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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS burn and trauma patients. Increasing multi-drug resistance of pathogens compounds this injury. While much has been learnt from host immune response to infection, a better understanding of the bacterial response to the host immune system is essential to develop new and better antimicrobials against target nosocomial pathogens. A nosocomial pathogen, Pseudomonas aeruginosa (PA), combats host-initiated oxidant stress through expression of the transactivating factor, OxyR, which regulates the production of catalases and hydroperoxidases. In addition, OxyR expression plays an essential role in the virulence of PA. We have shown that OxyR expression delays the maturation and function of host dendritic cells. Here we have tested the premise that OxyR also regulates PA virulence through secreted exotoxin production. Methods: Wild type PA (PAO1) and a deletion mutant lacking the oxyR gene (⌬OxyR) were grown in the log phase for 18 hours in Luria broth and the supernatant containing the secreted products was removed using centrifugation. Secreted proteins were then precipitated with ammonium sulfate and dialyzed against 0.9% NaCl. The proteins were then filter sterilized using a polysulfonate filter to remove LPS. ER-MP20 myeloid progenitor cells were harvested from the bone marrow of C57Blk/6J mice. These cells were differentiated into dendritic cells by the addition GM-CSF and TNF-␣. Various concentrations up to 20 ␮g of the bacterial proteins were added to the medium and cells were allowed to differentiate for 7 days. Cellular viability was then assayed using a cellular proliferation assay. These studies were repeated on ER-MP20 derived macrophages, the immortalized human monocyte cell line U937, and the murine macrophage P388D-1 cell line. Results: At a protein concentration of 5 ␮g PA supernatant protein, cellular proliferation was significantly reduced by 98.8⫾0.1% compared to untreated controls, while the ⌬OxyR supernatant protein decreased cell proliferation by only 13.7⫾7.0% (P ⬍ 0.05). Similar significant results were seen in the U937, P388D-1, and ER-MP20 derived macrophage cells. All reductions in cellular proliferation were abrogated when the proteins were denatured at 85 °C for 10 minutes. Conclusion: Taken together, our data indicates that OxyR controls the secretion of potent cytotoxic factors by PA. This gives credence to the probability that OxyR not only controls anti-oxidant response to host oxidant stress, but also controls a major portion of the organism⬘s virulence through the regulation of secreted exotoxins.

201. STAT1 ACETYLATION INHIBITS INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) EXPRESSION IN INTERFERON (IFN-⌫) TREATED RAW264.7 MURINE MACROPHAGES. L. Guo, H. Guo, C. Gao, Z. Mi, W. B. Russell, P. C. Kuo; Duke University, Durham, NC Introduction: Stat1 and NF-␬N are vital transcription factors for the induction of iNOS by IFN-␥. However, little is known of the counter-regulatory pathways which inhibit iNOS expression in this setting. Gene transcription can be regulated by acetylation of key transcription factor proteins. The role of Stat1 acetylation in this pathway has not been previously examined. We hypothesized that acetylation of the Stat1 protein may regulate macrophage iNOS expression by altering Stat1 binding to the iNOS promoter. Methods: RAW 264.7 murine macrophage cells were treated with IFN-␥ (500ng). In selected instances, deacetylase inhibitors trichostatin A (TSA; 200␮M) or valproic acid (VPA; 1.5mM) were added. Unstimulated cells served as Controls. Following 12 hours of incubation, media were analyzed for nitrite, an NO metabolite. Levels of iNOS and Stat1 mRNA were assayed by reverse transcriptase-PCR, and protein expression of cellular iNOS and Stat1 p91/p84 was measured by immunoblot analysis. Acetylated Stat1 was determined by immunoprecipitation using antiacetylated-lysine antibody followed by western blotting with Stat1 p91/p84 antibody. Cycloheximide (CHX), an inhibitor of protein synthesis, was used to assess degradation of Stat1 after treatment

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with TSA⫹IFN-␥ compared to treatment with IFN-␥ alone. Gel shift and chromatin immunoprecipitation (ChIP) assays tested in vitro and in vivo binding of Stat1 and NF-␬B to the murine iNOS promoter. (Data are presented mean ⫾ SEM; statistical analysis was performed using the Students t test.) Results: Nitrite, iNOS protein and iNOS mRNA expression increased by 15-, 5- and 5fold after treatment with IFN-␥ (p⬍0.01 vs Control for nitrite, protein and mRNA). In TSA⫹IFN-␥, TSA mediated acetylation ablated IFN-␥ induced changes such that nitrite, iNOS protein and iNOS mRNA expression were not statistically different from Controls or TSA alone. None of the treatments induced a change in the amount of Stat1 protein or mRNA, and they had no effect on the Stat1 protein half-life. Co-IP with an anti-acetylated-lysine antibody followed by probing with an anti-Stat1 antibody (p84/p91) showed that IFN-␥ alone was associated with a 2-fold increase in acetylated Stat1 (p⬍0.02 vs. Control and TSA alone). TSA⫹IFN-␥ caused an additional 4-fold increase in acetylated Stat1 (p⬍0.05 vs IFN alone). Gelshift and ChIP studies demonstrated an 8-fold increase in Stat1 binding in the presence of IFN alone (p⬍0.01 vs Control and TSA alone) However, in TSA⫹IFN-␥, Stat1 binding was decreased to levels not different from Control. Similar studies examing NF-␬B found no TSA-dependent alteration in in vivo or in vitro binding to either of its two elements in the murine iNOS promoter. Valproic acid was less potent than TSA, but VPA⫹IFN-␥ was still associated with significant (albeit less dramatic than TSA⫹ IFN-␥) alterations in nitrite, iNOS protein, iNOS mRNA, Stat1 acetylation and Stat1 binding. Conclusions: Acetylation of Stat1 protein correlates with decreased in Stat1 binding to the iNOS promoter with resultant inhibition of IFN-␥ mediated iNOS expression and nitric oxide production in RAW264.7 murine macrophages. Acetylation of the Stat1 protein may serve to down regulate iNOS expression in pro-inflammatory states.

202. SERUM OSMOLALITY, DESPITE TIGHT GLYCEMIC CONTROL, PREDICTS MORTALITY: A STUDY OF 1037 TRAUMA PATIENTS. D. Heffernan, P. R. Norris, J. A. Morris, Jr.; Vanderbilt University Medical Center, Nashville, TN Introduction: Hyperglycemia and hyperosmolality disrupt the immune system, reduce neutrophil activity, alter cytokine patterns, and impair complement function. Tight control of glucose has been shown to improve ICU mortality. Hypothesis: We hypothesized that abnormal glucose would be a major contributor to elevated serum osmolality, and that any effect of serum osmolality on mortality would be attenuated by tight glucose control. Methods: Out of 2,389 consecutive ICU admissions, 1,037 were admitted directly to the ICU, had an ICU length of stay ⱖ 3 days, and had basic metabolic panel laboratory results (BMP, N⫽3,824) including serum sodium (Na ⫹), glucose (GLU), and blood urea nitrogen (BUN). Patients were stratified based on outcome (live or die). Osmolality was computed from the first BMP in each day as: (Na ⫹ in mEq/l * 2) ⫹ (BUN in mg/dl ⫼ 2.8) ⫹ (GLU in mg/dl ⫼ 18) Differences in GLU, Na ⫹, BUN, and calculated osmolality between outcome groups were assessed using Wilcoxon’s Rank Sum test at each day. Statistical significance was defined as P⬍.01. Clinical significance was defined as a median value outside the laboratory range of normal. Results: Patients’ admission hyperglycemia was returned to the normal range according to protocol in the first 24 hours (figure, top). Glucose was not significantly different in survivors and non-survivors over the first 96 hours. Serum sodium and BUN stratified patients by outcome statistically (P ⬍ .01) but not clinically (not shown). Calculated serum osmolality stratified patients both statistically and clinically by outcome (figure, bottom). Conclusions: 1. Serum osmolality in patients who die is statistically and clinically elevated over the first 96 hours of ICU stay; 2. Clinically significant aberrations in serum osmolality persist despite tight glucose control; 3. Serum osmolality is a potential new marker for therapeutic intervention.

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS tivation in Kupffer cells and SM⌽ to the same extent. Coadministration of ICI 182,780 abolished the effects of E2-BSA. Moreover, IL-6 production was significantly suppressed by p38 inhibitor in Kupffer cells and by ERK-1/2 or JNK inhibitors in SM⌽. TNF-␣ production, on the other hand, was decreased by p38 or JNK inhibitors in both Kupffer cells and SM⌽. These results suggest that the salutary effects of E2 on KC and SM⌽ cytokine production are mediated, at least in part, via non-genomic pathway and that these non-genomic effects are likely mediated via MAPK pathways following T-H.

203. ESTROGEN-MEDIATED ACTIVATION OF NON-GENOMIC PATHWAY IMPROVES MACROPHAGES CYTOKINE PRODUCTION FOLLOWING TRAUMA-HEMORRHAGE. T. Suzuki, H. Yu, Y. Hsieh, M. A. Choudhry, K. I. Bland, I. H. Chaudry; University of Alabama at Birmingham, Birmingham, AL Although studies have shown that 17␤-estradiol (E2) administration following trauma-hemorrhage (T-H) restores the altered Kupffer cell and splenic macrophage (SM⌽) cytokine production, it remains unknown if E2 mediates those effects via genomic or non-genomic pathway. To determine this, we used E2 conjugated with BSA (E2-BSA) which acts on only cell surface membrane E2 receptors (ER) to determine the role of non-genomic pathway in E2-mediated restoration of Kupffer cells and SM⌽ cytokine production following T-H. We also examined if mitogen activated protein kinases (MAPK) play a role in mediating the non-genomic effects of E2. Male Sprague-Dawley rats underwent T-H (mean BP 40 mmHg for 90 min, then resuscitation). E2, E2-BSA (1 mg/kg E2) with or without an ER antagonist (ICI 182,780), or vehicle was injected intravenously during resuscitation. At 2 h after T-H or sham operation, Kupffer cells and SM⌽ were isolated and their production of IL-6 and TNF-␣ in response to LPS (1 ␮g/ml) was determined. Moreover, the activation of MAPK (e.g., p38, ERK-1/2 and JNK) in Kupffer cells and SM⌽ was determined. To delineate further the role of MAPK, isolated Kupffer cells and SM⌽ were cultured with selective inhibitors of p38 (SB203580 5 ␮M), ERK1/2 (PD98059 20 ␮M) or JNK (SP600125 20 ␮M) and the effects of these treatments on cytokine production was evaluated in vitro. One-way ANOVA and Student-Newman-Keuls method were used for analysis. Values are mean ⫾ SEM of n ⫽ 5-6/group. T-H produced a significant increase in Kupffer cells cytokine (IL-6 and TNF-␣) production and MAPK activation. In contrast, both cytokine and MAPK activation were decreased in SM⌽ following T-H. Although E2-BSA administration significantly attenuated alterations in Kupffer cells/SM⌽ cytokine production, the levels of these cytokines remained higher in KC and lower in SM⌽ than shams. Administration of E2, on the other hand, normalized the cytokine production similar to that observed in shams. In contrast E2 or E2-BSA prevented T-H-mediated alterations in MAPK ac-

204. ACID IS THE PRIMING INSULT FOR THE DOUBLE HIT MODEL OF ASPIRATION INDUCED LUNG INJURY. K. Raghavendran, B. A. Davidson, J. Helinski, M. T. Dayton, P. R. Knight; University at Buffalo, SUNY, Buffalo, NY Introduction: Aspiration induced lung injury is the third commonest cause of ARDS. However, only 30% of witnessed gastric aspiration lead to ARDS. Previous data from our laboratory has indicated a synergistic lung injury from CASP (combination of acid and particulate matter) compared to either gastric acid alone(ACID) or small non-acidified gastric particles (SNAP). The following experiments were conducted to study the pathogenesis of the synergistic lung injury in CASP by varying the sequential timing and order of ACID and SNAP components. Materials and Methods: Groups of long-Evans rats (n⫽9-12) were subjected to intra-tracheal ACID, SNAP, ACID followed by SNAP and SNAP followed by ACID with varied intervals of simultaneous administration and 4/ 24hr intervals between the two insults. The rats were then sacrificed at 4 and 24 hr following the last insult. A qualitative, quantitative cellular analysis of the broncho-alveolar lavage (BAL) and an arterial blood gas were performed. Additionally, BAL fluid concentrations of for albumin and cytokines (TNF-␣, IL-1␤, IL-10, CINC-1, MIP-2, MCP-1) were determined by ELISA. Statistical analysis was performed using student’s T- test with bonferroni correction (*p values⬍ 0.01). Results: Acid followed by SNAP, but not SNAP followed by acid aspiration causes a synergistic lung injury as assessed by leakage of albumin into the airspaces. This synergistic increase in lung injury is lost when SNAP follows ACID by more than 8 hr (Figure 1). The decrease in PaO2/FiO2(Figure 2) was also greater, BAL neutrophils and CINC-1(Figure 3) higher when ACID was followed by SNAP compared to SNAP followed by acid. Levels of MCP-1, MIP-2 were elevated in the group of animals that received SNAP followed by ACID. There were no significant differences in the levels of IL-1, TNF and IL-10. Conclusions: For a synergistic lung injury to be produced ACID has to be the priming insult followed by SNAP. If particulate injury follows ACID byⱖ 8 hr, this synergism is not seen.