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Biomedical subjects

Thomas P Shanley

Publications and source records attributed to Thomas P Shanley.

At least 19 recordsLinked to original sources
PubMed2006

Extracellular heat shock protein-70 induces endotoxin tolerance in THP-1 cells.

Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-kappaB activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 microg/ml HSP-70) for 18 h, followed by LPS stimulation (1 microg/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-kappaB-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP-70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100 degrees C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor kappaB-alpha and inhibited activation of inhibitor kappaB kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-kappaB following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.

Antibodies, Blocking↗
PubMed2006

Approach to management of malignant hyperthermia-like syndrome in pediatric diabetes mellitus.

BACKGROUND: Hyperglycemic hyperosmolar nonketotic syndrome (HHNS) is usually associated with type 2 diabetes mellitus and is rare in children. However, a fatal malignant hyperthermia-like syndrome (MHLS) with rhabdomyolysis associated with new-onset diabetes mellitus and HHNS in adolescents has been described. DESIGN/METHODS: Case series. RESULTS: A 16-yr-old obese male (case A) and a 10-yr-old mid-pubertal nonobese female (case B) presented within a 6-month period with emesis, altered mental status, blood glucose >1600 mg/dL, and laboratory evidence of rhabdomyolysis. Case A developed fever after initiation of insulin therapy, along with refractory hypotension and multiorgan failure. He died 14 hrs after admission. Case B developed fever before insulin therapy, was treated with dantrolene, and made a full recovery. Metabolic workup showed evidence of short-chain acyl-CoA dehydrogenase (SCAD) deficiency. CONCLUSIONS: We report two cases of malignant hyperthermia-like syndrome associated with HHNS in adolescents. Their respective fluid management and clinical courses are described. Dantrolene therapy should be initiated immediately after this syndrome is recognized. We believe it is unlikely insulin is the sole trigger for MHLS. Case B is unique in that there was evidence of SCAD deficiency, a metabolic defect that we propose could lead to MHLS. We recommend that all patients with HHNS and MHLS be evaluated for an underlying metabolic disorder.

Adolescent↗
PubMed2006

Characteristics and outcomes of interhospital transfers from level II to level I pediatric intensive care units.

OBJECTIVE: To examine the characteristics, resource utilization, and outcomes for transfer admissions from level II to level I pediatric intensive care units (PICUs). DESIGN: Retrospective study. SETTING: A 16-bed level I PICU in a tertiary care children's hospital. PATIENTS: All transfer admissions from level II PICUs from January 1, 1997, through December 31, 2003; admissions for cardiac surgery were excluded. Patient characteristics, resource utilization, and outcomes were described and then compared across predefined strata (low <5%, moderate 5-30%, and high >30%) of predicted probability of death. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 168 transfer admissions, 45%, 30%, and 25% were in the low, moderate, and high mortality risk groups, respectively. Length of stay at the referring PICU was shortest for the high-risk admissions. The most frequent diagnoses among all risk groups were respiratory failure (49%) and sepsis (14%). High-risk admissions were more likely to receive advanced therapies such as extracorporeal membrane oxygenation (41.5% high risk vs. 39.2% moderate vs. 6.6% low risk, p < .01) and renal replacement therapy (34.2% vs. 17.7% vs. 2.6%, p < .01). The high-risk admissions had longer PICU length of stay and the highest death rates (34% vs. 10% vs. 4%, p < .01) when compared with the moderate- and low-risk admissions, respectively. CONCLUSIONS: This study highlights significant differences in patient characteristics, resource utilization, and outcomes across mortality risk-stratified groups of critically ill and injured children transferred from level II to level I PICU care. Further studies are warranted to investigate decision making that prompt inter-PICU transfers.

Female↗
PubMed2005

Heat shock-mediated regulation of MKP-1.

Heat shock modulates cellular proinflammatory responses, and we have been interested in elucidating the mechanisms that govern this modulation. The dual specific phosphatase, MAP kinase phosphatase-1 (MKP-1), is an important modulator of cellular inflammatory responses, and we recently reported that heat shock increases expression of MKP-1. Herein we sought to elucidate the mechanisms by which heat shock modulates MKP-1 gene expression. Subjecting RAW264.7 macrophages to heat shock increased MKP-1 gene expression in a time-dependent manner. Transfection with a wild-type murine MKP-1 promoter luciferase reporter plasmid demonstrated that heat shock activates the MKP-1 promoter. When the reporter plasmid was transfected into heat shock factor-1 (HSF-1)-null fibroblasts, the MKP-1 promoter was activated in response to heat shock in a manner similar to that of wild-type fibroblasts with intact HSF-1. Site-directed mutagenesis of two potential heat shock elements in the MKP-1 promoter demonstrated that both sites are required for basal promoter activity. mRNA stability assays demonstrated that heat shock increased MKP-1 mRNA stability compared with cells maintained at 37 degrees C. Inhibition of p38 MAP kinase activity inhibited heat shock-mediated expression of MKP-1. These data demonstrate that heat shock regulates MKP-1 gene expression at both the transcriptional and posttranscriptional levels. Transcriptional mechanisms are HSF-1 independent but are dependent on putative heat shock elements in the MKP-1 promoter. Posttranscriptional mechanisms involve increased stability of MKP-1 mRNA that is partially dependent on p38 MAP kinase activity. These data demonstrate another potential mechanism by which heat shock can modulate inflammation-related signal transduction.

Animals↗
PubMed2005

Contribution of MKP-1 regulation of p38 to endotoxin tolerance.

Endotoxin tolerance has been characterized as diminished TNF-alpha expression after a second LPS stimulus and is dependent on new protein synthesis. LPS-induced expression of TNF-alpha is partly regulated by the p38 mitogen-activated protein (MAP) kinase, which post-transcriptionally stabilizes TNF-alpha mRNA. The dual-specific phosphatase, MKP-1, has been shown to negatively regulate p38 via dephosphorylation. We hypothesized that MKP-1 expression induced during tolerance regulates TNF-alpha expression by inhibiting p38 activity. To test this hypothesis, tolerance was induced in THP-1 cells, and naive or tolerized cells were rechallenged 18 h later with LPS (1 microg/mL) and TNF-alpha production was measured. Under similar conditions, nuclear proteins were isolated after LPS stimulation and were analyzed for phospho-p38 and MKP-1 by Western blot. Transient overexpression of MKP-1 was achieved using an adenoviral expression strategy and infected cells subsequently treated with LPS for TNF-alpha production and p38 activation. Results showed that LPS tolerance was induced as reflected by decreased TNF-alpha. Induction of LPS hyporesponsiveness could be mimicked by overexpression of MKP-1 but not beta-gal. MKP-1 expression was noted only in LPS-tolerized or Ad-MKP-1 infected cells. In the canonical and Ad-MKP-1-mediated tolerance models, decreased phospho-p38 activity was observed. MKP-1s role in mediating endotoxin tolerance was further confirmed by demonstrating the inability to fully tolerize peritoneal macrophages isolated from MKP-1 null mutant (vs. wild type) mice (24% vs. 72% reductions, respectively). These data demonstrate that the dual specific phosphatase MKP-1 is an important mediator of endotoxin tolerance via p38 regulation.

Adenoviridae↗
PubMed2004

Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo.

Regulation of pulmonary inflammation involves an intricate balance of both pro- and anti-inflammatory mediators. Acute lung injury can result from direct pulmonary insults that activate alveolar macrophages to respond with increased cytokine expression. Such cytokine gene expression is mediated in part via NF-kappaB. IL-10 has been previously identified as an important endogenous anti-inflammatory cytokine in vivo on the basis of inhibiting NF-kappaB activation; however, the mechanism of this inhibition remains incompletely defined. We hypothesized that IL-10 regulated NF-kappaB activation in vivo via IkappaK inhibition. A bitransgenic mouse that allowed for externally regulated, lung-specific human IL-10 overexpression was generated. In the bitransgenic mice, introduction of doxycycline induced lung-specific, human IL-10 overexpression. Acute induction of IL-10 resulted in significant decreases in bronchoalveolar lavage fluid neutrophils (48%, P = 0.03) and TNF (62%, P < 0.01) following intratracheal LPS compared with bitransgenic negative mice. In vitro kinase assays showed this decrease to correlate to diminished lung IkappaK activity. Furthermore, we also examined the effect of chronic IL-10 overexpression in these transgenic mice. Results show that IL-10 overexpression in lungs of mature mice increased the number of intrapulmonary cells the phenotype of which was skewed toward increased B220+/CD45+ B cells and CD4+ T cells and was associated with increased CC chemokine expression. Thus regulated, lung-specific IL-10 overexpression may have a variety of complex immunologic effects depending on the timing and duration of expression.

Animals↗
PubMed2004

Heat shock inhibition of lipopolysaccharide-mediated tumor necrosis factor expression is associated with nuclear induction of MKP-1 and inhibition of mitogen-activated protein kinase activation.

OBJECTIVE: Application of heat shock before an inflammatory stimulus often results in an attenuated response to that stimulus. As a result, it has become increasingly appreciated that heat shock may induce cross-tolerance to a variety of stimuli based on in vitro and in vivo models. Circulating peripheral blood monocytes are key mediators of cytokine release following endotoxin challenge. The mitogen-activated protein kinases play a key role in the transcriptional regulation of this response including expression of tumor necrosis factor. As such, counterregulatory phosphatases that target mitogen-activated protein kinase may play a role in this heat shock-mediated effect. We hypothesized that prior heat shock to monocytes would induce a phosphatase, MKP-1, that regulated mitogen-activated protein kinase activity and subsequently conferred cross-tolerance to lipopolysaccharide stimulation. DESIGN: Experimental. SETTING: University research foundation laboratory. SUBJECTS: THP-1 human monocyte cell line. INTERVENTIONS: THP-1 cells were exposed to either heat shock (43 degrees C, 1 hr) or normothermia (37 degrees C, 1 hr) and allowed to recover before stimulation with endotoxin (lipopolysaccharide). MEASUREMENTS AND MAIN RESULTS: Induction of a heat shock response was determined by heat shock protein-70 expression. Tumor necrosis factor and interleukin-10 were measured by enzyme-linked immunosorbent assay to assess heat shock inhibition of lipopolysaccharide-induced gene expression. The effect of heat shock on lipopolysaccharide-mediated activation of the p38 and ERK kinases was examined by measuring phospho-specific isoforms of p38 and ERK1/2 and correlated to in vitro kinase activity. Confirmatory data were generated from experiments employing either pharmacologic inhibition or genetic deletion of MKP-1. Heat shock induced the nuclear localized phosphatase, MKP-1, that attenuated p38 and ERK kinase activity resulting in significantly diminished tumor necrosis factor expression in response to lipopolysaccharide. CONCLUSIONS: The effect of heat shock on decreasing the tumor necrosis factor response to lipopolysaccharide is conferred by induction of MKP-1, which negatively regulates p38 and ERK kinases. Modulation of phosphatase activity may be a potential strategy for attenuating acute inflammatory responses.

Animals↗
PubMed2003

Short-term modulation of interleukin-1beta signaling by hyperoxia: uncoupling of IkappaB kinase activation and NF-kappaB-dependent gene expression.

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-alpha (TNF-alpha)-mediated activation of NF-kappaB and NF-kappaB-dependent gene expression by a mechanism involving increased activation of IkappaB kinase (IKK). Because the signal transduction mechanisms induced by IL-1beta are distinct to that of TNF-alpha, herein we sought to determine whether hyperoxia modulates IL-1beta-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1beta caused increased activation of IKK, prolonged the degradation of IkappaBalpha, and prolonged the nuclear translocation and DNA binding of NF-kappaB compared with cells treated with IL-1beta alone in room air. Hyperoxia did not affect IL-1beta-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-beta alone. In contrast to the effects on the IKK/IkappaBalpha/NF-kappaB pathway, simultaneous treatment with hyperoxia and IL-1beta did not augment NF-kappaB-dependent gene expression compared with treatment with IL-1beta alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1beta caused hyperphosphorlyation of the NF-kappaB p65 subunit compared with treatment with IL-1beta alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1beta augments activation of IKK, prolongs degradation of IkappaBalpha, and prolongs nuclear translocation and DNA binding of NF-kappaB. This activation, however, is not coupled to increased expression of NF-kappaB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.

Cell Line↗
PubMed2003

Combined steroid treatment for congenital heart surgery improves oxygen delivery and reduces postbypass inflammatory mediator expression.

BACKGROUND: Steroid administration during cardiopulmonary bypass is thought to improve cardiopulmonary function by modulating bypass-related inflammation. This study was designed to compare preoperative and intraoperative methylprednisolone (MP) to intraoperative MP alone with respect to postbypass inflammation and clinical outcome. METHODS AND RESULTS: Twenty-nine pediatric patients undergoing bypass procedures were randomly assigned to receive preoperative and intraoperative MP (30 mg/kg 4 hours before bypass and in bypass prime, n=14) or intraoperative MP only (30 mg/kg, n=15). Myocardial inflammatory mediator mRNA expression was determined in paired atrial biopsies (before and after bypass) by ribonuclease protection. Before and after bypass, serum IL-6 and IL-10 were measured by ELISA. Postoperative outcome was assessed by intubation time, CICU length of stay, fluid balance, arterio-venous O2 difference (DeltaA-VO2), and inotrope requirements. Compared with intraoperative MP alone, combined preoperative and intraoperative MP was associated with reduced myocardial mRNA expression for IL-6, MCP-1, and ICAM-1 both before and after bypass (P<0.05). Patients who received combined steroids had lower serum IL-6 and increased IL-10 at end-bypass (P<0.05), although differences were negligible by 24 hours. Combined MP treatment was associated with reduced fluid requirements, lower body temperature, and lower DeltaA-VO2 for the first 24 hours after surgery (P<0.05), along with trends toward improvement in other clinical outcomes. CONCLUSIONS: Compared with intraoperative steroid treatment, combined preoperative and intraoperative steroid administration attenuates inflammatory mediator expression more effectively and is associated with improved indexes of O2 delivery in the first 24 hours after congenital heart surgery. These findings need to be confirmed in a larger multicenter trial.

Body Temperature↗
PubMed2003

Molecular genetics in the pediatric intensive care unit.

Molecular genetics and genomics have become highly relevant tools in the field of pediatric critical care medicine. The most immediate examples involve the use of molecular genetics as routine diagnostic tests (eg, detection of herpes simplex virus by PCR). Perhaps the most exciting and promising examples involve the research efforts based in these fields, which hold the potential to gain novel insight regarding the complex mechanisms involved in critical illness. Ultimately, the generation of more comprehensive and fundamental knowledge can lead to the design of more effective and specific therapeutic strategies.

Critical Care↗
PubMed2002

Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages.

We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB-dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells.

Animals↗
PubMed2002

Heat shock inhibits activation of NF-kappaB in the absence of heat shock factor-1.

The heat shock response is known to inhibit NF-kappaB activation and NF-kappaB-dependent gene expression. Herein we determined if cells lacking heat shock factor-1 (HSF-1), the major transcription factor regulating heat shock protein gene expression, have an altered ability to modulate NF-kappaB activation. Embryonic fibroblasts from HSF-1-null mutant mice (HSF-1-/- cells) had a drastically reduced ability to express heat shock protein-70 in response to heat shock, compared to embryonic fibroblasts from wild-type mice (HSF+/+ cells). There was no difference, however, in the ability of heat shock to inhibit TNFalpha-mediated NF-kappaB activation, IkappaBalpha degradation, IkappaB kinase activation, and macrophage chemotactic protein-1 expression in the HSF-1-/- cells compared to the HSF-1+/+ cells. These data demonstrate that heat shock inhibits activation of the NF-kappaB/IkappaBalpha pathway and NF-kappaB-dependent gene expression in the absence of an intact heat shock response.

Animals↗
PubMed2002

Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages.

Previous studies demonstrated important interactions between the heat shock response and the IkappaBalpha/NF-kappaB pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IkappaBalpha in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IkappaBalpha degradation, and the proximal mechanisms by which heat shock inhibits degradation of IkappaBalpha in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IkappaBalpha degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-kappaB. Beyond these recovery periods, heat shock did not inhibit IkappaBalpha degradation. IkappaB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IkappaBalpha and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IkappaBalpha is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IkappaBalpha involves dual modulation of IKK and intracellular phosphatase activity.

Animals↗
PubMed2002

Inhaled nitric oxide increases endothelin-1 levels: a potential cause of rebound pulmonary hypertension.

OBJECTIVE: Inhaled nitric oxide (iNO) is front-line therapy for pulmonary hypertension after repair of congenital heart disease. However, little clinical data exists regarding the effects of iNO on regulators of pulmonary vascular resistance. An imbalance between primary vasodilators, such as NO, and vasoconstrictors, such as endothelin-1 (ET-1), has been implicated in rebound pulmonary hypertension upon iNO withdrawal. The objective of this study was to determine whether iNO therapy alters plasma ET-1 levels. DESIGN: This is a prospective study involving pediatric and adult patients at risk for pulmonary hypertension. SETTING: Pediatric patients were in the cardiac intensive care unit and adult patients were in a tertiary-care hospital. PATIENTS: Group 1 included children with congenital heart disease requiring iNO for treatment of pulmonary hypertension after cardiopulmonary bypass (n = 15), group 2 was adults receiving iNO (n = 10), and group 3 included children at risk for pulmonary hypertension after bypass that did not require iNO (n = 8). INTERVENTIONS: Dosages of iNO were 2-60 ppm. The duration of therapy ranged from 23 to 188 hrs in group 1 and 29 to 108 hrs in group 2. MEASUREMENTS AND MAIN RESULTS: Arterial blood was obtained for the measurement of ET-1 levels before and during iNO therapy and 24 hrs after iNO withdrawal. Group 1 mean ET-1 levels increased to 127% of baseline by 12 hrs of iNO, remained elevated at 48 hrs (p < .05), then decreased to 71% of iNO levels 24 hrs after withdrawal (p < .01). Group 2 ET-1 levels increased to 147%, and 137% of baseline at 12 and 24 hrs of iNO therapy, then fell to 68% of baseline within 24 hrs of discontinuing iNO. ET-1 levels in group 3 decreased after surgery (p < .05). CONCLUSIONS: These data suggest that iNO increased plasma ET-1 levels, which subsequently decreased when iNO was discontinued. Increased circulating ET-1 levels might contribute to rebound pulmonary hypertension upon iNO withdrawal.

Administration, Inhalation↗
PubMed2002

Role of nitric oxide in acute lung inflammation: lessons learned from the inducible nitric oxide synthase knockout mouse.

OBJECTIVE: Acute lung inflammation is characterized by complex interactions among cytokines, chemokines, adhesion molecules, leukocytes, and other mediators. Proinflammatory cytokines have been implicated in the up-regulation of the inducible form of nitric oxide synthase (iNOS), which produces large amounts of nitric oxide (NO). Conversely, in some systems, NO regulates the expression of cytokines to affect leukocyte recruitment. Thus, the role of NO both exogenously administered and endogenously produced by iNOS in acute lung inflammation has not been fully elucidated. The current studies suggest a proinflammatory role for inhaled NO in a compartmentalized model of lung injury, whereas blocking of iNOS afforded protection. These results and other previous investigations have been complicated by the use of nonselective blockers of the iNOS isoform. MEASUREMENTS AND MAIN RESULTS: In an attempt to circumvent this, we examined the response of the lung to direct endotoxin challenge in mice in which iNOS had been genetically deleted (iNOS-/-). We observed a significant decrease in the inflammatory response in the iNOS-/- mice compared with wild-type mice as characterized by decreases in neutrophil accumulation and cytokine expression. Additionally, the lung cytokine response in the iNOS-/- mice was characterized by a significant increase in interleukin-12 and an inability to up-regulate interleukin-10. CONCLUSIONS: Induction of NO may be a key mediator in driving the cytokine response to endotoxin toward an increased type-2 (interleukin-10) response and a diminished type-1 (interleukin-12) response.

Animals↗