Biomedical subjects

David Gourlay

Publications and source records attributed to David Gourlay.

10 recordsLinked to original sources

PubMed2005

Pelvic angiography for recurrent traumatic pelvic arterial hemorrhage.

BACKGROUND: Angiography is the gold standard for the diagnosis and treatment of pelvic arterial hemorrhage associated with pelvic fractures. In most cases, a single angiogram with embolization is adequate to control pelvic arterial hemorrhage. However, a small subset of patients, require repeat pelvic angiogram to evaluate and treat recurrent hemorrhage. This study seeks to define this population and determine clinical predictors of recurrent hemorrhage. METHODS: We conducted a retrospective case control study comparing patients with traumatic pelvic fracture undergoing repeated pelvic angiogram versus a single angiogram between the years 1995 and 2000. Stepwise logistic regression was used to identify the independent predictors of recurrent hemorrhage. RESULTS: In the years studied, 556 patients underwent a pelvic angiogram to evaluate for pelvic arterial hemorrhage associated with pelvic fractures. Among these, 42 (7.5%) patients underwent a second angiogram for suspected recurrent hemorrhage. In comparison to the initial angiogram, the source of bleeding on the repeat angiogram occurred at a new bleeding site in 68%, at a previously embolized site in 18%, and both in 14%. Significant risk factors for recurrent pelvic arterial hemorrhage included hypotension or transfusion of >2 U of blood per hour before the initial angiogram, pubic symphysis widening, and more than two injured arteries requiring embolization (p < 0.05). Of these, more than two injured arteries requiring embolization (odds ratio, 16.0; 95% confidence interval, 2.9-88) and transfusion of >2 U of blood per hour (odds ratio, 6.9; 95% confidence interval,1.9-25) were independent predictors of recurrent hemorrhage. CONCLUSION: Angiographic control of traumatic pelvic arterial hemorrhage is highly successful. However, recurrent pelvic arterial hemorrhage does occur. We identified a subgroup of patients with pelvic fractures who are at increased risk of recurrent pelvic arterial hemorrhage and should be considered for early repeat angiography for signs of ongoing hemorrhage.

Acetabulum↗

PubMed2004

Implications of proteasome inhibition: an enhanced macrophage phenotype.

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.

Cell Line↗

PubMed2004

Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation.

Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections. Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury. Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role. Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1-100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells. Within the sense group, 1-1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-kappaB and AP-1. This activation was associated with proinflammatory cytokine production and cellular spreading. Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group. Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group. Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS. These data suggest that redundant pathways exist that are functional at higher concentrations of LPS. Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.

Blotting, Western↗

PubMed2003

Modulation of endotoxin-induced endothelial activity by microtubule depolymerization.

BACKGROUND: Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules. The potential role of this polymerization event, however, is unknown. METHODS: Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine. Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay. Finally, neutrophil adhesion was assayed fluorometrically under the various conditions. RESULTS: Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion. Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB. This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression. As a result, microtubule disruption led to a significant reduction in neutrophil adhesion. CONCLUSION: Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1. Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.

Adaptor Protein Complex 1↗

PubMed2003

Endotoxin-induced endothelial cell proinflammatory phenotypic differentiation requires stress fiber polymerization.

Endotoxin-induced intercellular adhesion molecule-1 (ICAM-1) and interleukin 8 (IL-8) production in endothelial cells, which is mediated by Toll-receptor signaling, is essential for optimal neutrophil recruitment and migration during sepsis. Endotoxin also causes stress fiber polymerization that has recently been shown to affect intracellular signaling. However, the role of this polymerization process on endothelial-induced neutrophil adhesion and migration is unknown. Human umbilical vein endothelial cells (HUVEC) were stimulated with lipopolysaccharide (LPS). Selected cells were pretreated with cytochalasin D (CD) or lactrunculin A (LA), agents that disrupt actin polymerization. Cellular protein was extracted and analyzed by Westem blot for the phosphorylated form of IL-1-associated kinase (IRAK) and production of ICAM-1. Extracted nuclear protein was analyzed by Western blot and electrophoretic mobility shift assay (EMSA) for nuclear translocation and activity of NF-kappaB. IL-8 production was determined by enzyme-linked immunoabsorbant assay (ELISA). Neutrophil adhesion was assayed fluorometrically using calcein-AM-labeled neutrophils on treated endothelial cells. LPS treatment led to phosphorylation of IRAK, and subsequent NF-kappaB translocation and activation. This cellular signaling was followed by ICAM-1 expression and IL-8 production. Pretreatment of cells with CD or LA led to a significant inhibition of IRAK phosphorylation, and NF-kappaB nuclear translocation and activation. Actin depolymerization also significantly inhibited LPS-induced ICAM-1 and IL-8 production. HUVEC pretreated with CD or LA demonstrated significant inhibition of LPS-induced neutrophil adhesion. Endotoxin-induced actin polymerization is essential for optimal intracellular signaling through IRAK and NF-kappaB. Failure of these signaling events is associated with a marked reduction in adhesion molecule production, IL-8 production, and neutrophil adhesion. These findings support the necessity of stress fiber polymerization for optimal recruitment of neutrophils during sepsis.

Actins↗

PubMed2003

Platelet-activating factor acetylhydrolase inhibits alveolar macrophage activation in vivo.

Platelet-activating factor (PAF) is an important proinflammatory mediator of septic shock. PAF is produced by activated macrophages and acts to perpetuate the response to endotoxin. PAF is metabolized by an endogenous PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of postinjury multiple organ failure. We have previously shown that PAF-AH significantly inhibits macrophage activation by lipopolysaccharide (LPS) in vitro. The purpose of these studies was to determine whether this effect would translate to an in vivo model of remote lung injury. Wistar rats were administered a single intravenous dose of PAF-AH (5 mg/kg) or its carrier solution simultaneous with the induction of zymosan peritonitis. After 24 h, alveolar macrophages were obtained by bronchoalveolar lavage and stimulated in vitro with LPS (1 microg/mL). Supernatants were collected at 18 h for cytokine production and cellular and nuclear protein extractions were performed at 30 and 60 min to assess the activation of p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases and the nuclear translocation of nuclear factor (NF)-kappaB. Administration of PAF-AH significantly inhibited LPS-induced tumor necrosis factor alpha and interleukin-1beta production by alveolar macrophages from zymosan-treated animals. This functional inhibition was associated with inhibition of ERK 1/2 kinase and NF-kappaB activation but not p38 kinase activation. Interleukin 6 production was depressed in the macrophages from zymosan-treated animals but no additional inhibition resulted from PAF-AH treatment. In conclusion, zymosan peritonitis leads to priming of alveolar macrophages such that their subsequent tumor necrosis factor alpha response to LPS is enhanced. In vivo administration of PAF-AH abrogates this response, suggesting that this priming may be PAF dependent. This effect of PAF-AH may be mediated by the inhibition of intracellular signaling via inhibition of ERK kinase and NF-kappaB activation.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2003

Modulation of endotoxin-induced endothelial function by calcium/calmodulin-dependent protein kinase.

Endothelial cells facilitate sepsis-induced neutrophil adherence through the production of adhesion molecules and proinflammatory cytokines. The production of these factors requires coordinated intracellular inflammatory signaling. Recently, patients prone to sepsis-induced complications have been shown to have derangements in intracellular calcium and potentially calcium/calmodulin-dependent protein kinase (CaMK) activity, but the impact of these impairments is unknown. Human umbilical vein endothelial vein endothelial cells (HUVECs) were exposed to lipopolysaccharide (LPS) for various periods of time. Select HUVECs were pretreated with an inhibitor of CaMK II, KN62. Total cellular and nuclear proteins were extracted and analyzed for various components of the Toll-mediated signal cascade. Neutrophil adhesion was assayed fluorometrically using calcein-labeled neutrophils on treated HUVECs. LPS stimulation led to mitogen-activated protein kinase activation and translocation of activator protein-1 (AP-1) and nuclear factor (NF)-kappaB. CaMK blockade inhibited LPS induced ERK 1/2 and JNK but enhanced p38 activity. This selective MAPK inhibition was associated with a reduction in AP-1 activity, with no affect on NF-kappaB activity. Associated with this altered cell signaling was increased ICAM-1 production and enhanced neutrophil adhesion. Altered CaMK activity resulted in dysregulated mitogen-activated protein kinase signaling, demonstrated by reduced ERK 1/2 and JNK activity but enhanced p38 activity. This altered signaling is associated with reduced AP-1 activation and unaffected NF-kappaB activation. Neutrophil adhesion, however, is enhanced presumably through increased ICAM-1 production. Therefore, CaMK inhibition of endothelial cells, characteristic of sustained increases in intracellular calcium, appears to result in a dysregulated proadhesive phenotype.

Blotting, Western↗

PubMed2002

Hypertonic preconditioning inhibits macrophage responsiveness to endotoxin.

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40-100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.

Animals↗

PubMed2002

Platelet-activating factor priming of inflammatory cell activity requires cellular adherence.

BACKGROUND: Platelet-activating factor (PAF) primes tissue-fixed inflammatory cells, but has no effect on circulating cells. Adherence of inflammatory cells leads to cytoskeletal reorganization, which is essential for optimal inflammatory function. The purpose of this study was to investigate whether cellular adherence plays a role in PAF priming of inflammatory cells. METHODS: Differentiated THP-1 cells were maintained under adherent and nonadherent conditions. Selected cells were pretreated with PAF, followed by endotoxin stimulation. Cellular and nuclear proteins were analyzed by Western blot for components of the Toll-like receptor-mediated signaling cascade. Cytokine analysis was performed by enzyme-linked immunosorbent assay. RESULTS: Endotoxin led to activation of interleukin (IL)-1-associated kinase, extracellular signal-regulated kinase 1/2 and p38, and nuclear translocation of nuclear factor-kappaB, all of which were significantly enhanced by previous cellular adherence. PAF led to priming only under adherent conditions, demonstrated by increased IL-1-associated kinase and extracellular signal-regulated kinase 1/2 activity; nuclear factor-kappaB translocation; and IL-6, IL-8, and tumor necrosis factor-alpha production over non-PAF-treated cells. PAF had no significant effect on p38 activity or IL-10 production under any condition. CONCLUSIONS: PAF primes mononuclear cells by increasing Toll-mediated signaling only under adherent conditions. This, therefore, would limit PAF-induced priming in vivo to foci of stimulated adherent inflammatory cells with little effect systemically on circulating cells.

Calcium↗

PubMed2002

Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness.

BACKGROUND: This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype. METHODS: Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Student's t tests. RESULTS: Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA. CONCLUSION: Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.

Analysis of Variance↗