods: Sham or cecal ligation and puncture (CLP) surgeries were performed on 6-week-old male ICR mice. High-dose cyclophosphamide (70 mg/kg, Hi-CY) or saline was administered every 24 h for 4 days beginning 24 h after surgery, when the animals were already demonstrating signs of sepsis. Analgesics were administered at 12 and 18 h post-operatively along with a broad-spectrum antibiotic (imipenem) at 2 h and then every 12 h for four days. Complete leukocyte counts and survival were measured for 28 days.

Results: Neutrophil counts in Hi-CY, sham-operated mice reached an early nadir of 100 cells/uL blood, followed by a marked rebound neutrophilia which peaked at 10,000 cells/uL before returning to normal by day 28. The pattern for other leukocytes was similar. Hi-CY treated mice had an increased survival compared to sham-operated controls at 4 days. (60% versus 30%, p<0.05) However, this survival difference was lost by day 7, with no animals surviving in either group.

Conclusions: Neutrophil counts in Hi-CY treated sham mice demonstrated a significant early neutropenia followed by rebound neutrophilia. Importantly, Hi-CY therapy increased survival during the early stages of CLP-induced sepsis. But, once the cyclophosphamide therapy was stopped, a neutrophilia followed and the protective effect was lost. These data demonstrate a correlation between increased early survival after sepsis and cyclophosphamide-induced leukopenia and support further study of the potential protective effects of immunoablative therapy for sepsis.



Systemic inflammatory responses can be regulated through brain muscarinic M1 receptor-mediated mechanisms and efferent vagus nerve signaling to the spleen (Proc Natl Acad Sci USA, 2006, 5219; Proc Natl Acad Sci USA, 2008, 11008). Here we evaluated the anti-inflammatory efficacy of the centrally-acting M1 muscarinic receptor agonist xanomeline in lethal endotoxemia. Single drug (5 or 20 mg/kg), i.p. administration 1h prior to endotoxin (6 mg/kg, i.p.) dose-dependently reduced serum TNF levels and significantly (up to 100%) improved survival in mice. Surprisingly, xanomeline retained its TNF-suppressing and survival-improving effects even when administered as long as 20h prior to endotoxin. The survival-improving effect of xanomeline when given 1h and 20h prior to endotoxin was abolished by pharmacological blockade of brain muscarinic receptors, but not peripheral muscarinic receptors. Xanomeline early (-20h) administration resulted in significant suppression of ex vivo TNF, IFN-γ and IL-6 production from endotoxin-stimulated total spleen cells, an effect accompanied by significantly increased T cell number, and decreased numbers of B cells and dendritic cells in spleen. Together these results demonstrate a long-lasting anti-inflammatory effect of xanomeline and indicate that drug-induced central muscarinic receptor-dependent signaling is associated with reduced spleen cell cytokine production and altered number of splenic leukocyte subpopulations. Our findings are of interest for further development of xanomeline and other centrally-acting muscarinic M1 agonists as a novel class of experimental anti-inflammatory therapeutics. (This study was funded in part by NIH/NIGMS).


EFFECTS OF LXA4 ON BACTERIAL LOAD AND INFLAMMATORY RESPONSE IN SEPSISK. Yin, J. Walker*, E. Dichter*, G. Lacorte*, B. Spur*, A. Rodriguez*. Department of Cell Biology, UMDNJ-SOM, Stratford, NJ 08084

Sepsis is characterized by dysregulated systemic inflammation with release of large amounts of inflammatory mediators. This dysregulation can lead to multiple organ failure and/or immunosuppression. In the latter condition, the host may be susceptible to opportunistic infections or be unable to clear an existing infection. Lipoxin A4 (LXA4) is an endogenously produced metabolite of arachidonic acid, with the novel action of inflammation resolution. With respect to this, LXA4 increases macrophage recruitment to site of injury while decreasing inflammatory mediator release in various models of inflammation. Thus, we hypothesized that LXA4 could resolve inflammation in the cecal ligation and puncture (CLP) model of polymicrobial sepsis by increasing macrophage recruitment to prevent bacterial spread while decreasing systemic inflammatory response. CLP rats were given either saline or LXA4 (40 μg/kg, i.p.) 5h after surgery. All rats were sacrificed 48h after CLP surgery. Administration of LXA4 increased the number of peritoneal cells by approximately 2-fold (P < 0.05). This increase was due to an increase in macrophage number while neutrophils numbers were not significantly altered. This change in inflammatory cell number resulted in reduced blood bacterial load compared to CLP rats given vehicle saline. Plasma IL-6 and MCP-1 levels were significantly reduced by approximately 2-fold in LXA4 treated rats compared to CLP rats given saline vehicle (P < 0.05). In addition, plasma LXA4 levels were significantly higher in CLP rats given LXA4. These results strongly suggest that LXA4 can reduce the systemic inflammatory response while preventing bacterial spread, in the CLP model of polymicrobial sepsis. The results indicate that LXA4 can reduce systemic inflammation without compromising host response.


ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)-α IN SEPSISS. Standage, K. Odoms*, A. Denenberg*, B. Zingarelli,, H. Wong. Cincinnati Children's Hospital Medical Center Research Foundation, Cincinnati, OH 45229

Objective: PPAR-α, a member of the nuclear receptor superfamily, modulates metabolism and inflammation at many levels. Our previous work using genome-wide expression profiling in septic children identified repression of the PPAR-α signaling pathway as a marker for increased illness severity and mortality.1 We hypothesized that PPAR-α plays an integral part in the host response to sepsis and sought to elucidate that role.

Methods: Sepsis was induced in 5-6 week old C57BL/6 wild-type (WT) and PPAR-α null mice by intraperitoneal injection of a fecal slurry (1.3 mg/kg). Survival was monitored for 72 hours (n = 26/group). Other mice (n = 5/group) were sacrificed at 6 hours after slurry injection, and lung and liver tissue analyzed for myeloperoxidase (MPO) levels, a measure of neutrophil infiltration. Serum from these groups was also assayed for cytokine levels (TNFα, IL1β, IL6, IL10, KC, MIP1α) using a multiplex bead array.

Results: PPAR-α null mice had a significantly lower probability of survival over the 72 hour observation period (p = 0.01; Kaplan-Meier) and also demonstrated increased early death (24 hr survival probability = 0.3 in null mice vs. 0.8 in WT mice). Lung and liver MPO levels were significantly increased in PPAR-α null mice compared with WT (p = <0.001, p = 0.009 respectively). Serum cytokine levels demonstrated a trend towards increased inflammation in null mice with IL-6 levels dramatically higher in the null mice compared to WT (median values 23,327 vs. 7,904 pg/ml, IQR 22,868-23,776 & 6,229-9,118 respectively, p =.008).

Conclusions: These data suggest that functional PPAR-α promotes survival and exerts an anti-inflammatory effect in this murine model of polymicrobial sepsis. (Supported by R01GM064619)

1BMC Medicine. 7:34, 2009.


PLASMA ATP CONCENTRATIONS CORRELATE WITH NEUTROPHIL ACTIVATION AFTER SEPSISY. Sumi*, Y. Chen, Y. Yao*, T. Woehrle*, A. El Khal*, W.G. Junger. Department of Surgery, BIDMC, Harvard Medical School, Boston, MA 02215

We have previously shown that extracellular ATP is required for neutrophil activation by stimulating P2Y2-type purinergic receptors. Changes in extracellular ATP concentrations could thus affect neutrophil activation and neutrophil-induced organ damage. Here we investigated in a mouse sepsis model if plasma ATP levels may influence neutrophil activation. Wild type (WT) or P2Y2 knockout (KO) mice were subjected to cecal ligation and puncture (CLP) and blood was drawn via axial vessels. ATP concentration in heparinized plasma (HPLC) and neutrophil activation (CD11b expression) were determined. In WT CLP mice, ATP levels were significantly higher than in shams (Fig; n=5/group, mean ± SD, Student's t test, *p<0.05, #p<0.01). CLP increased CD11b expression in WT mice to 154±30 (mean channel fluorescence), compared to 76±5 in WT shams and 47±9 in P2Y2KO mice with CLP. Treatment with the P2Y2 receptor antagonist suramin educed CLP-induced CD11b expression by 90%. We conclude that ATP released into plasma and feedback via P2Y2 receptors are involved in neutrophil activation, suggesting that P2 receptor antagonists could be used to reduce neutrophil-induced host tissue damage after sepsis. (Support: Novo Nordisk Shock Society, NIGMS, NIAID, DOD, & Boehringer Ingelheim).




Objective: Glutamine (GLN) is a key "pharmacologically-acting nutrient" and pharmacologic doses of GLN given via 'bolus' administration demonstrate potent gut-protective effects in various critical illness conditions. A key mechanism of GLN's protection involves enhanced expression of heat shock proteins (HSP). However, mechanisms by which GLN upregulates HSP remain unclear. The heat shock transcription factor (HSF-1) is the master regulator for heat shock gene expression. This study is aimed to investigate whether GLN's action is mediated by modulation of HSF-1's transactivation activity or/and expression.

Methods: Dextran sulfate sodium (DSS)-colitis was induced in Sprague-Dawley rats. GLN (0.75g/kg/day) or sham was administered to rats by oral gavage during the 7-day DSS treatment. In Vitro YAMC colonic epithelial cells received concurrent treatment of varying concentrations of GLN and cytokines (TNF-α/IFN-γ).

Results:In vivo, bolus GLN mitigated disease activity, preserved gut integrity and blunted over-produced TNF-α in rats with colitis, associated with enhanced HSP25 and HSP70 in colonic mucosa. In vitro, GLN also led to a dose-dependent increase in YAMC HSP expression following the cytokine insults. Both in vivo and in vitro, GLN treatment led to a concordant increase in HSF-1's expression and its nuclear translocation and phosphorylation. Further, enhancement of HSF-1 expression by GLN could be abrogated via inhibition of double-stranded (ds) RNA-dependent protein kinase (PKR). This is independent of PKR's well recognized role as a translational inhibitor via phosphorylating eukaryotic initiation factor 2α.

Conclusions: This study provides the first evidence that GLN may not only be an activator of HSF-1's transactivation activity, but also enhances HSF-1 expression. Furthermore, PKR plays an essential role in GLN's modulation of heat shock response.


MECHANISMS THROUGH WHICH GUT BCL-2 OVER-EXPRESSION IMPROVES SURVIVAL IN MICE GIVEN IRRADIATION FOLLOWED BY MRSA PNEUMONIAE. Jung*, E. Perrone*, P. Brahmamdan*, J. McDonough*, C. Davis, J. Dominguez, R. Hotchkiss, C. Coopersmith. Washington Univ. Sch. of Medicine, St. Louis, MO 63110

Radiation exposure is a risk due to terrorism. Methicillin-resistant S. aureus (MRSA) pneumonia is common. These threats together could be synergistically lethal. Wild type (WT) Fvb/n mice and mice that overexpress gut Bcl-2 were given whole-body irradiation (250 rads, IR) or not irradiated (NR). Four days later, mice were given 1×107 CFU of intra-tracheal MRSA or saline (S), then sacrificed at 24 hours or followed 7 days for survival. Gut epithelial apoptosis was measured by caspase-3 (c-3) and H&E staining (n=9-15). MRSA was cultured in blood and BAL fluid. ANOVA or log rank test was used for data analysis. WT mice subjected to either IR or MRSA alone had 100% survival, but IR+MRSA resulted in 53% survival (n=8-15; p=0.01). IR or MRSA alone caused a slight increase in gut apoptosis compared to NR+S (c-3: IR 14±2, MRSA 19±6, vs 6±2 apoptotic cells /100 crypts; H&E: IR 17±2, MRSA 16±3, vs 7±1; p<0.05) but IR+MRSA caused a further synergistic increase in gut apoptosis (c-3: 79±27; H&E: 100±34; p<0.05). Compared to NR+MRSA, IR+MRSA had higher blood (4±0.5 vs 2±0.3 log CFU/ml MRSA; p<0.05) and BAL (5±0.3 vs 2±0.2; p<0.01) bacterial counts. When subjected to the same IR+ MRSA insult, mice with Bcl-2 over-expression had lower levels of gut apoptosis (c-3: 13±3; H&E: 14±3; p<0.01 vs WT IR+ MRSA) but similar bacterial counts (blood: 4±0.2; BAL: 5±0.3) as their WT littermates. IR+MRSA in Bcl-2 mice resulted in 92% survival compared to 45% in their WT IR+MRSA littermates (n=12-13; p<0.01). In conclusion, either low level irradiation or MRSA pneumonia alone is nonlethal but together significantly worsen survival. Gut Bcl-2 overexpression significantly decreases gut apoptosis and increases survival, but does not alter local or systemic infection. Gut apoptosis may be a therapeutic target in the treatment of sepsis in an irradiated host.


INTRAVENOUS INFUSION OF MESENCHYMAL STEM CELLS IS ASSOCIATED WITH IMPROVED MYOCARDIAL FUNCTION DURING ENDOTOXEMIAB. Weil*, J. Herrmann*, A. Abarbanell*, M. Manukyan*, J. Poynter*, D. Meldrum. Indiana University School of Medicine, Indianapolis, IN 46202

Objective: To determine whether infusion of mesenchymal stem cells (MSCs) is associated with improved myocardial function in a rat model of endotoxemia.

Methods: Adult Sprague-Dawley rats were administered saline (vehicle) or lipopolysaccharide (LPS; 20 mg/kg) via tail vein injection. Treatments, either vehicle or 2 × 106 MSCs, were infused 1 hour later via tail vein. Animals were divided into the following groups: 1) vehicle + vehicle (control; n = 6), 2) LPS + vehicle (n = 6), and 3) LPS + MSCs (n = 6). 6 hours after induction of endotoxemia, left ventricular function was assessed via parasternal short-axis M-mode echocardiography. Left ventricular ejection fraction (EF) and fractional shortening (FS) were determined over 3 adjacent cardiac cycles. Animals were then sacrificed and hearts and serum were collected for determination of cytokine levels via ELISA.

Results: Animals injected with LPS + vehicle exhibited depressed cardiac function as indicated by a 26% and 37% reduction in EF and FS from baseline, respectively. Treatment with MSCs was associated with improved cardiac function compared with vehicle treatment as indicated by a reduction in EF and FS of only 10% and 17%, respectively (P < 0.05). Myocardial levels of TNF-α and IL-1β were nearly tripled in LPS treated animals versus control. Similarly, serum levels of IL-1β, IL-6, and IL-10 were substantially increased in LPS treated animals. Treatment with MSCs, however, was associated with significant reductions in myocardial levels of TNF-α and IL-1β and in serum levels of IL-1β and IL-6. Additionally, treatment with MSCs was associated with an increase in serum IL-10.

Conclusions: Infusion of MSCs modulates the systemic inflammatory response and is associated with improved myocardial function during endotoxemia.


Abstract withdrawn.


ORAL GLUTAMINE REDUCES MYOCARDIAL INJURY IN PATIENTS UNDERGOING CARDIAC SURGERYA. Sufit*, C. Hamiel*, A. Baer*, K. Queensland*, P.E. Wischmeyer. U. of Colorado, Denver Aurora, CO 80045

Objectives: Glutamine (GLN) can protect against in vitro and in vivo myocardial cell injury following ischemia and reperfusion injury. This effect may be mediated by increased heat shock protein (HSP) expression. However, the potential for GLN to protect the myocardium in patients undergoing cardiac surgery is unknown. Thus, our objective was to perform a proof-of-concept pilot trial to determine if pre-operative GLN therapy could attenuate myocardial injury as measured by serum injury markers in patients undergoing cardiac surgery.

Methods: Subjects undergoing elective cardiac surgery (CABG or heart valve replacement/repair), in which cardiopulmonary bypass was required were enrolled in a randomized, double-blinded trial to receive oral GLN (25mg BID) (n=7) or maltodextran control (C) (n=7) daily for 3 days prior to surgery. Serum troponin (TROP I), creatine kinase (CK-MB), myoglobin, and white cell HSP70 level was obtained at: baseline, pre-anesthesia, and at 6, 24, 48, and 72 h post-surgery.

Results: Of enrolled patients, 3 withdrew (2 GLN, 1 C), 1 expired in OR (C pt), and 1 underwent non-qualifying surgery (GLN pt). No adverse effects were noted in either group. Thus, 10 patients (4 GLN and 6 C) had samples available for analysis. Our data reveals patients receiving GLN had significantly decreased TROP I, at 24, 48, and 72 h post-surgery (all p < 0.05) versus C. GLN therapy also reduced CK-MB at 24 and 48 h (p < 0.05 and p < 0.001 respectively) and myoglobin 24 h post-surgery (p<0.05) versus C. GLN treated patients showed a non-statistically significant trend towards increased cellular HSP70 content.

Conclusions: In this pilot study, GLN therapy prior to cardiac surgery reduces myocardial injury as measured by serum injury markers. This may be due to increased cellular HSP70 expression. This data indicates a larger trial of GLN therapy in cardiac surgery powered on clinical outcomes is warranted. (Supported by NIH K23 RR018379).


LEUKOCYTIC AND ENDOTHELIAL MYOSIN LIGHT CHAIN KINASE CONTRIBUTE TO GUT MICROVASCULAR HYPERPERMEABILITY AND BARRIER DYSFUNCTION IN THERMAL INJURYM. Guo, C. Sun, Q. Shen, B. J. Frederich, S. Y. Yuan, M. H. Wu. Division of Research, Department of Surgery, University of California Davis School of Medicine, Sacramento, CA 95817

Non-muscle myosin light chain kinase (nmMLCK) has recently been identified to express mainly in endothelial/epithelial cells and leukocytes. The aim of this study was to characterize the role of nmMLCK in gut barrier dysfunction during systemic inflammation caused by 25-40% TBSA full-thickness burns. The injury caused intestinal mucosa inflammation and neutrophil infiltration. Intravital microscopic studies showed significant leukocyte adhesion and plasma leakage in intestinal microvessels coupled with increased gut lumen-to-blood transport of small molecules. The abnormalities were significantly attenuated by either neutrophil depletion or nmMLCK deficiency. To examine the relative importance of leukocytic and endothelial nmMLCK, we generated chimeric mice (nmMLCKPMN-/EC+ and nmMLCKPMN+/EC-) via bone marrow irradiation and transplant. While there was no difference in basal permeability between wild type and nmMLCK-/- mice, the burn-induced hyperpermeability was reduced to the same extent in the chimeric mice compared to the wild type. Additionally, in vitro assays showed that stimulation of Caco-2 with neutrophils decreased transcellular electric resistance and increased myosin light chain phosphorylation and claudin-1 internalization; the responses were prevented by MLCK inhibition. Taken together, the results suggest that both endothelial and leukocytic nmMLCK contribute to gut barrier injury. (Supported by NIH RO1 HL96640, HL61507, and HL70752).


VAGAL STIMULATION DECREASES INFLAMMATION THROUGH A GHRELIN MEDIATED MECHANISM IN TRAUMATIC BRAIN INJURYV. Bansal, S. Ryu*, C. Blow*, M. Krzyzaniak*, C. Peterson*, W. Loomis*, B. Eliceiri*, A. Baird*, R. Coimbra. UCSD, San Diego, CA 92103

Background: TBI can cause a release of inflammatory cytokines that may result in sepsis and death. Vagal stimulation (VS) decreases pro-inflammatory cytokines in sepsis. The hormone ghrelin has potent anti-inflammatory effects; furthermore, ghrelin release is stimulated by acetylcholine. We hypothesized that VS decreases post-TBI inflammation through a ghrelin mediated mechanism.

Methods: A weight drop model created TBI in five groups of Balb/c mice. Group TBI: TBI only; Group TBI/Ghrelin: animals were given 10 μg of intraperitoneal ghrelin prior to and 1 hour after TBI; Group TBI/VS: animals underwent cervical electric VS before TBI +/− ghrelin receptor antagonist (GRa) (6mg/kg). Group Sham: no TBI, VS or ghrelin. Blood was drawn post-TBI for measurements of serum TNF-α, and ghrelin levels (ELISA). Stomach, intestine, brain, lung, liver, and kidney were harvested 2 hrs post-TBI and were assayed for ghrelin.

Results: Ghrelin increased following VS 2 hrs post-TBI compared to sham or TBI alone (1200±162 pg/ml vs. 657±106 pg/ml vs. 652±72 pg/ml; p<.01). An increase in TNF-α was observed in TBI at 6 hours compared to sham (34.4 ± 1.9 pg/ml vs. 22.9 ± 1.4 pg/ml;p<.001). VS+TBI and ghrelin+TBI had levels comparable to sham (34.4±1.9 pg/ml vs. 22.9 ±1.4 pg/ml; 21.8±1.0 pg/ml; 24.0±1.5 pg/ml p<.03). In contrast, TNF-α levels remained elevated in VS+GRa (34.9±2.9 pg/ml p<.01). The highest levels of ghrelin was in the stomach and TBI decreased stomach ghrelin levels in contrast to an increase by VS (4037±137 pg/ml vs. 5223±314 pg/ml;p<.01).

Conclusions: In TBI, VS increased serum ghrelin and decreased TNF-α which was lost with GHr. Our data suggests the release of ghrelin plays an important role in the anti-inflammatory effects of VS following TBI.


FEMALE X-CHROMOSOME MOSAICISM CONTRIBUTES TO THE GENDER DIMORPHIC CHARACTER OF THE INFLAMMATORY RESPONSEZ. Spolarics, S. Federici, Z. Németh, G. Haskó, E. A. Deitch, R. Chandra. Department of Surgery, New Jersey Medical School, Newark NJ

X-linked genetic polymorphisms present a unique condition because, in females, either the maternal or the paternal X chromosomes is inactivated in each individual cells resulting in cellular mosaicism. In contrast, males carry exclusively the maternal X chromosome. Thus, female X-chromosome mosaicism may contribute to the gender dimorphic character of the inflammatory response. To test this hypothesis we employed mice deficient of X-linked gp91phox (Nox4) and compared the inflammatory response to LPS in vivo or sepsis (CLP) among heterozygous mosaics, WT and homozygous animals. Mosaic subpopulations of neutrophils (PMN) were analyzed by our flow cytometry assay, which identifies mosaic subpopulations by gp91 expression. LPS treated gp91 mosaic mice presented white blood cell trafficking patterns similar to that of observed in WT mice despite the fact that the deficient PMN subpopulation in mosaic animals displayed increased cell activation as reflected in elevated CD11b expression and increased splenic PMN infiltration. Observations on splenic T-cell depletion and blood cytokine responses after LPS or CLP also indicated that mosaic animals present an inflammatory response that is not simply an intermediary, mixed response between deficient and WT but uniquely characteristic to mosaics. Gp91 mosaic animals also showed improved survival and efficient bacterial clearance after CLP, which indicates the potential clinical benefit of mosaic status. The study supports the notion that female X chromosome mosaicism could contribute to the gender dimorphic character of the inflammatory response and therefore it is an important confounder in genetic association or gender related clinical studies. (Supported by NIH NIGMS GM084932).



Rationale: Excess ROS is generated after burn injury and is implicated in priming neutrophils, systemic inflammatory response syndrome, tissue damage and multiple organ failure. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), a bZIP transcription factor is a master regulator of a robust cytoprotective defenses that includes antioxidant defenses. The present study was designed to investigate if role of Nrf2 in the pathophysiology of burn injury.

Methods: Wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) mice were subjected to 15% and or 30% total body surface area (TBSA) burn or sham injury. Intestinal damage, systemic inflammation and mortality were assessed after burn injury. Macrophages were used to investigate ROS generation, TLR4 activation and cytokine secretion.

Results: i) Burn injury caused significantly higher mortality in Nrf2−/− mice when compared to Nrf2+/+ mice; ii) Systemic inflammation as assessed by serum cytokine levels (IL-6, TNF-α, IL-1β and HMGB1) were markedly elevated in Nrf2−/− compared to Nrf2+/+ mice; iii) Relative to Nrf2+/+, Nrf2−/− mice showed greater intestinal injury as evidenced by intestinal permeability, apoptosis and histopathology; iv) mRNA expression of pro-inflammatory cytokines in intestine was significantly higher in Nrf2−/− mice compared to Nrf2+/+ mice; v) Relative to Nrf2+/+, macrophages from burn Nrf2−/− mice showed greater levels of ROS, TLR4 surface expression and secretion of inflammatory cytokines at baseline and after LPS stimulation.

Conclusions: Nrf2 is critical host factor to limit immune dysregulation, intestinal damage and mortality after burn injury. (Supported by NIGMS R01GM079239).


T-CELL IMMUNOSUPPRESSION FOLLOWING SURGERY IS NOT MEDIATED VIA DYSFUNCTIONAL MACROPHAGESD.S. Quaiser*, M. Albertsmeier*, S. Zedler*, C.J. Bruns*, H. Winter*, E. Faist, M.K. Angele. Department of Surgery, LMU Klinikum Großhadern, Munich, Germany

Introduction: Recent experimental studies in a mouse trauma-hemorrhage model suggest a pivotal role of T-cells in maintaining immunosuppression. Here we investigated the interaction of T-cells and monocytes (Mφ) in humans following major surgery.

Materials and Methods: PBMC were harvested from 12 surgical patients preoperatively, 2 hr and 24 hr post-operatively and separated into T-cell (CD3+) and Mφ (CD14+). These isolated cells were co-incubated with each other and with preoperatively harvested frozen immune cells stimulated with anti-CD3, anti-CD28 and LPS for 24 hr. IFN-y, IL-12, IL-6, GM-CSF were measured in the supernatants by enzyme immunoassay. For characterization of non-stimulated T-cell and Mφ surface markers were analyzed by flow-cytometry.

Results: Surface markers on T-cells: CD3 (−30 %), CD4 (−41 %), CD25 (−35 %), CD28 (−35 %), CD127 (−29 %) were significantly suppressed 2 hr postoperatively. In contrast, CD40, CD80, CD86 and HLA-DR on Mφ were maintained. Cytokine release capacities of Mφ for TNF (+150%), IL-6 (+173%) and GM-CSF (+191%) were increased post-operatively irrespective whether pre- or postoperative T-cells were co-incubated. In contrast, T-cells harvested 2 or 24 hr postoperatively released significantly decreased amounts of IFN-γ. Co-incubation of T-cells with frozen Mφ, releasing non-detectable levels of IL-12, did not affect T-cell responses.

Conclusions: Early after major abdominal surgery suppressed T-cell as opposed to maintained Mφ responses are evident. Moreover, diminished Mφ release capacities for IL-12 do not affect T-cell function. Those results collectively suggest a minor role of Mφ in postoperative suppression of T cell function. Studies using alternative stimulation patterns, however, are required to further elucidate the complex interaction of T-cells and Mφ.


GLUCAN PHOSPHATE DECREASES HMGB-1 TRANSLOCATION IN THE MYOCARDIUM OF SEPTIC MICE VIA INHIBITION OF NFκB ACTIVATIONT. Ha*, X. Liu*, C. Lu*, J. Kelley*, J. Kalbfleisch*, R.L. Kao*, D.L. Williams, C. Li. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. HMGB1 serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) significantly attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of CLP septic mice. GP was administered to mice 1 hr before induction of CLP. Sham surgically operated mice served as control. The levels of HMGB1 and TLR4 in the myocardium were examined by Western blot. NFκB binding activity was assessed by EMSA. In a parallel study, H9C2 cardiomyoblasts were treated with LPS in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NFκB activation, before LPS stimulation. CLP significantly increased the levels of HMGB1 and NFκB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-increased NFκB binding activity in the myocardium. In vitro studies indicated that GP significantly prevented LPS-induced HMGB1 translocation and NFκB binding activity. Blockade of NFκB activation significantly attenuated LPS-induced HMGB1 translocation. GP administration also reduced LPS stimulated association between TLR4 and HMGB1. These data suggest that GP attenuates septic cardiomyopathy, in part, by preventing NFκB activation and subsequent HMGB-1 translocation.

©2010The Shock Society