Injury Severity is a Key Contributor to Coagulation Dysregulation and Fibrinogen Consumption.

Background: Traumatic injury is a leading cause of death for those under the age of 45, with 40% occurring due to hemorrhage. Severe tissue injury and hypoperfusion lead to marked changes in coagulation, thereby preventing formation of a stable blood clot and increasing hemorrhage associated mortality. Objectives: We aimed to quantify changes in clot formation and mechanics occurring after traumatic injury and the relationship to coagulation kinetics, and fibrinolysis. Methods: Plasma was isolated from injured patients upon arrival to the emergency department. Coagulation kinetics and mechanics of healthy donors and patient plasma were compared with rheological, turbidimetric and thrombin generation assays. ELISA's were performed to determine tissue plasminogen activator (tPA) and D-dimer concentration, as fibrinolytic markers. Results: Sixty-three patients were included in the study. The median injury severity score (ISS) was 17, median age was 37.5 years old, and mortality rate was 30%. Rheological, turbidimetric and thrombin generation assays indicated that trauma patients on average, and especially deceased patients, exhibited reduced clot stiffness, increased fibrinolysis and reduced thrombin generation compared to healthy donors. Fibrinogen concentration, clot stiffness, D-dimer and tPA all demonstrated significant direct correlation to increasing ISS. Machine learning algorithms identified and highlighted the importance of clinical factors on determining patient outcomes. Conclusions: Viscoelastic and biochemical assays indicate significant contributors and predictors of mortality for improved patient treatment and therapeutic target detection. ESSENTIALS: Traumatic injury may lead to alterations in a patient's ability to form stable blood clotsA study was performed to assess how trauma severity affects coagulation kineticsKey alterations were observed in trauma patients, who exhibit weaker and slower forming clotsPaired with machine learning methods, the results indicate key aspects contributing to mortality.

Insulin-Dependent Regulation of mTORC2-Akt-FoxO Suppresses TLR4 Signaling in Human Leukocytes: Relevance to Type 2 Diabetes.

Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) signaling intermediates in leukocytes from patients with type 2 diabetes or acute insulin resistance associated with cardiopulmonary bypass surgery. We extend this work to show that in addition to matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient leukocytes also express IRS-1 phosphorylated on Ser(312), Akt phosphorylated on Thr(308), and elevated TLR4 expression. Similar signaling intermediates were detected in leukocytes and neutrophils treated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. In contrast, insulin, but not LPS, induced mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt on Ser(473) and FoxO1/O3a on Thr(24/32) in leukocytes and neutrophils. Insulin suppressed LPS-induced responses in a dose- and time-dependent manner. AS1842856, a FoxO1 inhibitor, also suppressed TLR4 signaling. We propose that insulin is a homeostatic regulator of leukocyte responses to LPS/TLR4 and that the signaling intermediates expressed in leukocytes of patients with type 2 diabetes indicate TLR4 signaling dominance and deficient insulin signaling. The data suggest that insulin suppresses LPS/TLR4 signals in leukocytes through the mTORC2-Akt-FoxO signaling axis. Better understanding of leukocyte signaling in patients with type 2 diabetes may shed new light on disease causation and progression.

Proteolytic Cleavage of AMPKα and Intracellular MMP9 Expression Are Both Required for TLR4-Mediated mTORC1 Activation and HIF-1α Expression in Leukocytes.

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.

Human metabolic response to systemic inflammation: assessment of the concordance between experimental endotoxemia and clinical cases of sepsis/SIRS.

INTRODUCTION: Two recent, independent, studies conducted novel metabolomics analyses relevant to human sepsis progression; one was a human model of endotoxin (lipopolysaccharide (LPS)) challenge (experimental endotoxemia) and the other was community acquired pneumonia and sepsis outcome diagnostic study (CAPSOD). The purpose of the present study was to assess the concordance of metabolic responses to LPS and community-acquired sepsis. METHODS: We tested the hypothesis that the patterns of metabolic response elicited by endotoxin would agree with those in clinical sepsis. Alterations in the plasma metabolome of the subjects challenged with LPS were compared with those of sepsis patients who had been stratified into two groups: sepsis patients with confirmed infection and non-infected patients who exhibited systemic inflammatory response syndrome (SIRS) criteria. Common metabolites between endotoxemia and both these groups were individually identified, together with their direction of change and functional classifications. RESULTS: Response to endotoxemia at the metabolome level elicited characteristics that agree well with those observed in sepsis patients despite the high degree of variability in the response of these patients. Moreover, some distinct features of SIRS have been identified. Upon stratification of sepsis patients based on 28-day survival, the direction of change in 21 of 23 metabolites was the same in endotoxemia and sepsis survival groups. CONCLUSIONS: The observed concordance in plasma metabolomes of LPS-treated subjects and sepsis survivors strengthens the relevance of endotoxemia to clinical research as a physiological model of community-acquired sepsis, and gives valuable insights into the metabolic changes that constitute a homeostatic response. Furthermore, recapitulation of metabolic differences between sepsis non-survivors and survivors in LPS-treated subjects can enable further research on the development and assessment of rational clinical therapies to prevent sepsis mortality. Compared with earlier studies which focused exclusively on comparing transcriptional dynamics, the distinct metabolomic responses to systemic inflammation with or without confirmed infection, suggest that the metabolome is much better at differentiating these pathophysiologies. Finally, the metabolic changes in the recovering patients shift towards the LPS-induced response pattern strengthening the notion that the metabolic, as well as transcriptional responses, characteristic to the endotoxemia model represent necessary and "healthy" responses to infectious stimuli.

Physiologic variability at the verge of systemic inflammation: multiscale entropy of heart rate variability is affected by very low doses of endotoxin.

INTRODUCTION: Human injury or infection induces systemic inflammation with characteristic neuroendocrine responses. Fluctuations in autonomic function during inflammation are reflected by beat-to-beat variation in heart rate, termed heart rate variability (HRV). In the present study, we determine threshold doses of endotoxin needed to induce observable changes in markers of systemic inflammation, investigate whether metrics of HRV exhibit a differing threshold dose from other inflammatory markers, and investigate the size of data sets required for meaningful use of multiscale entropy (MSE) analysis of HRV. METHODS: Healthy human volunteers (n = 25) were randomized to receive placebo (normal saline) or endotoxin/lipopolysaccharide (LPS): 0.1, 0.25, 0.5, 1.0, or 2.0 ng/kg administered intravenously. Vital signs were recorded every 30 min for 6 h and then at 9, 12, and 24 h after LPS. Blood samples were drawn at specific time points for cytokine measurements. Heart rate variability analysis was performed using electrocardiogram epochs of 5 min. Multiscale entropy for HRV was calculated for all dose groups to scale factor 40. RESULTS: The lowest significant threshold dose was noted in core temperature at 0.25 ng/kg. Endogenous tumor necrosis factor α and interleukin 6 were significantly responsive at the next dosage level (0.5 ng/kg) along with elevations in circulating leukocytes and heart rate. Responses were exaggerated at higher doses (1 and 2 ng/kg). Time domain and frequency domain HRV metrics similarly suggested a threshold dose, differing from placebo at 1.0 and 2.0 ng/kg, below which no clear pattern in response was evident. By applying repeated-measures analysis of variance across scale factors, a significant decrease in MSE was seen at 1.0 and 2.0 ng/kg by 2 h after exposure to LPS. Although not statistically significant below 1.0 ng/kg, MSE unexpectedly decreased across all groups in an orderly dose-response pattern not seen in the other outcomes. CONCLUSIONS: By using repeated-measures analysis of variance across scale factors, MSE can detect autonomic change after LPS challenge in a group of 25 subjects using electrocardiogram epochs of only 5 min and entropy analysis to scale factor of only 40, potentially facilitating MSE's wider use as a research tool or bedside monitor. Traditional markers of inflammation generally exhibit threshold dose behavior. In contrast, MSE's apparent continuous dose-response pattern, although not statistically verifiable in this study, suggests a potential subclinical harbinger of infectious or other insult. The possible derangement of autonomic complexity prior to or independent of the cytokine surge cannot be ruled out. Future investigation should focus on confirmation of overt inflammation following observed decreases in MSE in a clinical setting.

Integrated transcriptional and metabolic profiling in human endotoxemia.

In this meta-study, we aimed to integrate biological insights gained from two levels of -omics analyses on the response to systemic inflammation induced by lipopolysaccharide in humans. We characterized the interplay between plasma metabolite compositions and transcriptional response of leukocytes through integration of transcriptomics with plasma metabonomics. We hypothesized that the drastic changes in the immediate environment of the leukocytes might have an adaptive effect on shaping their transcriptional response in conjunction with the initial inflammatory stimuli. Indeed, we observed that leukocytes, most notably, tune the activity of lipid- and protein-associated processes at the transcriptional level in accordance with the fluctuations in metabolite compositions of surrounding plasma. A closer look into the transcriptional control of only metabolic pathways uncovered alterations in bioenergetics and defenses against oxidative stress closely associated with mitochondrial dysfunction and shifts in energy production observed during inflammatory processes.

APOε4 is associated with enhanced in vivo innate immune responses in human subjects.

BACKGROUND: The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (Apo) E, a lipid-trafficking protein that affects inflammation, has well-described wild-type (ε3) and disease-associated (ε2 and ε4) alleles, but its connection to human innate immunity is undefined. OBJECTIVE: We sought to define the relationship of APOε4 to the human innate immune response. METHODS: We evaluated APOε4 in several functional models of the human innate immune response, including intravenous LPS challenge in human subjects, and assessed APOε4 association to organ injury in patients with severe sepsis, a disease driven by dysregulated innate immunity. RESULTS: Whole blood from healthy APOε3/APOε4 volunteers induced higher cytokine levels on ex vivo stimulation with Toll-like receptor (TLR) 2, TLR4, or TLR5 ligands than blood from APOε3/APOε3 patients, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in APOε3/APOε4 monocytes. By contrast, APOε3/APOε3 and APOε3/APOε4 serum neutralized LPS equivalently and supported similar LPS responses in Apoe-deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous LPS, APOε3/APOε4 patients had higher hyperthermia and plasma TNF-α levels and earlier plasma IL-6 than APOε3/APOε3 patients. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury and altered splenic lymphocyte apoptosis after systemic LPS compared with APOE3 counterparts. In a cohort of 828 patients with severe sepsis, APOε4 was associated with increased coagulation system failure among European American patients. CONCLUSIONS: APOε4 is a determinant of the human innate immune response to multiple TLR ligands and associates with altered patterns of organ injury in human sepsis.

Natural history of retained surgical items supports the need for team training, early recognition, and prompt retrieval.

BACKGROUND: Unintentionally retained items feature prominently among surgical "never events." Our knowledge of these rare occurrences, including natural history and intraoperative safety omission or variance (SOV) profile, is limited. We sought to bridge existing knowledge gaps by presenting a secondary analysis of a multicenter study focused on these important aspects of retained surgical items (RSIs). METHODS: This is a post hoc analysis of results from a multicenter retrospective study of RSIs between January 2003 and December 2009. After excluding previously reported intravascular RSIs (n = 13), a total of 71 occurrences were analyzed for (1) item location and type; (2) time to presentation and/or discovery; (3) presenting signs and symptoms; (4) procedure and incision characteristics; (5) pathology reports; and (6) patterns of SOVs abstracted from medical and operative records. These SOV were then grouped into individual vs team errors and single- vs multifactorial occurrences. RESULTS: Among 71 cases, there were 48 women and 23 men. Mean patient age was 49.7 ± 17.5 years (range 19 to 83 years). Mortality was 4 of 71 (5.63%, only 1 attributable to RSI). Twelve cases (16.9%) occurred at nonparticipating referring hospitals. Most RSI procedures (62%) occurred on the day of hospital admission. The median time from index RSI case to retained item removal was 2 days (range <1 to >3,600 days, n = 63). Abdominal RSIs predominated, and plain radiography was the most common identification method. Most RSIs removed early (<24 hours, n = 23) were asymptomatic. The most common clinical/diagnostic findings in the remaining group were focal pain (n = 22), abscess/fluid collection (n = 18), and mass (n = 8). Most common pathology findings included exudative reaction (n = 22), fibrosis (n = 17), and purulence/abscess (n = 15). On detailed review of intraprocedural events, most RSI cases were found to involve team/system errors (50 of 71) and 2 or more SOVs (37 of 71). Isolated human error was seen in less than 10% of cases. CONCLUSIONS: The finding that most operations complicated by RSIs were found to involve team/system errors and 2 or more SOVs emphasizes the importance of team safety training. The observation that early RSI removal minimizes patient morbidity and symptoms highlights the need for prompt RSI identification and treatment. The incidence of inflammation-related findings increases significantly with longer retention periods.

Effects of coupled dose and rhythm manipulation of plasma cortisol levels on leukocyte transcriptional response to endotoxin challenge in humans.

Severe traumas are associated with hypercortisolemia due to both disruption of cortisol secretion rhythm and increase in its total concentration. Understanding the effects of altered cortisol levels and rhythms on immune function is of great clinical interest, to prevent conditions such as sepsis from complicating the recovery. This in vivo study assesses the responses of circulating leukocytes to coupled dose and rhythm manipulation of cortisol, preceding an immune challenge induced by endotoxin administration. Through continuous infusion, plasma cortisol concentration was increased to and kept constant at a level associated with major physiologic stress. In response, transcriptional programming of leukocytes was altered to display a priming response before endotoxin exposure. Enhanced expression of a number of receptors and signaling proteins, as well as lowered protein translation and mitochondrial function indicated a sensitization against potential infectious threats. Despite these changes, response to endotoxin followed very similar patterns in both cortisol and saline pre-treated groups except one cluster including probe sets associated with major players regulating inflammatory response. In sum, altered dose and rhythm of plasma cortisol levels engendered priming of circulating leukocytes when preceded an immune challenge. This transcriptional program change associated with stimulated surveillance function and suppressed energy-intensive processes, emphasized permissive actions of cortisol on immune function.

Cellular metabolic regulators: novel indicators of low-grade inflammation in humans.

OBJECTIVE: The Toll-like receptor 4 (TLR4) ligand endotoxin triggers robust systemic inflammatory responses in humans at doses equal to or greater than 1 ng/kg. In this study, we tested the hypothesis that evidence of TLR4-induced responses would be detectable in leukocytes challenged with endotoxin doses that are below the threshold needed to trigger a characteristic systemic inflammatory phenotype in humans. METHODS: Subjects were challenged with endotoxin at 1, 0.5, or 0.1 ng/kg (n = 5 per dose). Systemic responses were monitored for 24 hours. Blood samples, collected at designated intervals, were used to determine plasma cytokines levels, total and differential leukocyte counts, expression of leukocyte cell surface receptors, and changes in the leukocyte transcriptome. Western blotting was used to determine changes in leukocyte protein expression. RESULTS: We found that in vivo endotoxin at doses below 1.0 ng/kg triggers weak and variable responses in humans. In marked contrast, we show that endotoxin at a concentration as low as 0.1 ng/kg triggers a transient decline in cellular ATP levels in leukocytes. This is associated with the appearance of a unique protein expression signature in leukocytes. The protein expression signature includes 3 prominent features: (i) AMP-activated protein kinase subunit α (AMPKα) degradation, (ii) increased hypoxia inducible factor-1 (HIF-1) α expression, and (iii) autophagy, collectively indicative of a regulated metabolic response. An indistinguishable response phenotype was observed in human leukocytes treated with endotoxin in vitro. CONCLUSIONS: These data demonstrate for the first time in humans that a TLR4 ligand concentration that is below the threshold needed to trigger clinically evident systemic inflammatory manifestations initiates a transient decline in ATP levels, AMPKα degradation, HIF-1α expression, and autophagy in leukocytes. This establishes that low-grade TLR4 activation exerts control over leukocyte metabolism in the absence of systemic inflammatory indicators.

Temporal metabolic profiling of plasma during endotoxemia in humans.

Endotoxemia induced by the administration of low-dose lipopolysaccharide (LPS) to healthy human volunteers is a well-established experimental protocol and has served as a reproducible platform for investigating the responses to systemic inflammation. Because metabolic composition of a tissue or body fluid is uniquely altered by stimuli and provides information about the dominant regulatory mechanisms at various cellular processes, understanding the global metabolic response to systemic inflammation constitutes a major part in this investigation complementing the studies undertaken so far in both clinical and systems biology fields. This article communicates the first proof-of-principle metabonomic analysis, which comprised global biochemical profiles in human plasma samples from healthy subjects given intravenous endotoxin at 2 ng/kg. Concentrations of a total of 366 plasma biochemicals were determined in archived blood samples collected from 15 endotoxin-treated subjects at five time points within 24 h after treatment and compared with control samples collected from four saline-treated subjects. Principal component analysis within this data set determined the sixth hour as a critical time point separating development and recovery phases of the LPS-induced metabolic changes. Consensus clustering of the differential metabolites identified two distinct subsets of metabolites that displayed common coherent profiles with opposing directionality. The first group of metabolites, which were mostly associated with pathways related to lipid metabolism, was upregulated within the first 6 h and downregulated by the 24th hour following LPS administration. The second group of metabolites, in contrast, was first downregulated until the sixth hour, then upregulated. Metabolites in this group were predominantly amino acids or their derivatives. In summary, nontargeted biochemical profiling and unsupervised multivariate analyses highlighted the prominent roles of lipid and protein metabolism in regulating the response to systemic inflammation while also revealing their dynamics in opposite directions.

Retained surgical items: a problem yet to be solved.

BACKGROUND: Retained surgical items (RSI) continue to occur. Large RSI studies are few due to low RSI frequency in single institutions and the medicolegal implications. Consequently, RSI risks are not fully defined, with discrepancies persisting among published studies. The goals of this study were to better define risk factors for RSI, to clarify previously discrepant risk factors, and to evaluate other potential contributors to RSI occurrence, such as trainee presence during an operation. STUDY DESIGN: Multicenter case-match study of RSI risk factors was conducted between January 2003 and December 2009. Cases complicated by RSI were identified at participating centers using clinical quality improvement and adverse event reporting data. Case match controls (non-RSI) were selected from same or similar-type cases performed at each respective institution. Retained surgical item risk factors were evaluated by univariate and multivariate conditional logistic regression. RESULTS: Fifty-nine RSIs and 118 matched controls were analyzed (RSI incidence 1 in 6,975 or 59 in 411,526). Retained surgical items occurred despite use of confirmatory x-rays (13 of 27 instances) and/or radiofrequency tagging (2 of 32 instances). Among previously discrepant results, we confirmed that body mass index, unexpected intraoperative events, and procedure duration were associated with increased RSI risk. The occurrence of any safety variance, and specifically an incorrect count at any time during the procedure, was associated with elevated RSI risk. Trainee presence was associated with 70% lower RSI risk compared with trainee absence. CONCLUSIONS: Longer duration of surgery, safety variances, and incorrect counts during the procedure result in elevated RSI risk. The possible positive influence of trainee presence on RSI risk deserves additional study. Our findings highlight the need for zero tolerance for safety omissions, continued study and development of novel approaches to RSI reduction, and establishing anonymous RSI reporting systems to better track both the incidence and risks associated with this problem, which has yet to be solved.

Experimental human endotoxemia: a model of the systemic inflammatory response syndrome?

BACKGROUND: The normal human intravenous endotoxin model has been used for more than 50 years. It was once considered a possible model of sepsis, but, because no infection is present, it is better described as a model of systemic inflammation. We demonstrate herein that at least three of four systemic inflammatory response syndrome (SIRS) criteria are achieved with the model. METHODS: Otherwise healthy human volunteers were given Escherichia coli endotoxin 2 ng/kg intravenously. Vital signs were monitored, and blood samples were collected over time for assessment of white blood cells (WBCs), cytokines, counter-regulatory hormones, and monocyte receptors. RESULTS: The means of three variables (core temperature, heart rate, WBC) met the SIRS criteria. Compared with baseline, cytokines were elevated acutely, with tumor necrosis factor-alpha (TNFα) exhibiting temporal primacy over the other cytokines. Counter-regulatory hormones (cortisol, epinephrine) also were elevated acutely. Finally, the monocyte cell-surface receptors cluster of differentiation molecule (CD) 11b and TNF receptor-II were elevated and decreased, respectively. CONCLUSIONS: The experimental human endotoxin model satisfies SIRS criteria and probably is best described as a model of Toll-like receptor 4 agonist-induced systemic inflammation.

Continuous enteral and parenteral feeding each reduces heart rate variability but differentially influences monocyte gene expression in humans.

Enteral (EN) or parenteral (PN) nutrition is used to support critically ill patients until oral feeding resumes. Enteral nutrition is assumed preferable to PN, but the differential influence on immune function is not well defined. Autonomic nervous activity is known to influence innate immune responses, and we hypothesized that EN and PN could influence both autonomic signaling and gene activation in peripheral blood monocytes (PBMs). Ten subjects (aged 18-36 years) received continuous EN or PN for 72 h. Peripheral blood monocytes were isolated from whole blood before and after continuous feeding and were analyzed for gene expression using a microarray platform. Gene expression after feeding was compared from baseline and between groups. To measure autonomic outflow, subjects also underwent heart rate variability (HRV) monitoring during feeding. Time and frequency domain HRV data were compared between groups and five orally fed subjects for changes from baseline and changes over time. During continuous EN and PN, subjects exhibited declines in both time and frequency domain HRV parameters compared with baseline and with PO subjects, indicating a loss of vagal/parasympathetic tone. However, PN feeding had a much greater influence on PBM gene expression compared with baseline than EN, including genes important to innate immunity. Continuous EN and PN are both associated with decreasing vagal tone over time, yet contribute differently to PBM gene expression, in humans. These preliminary findings support assumptions that PN imposes a systemic inflammatory risk but also imply that continuous feeding, independent of route, may impart additional risk through different mechanisms.

A novel model of common Toll-like receptor 4- and injury-induced transcriptional themes in human leukocytes.

INTRODUCTION: An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that the Toll-like receptor 4 (TLR4) induced transcription patterns elicited in humans exposed to in vivo endotoxin would parallel gene expression patterns observed in trauma patients with initial non-infectious injury. In addition, we sought to identify functional modules that are commonly affected by these two insults of differing magnitude and duration. METHODS: PBL were obtained from seven adult human subject experimental groups. The groups included a group of healthy, hospitalized volunteers (n = 15), that comprised four study groups of subjects challenged with intravenous endotoxin, without or with cortisol, and two serial samplings of trauma patients (n = 5). The PBL were analyzed for gene expression using a 8,793 probe microarray platform (Gene Chip® Focus, Affymetrix). The expression of a subset of genes was determined using qPCR. RESULTS: We describe sequential selection criteria of gene expression data that identifies 445 genes that are significantly differentially expressed (both P ≤ 0.05 and > 1.2 fold-change) in PBL derived from human subjects during the peak of systemic inflammatory responses induced by in vivo endotoxin, as well as in PBL obtained from trauma patients at 1 to 12 days after admission. We identified two functional modules that are commonly represented by this analysis. The first module includes more than 50 suppressed genes that encode ribosomal proteins or translation regulators. The second module includes up-regulated genes encoding key enzymes associated with glycolysis. Finally, we show that several circadian clock genes are also suppressed in PBL of surgical ICU patients. CONCLUSIONS: We identified a group of > 400 genes that exhibit similar expression trends in PBL derived from either endotoxin-challenged subjects or trauma patients. The suppressed translational and circadian clock modules, and the upregulated glycolytic module, constitute a robust and long lasting PBL gene expression signature that may provide a tool for monitoring systemic inflammation and injury.

Voluntarily produced increases in heart rate variability modulate autonomic effects of endotoxin induced systemic inflammation: an exploratory study.

Exposure of healthy people to lipopolysaccharide (LPS; endotoxin) produces a pro-inflammatory response, subjective symptoms, and decreased heart rate variability (HRV). Given the efficacy of HRV biofeedback (BF) for treating asthma, the large autonomic effects of HRV BF, and the link between vagus nerve activity and inflammation, we hypothesized that HRV BF would dampen the acute manifestations of systemic inflammation induced by LPS challenge. Healthy participants age 18-40 were randomly assigned to four-one-hour training sessions of either HRV BF (n = 6) or a control 15/min paced breathing condition (n = 5) prior to acute experimentally induced LPS exposure. Participants were coached to do the procedures for 10 min each at five hourly time points after LPS injection, and then 2 h later. Subjective symptoms, HRV parameters, and plasma cytokine levels were measured at each time point, 2 h afterward, and the following morning. Participants were able to perform the procedures both during four pre-exposure training sessions and while experiencing LPS-induced symptoms. The HRV BF group showed significant attenuation of the LPS-induced decline in HRV for the 6 h following LPS exposure, suggesting that HRV BF decreased autonomic dysfunction produced by LPS-induced inflammation. HRV BF also reduced symptoms of headache and eye sensitivity to light, but did not affect LPS-induced levels of pro-inflammatory cytokines or symptoms of nausea, muscle aches, or feverishness. Further evaluation of HRV BF appears to be warranted among patients with inflammatory conditions.

Relationship of basal heart rate variability to in vivo cytokine responses after endotoxin exposure.

Autonomic inputs from the sympathetic and parasympathetic nervous systems, as measured by heart rate variability (HRV), have been reported to correlate to the severity injury and responses to infectious challenge among critically ill patients. In addition, parasympathetic/vagal activity has been shown experimentally to exert anti-inflammatory effects via attenuation of splanchnic tissue TNF-alpha production. We sought to define the influence of gender on HRV responses to in vivo endotoxin challenge in healthy humans and to determine if baseline HRV parameters correlated with endotoxin-mediated circulating cytokine responses. Young (<30 years of age), healthy subjects (n = 30) received endotoxin (2 ng/kg), and HRV and blood samples were obtained serially thereafter. Plasma cytokines were measured by enzyme-linked immunosorbent assay, and HRV parameters were determined by analysis of serial 5-min epochs of heart rate monitoring. In addition, calculation of multiscale entropy deriving from cardiac monitoring data was performed. The influence of factors such as gender, body mass index, and resting heart rate on HRV after endotoxin exposure was assessed. We found that gender, body mass index, or resting heart rate did not significantly alter the HRV response after endotoxin exposure. Using entropy analysis, we observed that females had significantly higher entropy values at 24 h after endotoxin exposure. Using a serially sampling protocol for cytokine determination, we found a significant correlation of several baseline HRV parameters (percentage of interval differences of successive interbeat intervals more than 50 ms, r = 0.42, P < 0.05; high-frequency variability, r = 0.4, P < 0.05; and low-frequency/high-frequency ratio, r = -0.43, P < 0.05) on TNF-alpha release after endotoxin exposure.

In vivo endotoxin synchronizes and suppresses clock gene expression in human peripheral blood leukocytes.

OBJECTIVES: The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. DESIGN: Clinical and laboratory investigation. SETTING: University-based research laboratory and clinical research center. SUBJECTS: Human volunteers. INTERVENTIONS: Human subjects were administered a standard dose of endotoxin (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion. MEASUREMENTS AND MAIN RESULTS: Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2 method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1epsilon, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. CONCLUSIONS: Circadian clock gene expression in peripheral blood leukocytes is dramatically altered and possibly uncoupled from the activity of the central clock during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.

Influence of acute epinephrine infusion on endotoxin-induced parameters of heart rate variability: a randomized controlled trial.

OBJECTIVE: To determine whether the acute anti-inflammatory influence of epinephrine (EPI) extends to changes in heart rate variability (HRV) induced by the prototypical inflammatory stimulus, endotoxin (lipopolysaccharide [LPS]). SUMMARY BACKGROUND DATA: HRV reflects fluctuating cardiac autonomic inputs and is acutely reduced during the systemic inflammation induced by LPS as well as during severe critical illnesses such as sepsis and traumatic injury. While EPI may diminish proinflammatory cytokine release, it is unknown whether this net anti-inflammatory activity extends to HRV. METHODS: Healthy volunteers (n = 17) were randomized to either saline + LPS (2 ng/kg) or LPS + antecedent EPI infusion (30 ng/kg/min) from -3 to 6 hours relative to LPS. HRV and blood samples were obtained before EPI and LPS as well as hourly afterward. Plasma cytokines were measured by ELISA. Statistical analysis was by repeated measures analysis of variance. This study was registered at Clinicaltrials.gov and is listed under the following ID number: NCT00753402. RESULTS: LPS acutely influenced all measured parameters of HRV including standard deviation of the average beat to beat intervals over a 5-minute period, percentage of interval differences of successive interbeat intervals greater than 50 milliseconds and square root of the mean squared differences, high frequency (HF), low frequency, low frequency/HF, and very low frequency (all P < 0.01). EPI infusion reduced the inflammatory cytokine response to LPS as measured by decreased TNFalpha, IL-6, and IL-8 (P < 0.01). Relative to the saline + LPS group, antecedent EPI infusion was associated with further reductions in parameters of HRV measuring vagal/parasympathetic activity including, percentage of interval differences of successive interbeat intervals greater than 50 milliseconds, square root of the mean squared differences, and HF (P < 0.05). CONCLUSION: Prior EPI exposure exerts anti-inflammatory influences but also may reduce vagus nerve activity. Hence, acute EPI administration may be protective against early inflammatory challenges but diminish vagal nerve responsiveness to subsequent stimuli.

A single nucleotide polymorphism in the Mdm2 promoter and risk of sepsis.

BACKGROUND: The Mdm2-SNP309(T/G) polymorphism has been shown to upregulate transcription of Mdm2 and subsequently attenuate the p53 pathway. Its role in regulating the human response to acute illness has not been reported. METHODS: Patients from the surgical intensive care unit were prospectively enrolled. SNP309 genotype was determined, and a genotype-based comparison of clinical outcomes was performed. RESULTS: Of the 85 enrolled patients, 41 had wild type (T/T) and 44 had mutant (32 T/G and 12 G/G) genotypes. The mutant-genotype group tended to have a longer LOS in both the surgical intensive care unit (P = .40) and the hospital (P = .08), but these trends did not reach significance. No observable genotype-based differences were noted in any other measured parameters. CONCLUSIONS: The Mdm2-SNP309(G) allele may be associated with longer LOS. However, it does not appear to influence any other clinical characteristics, nor can it be used to predict clinical outcome.