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The gap junction protein connexin 43 is delivered to macrophage phagosomes and is required for efficient phagocytosis to occur
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Sinclair SB, Majetschak M; University of Miami, Department of Surgery
172. PENTOXIFYLLINE DOWNREGULATES STORED BLOOD INDUCED NEUTORPHIL ACTIVATION: A NEW ROLE FOR AN OLD DRUG. J. Deree, R. Lal l, H. Melbostad, W. Loomis, M. Grant, D. B. Hoyt, J. Martins, R. Coimbra; University of California at San Diego, San Diego, CA. Transfusion of packed red blood cells (PRBCs) is an independent risk factor for the Adult Respiratory Distress Syndrome (ARDS) and multisystem organ failure (MOF). Leukoreduction of stored blood has led to the idea that factors present in the supernatant of PRBCs activate neutrophils. Neutrophils normally serve as the first line of defense in the host. However, an exacerbated inflammatory response characterized by exaggerated neutrophil activation and proinflammatory mediator synthesis is deleterious to the host. We investigated the effects of Pentoxifylline (PTX), a phosphodiesterase inhibitor known to downregulate neutrophil superoxide production and proinflammatory mediator synthesis, on stored blood induced inflammation. We hypothesized that PTX would attenuate stored bloodinduced neutrophil activation and would decrease proinflammatory mediator synthesis using a whole blood preparation. Neutrophil activation was studied via the expression of a known marker of degranulation (CD66b) and by assessing MMP-9 levels, a proteolytic enzyme released by activated neutrophils. In addition, TNF-␣ levels were also measured. Whole blood was incubated with HBSS (control), LPS (100 g/mL) alone, LPS ⫹ 42-day old leukoreduced PRBC supernatant, and LPS ⫹ supernatant ⫹ PTX (2mM) at 37° C. TNF levels were measured for each group by ELISA. Active MMP9 levels were measured by zymography. Neutrophil CD66b expression was evaluated by flow cytometry in whole blood incubated with HBSS (control), fMLP (1M) alone, fMLP ⫹ supernatant, and fMLP ⫹ supernatant ⫹ PTX (2mM). LPS caused a significant increase in TNF-␣ levels when compared to control (100%). When PRBC supernatant was added, a 20% increase in TNF was seen (p⬍0.01). The addition of PTX to supernatant and whole blood caused a 106% decrease in TNF-␣ levels (p⬍0.0001). MMP9 levels were increased in all groups compared to control. PTX exerted no effects on MMP9 levels. CD66b expression was markedly increased by fMLP (100%), which was also observed after the addition of PRBC supernatant. PTX led to a 47% decrease in CD66b expression compared to fMLP⫹supernatant (p⬍0.01). Collectively, our data demonstrates that PTX selectively downregulates neutrophil CD66b expression and significantly decreases TNF-␣ levels in whole blood treated with agonists and supernatant from stored blood. Because the deleterious effects of blood transfusion on ARDS and MOF are associated with increased inflammatory mediator synthesis and neutrophil activation, the adjunctive use of PTX with blood transfusions may have therapeutic potential. 173. EXTRACELLULAR UBIQUITIN INCREASES IN HUMAN PACKED RED BLOOD CELL UNITS DURING STORAGE. Patel MB, Proctor KG, Veltman Y, Nguyen M,
Objective: Ubiquitin (Ub) is a highly conserved heat-stable 76 amino acid protein in all eukaryotic cells. It has multiple intracellular roles in protein degradation and posttranslational modification. In vitro data suggest that extracellular Ub has also pleiotropic effects on host defense mechanisms, including effects on apoptosis, cytokine secretion and growth regulation, and in vivo data indicate antiinflammatory actions. Erythrocytes contain 10-20 ng Ub/10 6 cells. Since hemolysis is known to occur with prolonged storage, we hypothesized that Ub is released over time into pRBC units and that this increase may correlate with the immunological properties of stored blood. Methods: After IRB approval volunteers (n⫽3) donated blood, which was processed by the blood bank into packed red blood cell units (pRBC). Blood units were stored at 0-6°C for 42 days. Daily aliquots were drawn and cell supernatants isolated by centrifugation at 1000 g for 20 min. Ubiquitin concentrations in the supernatants were measured by ELISA. The immunomodulatory effects of the supernatants were assessed by measuring lipopolysaccharide (LPS) evoked TNF-␣ production of normal whole blood in the presence of 33% supernatant obtained from different time points (n⫽9 per group). TNF-␣ was measured by ELISA. Data are expressed as mean ⫾ SEM and were analyzed by regression analysis, Spearman correlation (r s) and ANOVA (post hoc LSD). A 2-tailed p ⬍0.05 was considered significant. Results: Extracellular Ub linearly increased in pRBC units by 49 ⫾ 2 ng/mL per day (r 2⫽0.82, p ⬍0.001, Fig. A). PRBC supernatants time dependently inhibited LPS evoked TNF-␣ production of normal whole blood (n⫽9, Fig. B). The inhibitory effect of pRBC supernatants correlated significantly with the Ub concentration (r s⫽-0.53, p⬍ 0.001). Addition of exogenous Ub (equaling day 42 Ub concentration of 2170 ⫾ 268 ng/mL) to day 0-2 pRBC supernatants showed 31⫾15% of the inhibitory effect of day 42 supernatants on LPS evoked TNF-␣ response of normal whole blood (p ⬍0.05). Conclusions: Extracellular Ub concentrations in pRBC units linearly increase with duration of storage and may partially contribute to the immunological effects of stored blood. Furthermore, determination of Ub in pRBC units appears to be a sensitive testing technique for quality control purposes of blood transfusions and may be helpful for the assessment of novel blood preservation methods.
174. THE GAP JUNCTION PROTEIN CONNEXIN 43 IS DELIVERED TO MACROPHAGE PHAGOSOMES AND IS REQUIRED FOR EFFICIENT PHAGOCYTOSIS TO OCCUR. Anand RJ, Rippel CA, Leaphart CL, Li J, Hackam DJ; Childrens Hospital of Pittsburgh Introduction: The mechanisms governing phagocytosis by macrophages remain incompletely understood, although impaired phagocytosis may occur during advanced sepsis. Gap junctions are hexameric arrays of the protein connexin 43 (Cx43) which are localized on the cell membrane, and allow for the transfer of molecules between adjacent cells. The expression and possible role for Cx43 in macrophages remain largely unknown, although transfer of mole-
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS cules between the phagosome and the cytosol may be required for efficient phagocytosis to occur. We now hypothesize that Cx43 is expressed on macrophages, and that gap junctions participate in the process of phagocytosis. Methods: The expression of Cx43 in J774 macrophages was assessed by SDS-PAGE and laser confocal immunofluorescence using affinity purified polyclonal antibodies. Phagocytosis was determined by feeding J774 macrophages a meal of 0.8m latex particles or opsonized sheep erythrocytes (3h, 37°C), and removing the unbound particles by washing or hypotonic lysis. Intracellular particles were quantified using confocal microscopy in the x-z plane. Distribution of Cx43 around internalized particles was assessed by confocal microscopy and verified using sucrose density ultracentrifugation. Gap junction function was inhibited using oleamide (10M, 1h). Results: J774 macrophages were found to express the gap junction protein Cx43, which was localized at focal adhesions as well as on intracellular vesicles. J774 macrophages were found to bind particles to adhesion sites as well as to phagocytose them in a time and concentration dependent manner. Cx43 was found to accumulate strongly around internalized particles, and to persist over time on the phagosomal membrane. Strikingly, inhibition of gap junction function led to a significant reduction in the rate and extent of phagocytosis (control: 45⫾5% vs. olea 17⫾5%, p⬍0.05). Conclusions: These findings indicate a potential requirement for the gap junction protein connexin 43 in the regulation of phagocytosis and phagosomal maturation. Studies into the role of gap junctions on phagocytosis may provide novel therapeutic insights into diseases characterized by impaired microbial internalization, such as occurs during the late stages of sepsis. 175. ACTIVATED MACROPHAGES INHIBIT ENTEROCYTE GAP JUNCTIONS DURING INTESTINAL INFLAMMATION THROUGH THE RELEASE OF NITRIC OXIDE. R. J. Anand 1, C. A. Rippel 2, C. L. Leaphart 1, J. Li 1, D. J. Hackam 1; 1Childrens Hospital of Pittsburgh, Pittsburgh, PA, 2 University of Pittsburgh School of Medicine, Pittsburgh, PA. Introduction: Maintenance of barrier integrity requires communication between adjacent enterocytes through gap junctions composed of connexin43 (Cx43), which must be phosphorylated in order to function. Necrotizing enterocolitis (NEC) is characterized by an influx of activated macrophages and the release of nitric oxide (NO), although the mechanisms by which this leads to barrier dysfunction remain unknown. We therefore hypothesized that activated macrophages directly impair enterocyte gap junctions, and sought to define the mechanisms involved. Methods: J774 macrophages were activated (LPS 10 ng/ml ⫹ IFN␥ 100 U/ml, 12h), and NO release was assessed by Griess. Gap junction communication was assessed by fluorescence recovery after photobleaching (FRAP) ⫾ detaNONOate (50M). To simulate NEC, IEC-6 enterocytes were co-cultured with J774 on transwells, and the expression, distribution and phosphorylation of Cx43 in IEC-6 cells were measured by SDS-PAGE and immunofluorescence. Results: Activation of J774 macrophages led to the sustained release of NO (control J774: 1.3 ⫹/⫺ 0.1 M, activated J774 7.1⫹/1 1.7 M , p⬍0.05). NO significantly decreased gap junction function in IEC-6 cells (% recovery: control: 70% , NO: 35%, p⬍0.05). Under co-culture conditions, activated macrophages significantly decreased the phosphorylation of Cx43 in the adjacent enterocytes, which was not observed in the presence of non-activated macrophages, or cytokine stimulated IEC-6 cells alone. Activated macrophages also caused the rapid internalization of Cx43 within the enterocyte, providing a mechanism by which inter-enterocyte communication would be impaired. This effect was maintained when IEC-6 cells where treated with supernatants obtained from activated macrophages, suggesting that a soluble factor was involved. Strikingly, pre-incubation of J774 cells with the iNOS inhibitor L-Nil reversed the effects of activated macrophages on Cx43 phosphorylation and restored Cx43 localization to the enterocyte plasma membrane. Conclusions: Activated macrophages impair inter-
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enterocyte communication through de-phosphorylation and internalization of Cx43 via NO. These findings shed light into the pathogenesis of NEC, and identify intestinal Cx43 as a potential therapeutic target for this disease. 176. THE INFLUENCE OF ANGIOTENSIN II AND CYCLIC NUCLEOTIDE SECOND MESSENGER SIGNAL TRANSDUCTION ON ISCHEMIA-REPERFUSION-INDUCED ELEVATIONS IN MICROVASCULAR HYDRAULIC PERMEABILITY. Ramirez R, Sadjadi J, Curran B, Victorino GP; UCSF--East Bay Introduction: Intravascular volume loss from ischemia-reperfusion injury is a major clinical concern. We hypothesize that angiotensin II influences the ischemia-reperfusion-associated microvascular fluid leak through cyclic nucleotide second messenger signal transduction mechanisms. The purposes are to determine hydraulic permeability after ischemia-reperfusion of venules treated with 1) angiotensin II, 2) a cAMP synthesis inhibitor, 3) a cGMP inhibitor, and 4) simultaneous administration of angiotensin II and either a cAMP synthesis inhibitor or a cGMP synthesis inhibitor. Methods: Rat mesenteric postcapillary venules were micro-cannulated to measure hydraulic permeability (Lp). Ischemia-reperfusion was achieved by placing animals in a 5% oxygen environment and preventing venular flow followed by allowing blood flow to resume. Lp was measured after ischemia-reperfusion and treatment with 1) angiotensin II (20nM), 2) cAMP synthesis inhibitor (DDA,10uM), 3) cGMP synthesis inhibitor (LY83583, 10uM), and 4) angiotensin II plus either cAMP inhibition or cGMP inhibition, (n⫽6 in each group). Results: Compared to the 7-fold increase in Lp due to ischemia-reperfusion alone: 1) angiotensin II attenuated the 7-fold increase by 50% (p⬍0.001), 2) cAMP inhibition attenuated the 7-fold increase by 45% (p⬍0.001), 3) cGMP inhibition completed blocked any elevation in Lp due to ischemia-reperfusion (p⬍0.001), and 4) angiotensin II ⫹ cAMP inhibition was not statistically different from angiotensin II alone (p⫽0.16) while angiotensin II ⫹ cGMP inhibition attenuated the 7-fold increase in Lp due to ischemia-reperfusion by 65% (p⬍0.001). Conclusion: Treatment with angiotensin II attenuated increases in hydraulic permeability due to ischemia-reperfusion by 50%. Inhibition of cGMP synthesis completely blocked any increase in permeability due to ischemia-reperfusion while cAMP inhibition appears to play a lesser role. This emphasizes the major impact that cyclic nucleotide second messengers play in ischemia-reperfusion. A better understanding of mediators that reduce intravascular fluid loss from IR-induced microvascular dysfunction may help clinicians treat uncontrolled fluid extravasation that occurs during shock and sepsis. 177. CORRELATION OF SF-36 AND SF-12 IN A TRAUMA POPULATION. Kiely M, Weidner K, Weigelt J; Medical College of Wisconsin Background: The SF-36 is a commonly used general measure of health-related quality of life (QOL). The SF-12 is a related tool with less response burden, but its performance in a general trauma population is unknown. Hypothesis: The SF-12 would provide similar information to the SF-36 in blunt trauma patients. Patients and Methods: Adults with non-neurologic blunt injury were prospectively enrolled. Demographic, injury, social support, and socioeconomic data were collected. Patients responded to QOL, functional, PTSD, and depression questionnaires 1 and 6 months after injury. Physical (PCS) and mental (MCS) component scores of the SF-36 and SF12 were compared using Pearson’s correlation coefficient. Linear regression identified factors associated with the SF-12 and SF-36 PCS and MCS. Responsiveness to change was assessed using the standardized response mean. Results: Data from 185 patients were analyzed. Correlation of the PCS was 0.924 and MCS was 0.925 (both p⬍0.001). QOL remained below population norms at 6 months. ⴱp⬍0.05 compared to population norms, SF-12 and SF-36 PCS and MCS. PCS was moderately responsive to change, and was equivalent using either the SF-12 or SF-36. MCS was
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Sinclair SB, Majetschak M; University of Miami, Department of Surgery
172. PENTOXIFYLLINE DOWNREGULATES STORED BLOOD INDUCED NEUTORPHIL ACTIVATION: A NEW ROLE FOR AN OLD DRUG. J. Deree, R. Lal l, H. Melbostad, W. Loomis, M. Grant, D. B. Hoyt, J. Martins, R. Coimbra; University of California at San Diego, San Diego, CA. Transfusion of packed red blood cells (PRBCs) is an independent risk factor for the Adult Respiratory Distress Syndrome (ARDS) and multisystem organ failure (MOF). Leukoreduction of stored blood has led to the idea that factors present in the supernatant of PRBCs activate neutrophils. Neutrophils normally serve as the first line of defense in the host. However, an exacerbated inflammatory response characterized by exaggerated neutrophil activation and proinflammatory mediator synthesis is deleterious to the host. We investigated the effects of Pentoxifylline (PTX), a phosphodiesterase inhibitor known to downregulate neutrophil superoxide production and proinflammatory mediator synthesis, on stored blood induced inflammation. We hypothesized that PTX would attenuate stored bloodinduced neutrophil activation and would decrease proinflammatory mediator synthesis using a whole blood preparation. Neutrophil activation was studied via the expression of a known marker of degranulation (CD66b) and by assessing MMP-9 levels, a proteolytic enzyme released by activated neutrophils. In addition, TNF-␣ levels were also measured. Whole blood was incubated with HBSS (control), LPS (100 g/mL) alone, LPS ⫹ 42-day old leukoreduced PRBC supernatant, and LPS ⫹ supernatant ⫹ PTX (2mM) at 37° C. TNF levels were measured for each group by ELISA. Active MMP9 levels were measured by zymography. Neutrophil CD66b expression was evaluated by flow cytometry in whole blood incubated with HBSS (control), fMLP (1M) alone, fMLP ⫹ supernatant, and fMLP ⫹ supernatant ⫹ PTX (2mM). LPS caused a significant increase in TNF-␣ levels when compared to control (100%). When PRBC supernatant was added, a 20% increase in TNF was seen (p⬍0.01). The addition of PTX to supernatant and whole blood caused a 106% decrease in TNF-␣ levels (p⬍0.0001). MMP9 levels were increased in all groups compared to control. PTX exerted no effects on MMP9 levels. CD66b expression was markedly increased by fMLP (100%), which was also observed after the addition of PRBC supernatant. PTX led to a 47% decrease in CD66b expression compared to fMLP⫹supernatant (p⬍0.01). Collectively, our data demonstrates that PTX selectively downregulates neutrophil CD66b expression and significantly decreases TNF-␣ levels in whole blood treated with agonists and supernatant from stored blood. Because the deleterious effects of blood transfusion on ARDS and MOF are associated with increased inflammatory mediator synthesis and neutrophil activation, the adjunctive use of PTX with blood transfusions may have therapeutic potential. 173. EXTRACELLULAR UBIQUITIN INCREASES IN HUMAN PACKED RED BLOOD CELL UNITS DURING STORAGE. Patel MB, Proctor KG, Veltman Y, Nguyen M,
Objective: Ubiquitin (Ub) is a highly conserved heat-stable 76 amino acid protein in all eukaryotic cells. It has multiple intracellular roles in protein degradation and posttranslational modification. In vitro data suggest that extracellular Ub has also pleiotropic effects on host defense mechanisms, including effects on apoptosis, cytokine secretion and growth regulation, and in vivo data indicate antiinflammatory actions. Erythrocytes contain 10-20 ng Ub/10 6 cells. Since hemolysis is known to occur with prolonged storage, we hypothesized that Ub is released over time into pRBC units and that this increase may correlate with the immunological properties of stored blood. Methods: After IRB approval volunteers (n⫽3) donated blood, which was processed by the blood bank into packed red blood cell units (pRBC). Blood units were stored at 0-6°C for 42 days. Daily aliquots were drawn and cell supernatants isolated by centrifugation at 1000 g for 20 min. Ubiquitin concentrations in the supernatants were measured by ELISA. The immunomodulatory effects of the supernatants were assessed by measuring lipopolysaccharide (LPS) evoked TNF-␣ production of normal whole blood in the presence of 33% supernatant obtained from different time points (n⫽9 per group). TNF-␣ was measured by ELISA. Data are expressed as mean ⫾ SEM and were analyzed by regression analysis, Spearman correlation (r s) and ANOVA (post hoc LSD). A 2-tailed p ⬍0.05 was considered significant. Results: Extracellular Ub linearly increased in pRBC units by 49 ⫾ 2 ng/mL per day (r 2⫽0.82, p ⬍0.001, Fig. A). PRBC supernatants time dependently inhibited LPS evoked TNF-␣ production of normal whole blood (n⫽9, Fig. B). The inhibitory effect of pRBC supernatants correlated significantly with the Ub concentration (r s⫽-0.53, p⬍ 0.001). Addition of exogenous Ub (equaling day 42 Ub concentration of 2170 ⫾ 268 ng/mL) to day 0-2 pRBC supernatants showed 31⫾15% of the inhibitory effect of day 42 supernatants on LPS evoked TNF-␣ response of normal whole blood (p ⬍0.05). Conclusions: Extracellular Ub concentrations in pRBC units linearly increase with duration of storage and may partially contribute to the immunological effects of stored blood. Furthermore, determination of Ub in pRBC units appears to be a sensitive testing technique for quality control purposes of blood transfusions and may be helpful for the assessment of novel blood preservation methods.
174. THE GAP JUNCTION PROTEIN CONNEXIN 43 IS DELIVERED TO MACROPHAGE PHAGOSOMES AND IS REQUIRED FOR EFFICIENT PHAGOCYTOSIS TO OCCUR. Anand RJ, Rippel CA, Leaphart CL, Li J, Hackam DJ; Childrens Hospital of Pittsburgh Introduction: The mechanisms governing phagocytosis by macrophages remain incompletely understood, although impaired phagocytosis may occur during advanced sepsis. Gap junctions are hexameric arrays of the protein connexin 43 (Cx43) which are localized on the cell membrane, and allow for the transfer of molecules between adjacent cells. The expression and possible role for Cx43 in macrophages remain largely unknown, although transfer of mole-
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS cules between the phagosome and the cytosol may be required for efficient phagocytosis to occur. We now hypothesize that Cx43 is expressed on macrophages, and that gap junctions participate in the process of phagocytosis. Methods: The expression of Cx43 in J774 macrophages was assessed by SDS-PAGE and laser confocal immunofluorescence using affinity purified polyclonal antibodies. Phagocytosis was determined by feeding J774 macrophages a meal of 0.8m latex particles or opsonized sheep erythrocytes (3h, 37°C), and removing the unbound particles by washing or hypotonic lysis. Intracellular particles were quantified using confocal microscopy in the x-z plane. Distribution of Cx43 around internalized particles was assessed by confocal microscopy and verified using sucrose density ultracentrifugation. Gap junction function was inhibited using oleamide (10M, 1h). Results: J774 macrophages were found to express the gap junction protein Cx43, which was localized at focal adhesions as well as on intracellular vesicles. J774 macrophages were found to bind particles to adhesion sites as well as to phagocytose them in a time and concentration dependent manner. Cx43 was found to accumulate strongly around internalized particles, and to persist over time on the phagosomal membrane. Strikingly, inhibition of gap junction function led to a significant reduction in the rate and extent of phagocytosis (control: 45⫾5% vs. olea 17⫾5%, p⬍0.05). Conclusions: These findings indicate a potential requirement for the gap junction protein connexin 43 in the regulation of phagocytosis and phagosomal maturation. Studies into the role of gap junctions on phagocytosis may provide novel therapeutic insights into diseases characterized by impaired microbial internalization, such as occurs during the late stages of sepsis. 175. ACTIVATED MACROPHAGES INHIBIT ENTEROCYTE GAP JUNCTIONS DURING INTESTINAL INFLAMMATION THROUGH THE RELEASE OF NITRIC OXIDE. R. J. Anand 1, C. A. Rippel 2, C. L. Leaphart 1, J. Li 1, D. J. Hackam 1; 1Childrens Hospital of Pittsburgh, Pittsburgh, PA, 2 University of Pittsburgh School of Medicine, Pittsburgh, PA. Introduction: Maintenance of barrier integrity requires communication between adjacent enterocytes through gap junctions composed of connexin43 (Cx43), which must be phosphorylated in order to function. Necrotizing enterocolitis (NEC) is characterized by an influx of activated macrophages and the release of nitric oxide (NO), although the mechanisms by which this leads to barrier dysfunction remain unknown. We therefore hypothesized that activated macrophages directly impair enterocyte gap junctions, and sought to define the mechanisms involved. Methods: J774 macrophages were activated (LPS 10 ng/ml ⫹ IFN␥ 100 U/ml, 12h), and NO release was assessed by Griess. Gap junction communication was assessed by fluorescence recovery after photobleaching (FRAP) ⫾ detaNONOate (50M). To simulate NEC, IEC-6 enterocytes were co-cultured with J774 on transwells, and the expression, distribution and phosphorylation of Cx43 in IEC-6 cells were measured by SDS-PAGE and immunofluorescence. Results: Activation of J774 macrophages led to the sustained release of NO (control J774: 1.3 ⫹/⫺ 0.1 M, activated J774 7.1⫹/1 1.7 M , p⬍0.05). NO significantly decreased gap junction function in IEC-6 cells (% recovery: control: 70% , NO: 35%, p⬍0.05). Under co-culture conditions, activated macrophages significantly decreased the phosphorylation of Cx43 in the adjacent enterocytes, which was not observed in the presence of non-activated macrophages, or cytokine stimulated IEC-6 cells alone. Activated macrophages also caused the rapid internalization of Cx43 within the enterocyte, providing a mechanism by which inter-enterocyte communication would be impaired. This effect was maintained when IEC-6 cells where treated with supernatants obtained from activated macrophages, suggesting that a soluble factor was involved. Strikingly, pre-incubation of J774 cells with the iNOS inhibitor L-Nil reversed the effects of activated macrophages on Cx43 phosphorylation and restored Cx43 localization to the enterocyte plasma membrane. Conclusions: Activated macrophages impair inter-
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enterocyte communication through de-phosphorylation and internalization of Cx43 via NO. These findings shed light into the pathogenesis of NEC, and identify intestinal Cx43 as a potential therapeutic target for this disease. 176. THE INFLUENCE OF ANGIOTENSIN II AND CYCLIC NUCLEOTIDE SECOND MESSENGER SIGNAL TRANSDUCTION ON ISCHEMIA-REPERFUSION-INDUCED ELEVATIONS IN MICROVASCULAR HYDRAULIC PERMEABILITY. Ramirez R, Sadjadi J, Curran B, Victorino GP; UCSF--East Bay Introduction: Intravascular volume loss from ischemia-reperfusion injury is a major clinical concern. We hypothesize that angiotensin II influences the ischemia-reperfusion-associated microvascular fluid leak through cyclic nucleotide second messenger signal transduction mechanisms. The purposes are to determine hydraulic permeability after ischemia-reperfusion of venules treated with 1) angiotensin II, 2) a cAMP synthesis inhibitor, 3) a cGMP inhibitor, and 4) simultaneous administration of angiotensin II and either a cAMP synthesis inhibitor or a cGMP synthesis inhibitor. Methods: Rat mesenteric postcapillary venules were micro-cannulated to measure hydraulic permeability (Lp). Ischemia-reperfusion was achieved by placing animals in a 5% oxygen environment and preventing venular flow followed by allowing blood flow to resume. Lp was measured after ischemia-reperfusion and treatment with 1) angiotensin II (20nM), 2) cAMP synthesis inhibitor (DDA,10uM), 3) cGMP synthesis inhibitor (LY83583, 10uM), and 4) angiotensin II plus either cAMP inhibition or cGMP inhibition, (n⫽6 in each group). Results: Compared to the 7-fold increase in Lp due to ischemia-reperfusion alone: 1) angiotensin II attenuated the 7-fold increase by 50% (p⬍0.001), 2) cAMP inhibition attenuated the 7-fold increase by 45% (p⬍0.001), 3) cGMP inhibition completed blocked any elevation in Lp due to ischemia-reperfusion (p⬍0.001), and 4) angiotensin II ⫹ cAMP inhibition was not statistically different from angiotensin II alone (p⫽0.16) while angiotensin II ⫹ cGMP inhibition attenuated the 7-fold increase in Lp due to ischemia-reperfusion by 65% (p⬍0.001). Conclusion: Treatment with angiotensin II attenuated increases in hydraulic permeability due to ischemia-reperfusion by 50%. Inhibition of cGMP synthesis completely blocked any increase in permeability due to ischemia-reperfusion while cAMP inhibition appears to play a lesser role. This emphasizes the major impact that cyclic nucleotide second messengers play in ischemia-reperfusion. A better understanding of mediators that reduce intravascular fluid loss from IR-induced microvascular dysfunction may help clinicians treat uncontrolled fluid extravasation that occurs during shock and sepsis. 177. CORRELATION OF SF-36 AND SF-12 IN A TRAUMA POPULATION. Kiely M, Weidner K, Weigelt J; Medical College of Wisconsin Background: The SF-36 is a commonly used general measure of health-related quality of life (QOL). The SF-12 is a related tool with less response burden, but its performance in a general trauma population is unknown. Hypothesis: The SF-12 would provide similar information to the SF-36 in blunt trauma patients. Patients and Methods: Adults with non-neurologic blunt injury were prospectively enrolled. Demographic, injury, social support, and socioeconomic data were collected. Patients responded to QOL, functional, PTSD, and depression questionnaires 1 and 6 months after injury. Physical (PCS) and mental (MCS) component scores of the SF-36 and SF12 were compared using Pearson’s correlation coefficient. Linear regression identified factors associated with the SF-12 and SF-36 PCS and MCS. Responsiveness to change was assessed using the standardized response mean. Results: Data from 185 patients were analyzed. Correlation of the PCS was 0.924 and MCS was 0.925 (both p⬍0.001). QOL remained below population norms at 6 months. ⴱp⬍0.05 compared to population norms, SF-12 and SF-36 PCS and MCS. PCS was moderately responsive to change, and was equivalent using either the SF-12 or SF-36. MCS was