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High-mobility group box 1 inhibition protects against hepatic ischemia-reperfusion injury
298
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
80. Intestinal Epithelial Cells Exposed to Heat Shock Stress Release a Soluble Compound That Activates the Virulence of P. aeruginosa. J. Kohler, MD, O. Zaborina, MD, L. Wu, MD, J. Alverdy, MD. University of Chicago. Introduction. Although there is a significant body of evidence that bacteria activate a variety of pro-inflammatory signals following their interaction with human epithelial cells, there is virtually no information on signaling in the opposite direction. In this study we sought to determine whether host cell factors released by intestinal epithelial cells subjected to stress could signal the opportunistic pathogen, P. aeruginosa, to express a key virulence related protein, the PA-I lectin. Methods. Human intestinal epithelia (Caco-2 bbe ) were grown to confluence in HDMEM media. To expose cells to a clinically relevant stress, hyperthermic conditions were created by immersing sealed culture dishes in a 42°C water bath for 23 min, followed by 2 h of recovery at 37°C and 5% CO 2 . Control cells were maintained in 37°C at 5% CO 2. . Media were then filtered through a 0.22-m filter before serial centrifugal membrane filtration using 100, 50, 30, 10, and 3 kDa filters and resuspended in HDMEM. GFP reporter strains of P. aeruginosa carrying a fusion construct for the PA-I lectin were then exposed to the epithelial cell derived filtrates from heat shock stressed Caco-2 cells. Dynamic fluorimetry was used to measure fluorescence (i.e. PA-I activity) over time. To determine if inducing compounds were proteins, fractions capable of activating PA-I were then heat inactivated by heating to 70°C for 30 min and tested against active fractions by the same method. Results. A 30to 50-kDa media fraction from heat-stressed cells induced a 10fold increase in PA-I promoter activity over controls, as measured by fluorescence (1082 ⫾ 216%, P ⬍ 0.01), while all other fractions showed no effect. Heat inactivation completely eliminated this effect (⫺156% of control ⫾ 293%, P ⫽ NS). Conclusion. Human intestinal epithelial cells release a soluble 30- to 50-kDa compound(s) that can activate a key virulence protein in P. aeruginosa. We speculate that the molecular dialogue between a pathogen and the intestinal epithelium is bidirectional and can be initiated by host stress conditions such as hyperthermia. 81. High-Mobility Group Box 1 Inhibition Protects Against Hepatic Ischemia-Reperfusion Injury. A. Tsung, MD, H. Tanaka, MD, K.G. Raman, MD, K.J. Tracey, MD, T.R. Billiar, MD, D.A. Geller, MD. University of Pittsburgh, Pittsburgh, PA. Introduction. Hepatic ischemia followed by reperfusion (I/R) occurs in the settings of trauma, transplantation, and elective liver resections. The mechanisms that account for local organ damage are only partially understood. High-mobility group box 1 (HMGB1) has been identified as a late mediator of lethality in sepsis and is released by damaged tissues where it could also act as a mediator of inflammation and organ injury. We hypothesized that HMGB1 would contribute to organ injury following hepatic I/R. Methods. HMGB1 protein expression and hepatocellular secretion were determined in rat hepatocytes subjected to hypoxia (1% O 2) versus normoxic cells. The role of HMGB1 in mediating hepatic I/R injury was examined in C57Bl/6 mice that underwent 90 min of partial I/R injury. These mice were pretreated with neutralizing HMGB1 antibody (600 g) or control normal saline 1 h prior to ischemia. Results. Basal HMGB1 expression was observed in normoxic hepatocytes and was dramatically up regulated 12–24 h after hypoxia. This corresponded to increased HMGB1 secretion 18 –24 h after hypoxia. In mice undergoing partial warm liver I/R injury, HMGB1 protein expression is increased as early as 1 h after reperfusion and then increases in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after warm ischemia compared to control animals. Conclusion. This study demonstrates that HMGB1 is up regulated in hepatocytes by simple hypoxia. The same was seen to occur in vivo
TABLE—ABSTRACT 81 Group
ALT-1 h
ALT-6 h
ALT-24h
Sham Control Anti-HMGB1
67 ⫾ 24 3101 ⫾ 1167 662 ⫾ 120*
57 ⫾ 8 8140 ⫾ 1656 2382 ⫾ 687*
51 ⫾ 2 905 ⫾ 65 265 ⫾ 16*
Note. Data are mean ⫾ SEM, n ⫽ 3– 4 per group; * indicates P ⬍ 0.05 versus control.
in liver I/R as early as 1 h. The reduction in liver damage by a neutralizing antibody shows that HMGB1 is a key mediator in the pathways that lead to organ damage in warm liver I/R. Thus, unlike sepsis where HMGB1 is late mediator, this danger protein appears to be an early mediator in I/R. 82. Direct Intracellular ATP Delivery for Treatment of Cyanide Intoxication. B.B. Chiang, MD, W.D. Ehringer, PhD, S.H. Su, MD, S. Chien, MD. University of Louisville School of Medicine. Cyanaide directly poisons the cellular respiratory mechanism, leading to cellular hypoxia and death. The aim of this study was to determine the effect of a newly developed direct intracellular ATP delivery technique, ATP encapsulated unilamellar fusogenic lipid vesicles (ATP-Vesicle), on rat with cyanide poisoning. Methods. ATP-Vesicle was prepared by lecithin, fusogenic agents, and sonication. There were five groups to be studied: 1, (ATP-Vesicle); 2, (lipid vesicles); 3, (ATP); 4, (saline); and 5, (lipid vesicle ⫹ ATP). Adult Sprague Dawley rats were anesthetized. The carotid artery and jugular vein were cannulated. ATP-Vesicle or other solutions were injected intraperitoneally 3 min prior to intravenous potassium cyanide (KCN) infusion (10 mg/kg/h).The survival time and dose of KCN injected were recorded. Results. The survival time and the fatal dose of KCN in rats treated with ATP-Vesicle were significantly longer (47 min) and higher (7.6 mg/kg/h) than that of other groups (Table). Discussion. Humans may encounter cya-
TABLE—ABSTRACT 82 Group
ATP-vesicle
Vesicle
ATP
Saline
Vesicle ⫹ ATP
Surv. T (min) 46.6 ⫾ 4.9* 33 ⫾ 2.8 33.4 ⫾ 2.8 32.4 ⫾ 2.7 27.3 ⫾ 1.6 KCN (mg) 7.6 ⫾ 0.8* 5.5 ⫾ 0.5 5.6 ⫾ 0.5 5.4 ⫾ 0.6 4.6 ⫾ 0.3 Note. Table n ⫽ 10; * p ⬍ 0.05 as compared to other groups.
nide in industrial chemicals, natural products, medications, combustion products, and terrorist attacks. Cyanide induced threatening symptoms resulted in permanent sequelae, and even death has been reported. We developed a new intracellular ATP delivery system, in which ATP is encapsulated in small unilemellar fusogenic lipid vesicles (ATP-Vesicle). This system can deliver ATP directly into the cytosol at sufficient rates to meet the energy demand of the cells. Our study demonstrated that animals treated with ATP-Vesicle could survive longer under higher KCN intoxication. It indicated that ATP-Vesicle could deliver ATP directly into cells to keep animals alive during chemical hypoxia induced by KCN. Our study may open a new field in the treatment of ischemic or hypoxic diseases.
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS
80. Intestinal Epithelial Cells Exposed to Heat Shock Stress Release a Soluble Compound That Activates the Virulence of P. aeruginosa. J. Kohler, MD, O. Zaborina, MD, L. Wu, MD, J. Alverdy, MD. University of Chicago. Introduction. Although there is a significant body of evidence that bacteria activate a variety of pro-inflammatory signals following their interaction with human epithelial cells, there is virtually no information on signaling in the opposite direction. In this study we sought to determine whether host cell factors released by intestinal epithelial cells subjected to stress could signal the opportunistic pathogen, P. aeruginosa, to express a key virulence related protein, the PA-I lectin. Methods. Human intestinal epithelia (Caco-2 bbe ) were grown to confluence in HDMEM media. To expose cells to a clinically relevant stress, hyperthermic conditions were created by immersing sealed culture dishes in a 42°C water bath for 23 min, followed by 2 h of recovery at 37°C and 5% CO 2 . Control cells were maintained in 37°C at 5% CO 2. . Media were then filtered through a 0.22-m filter before serial centrifugal membrane filtration using 100, 50, 30, 10, and 3 kDa filters and resuspended in HDMEM. GFP reporter strains of P. aeruginosa carrying a fusion construct for the PA-I lectin were then exposed to the epithelial cell derived filtrates from heat shock stressed Caco-2 cells. Dynamic fluorimetry was used to measure fluorescence (i.e. PA-I activity) over time. To determine if inducing compounds were proteins, fractions capable of activating PA-I were then heat inactivated by heating to 70°C for 30 min and tested against active fractions by the same method. Results. A 30to 50-kDa media fraction from heat-stressed cells induced a 10fold increase in PA-I promoter activity over controls, as measured by fluorescence (1082 ⫾ 216%, P ⬍ 0.01), while all other fractions showed no effect. Heat inactivation completely eliminated this effect (⫺156% of control ⫾ 293%, P ⫽ NS). Conclusion. Human intestinal epithelial cells release a soluble 30- to 50-kDa compound(s) that can activate a key virulence protein in P. aeruginosa. We speculate that the molecular dialogue between a pathogen and the intestinal epithelium is bidirectional and can be initiated by host stress conditions such as hyperthermia. 81. High-Mobility Group Box 1 Inhibition Protects Against Hepatic Ischemia-Reperfusion Injury. A. Tsung, MD, H. Tanaka, MD, K.G. Raman, MD, K.J. Tracey, MD, T.R. Billiar, MD, D.A. Geller, MD. University of Pittsburgh, Pittsburgh, PA. Introduction. Hepatic ischemia followed by reperfusion (I/R) occurs in the settings of trauma, transplantation, and elective liver resections. The mechanisms that account for local organ damage are only partially understood. High-mobility group box 1 (HMGB1) has been identified as a late mediator of lethality in sepsis and is released by damaged tissues where it could also act as a mediator of inflammation and organ injury. We hypothesized that HMGB1 would contribute to organ injury following hepatic I/R. Methods. HMGB1 protein expression and hepatocellular secretion were determined in rat hepatocytes subjected to hypoxia (1% O 2) versus normoxic cells. The role of HMGB1 in mediating hepatic I/R injury was examined in C57Bl/6 mice that underwent 90 min of partial I/R injury. These mice were pretreated with neutralizing HMGB1 antibody (600 g) or control normal saline 1 h prior to ischemia. Results. Basal HMGB1 expression was observed in normoxic hepatocytes and was dramatically up regulated 12–24 h after hypoxia. This corresponded to increased HMGB1 secretion 18 –24 h after hypoxia. In mice undergoing partial warm liver I/R injury, HMGB1 protein expression is increased as early as 1 h after reperfusion and then increases in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after warm ischemia compared to control animals. Conclusion. This study demonstrates that HMGB1 is up regulated in hepatocytes by simple hypoxia. The same was seen to occur in vivo
TABLE—ABSTRACT 81 Group
ALT-1 h
ALT-6 h
ALT-24h
Sham Control Anti-HMGB1
67 ⫾ 24 3101 ⫾ 1167 662 ⫾ 120*
57 ⫾ 8 8140 ⫾ 1656 2382 ⫾ 687*
51 ⫾ 2 905 ⫾ 65 265 ⫾ 16*
Note. Data are mean ⫾ SEM, n ⫽ 3– 4 per group; * indicates P ⬍ 0.05 versus control.
in liver I/R as early as 1 h. The reduction in liver damage by a neutralizing antibody shows that HMGB1 is a key mediator in the pathways that lead to organ damage in warm liver I/R. Thus, unlike sepsis where HMGB1 is late mediator, this danger protein appears to be an early mediator in I/R. 82. Direct Intracellular ATP Delivery for Treatment of Cyanide Intoxication. B.B. Chiang, MD, W.D. Ehringer, PhD, S.H. Su, MD, S. Chien, MD. University of Louisville School of Medicine. Cyanaide directly poisons the cellular respiratory mechanism, leading to cellular hypoxia and death. The aim of this study was to determine the effect of a newly developed direct intracellular ATP delivery technique, ATP encapsulated unilamellar fusogenic lipid vesicles (ATP-Vesicle), on rat with cyanide poisoning. Methods. ATP-Vesicle was prepared by lecithin, fusogenic agents, and sonication. There were five groups to be studied: 1, (ATP-Vesicle); 2, (lipid vesicles); 3, (ATP); 4, (saline); and 5, (lipid vesicle ⫹ ATP). Adult Sprague Dawley rats were anesthetized. The carotid artery and jugular vein were cannulated. ATP-Vesicle or other solutions were injected intraperitoneally 3 min prior to intravenous potassium cyanide (KCN) infusion (10 mg/kg/h).The survival time and dose of KCN injected were recorded. Results. The survival time and the fatal dose of KCN in rats treated with ATP-Vesicle were significantly longer (47 min) and higher (7.6 mg/kg/h) than that of other groups (Table). Discussion. Humans may encounter cya-
TABLE—ABSTRACT 82 Group
ATP-vesicle
Vesicle
ATP
Saline
Vesicle ⫹ ATP
Surv. T (min) 46.6 ⫾ 4.9* 33 ⫾ 2.8 33.4 ⫾ 2.8 32.4 ⫾ 2.7 27.3 ⫾ 1.6 KCN (mg) 7.6 ⫾ 0.8* 5.5 ⫾ 0.5 5.6 ⫾ 0.5 5.4 ⫾ 0.6 4.6 ⫾ 0.3 Note. Table n ⫽ 10; * p ⬍ 0.05 as compared to other groups.
nide in industrial chemicals, natural products, medications, combustion products, and terrorist attacks. Cyanide induced threatening symptoms resulted in permanent sequelae, and even death has been reported. We developed a new intracellular ATP delivery system, in which ATP is encapsulated in small unilemellar fusogenic lipid vesicles (ATP-Vesicle). This system can deliver ATP directly into the cytosol at sufficient rates to meet the energy demand of the cells. Our study demonstrated that animals treated with ATP-Vesicle could survive longer under higher KCN intoxication. It indicated that ATP-Vesicle could deliver ATP directly into cells to keep animals alive during chemical hypoxia induced by KCN. Our study may open a new field in the treatment of ischemic or hypoxic diseases.