Biomedical subjects

Iris Garcia

Publications and source records attributed to Iris Garcia.

At least 19 recordsLinked to original sources

PubMed2007

Vitamin E inhibits endotoxin-mediated transport of phosphatases to lipid rafts.

The production and release of inflammatory mediators is regulated by the coordinated activity of kinases and phosphatases. These proteins are known to regulate one another through an unknown mechanism. Previously, we have demonstrated that autocrine release of oxidants regulates macrophage activation in a similar fashion. The purpose of this study is to determine if attenuated oxidant activity by antioxidant exposure can regulate endotoxin-mediated kinase and phosphatase activity. Human promonocytic THP-1 cells were stimulated with lipopolysaccharide. Selected cells were pretreated with alpha-tocopherol succinate, LY294002, or an AKT inhibitor (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). Lipid raft and cellular protein were analyzed for lipid raft toll-like receptor 4 (TLR4) receptor formation and mitogen-activated protein kinase (MAPK) activation. Harvested supernatants were analyzed for tumor necrosis factor (TNF)-alpha production. Lipopolysaccharide stimulation led to the lipid raft mobilization of TLR4 and heat shock protein 70. This was followed by lipid raft mobilization of SH related complex homology 2 domain-containing inositol-5-phosphate (SHIP), activation of the MAPK, and production of TNF-alpha. Pretreatment with alpha-tocopherol succinate did not affect mobilization of TLR4 or heat shock protein 70, but did result in attenuated mobilization of SHIP, activation of the MAPK, and production of TNF-alpha. In addition, alpha-tocopherol succinate was associated with increased activation of the counter-regulatory kinase protein kinase B. Pretreatment with LY294002 or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate reversed the effects of alpha-tocopherol succinate. Thus, it seems that endotoxin-mediated activation requires the coordinated activity of kinases and phosphatases. Antioxidant exposure in the form of vitamin E seems to attenuate endotoxin-mediated SHIP activation resulting in increased AKT activity, and attenuated MAPK activation and TNF-alpha production.

Cell Differentiation↗

PubMed2005

Oxidative-induced calcium mobilization is dependent on annexin VI release from lipid rafts.

BACKGROUND: Oxidative stress results in macrophage reprogramming through the formation of focal adhesion-like complexes on lipid rafts. Although the cellular mechanisms responsible for this reprogramming remain unknown, oxidative stress is known to result in a transient increase in intracellular calcium. This transient flux is thought to occur through the membrane dissociation of the calcium-bound protein annexin VI. The purpose of this study is to clarify the source of the calcium, and determine if it is responsible for the formation of focal adhesion-like complexes during oxidative stress through the activation of calcium/calmodulin dependent protein kinase II (CaMK II). METHODS: THP-1 cells were stimulated with hydrogen peroxide. Selected cells were pretreated with methyl beta-cyclodextrin (MbetaCD), a cholesterol-depleting agent; 1,2-bis aminophenoxy ethane-N,N,N',N'-tetraacetic acid, an intracellular calcium chelator; or autocamtide 2-related inhibitory peptide, a CaMK II inhibitor. Intracellular calcium flux was determined by a Fluo-3 technique. Lipid raft and cellular protein were extracted and analyzed for active CaMK II, annexin VI, and components of focal adhesion-like complexes. RESULTS: Hydrogen peroxide exposure led to mobilization of annexin VI from lipid rafts to the cytosol, which was followed by an increase in cytosolic calcium, phosphorylation of CaMK II, and formation of focal adhesion-like complexes. Cholesterol depletion from lipid rafts attenuated all of these effects. 1,2-bis Aminophenoxy ethane-N,N,N',N'-tetraacetic acid and autocamtide 2-related inhibitory peptide pretreatment attenuated CaMK II phopshorylation and formation of focal ahdesionlike complexes. CONCLUSIONS: Macrophage reprogramming during oxidative stress occurs through the cytosolic mobilization of annexin VI from lipid rafts. As a result, bound calcium dissociates, resulting in the activation of CaMK II and the formation of focal adhesion-like complexes.

Annexin A6↗

PubMed2005

Calcium/calmodulin-dependent kinase II is required for platelet-activating factor priming.

Platelet-activating factor (PAF) primes the macrophage proinflammatory response to inflammatory stimuli, such as lipopolysaccharide (LPS). The cellular events responsible for this priming or reprogramming remain unresolved, but may occur through an increase in cytosolic calcium, inducing calcium/calmodulin-dependent kinase (CaMK) activation. To study this, differentiated THP-1 cells were used to study the effect of CaMK II and IV inhibition on PAF-induced reprogramming of TLR4-mediated events. LPS induced p38, ERK 1/2, and JNK/SAPK phosphorylation, NF-kappaB and AP-1 activation, and TNF-alpha and IL-10 production. PAF pretreatment selectively increased LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production. Inhibition of CaMK II prevented PAF-induced priming of these events. Inhibition of CaMK IV prevented LPS-induced ERK 1/2, JNK/SAPK, NF-kappaB and AP-1 activation, and TNF-alpha production, but increased IL-10 production with or without PAF pretreatment. Neither CaMK II nor IV inhibition had any affect on p38 activity. These data suggest that the function of CaMK II is essential for PAF-induced macrophage priming. This priming event is mediated in part by modulation of ERK 1/2, JNK/SAPK, NF-kappaB, and AP-1 activation. CaMK IV, on the other hand, is not specific for priming by PAF and appears to have a direct link in TLR4-mediated events.

Blood Platelets↗

PubMed2004

Effects of adsorbed proteins and surface chemistry on foreign body giant cell formation, tumor necrosis factor alpha release and procoagulant activity of monocytes.

The adhesion and activation of monocytes and macrophages are thought to affect the foreign body response to implanted medical devices. However, these cells interact with devices indirectly, because of the prior adsorption of proteins. Therefore, we preadsorbed several "model" biomaterial surfaces with proteins and then measured foreign body giant cell (FBGC) formation, tumor necrosis factor alpha (TNFalpha) release, and procoagulant activity. The model surfaces were tissue culture polystyrene (TCPS), untreated polystyrene (PS), and Primaria, whereas the proteins used were albumin, fibronectin, fibrinogen, and immunoglobulin. FBGC formation, TNFalpha release, and procoagulant activity of monocytes were the highest for surfaces preadsorbed with IgG. FBGC formation was lower on surfaces with adsorbed fibrinogen and fibronectin than on uncoated surfaces. TNFalpha release and procoagulant activity of monocytes were similar on surface adsorbed with fibrinogen, fibronectin, or albumin. Monocyte activation was also affected by the surface chemistry of the substrates, because FBGC formation was the highest on PS and the lowest on TCPS. Monocyte procoagulant activity was the highest on Primaria. Adsorbed proteins and surface chemistry were found to have strong effects on FBGC formation, monocyte TNFalpha release, and procoagulant activity in vitro, providing support for the idea that these same variables could affect macrophage-mediated foreign body response to biomaterials in vivo.

Adsorption↗

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

Induction of heme-oxygenase 1 inhibits endothelial cell activation by endotoxin and oxidant stress.

BACKGROUND: Increased oxidant stress has been implicated in a number of disease states, including systemic inflammation caused by ischemia/reperfusion injury or sepsis. We have demonstrated previously that oxidants enhance the proinflammatory response to endotoxin (lipopolysaccharide), although antioxidants inhibit this response. Heme-oxygenase 1 (HO-1) is an inducible, cytoprotective enzyme, which is up-regulated under conditions of oxidant stress. We hypothesized that the induction of HO-1 protein would attenuate the proinflammatory response of endothelial cells to lipopolysaccharide and oxidant stress. METHODS: Human umbilical vein endothelial cells were pretreated with hemin (100 micromol/L) for 5 hours, and the induction of HO-1 was confirmed by Western blot. After hemin exposure, cells were treated for 1 hour with either diamide, buthione sulfoximine, xanthine oxidase, or glucose oxidase to induce oxidant stress or lipopolysaccharide to induce an inflammatory response. Interleukin 8 (IL-8) and prostaglandin I(2) (PGI(2)) production were measured by enzyme-linked immunosorbent assay; p38 kinase, p42/44 extracellular regulated kinase, and c-jun N terminal kinase activation were measured by Western blot. RESULTS: HO-1 protein was increased 3-fold by exposure to hemin under all conditions. IL-8 production in response to lipopolysaccharide and xanthine oxidase was inhibited significantly by hemin exposure, although PGI(2) production was not affected. The up-regulation of HO-1 protein levels resulted in the inhibition of the lipopolysaccharide- and oxidant-induced activation of all 3 mitogen-activated protein kinases: p38 kinase, p42/44 extracellular regulated kinase, and c-jun N terminal kinase. CONCLUSIONS: The induction of HO-1 by hemin results in inhibition of the proinflammatory response of endothelial cells, as evidenced by the inhibition of IL-8 production without affecting PGI(2) production. All 3 mitogen-activated protein kinase signaling cascades are affected, which suggests that the mechanism of this effect may be proximal in the cell signaling process.

Cells, Cultured↗

PubMed2003

Mitogen-activated protein kinases in the intensive care unit: prognostic potential.

OBJECTIVE: To evaluate the prognostic significance of the activational status of p38, specifically progression to multiple organ dysfunction syndrome (MODS), in a group of severely injured trauma patients. SUMMARY BACKGROUND DATA: To date, therapeutic manipulation of the host immunoinflammatory response has not affected the outcome of patients with MODS. A major concern is the inability to identify the patient most at risk so as to enable early intervention. METHODS: Nineteen trauma patients underwent bronchoalveolar lavage (BAL). Cells obtained were plated, stimulated with lipopolysaccharide (LPS), and then harvested at varying time points after stimulation. p38 was evaluated by Western blot. RESULTS: Nineteen patients were categorized into two groups according to baseline and LPS-stimulated p38 activation in cells obtained by BAL. Group 1 demonstrated a 10-fold increase in p38 activation with LPS treatment over unstimulated controls. Group 2 had high baseline levels of p38 that were unresponsive to subsequent LPS stimulation. Both groups were similar with respect to age, gender, shock (systolic blood pressure < 90), Injury Severity Score, APACHE II, lactate levels, base deficit, blood transfusions, and the cell differential of BAL fluid. However, patients in group 2 had a greater incidence of progression to MODS as defined by the Marshall MOD score, a longer duration of mechanical ventilation, a longer stay in the intensive care unit, and a longer overall hospital stay than group 1. CONCLUSIONS: These results demonstrate the prognostic significance of p38 activation in predicting outcome in critically ill trauma patients. Furthermore, these results demonstrate that trauma populations identical by current scoring systems contain a mixture of patients with markedly different outcomes as identified by p38 activation. Measurement of p38 may enable early identification of a subgroup of patients at increased risk for MODS to permit effective therapeutic intervention.

APACHE↗

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

Randomized, prospective trial of antioxidant supplementation in critically ill surgical patients.

OBJECTIVE: To determine the effectiveness of early, routine antioxidant supplementation using alpha-tocopherol and ascorbic acid in reducing the rate of pulmonary morbidity and organ dysfunction in critically ill surgical patients. SUMMARY BACKGROUND DATA: Oxidative stress has been associated with the development of the acute respiratory distress syndrome (ARDS) and organ failure through direct tissue injury and activation of genes integral to the inflammatory response. In addition, depletion of endogenous antioxidants has been associated with an increased risk of nosocomial infections. The authors postulated that antioxidant supplementation in critically ill surgical patients may reduce the incidence of ARDS, pneumonia, and organ dysfunction. METHODS: This randomized, prospective study was conducted to compare outcomes in patients receiving antioxidant supplementation (alpha-tocopherol and ascorbate) versus those receiving standard care. The primary endpoint for analysis was pulmonary morbidity (a composite measure of ARDS and nosocomial pneumonia). Secondary endpoints included the development of multiple organ failure, duration of mechanical ventilation, length of ICU stay, and mortality. RESULTS: Five hundred ninety-five patients were enrolled and analyzed, 91% of whom were victims of trauma. The relative risk of pulmonary morbidity was 0.81 (95% confidence interval 0.60-1.1) in patients receiving antioxidant supplementation. Multiple organ failure was significantly less likely to occur in patients receiving antioxidants than in patients receiving standard care, with a relative risk of 0.43 (95% confidence interval 0.19-0.96). Patients randomized to antioxidant supplementation also had a shorter duration of mechanical ventilation and length of ICU stay. CONCLUSIONS: The early administration of antioxidant supplementation using alpha-tocopherol and ascorbic acid reduces the incidence of organ failure and shortens ICU length of stay in this cohort of critically ill surgical patients.

Adolescent↗

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↗

PubMed2002

The actin cytoskeleton: an essential component for enhanced TNFalpha production by adherent monocytes.

Monocyte adherence induces the formation of focal adhesions, the interaction sites of intracellular signaling molecules and cytoskeletal proteins such as actin. We previously demonstrated that adherence potentiates human monocyte LPS-induced TNFalpha production. Hence, we hypothesized that the actin cytoskeleton is integral to adherence-induced priming for enhanced LPS-induced TNFalpha production. In contrast to nonadherent cells, LPS induced significant transcription of TNFalpha mRNA and production of TNFalpha in adherent monocytes. Disrupting the actin cytoskeleton with cytochalasin D (CD) in adherent monocytes inhibited LPS-induced TNFalpha production by 55%, thereby abrogating adherence-induced priming. Moreover, CD pretreatment abrogated adherence-induced activation of Pyk2, a major focal adhesion kinase, and ERK 1/2, a component of the mitogen-activated protein kinase (MAPK) signaling pathway, and it completely inhibited LPS-induced ERK 1/2 activation. However, CD treatment of nonadherent monocytes failed to inhibit cytokine production. In conclusion, the actin cytoskeleton is integral in the reprogramming of the monocyte for enhanced cytokine production and in maintaining this "primed" state.

Actins↗

PubMed2002

The macrophage response to endotoxin requires platelet activating factor.

Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗