Susette M. Coyle

Human Toll-Like Receptor 4 Mutations but Not CD14 Polymorphisms Are Associated with an Increased Risk of Gram-Negative Infections

Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex. Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis. By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers. There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 [79%] of 14), compared with that in the wild-type population (11 [17%] of 63; P=.004). No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed. These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.

Total parenteral nutrition and bowel rest modify the metabolic response to endotoxin in humans.

Intestinal mucosal atrophy, as induced by total parenteral nutrition (TPN) and/or prolonged bowel rest, is hypothesized to enhance bowel endotoxin (LPS) translocation and may alter host responses to infection. To examine the effect of TPN-induced bowel atrophy on the response to LPS, 12 healthy volunteers were randomized to receive either enteral feedings (ENT, n = 6) or seven days of TPN without oral intake (TPN, n = 6). Enteral or TPN feedings were terminated 12 hours before the study period when a constant dextrose infusion (50 mg/kg/hour) was initiated and continued throughout the subsequent study period. After placement of arterial, hepatic vein, and femoral vein catheters, metabolic parameters were determined before and for six hours after an intravenous E. coli LPS challenge (20 U/kg). Subsequent peak levels of arterial glucagon (ENT, 189 +/- 39 pg/mL; TPN, 428 +/- 48; p less than 0.01), arterial epinephrine (ENT, 236 +/- 52 pg/mL; TPN, 379 +/- 49; p less than 0.05) and hepatic venous cachectin/tumor necrosis factor (cachectin/TNF) (ENT, 250 +/- 56 pg/mL; TPN, 479 +/- 136; p less than 0.05) were significantly higher in the TPN group than in the ENT group. The extremity efflux of lactate (ENT, -16 +/- 4 micrograms/min-100cc tissue; TPN, -52 +/- 13; t = 2 hours; p less than 0.05) and of amino acids (ENT, -334 +/- 77 nmol/min-100cc tissue; TPN, -884 +/- 58; t = 4 hours; p less than 0.05) were higher in the TPN subjects after the endotoxin challenge. Circulating C-reactive Protein (CRP) levels measured 24 hours postendotoxin were also significantly higher in the TPN subjects (ENT, 1.7 +/- 0.2 mg/dL; TPN, 3.2 +/- 0.3; p less than 0.01). Hence the counter-regulatory hormone and splanchnic cytokine responses to LPS were enhanced after TPN and bowel rest. This is associated with a magnified acute-phase response, peripheral amino acid mobilization, and peripheral lactate production. Thus antecedent TPN may influence the metabolic alterations seen in infection and sepsis via both an exaggerated counter-regulatory hormone response as well as an enhanced systemic and splanchnic production of cytokines.

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−&Dgr;&Dgr;CT 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, CSNK1 ϵ, 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.

Gender influences in vivo human responses to endotoxin.

ABSTRACT 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°C) compared with the women (1.4 ± 0.1°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.

Nicotine exposure alters in vivo human responses to endotoxin

The alpha 7 nicotinic receptor is reportedly a key element in the cholinergic anti‐inflammatory pathway. Because a prototypical ligand for this receptor is nicotine, we studied the in vivo human response to bacterial endotoxin or lipopolysaccharide (LPS) in the context of nicotine or placebo pretreatment. Twelve adult male normal subjects were studied prospectively. Six received overnight transcutaneous nicotine administration by application of a standard patch (7 mg). Six hours later, all subjects were given an intravenous dose of endotoxin (2 ng/kg) and were evaluated for an additional 24 h for circulating levels of inflammatory biomarkers, vital signs and symptoms. The nicotine subjects had elevated blood levels of the nicotine metabolite, continine, prior to and throughout the 24‐h post‐endotoxin exposure phase. Subjects receiving nicotine exhibited a significantly lower temperature response as well as attenuated cardiovascular responses for 2·5–6 h after LPS exposure. In addition, increased circulating interkeukin (IL)‐10 and cortisol levels were also noted in nicotine subjects. These data indicate an alteration in LPS‐induced systemic inflammatory responses in normal subjects exposed to transcutaneous nicotine. In this model of abbreviated inflammation, nicotine exposure attenuates the febrile response to LPS and promotes a more prominent anti‐inflammatory phenotype.

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.

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-&agr; 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-&agr; release after endotoxin exposure.

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 µg/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-α (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.

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 TNF&agr;, 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.

Expression of tumour necrosis factor receptor and Toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating

Toll‐like receptors (TLRs) are involved in the recognition of bacterial products and thus participate in the induction of the inflammatory cascade. However, much less is known about the evolution of leucocyte TLR expression during human inflammatory stress. We hypothesized that a decrease in leucocyte TLRs could account for the so‐called tolerance or hyporesponsiveness state to subsequent stimulation with bacteria‐derived products. Because of the profound monocytopenia that ensues after in vivo lipopolysaccharide (LPS) challenge, we also compared monocyte TLR expression using two different techniques of flow cytometric gating. In a first set of experiments, 17 healthy volunteers underwent LPS challenge. Blood was drawn at different time‐points and analysed by flow cytometry using light scatter gating and one‐colour analysis to assess the expression of the tumour necrosis factor receptor (TNFR) and TLR2 and TLR4 on both monocytes and granulocytes. In a second set of experiments, the assessment of those receptors was made using a more specific gating method that utilized light scatter and CD14 immunofluorescence in a two‐colour analysis. This was performed using whole blood drawn from five healthy volunteers and incubated ex vivo for different time periods with or without LPS and in 12 volunteers who underwent LPS challenge in vivo. The pattern of expression for monocyte TNFR was similar for both types of gating. Using only the light scatter gating, an initial drop of TLR 2 and 4 was observed on monocytes. By contrast, when using light scatter × immunofluorescence gating, an up‐regulation of these two receptors following both in vivo and in vitro LPS exposure was observed. LPS up‐regulates the expression of TLRs on monocytes and granulocytes. Depending upon the methodology utilized, contrasting results were obtained with respect to TLR2 and TLR4 expression. The flow cytometric gating technique used is of importance in determining cellular TLR2 and TLR4 expression, especially in blood samples exhibiting significant monocytopenia.