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.

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.

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.

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.

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.

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.

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.

Increased Healthcare-Associated Infections in a Surgical Intensive Care Unit Related to Boarding Non-Surgical Patients.

Background: Hospital over-capacity often forces boarding patients outside of their designated intensive care unit (ICU). Anecdotal evidence suggested medical intensive care unit (MICU) patients boarding in the surgical intensive care unit (SICU) were responsible for increases in healthcare-associated infection (HAI) rates. We studied the effect of ICU boarding on rates of SICU HAIs. Methods: This single-center, retrospective two-year database study compared primary SICU patients (Home) to MICU patients boarding in the SICU (Boarders). Variables studied included age, gender, Acute Physiology and Chronic Health Evaluation III (APACHE III) scores, and comorbidities. Healthcare-associated infections included Clostridium difficile infection, catheter-associated urinary tract infections, central line-associated blood stream infection, and ventilator-associated pneumonia. Student t-test, Fisher exact testing, and a multivariable regression model were used to determine the significance of associations. Results: A total of 2,562 patients were included in the study; 328 (12.8%) were Boarders and 2,234 (87.2%) were Home. Univariable analysis demonstrated that Boarders were older (64.0 ± 16.9 vs. 60.2 ± 17.4), more severely ill (APACHE III score 70.5 ± 31.1 vs. 53.4 ± 21.9), more likely to have cirrhosis, coronary artery disease, and asthma/chronic obstructive pulmonary disease, but less likely to have hypertension. On univariable analysis boarding was associated with an increase HAI rate (19 HAI/1,000 patient days vs. 6.2, p < 0.001). Multivariable regression modeling demonstrated boarding status remained independently associated with HAI (odds ratio [OR] 1.83 95% confidence interval [CI] 1.02-3.27). Cost estimates demonstrated an additional cost of $83,303 per 1,000 patient days. Conclusion: The practice of hospital boarding is associated with development of HAI and increased hospital costs. Efforts at determining the cause of this increase and then reducing HAIs will improve patient care and help hospital budgets.

TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2.

TLR4 is implicated in diseases associated with chronic low-grade inflammation, yet homeostatic signaling mechanisms that prevent and/or are affected by chronic TLR4 activation are largely uncharacterized. We recently reported that LPS/TLR4 activates in human leukocytes signaling intermediates (SI), abbreviated TLR4-SI, which include mTORC1-specific effectors and targets, and that leukocytes of patients with T2D or after cardiopulmonary bypass (CPB) expressed similar SI. Extending these previous findings, here we show that TLR4-SI expression post-CPB was associated with low serum bilirubin and reduced preoperative expression of biliverdin reductase A (BVRA), the enzyme that converts biliverdin to bilirubin, in patient's leukocytes. Biliverdin inhibited TLR4 signaling in leukocytes and triggered phosphorylation of mTORC2-specific targets, including Akt, PKCζ, AMPKα-LKB1-TSC1/2, and their association with BVRA. Torin, PP242, and a PKCζ inhibitory peptide, but not rapamycin, prevented these biliverdin-induced responses and TLR4 inhibition. In contrast, LPS/TLR4 triggered decreases in BVRA, AMPKα and PKCζ expression, and an increase in haptoglobin, a heme binding protein, in leukocytes in vivo and in vitro, indicating that activated TLR4 may suppress biliverdin/BVRA signaling. Significantly, compared to non-diabetics, BVRA and PKCζ expression was low and haptoglobin was high in T2D patients leukocytes. Sustained TLR4 activation may deregulate homeostatic anti-inflammatory BVRA/mTORC2 signaling and thereby contribute to chronic inflammatory diseases.

Low-dose steroid alters in vivo endotoxin-induced systemic inflammation but does not influence autonomic dysfunction.

Severe injury and infection are associated with autonomic dysfunction. Diminished heart rate variability (HRV) is also observed as a component of autonomic dysfunction and is induced by endotoxin administration to healthy subjects. It is established that low-dose glucocorticoid administration diminishes the systemic inflammatory manifestations of endotoxinemia but the influence of this anti-inflammatory intervention on overall autonomic dysfunction and HRV responses to endotoxin is unknown. This study was designed to assess the influence of a low-dose hydrocortisone infusion upon endotoxin-elicited systemic inflammatory responses including phenotypic features, cytokine production, and parameters of HRV. Of 19 subjects studied, nine received a continuous infusion of hydrocortisone (3 microg/kg/min continuously over 6 h) prior to intravenous administration of Escherichia coli endotoxin (2 ng/kg, CC-RE, Lot #2) while 10 healthy subjects received only the endotoxin after a 6-h period of saline control infusion. Serial determinations of vital signs, heart rate variability assessments, and cytokine levels were obtained over the subsequent 24 h. Prior cortisol infusion diminished the peak TNF-alpha (P < 0.01) and IL-6 (P < 0.0001) responses after endotoxin challenge, as compared to saline infusion controls and diminished the peak core temperature response to endotoxin (P < 0.01). In contrast to the influence of cortisol on the above parameters of systemic inflammation, the significant endotoxin-induced decreases in HRV time and frequency domains were not influenced by prior hydrocortisone treatment. Hence, alterations in autonomic dysfunction occur despite hydrocortisone attenuation of other traditional systemic manifestations of endotoxinemia. The maintenance or restoration of autonomic balance is not influenced by glucocorticoid administration.

Understanding Physiology in the Continuum: Integration of Information from Multiple -Omics Levels.

In this paper, we discuss approaches for integrating biological information reflecting diverse physiologic levels. In particular, we explore statistical and model-based methods for integrating transcriptomic, proteomic and metabolomics data. Our case studies reflect responses to a systemic inflammatory stimulus and in response to an anti-inflammatory treatment. Our paper serves partly as a review of existing methods and partly as a means to demonstrate, using case studies related to human endotoxemia and response to methylprednisolone (MPL) treatment, how specific questions may require specific methods, thus emphasizing the non-uniqueness of the approaches. Finally, we explore novel ways for integrating -omics information with PKPD models, toward the development of more integrated pharmacology models.

Gender influences in vivo human responses to endotoxin.

Gender appears to influence systemic and organ-specific inflammatory sequelae of ischemia-reperfusion and infectious challenge in many animal models. Despite the protection provided by female gender, androgen blockade, and/or estrogen administration in such experimental studies, many questions remain regarding the influence of gender dimorphism upon human responses to injury. We hypothesized that the administration of low-dose lipopolysaccharide (LPS) to otherwise healthy, young adults would provide insights regarding the influence of gender upon physiological and innate immune system responses to a prototypic inflammatory stimulus. To this end, 72 adult subjects (48 men, aged 29 +/- 1.0 years; 24 women, aged 26 +/- 1.0 years) were prospectively evaluated before and after the i.v. administration of LPS (2 ng/kg). All subjects developed symptoms within 1.0 to 1.5 h after LPS, and the men exhibited a greater increase in core temperature (2.1 +/- 0.1 degrees C) compared with the women (1.4 +/- 0.1 degrees C) (P < 0.001). In addition, the men exhibited a greater maximum decrease in mean arterial pressure (-13.0 +/- 1.3 mmHg) compared with the women (-8 +/- 1.3 mmHg) (P < 0.02). The changes in temperature and mean arterial pressure occurred without detectable differences between the male and female cohort responses of circulating white blood cell count and cortisol or cytokine levels. These results suggest that soluble inflammatory mediators generated by in vivo endotoxin activation of the innate immune system are insufficient to explain the resultant gender-specific phenotypic differences observed in young, adult humans.

Polymorphisms of heat shock protein-70 (HSPA1B and HSPA1L loci) do not influence infection or outcome risk in critically ill surgical patients.

Heat shock proteins (HSP) are induced in various stress conditions and have many cytoprotective effects, including formation of protein complexes for antigen presentation, stabilizing intracellular proteins, and facilitating protein folding. The HSP-70 gene exhibits polymorphisms at the HSPA1B and HSPA1L loci that reportedly influence cytokine levels and clinical outcomes in critically ill patients. These HSP variations also have been linked to TNF-beta polymorphisms associated with poor outcomes. This study further evaluated outcomes and risk of infection of HSP polymorphisms in critically ill patients. Seventy-six consecutive surgical intensive care unit uninfected patients with established systemic inflammatory response features were prospectively enrolled. Genomic DNA was isolated from whole blood samples and specific fragments, including the relevant polymorphic sites, were amplified by PCR, and restriction digestions were performed. Genotypes were determined by electrophoresis and all were confirmed by direct sequencing. Plasma cytokine levels for TNF-alpha were assayed in a subset of patients by enzyme-linked immunoabsorbent assay. None of the HSP alleles bore a significant relationship to nosocomial infection rates, organ specific dysfunctions, or mortality. No linkage of HSP genotype to common TNF-alpha or TNF-beta genotypes could be demonstrated, although the HSPA1L CT polymorphism was associated with higher levels of TNF-alpha compared with the TT genotype. These data suggest that polymorphisms of the HSPA1L or HSPA1B loci do not influence infection or other highly morbid outcomes in surgical intensive care unit patients.

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.

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.

Effects of drotrecogin alfa (activated) in human endotoxemia.

In a phase III clinical trial, drotrecogin alfa (activated) was shown to improve survival and promote faster improvement of cardiovascular and respiratory dysfunction in patients with severe sepsis. To further examine mechanisms involved in the action of this drug, a healthy human endotoxin model was used. Healthy volunteers (eight per group) received drotrecogin alfa (activated) or placebo intravenously for 8 h in a randomized, double-blind, controlled manner. After 2 h of study drug infusion, endotoxin (2 ng/kg) was infused and measurement of physiologic responses and biomarkers continued for 24 h. Consistent with results from severe sepsis clinical trials, drotrecogin alfa (activated) improved mean arterial pressure during the period of infusion after endotoxin exposure. In contrast to severe sepsis clinical trials using drotrecogin alfa (activated) but similar to another human endotoxin study, no significant antithrombotic, profibrinolytic, or anti-inflammatory effects were observed. These results suggest a novel role for drotrecogin alfa (activated) in the human endotoxin model.

Modulation of the lipopolysaccharide receptor complex (CD14, TLR4, MD-2) and toll-like receptor 2 in systemic inflammatory response syndrome-positive patients with and without infection: relationship to tolerance.

The lipopolysaccharide (LPS) receptor complex consists of two interacting receptors (CD14 and TLR4) and an associated protein (MD-2). When engaged by LPS, as in gram-negative infection, this complex transduces a signal detected by MyD88 and passed onward by a cascade of the IRAKs, TRAF6, and NIK, resulting in activation of NF-kappaB. A similar cascade, mediated by TLR2, occurs with ligands derived from gram-positive bacteria. In vitro studies of human monocytes have shown that TLR4 mRNA is paradoxically upregulated in response to "tolerizing" doses of LPS. This study evaluated changes in vivo of blood monocyte CD14, TLR4, TLR2, and MD-2 mRNA by reverse transcription followed by real-time polymerase chain reaction in surgical intensive care unit patients and in normal controls. In addition cell-surface receptor expression of TLR2, TLR4, and CD14 was assessed by flow cytometry in patients and normal controls. Inflammation-induced acute tolerance to LPS was evaluated by ex vivo whole blood tumor necrosis factor alpha production and was significantly reduced in patients compared with controls, confirming LPS hyporesponsiveness. Monocyte mRNA and cell-surface receptor expression of TLR4 were increased 2.4-fold (P < 0.05) and 1.7-fold (P <.002), respectively, in patients compared with normal controls. Monocyte TLR2 mRNA, MD-2 mRNA and CD14 and TLR2 cell-surface expression were not significantly changed compared with controls. The present study suggests that the acute inflammatory condition associated with peripheral cellular LPS hyporesponsiveness is neither specific to prior infectious challenge nor can be ascribed to significant alterations in expression of the cell-surface LPS binding complex proteins.

Influence of the TNF-alpha and TNF-beta polymorphisms upon infectious risk and outcome in surgical intensive care patients.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a well-documented central inflammatory mediator in sepsis. Specific polymorphisms of the TNF-alpha and TNF-beta genes (TNF2 and LTA + 250, respectively) have been suggested to correlate with higher mortality in septic shock. This study sought to determine whether these polymorphisms of the TNF-alpha and -beta genes are associated with an increased risk of infection in an at-risk surgical intensive care population. MATERIALS AND METHODS: Forty-four consecutive patients with systemic inflammatory response syndrome were enrolled prospectively in the study. Genomic DNA was isolated from whole blood samples using standard phenol/chloroform extraction techniques. Specific fragments including the polymorphic sites of each gene were amplified by polymerase chain reaction, and restriction enzyme digestions were performed. Genotypes were determined by gel electrophoresis and confirmed by direct sequencing. RESULTS: Eighty-six percent of the patients were TNF1 homozygotes (G:G at -308 of the TNF-alpha promoter region), whereas 9% of the patients were homozygous for TNF2 (A:A). There was no difference in the incidence of sepsis, septic shock, or mortality between patients bearing the various alleles. Only 13.6% of the patients exhibited the G:G alleles for TNF-beta, whereas the homozygous A:A was present in 45.4% of the patients. CONCLUSION: The presence of the A allele at these polymorphic sites did not predispose critically ill surgical patients to either infection or septic shock.