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Gender dimorphisms in endotoxin-stimulated progenitor cell function
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
p⬍0.05. Conclusion: Liposomal cDNA gene transfer is a feasible approach to affect dermal and epidermal regeneration in pigs with a full excision wound. The efficacy of liposomal gene transfer of various growth factors can now be tested in this pig wound model.
⫺1111 holds promise for manipulating this fundamental biologic inflammatory response.
469. GENDER DIMORPHISMS IN ENDOTOXIN-STIMULATED PROGENITOR CELL FUNCTION. P. Chrisostomo, M. Wang, C. Herring, E. Morrell, M. Wairiuko, D. Meldrum; Indiana University, Indianapolis, IN. Background: Progenitor cell therapy has emerged as an exciting new area of medicine and surgery. The plasticity of progenitor cells has resulted in positive remodeling and the regeneration of viable tissues. Following endotoxin (LPS) exposure, progenitor cells release various anti-inflammatory factors. Indeed, the release of growth factors may limit apoptosis and inflammation. Thus, it has been proposed that those patients with higher circulating progenitor cell counts may be more resistant to septic and traumatic insults. There are clear gender differences in response to such insults; therefore, we hypothesized that sex differences in the progenitor cell response to LPS may exist. Methods: Bone marrow progenitor cells (BMSCs) were obtained from male and female mice (c57BL). 1 million BMSCs per well (triplicate wells per group, n⫽6/group) were stressed by increasing doses of LPS (50, 100, 200 ng/ml). BMSC activation was determined by measuring VEGF production by ELISA. Differences were considered significant if P⬍0.01 (ANOVA with Bonferroni’s). Results: LPS resulted in significant activation of both male and female BMSC; however, LPS provoked significantly more VEGF production in females vs. males at all LPS doses (890⫹/⫺9 vs. 799⫹/ ⫺13; 868⫹/⫺8 vs. 790⫹/⫺12; 864⫹/⫺6 vs. 789⫹/⫺11 pg/ml at 50, 100, and 200 ng/ml LPS, respectively). Conclusions: This study constitutes the first demonstration that sex differences exist in LPS stimulated progenitor cell function. Gender differences in progenitor cell function may have important implications in understanding the observed sex differences in the host’s response to LPS. 470. NF-B ELEMENTS REGULATE TRANSCRIPTIONAL ACTIVITY WITHIN THE MURINE MYD88 PROMOTER. K. R. Wasiluk, K. A. McCulloch, K. L. Banton, D. L. Dunn; University of Minnesota, Minneapolis, MN. Background: MyD88 acts as a central proximal intracellular signaling intermediate in the innate immune response to evolutionarily conserved pathogen-associated structures recognized by several Toll-like receptors (TLRs). The interaction of lipopolysaccharide (LPS), recognized by TLR4, has been implicated in the pathophysiology of gram-negative bacterial sepsis. Such interaction is mediated by a complex pathway resulting in the activation of the transcription factor NF-B and subsequent production of inflammatory cytokines. The precise mechanisms involved have not been delineated. We hypothesized that specific regulatory elements exist within the MyD88 promoter. Methods: In earlier work, we showed that murine MyD88 promoter-luciferase constructs pGL3-Basic/MyD88⫺1310/⫺1 and pGL3-Basic/MyD88⫺1111/⫺1 have increased promoter activity, suggesting the presence of at least one positive regulatory element. In our current study, we analyzed the promoter sequence and found several putative NF- sites. We plated 4 ⫻ 105 RAW 264.7 cells in 12-well plates; 16 hours later, we added a specific NF- inhibitor, helenalin, to the cells, then transfected them with 2 g of plasmid. After obtaining cell lysates 24 hours later, we quantitated relative luciferase units (RLUs) using the luciferase assay with a TD 20/20 luminometer. Statistics were by ANOVA, with the Student’s t-test for pairwise comparison. Results: Both pGL3-Basic/MyD88-1310/-1 and pGL3-Basic/ MyD88⫺1111/⫺1 showed decreased transcriptional activity (p⬍0.05), in a dose-dependent fashion in the presence of increasing concentrations of the specific NF-B inhibitor helenalin. Conclusion: Our demonstration of the presence of at least one NF-kB transcriptional regulatory element within the murine MyD88 promoter between ⫺1310 and
471. PURINERGIC RECEPTOR EXPRESSION IS ALTERED IN EXTRA-INTESTINAL ORGANS FOLLOWING INTESTINAL ISCHEMIA/REPERFUSION INJURY. P. M. Milano, C. D. Douillet, B. L. Zarzaur, W. P. Robinson, III, S. K. Beidler, P. B. Rich; University of North Carolina at Chapel Hill, Chapel Hill, NC. Background: Intestinal ischemia/reperfusion (IIR) injury is known to initiate the systemic inflammatory response syndrome, often progressing to multiple organ failure, resulting in significant morbidity and mortality. Pro-inflammatory cytokines released following IIR injury have been implicated as mediators in the associated extraintestinal manifestations of this disease process, specifically pulmonary and renal dysfunction. Extracellular nucleotides are known to be released in response to ischemic stress, and modulate a host of proinflammatory responses via interactions with purinergic receptors. In this study, we investigate the hypothesis that purinergic receptor expression is altered in clinically relevant extra-intestinal organs following IIR injury. Methods: Adult male BalbC mice (n⫽17; wt: 30.0 ⫹/⫺ 3.3 grams) were anesthetized and randomized to receive either sham laparotomy (control, n⫽5), or 15 minutes of superior mesenteric artery occlusion. Experimental ischemia was followed by a subsequent period of reperfusion [1 minute (n⫽6) or 1 hour (n⫽6)]. The mice were then sacrificed, and lung, kidney, and intestinal tissues were harvested for analysis. Following RNA extraction, purinergic receptor mRNA expression for P2⫻7, P2Y2, P2Y4, P2Y6, A2b and A3, was analyzed with real-time RT-PCR using previously optimized intron-spanning murine primers. Receptor gene patterns were normalized as a ratio of cyclophilin A expression. Results: Significant differences in tissue purinergic receptor expression were observed in both the lungs and kidneys of mice exposed to IIR injury when compared to controls. In the lung, P2Y2 receptor expression was increased in the 1 hour IIR group when compared to control (1.687 ⫹/⫺ 0.227 vs. 1.014 ⫹/⫺ 0.116; p⬍0.05). Pulmonary A3 receptor expression was incrementally elevated following intestinal ischemia/reperfusion injury (ctrl. 1.042 ⫹/⫺ 0.213 vs. IIR 1 hr. 2.093 ⫹/⫺ 0.142 and IIR 1 min. 1.239 ⫹/⫺ 0.306 vs. IIR 1 hr.; p⬍0.05). In the kidney, P2Y2 receptor expression was increased in the 1 hour IIR group compared to both 1 minute IIR and control (IIR 1 hr. 1.845 ⫹/⫺ 0.333 vs. IIR 1 min. 0.911 ⫹/⫺ 0.137 and vs. ctrl. 0.717 ⫹/⫺ 0.276; p⬍0.05). A3 receptor expression in the kidney was decreased in the 1 hour IIR group compared to the 1 minute IIR group (0.496 ⫹/⫺ 0.121 vs. 1.436 ⫹/⫺ 0.261; p⬍0.05). No significant changes were noted in the intestinal tissue purinergic receptor profiles. Conclusions: Purinergic receptor expression patterns are altered in the lung and kidney following intestinal ischemia/reperfusion injury. Intestinal purinergic receptor expression patterns are unchanged following experimental
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
p⬍0.05. Conclusion: Liposomal cDNA gene transfer is a feasible approach to affect dermal and epidermal regeneration in pigs with a full excision wound. The efficacy of liposomal gene transfer of various growth factors can now be tested in this pig wound model.
⫺1111 holds promise for manipulating this fundamental biologic inflammatory response.
469. GENDER DIMORPHISMS IN ENDOTOXIN-STIMULATED PROGENITOR CELL FUNCTION. P. Chrisostomo, M. Wang, C. Herring, E. Morrell, M. Wairiuko, D. Meldrum; Indiana University, Indianapolis, IN. Background: Progenitor cell therapy has emerged as an exciting new area of medicine and surgery. The plasticity of progenitor cells has resulted in positive remodeling and the regeneration of viable tissues. Following endotoxin (LPS) exposure, progenitor cells release various anti-inflammatory factors. Indeed, the release of growth factors may limit apoptosis and inflammation. Thus, it has been proposed that those patients with higher circulating progenitor cell counts may be more resistant to septic and traumatic insults. There are clear gender differences in response to such insults; therefore, we hypothesized that sex differences in the progenitor cell response to LPS may exist. Methods: Bone marrow progenitor cells (BMSCs) were obtained from male and female mice (c57BL). 1 million BMSCs per well (triplicate wells per group, n⫽6/group) were stressed by increasing doses of LPS (50, 100, 200 ng/ml). BMSC activation was determined by measuring VEGF production by ELISA. Differences were considered significant if P⬍0.01 (ANOVA with Bonferroni’s). Results: LPS resulted in significant activation of both male and female BMSC; however, LPS provoked significantly more VEGF production in females vs. males at all LPS doses (890⫹/⫺9 vs. 799⫹/ ⫺13; 868⫹/⫺8 vs. 790⫹/⫺12; 864⫹/⫺6 vs. 789⫹/⫺11 pg/ml at 50, 100, and 200 ng/ml LPS, respectively). Conclusions: This study constitutes the first demonstration that sex differences exist in LPS stimulated progenitor cell function. Gender differences in progenitor cell function may have important implications in understanding the observed sex differences in the host’s response to LPS. 470. NF-B ELEMENTS REGULATE TRANSCRIPTIONAL ACTIVITY WITHIN THE MURINE MYD88 PROMOTER. K. R. Wasiluk, K. A. McCulloch, K. L. Banton, D. L. Dunn; University of Minnesota, Minneapolis, MN. Background: MyD88 acts as a central proximal intracellular signaling intermediate in the innate immune response to evolutionarily conserved pathogen-associated structures recognized by several Toll-like receptors (TLRs). The interaction of lipopolysaccharide (LPS), recognized by TLR4, has been implicated in the pathophysiology of gram-negative bacterial sepsis. Such interaction is mediated by a complex pathway resulting in the activation of the transcription factor NF-B and subsequent production of inflammatory cytokines. The precise mechanisms involved have not been delineated. We hypothesized that specific regulatory elements exist within the MyD88 promoter. Methods: In earlier work, we showed that murine MyD88 promoter-luciferase constructs pGL3-Basic/MyD88⫺1310/⫺1 and pGL3-Basic/MyD88⫺1111/⫺1 have increased promoter activity, suggesting the presence of at least one positive regulatory element. In our current study, we analyzed the promoter sequence and found several putative NF- sites. We plated 4 ⫻ 105 RAW 264.7 cells in 12-well plates; 16 hours later, we added a specific NF- inhibitor, helenalin, to the cells, then transfected them with 2 g of plasmid. After obtaining cell lysates 24 hours later, we quantitated relative luciferase units (RLUs) using the luciferase assay with a TD 20/20 luminometer. Statistics were by ANOVA, with the Student’s t-test for pairwise comparison. Results: Both pGL3-Basic/MyD88-1310/-1 and pGL3-Basic/ MyD88⫺1111/⫺1 showed decreased transcriptional activity (p⬍0.05), in a dose-dependent fashion in the presence of increasing concentrations of the specific NF-B inhibitor helenalin. Conclusion: Our demonstration of the presence of at least one NF-kB transcriptional regulatory element within the murine MyD88 promoter between ⫺1310 and
471. PURINERGIC RECEPTOR EXPRESSION IS ALTERED IN EXTRA-INTESTINAL ORGANS FOLLOWING INTESTINAL ISCHEMIA/REPERFUSION INJURY. P. M. Milano, C. D. Douillet, B. L. Zarzaur, W. P. Robinson, III, S. K. Beidler, P. B. Rich; University of North Carolina at Chapel Hill, Chapel Hill, NC. Background: Intestinal ischemia/reperfusion (IIR) injury is known to initiate the systemic inflammatory response syndrome, often progressing to multiple organ failure, resulting in significant morbidity and mortality. Pro-inflammatory cytokines released following IIR injury have been implicated as mediators in the associated extraintestinal manifestations of this disease process, specifically pulmonary and renal dysfunction. Extracellular nucleotides are known to be released in response to ischemic stress, and modulate a host of proinflammatory responses via interactions with purinergic receptors. In this study, we investigate the hypothesis that purinergic receptor expression is altered in clinically relevant extra-intestinal organs following IIR injury. Methods: Adult male BalbC mice (n⫽17; wt: 30.0 ⫹/⫺ 3.3 grams) were anesthetized and randomized to receive either sham laparotomy (control, n⫽5), or 15 minutes of superior mesenteric artery occlusion. Experimental ischemia was followed by a subsequent period of reperfusion [1 minute (n⫽6) or 1 hour (n⫽6)]. The mice were then sacrificed, and lung, kidney, and intestinal tissues were harvested for analysis. Following RNA extraction, purinergic receptor mRNA expression for P2⫻7, P2Y2, P2Y4, P2Y6, A2b and A3, was analyzed with real-time RT-PCR using previously optimized intron-spanning murine primers. Receptor gene patterns were normalized as a ratio of cyclophilin A expression. Results: Significant differences in tissue purinergic receptor expression were observed in both the lungs and kidneys of mice exposed to IIR injury when compared to controls. In the lung, P2Y2 receptor expression was increased in the 1 hour IIR group when compared to control (1.687 ⫹/⫺ 0.227 vs. 1.014 ⫹/⫺ 0.116; p⬍0.05). Pulmonary A3 receptor expression was incrementally elevated following intestinal ischemia/reperfusion injury (ctrl. 1.042 ⫹/⫺ 0.213 vs. IIR 1 hr. 2.093 ⫹/⫺ 0.142 and IIR 1 min. 1.239 ⫹/⫺ 0.306 vs. IIR 1 hr.; p⬍0.05). In the kidney, P2Y2 receptor expression was increased in the 1 hour IIR group compared to both 1 minute IIR and control (IIR 1 hr. 1.845 ⫹/⫺ 0.333 vs. IIR 1 min. 0.911 ⫹/⫺ 0.137 and vs. ctrl. 0.717 ⫹/⫺ 0.276; p⬍0.05). A3 receptor expression in the kidney was decreased in the 1 hour IIR group compared to the 1 minute IIR group (0.496 ⫹/⫺ 0.121 vs. 1.436 ⫹/⫺ 0.261; p⬍0.05). No significant changes were noted in the intestinal tissue purinergic receptor profiles. Conclusions: Purinergic receptor expression patterns are altered in the lung and kidney following intestinal ischemia/reperfusion injury. Intestinal purinergic receptor expression patterns are unchanged following experimental