The Absence of Fever Is Associated With Higher Mortality and Decreased Antibiotic and IV Fluid Administration in Emergency Department Patients With Suspected Septic Shock.

OBJECTIVE: This study evaluates whether emergency department septic shock patients without a fever (reported or measured) receive less IV fluids, have decreased antibiotic administration, and suffer increased in-hospital mortality. DESIGN: This was a secondary analysis of a prospective, observational study of patients with shock. SETTING: The study was conducted in an urban, academic emergency department. PATIENTS: The original study enrolled consecutive adult (aged 18 yr or older) emergency department patients from November 11, 2012, to September 23, 2013, who met one of the following shock criteria: 1) systolic blood pressure less than 90 mm Hg after at least 1L IV fluids, 2) new vasopressor requirement, or 3) systolic blood pressure less than 90 mm Hg and IV fluids held for concern of fluid overload. The current study is limited to patients with septic shock. Patients were grouped as febrile if they had a subjective fever or a measured temperature >100.4°F documented in the emergency department; afebrile patients lacked both. MEASUREMENTS AND MAIN RESULTS: Among 378 patients with septic shock, 207 of 378 (55%; 50-60%) were febrile by history or measurement. Afebrile patients had lower rates of antibiotic administration in the emergency department (81% vs 94%; p < 0.01), lower mean volumes of IV fluids (2,607 vs 3,013 mL; p < 0.01), and higher in-hospital mortality rates (33% vs 11%; p < 0.01). After adjusting for bicarbonate less than 20 mEq/L, lactate concentration, respiratory rate greater than or equal to 24 breaths/min, emergency department antibiotics, and emergency department IV fluids volume, being afebrile remained a significant predictor of in-hospital mortality (odds ratio, 4.3; 95% CI, 2.2-8.2; area under the curve = 0.83). CONCLUSIONS: In emergency department patients with septic shock, afebrile patients received lower rates of emergency department antibiotic administration, lower mean IV fluids volume, and suffered higher in-hospital mortality.

Zonulin as prehaptoglobin2 regulates lung permeability and activates the complement system.

Zonulin is a protein involved in the regulation of tight junctions (TJ) in epithelial or endothelial cells. Zonulin is known to affect TJ in gut epithelial cells, but little is known about its influences in other organs. Prehaptoglobin2 has been identified as zonulin and is related to serine proteases (MASPs, C1qrs) that activate the complement system. The current study focused on the role of zonulin in development of acute lung injury (ALI) in C57BL/6 male mice following intrapulmonary deposition of IgG immune complexes. A zonulin antagonist (AT-1001) and a related peptide with permeability agonist activities (AT-1002) were employed and given intratracheally or intravenously. Also, zonulin was blocked in lung with a neutralizing antibody. In a dose-dependent manner, AT-1001 or zonulin neutralizing antibody attenuated the intensity of ALI (as quantitated by albumin leak, neutrophil accumulation, and proinflammatory cytokines). A similar pattern was found using the bacterial lipopolysaccharide model of ALI. Using confocal microscopy on sections of injured lungs, staining patterns for TJ proteins were discontinuous, reduced, and fragmented. As expected, the leak of blood products into the alveolar space confirmed the passage of 3 and 20 kDa dextran, and albumin. In contrast to AT-1001, application of the zonulin agonist AT-1002 intensified ALI. Zonulin both in vitro and in vivo induced generation of complement C3a and C5a. Collectively, these data suggest that zonulin facilitates development of ALI both by enhancing albumin leak and complement activation as well as increased buildup of neutrophils and cytokines during development of ALI.

Phagocyte-derived catecholamines enhance acute inflammatory injury.

It is becoming increasingly clear that the autonomic nervous system and the immune system demonstrate cross-talk during inflammation by means of sympathetic and parasympathetic pathways. We investigated whether phagocytes are capable of de novo production of catecholamines, suggesting an autocrine/paracrine self-regulatory mechanism by catecholamines during inflammation, as has been described for lymphocytes. Here we show that exposure of phagocytes to lipopolysaccharide led to a release of catecholamines and an induction of catecholamine-generating and degrading enzymes, indicating the presence of the complete intracellular machinery for the generation, release and inactivation of catecholamines. To assess the importance of these findings in vivo, we chose two models of acute lung injury. Blockade of alpha2-adrenoreceptors or catecholamine-generating enzymes greatly suppressed lung inflammation, whereas the opposite was the case either for an alpha2-adrenoreceptor agonist or for inhibition of catecholamine-degrading enzymes. We were able to exclude T cells or sympathetic nerve endings as sources of the injury-modulating catecholamines. Our studies identify phagocytes as a new source of catecholamines, which enhance the inflammatory response.

Cross-talk between TLR4 and FcgammaReceptorIII (CD16) pathways.

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcgammaR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcgammaRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRgamma-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4(-/-) mice. Unexpectedly, TLR4 mut and TLR4(-/-) mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcgammaRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcgammaRIII signaling via FcRgamma-subunit activation.

Functions of the complement components C3 and C5 during sepsis.

Activation of the complement system is a key event in the pathogenesis of sepsis. Nevertheless, the exact mechanisms remain inadequately understood. In the current study, we examined the role of complement C3 and C5 in sepsis in wild-type and C3- or C5-deficient mice induced by cecal ligation and puncture. When compared to wild-type mice, C5(-/-) showed identical survival, and C3(-/-) presented significantly reduced survival. Interestingly, this was associated with significant decreases in plasma levels of proinflammatory mediators. Moreover, although septic C3(-/-) animals displayed a 10-fold increase of blood-borne bacteria, C5(-/-) animals exhibited a 400-fold increase in bacteremia when compared to wild-type mice. These effects were linked to the inability of C5(-/-) mice to assemble the terminal membrane attack complex (MAC), as determined by complement hemolytic activity (CH-50). Surprisingly, although negative control C3(-/-) mice failed to generate the MAC, significant increases of MAC formation was found in septic C3(-/-) mice. In conclusion, our data corroborate that hemolytic complement activity is essential for control of bacteremia in septic mice. Thus, during sepsis, blockade of C5a or its receptors (rather than C5) seems a more promising strategy, because C5a-blockade still allows for MAC formation while the adverse effects of C5a are prevented.

Adverse functions of IL-17A in experimental sepsis.

IL-17A is a proinflammatory cytokine produced by a variety of cells. In the current study, we examined the role of IL-17A in sepsis induced in mice by cecal ligation and puncture (CLP). IL-17A levels, which rose time-dependently in plasma after CLP, were not affected in the absence of alphabeta T cells or neutrophils. In sharp contrast, gammadelta T cell-knockout or gammadelta T cell-depleted mice displayed baseline IL-17A plasma levels after CLP. Neutralization of IL-17A by two different antibodies improved sepsis (survival from approximately 10% to nearly 60%). Unexpectedly, antibody treatment was protective, even when administration of anti-IL-17A was delayed for up to 12 h after CLP. These protective effects of IL-17A blockade were associated with substantially reduced levels of bacteremia together with significant reductions of systemic proinflammatory cytokines and chemokines in plasma. In vitro incubation of mouse peritoneal macrophages with lipopolysaccharide (LPS) in the copresence of IL-17A substantially increased the production of TNF-alpha, IL-1beta, and IL-6 by these cells. These data suggest that, during experimental sepsis, gammadelta T cell-derived IL-17A promotes high levels of proinflammatory mediators and bacteremia, resulting in enhanced lethality. IL-17A may be a potential therapeutic target in sepsis.

Serum Lactate Predicts Adverse Outcomes in Emergency Department Patients With and Without Infection.

INTRODUCTION: Lactate levels are increasingly used to risk stratify emergency department (ED) patients with and without infection. Whether a serum lactate provides similar prognostic value across diseases is not fully elucidated. This study assesses the prognostic value of serum lactate in ED patients with and without infection to both report and compare relative predictive value across etiologies. METHODS: We conducted a prospective, observational study of ED patients displaying abnormal vital signs (AVS) (heart rate ≥130 bpm, respiratory rate ≥24 bpm, shock index ≥1, and/or systolic blood pressure <90 mmHg). The primary outcome, deterioration, was a composite of acute renal failure, non-elective intubation, vasopressor administration or in-hospital mortality. RESULTS: Of the 1,152 patients with AVS who were screened, 488 patients met the current study criteria: 34% deteriorated and 12.5% died. The deterioration rate was 88/342 (26%, 95% CI: 21 - 30%) for lactate < 2.5 mmol/L, 47/90 (52%, 42 - 63%) for lactate 2.5 - 4.0 mmol/L, and 33/46 (72%, 59 - 85%) for lactate >4.0mmol/L. Trended stratified lactate levels were associated with deterioration for both infected (p<0.01) and non-infected (p<0.01) patients. In the logistic regression models, lactate > 4mmol/L was an independent predictor of deterioration for patients with infection (OR 4.8, 95% CI: 1.7 - 14.1) and without infection (OR 4.4, 1.7 - 11.5). CONCLUSION: Lactate levels can risk stratify patients with AVS who have increased risk of adverse outcomes regardless of infection status.

Assessing The Predictive Value of Clinical Factors Used to Determine The Presence of Sepsis Causing Shock in the Emergency Department.

INTRODUCTION: Differentiating shock etiologies is a challenging task in the Emergency Department (ED); even the strongest clinical predictors leave some diagnostic uncertainty. This study sought to establish an evidence base for using clinical covariates in the diagnostic evaluation of septic shock. METHODS: We conducted a prospective, observational study of consecutive ED patients with shock from November 11, 2012 to September 23, 2013. We included all patients at least 18 years old with shock, defined as new vasopressor requirement, systolic blood pressure less than 90 mmHg after at least 1 L of crystalloid or 2 units packed red blood cells, or systolic blood pressure less than 90 mmHg and fluids withheld due to concern for fluid overload. Multivariate logistic regression and recursive partitioning models were constructed, predicting septic cause of shock. The logistic regression model was derived using first 500 patients, and validated with the subsequent 200 patients. RESULTS: In the derivation cohort, 55.6% (95% confidence interval: 51.2%-60.0%) were septic, and 20.8% (17.2%-24.4%) died during hospitalization. The multivariate model (derivation area under the curve = 0.88, validation area under the curve = 0.89) identified predictors of septic shock, including temperature more than 100.4°F (odds ratio 4.6, 2.3-9.2) and history of fever (odds ratio 9.2, 4.4-19.2); however, only 153 of 277 (55.3%, 49.5%-61.2%) patients with septic shock had either of these findings. In the recursive partitioning model, if all predictors were absent, the probability of sepsis causing shock was 21% (16.6%-25.6%). CONCLUSIONS: Clinical data can predict the presence of sepsis causing shock in the ED in most patients. The remaining diagnostic uncertainty provides an opportunity for adding novel diagnostic testing.

Derivation and Validation of Predictive Factors for Clinical Deterioration after Admission in Emergency Department Patients Presenting with Abnormal Vital Signs Without Shock.

INTRODUCTION: Strategies to identify high-risk emergency department (ED) patients often use markedly abnormal vital signs and serum lactate levels. Risk stratifying such patients without using the presence of shock is challenging. The objective of the study is to identify independent predictors of in-hospital adverse outcomes in ED patients with abnormal vital signs or lactate levels, but who are not in shock. METHODS: We performed a prospective observational study of patients with abnormal vital signs or lactate level defined as heart rate ≥130 beats/min, respiratory rate ≥24 breaths/min, shock index ≥1, systolic blood pressure <90 mm/Hg, or lactate ≥4 mmole/L. We excluded patients with isolated atrial tachycardia, seizure, intoxication, psychiatric agitation, or tachycardia due to pain (ie: extremity fracture). The primary outcome was deterioration, defined as development of acute renal failure (creatinine 2× baseline), non-elective intubation, vasopressor requirement, or mortality. Independent predictors of deterioration after hospitalization were determined using logistic regression. RESULTS: Of 1,152 consecutive patients identified with abnormal vital signs or lactate level, 620 were excluded, leaving 532 for analysis. Of these, 53/532 (9.9±2.5%) deteriorated after hospital admission. Independent predictors of in-hospital deterioration were: lactate >4.0 mmol/L (OR 5.1, 95% CI [2.1-12.2]), age ≥80 yrs (OR 1.9, CI [1.0-3.7]), bicarbonate <21 mEq/L (OR 2.5, CI [1.3-4.9]), and initial HR≥130 (OR 3.1, CI [1.5-6.1]). CONCLUSION: Patients exhibiting abnormal vital signs or elevated lactate levels without shock had significant rates of deterioration after hospitalization. ED clinical data predicted patients who suffered adverse outcomes with reasonable reliability.

The Utility of Inflammatory and Endothelial Markers to Identify Infection in Emergency Department Patients.

BACKGROUND: Identifying infection in emergency department (ED) patients can be challenging. This study assesses the value that inflammatory and endothelial biomarkers add to clinical data when predicting infectious etiologies of abnormal vital signs (AVSs) in ED patients. METHODS: This study was a prospective, observational cohort study of ED patients with AVSs at an urban, academic tertiary-care hospital, identified from March 1, 2013, to April 15, 2013. Collected blood samples were assayed for soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule 1, vascular cell adhesion molecule 1, plasminogen activator inhibitor 1, interleukin 6, sFlt-1, and procalcitonin. History and physical examination were abstracted from the ED documentation. The primary outcome, infectious etiology, was adjudicated by review of the hospital documentation. Three multivariate logistic regression models predicting infection were created using clinical data, biomarkers, and combined clinical data and biomarker assessments. Integrated discrimination improvement tested the discriminate value of the biomarker and combined models compared with the clinical data model. RESULTS: We enrolled 115 patients: 49 determined to have an infection (43%) and 66 without (57%). All biomarkers were significantly associated with infection in univariate analysis. The best clinical model (area under the curve [AUC] = 0.76) included initial temperature (odds ratio [OR], 1.6; confidence interval [CI], 1.1-2.2) and history of fever (OR, 5.0; CI, 1.4-14). The best biomarker model (AUC, 0.82) predicting infection included sE-selectin (OR, 11.0; 95% CI, 1.6-74) and interleukin 6 (OR, 5.1; CI, 2.3-11.6). The combined clinical and biomarker model had an AUC of 0.88, with integrated discrimination improvement = 0.21, compared with the clinical model alone. CONCLUSION: Inflammatory and endothelial markers can improve the clinical identification of infection in ED patients with AVSs.

Upregulation of phagocyte-derived catecholamines augments the acute inflammatory response.

Following our recent report that phagocytic cells (neutrophils, PMNs, and macrophages) are newly discovered sources of catecholamines, we now show that both epinephrine and norepinephrine directly activate NFkappaB in macrophages, causing enhanced release of proinflammatory cytokines (TNFalpha, IL-1beta, IL-6). Both adrenal-intact (AD+) and adrenalectomized (ADX) rodents were used, because ADX animals had greatly enhanced catecholamine release from phagocytes, facilitating our efforts to understand the role of catecholamines released from phagocytes. Phagocytes isolated from adrenalectomized rats displayed enhanced expression of tyrosine-hydroxylase and dopamine-beta-hydroxylase, two key enzymes for catecholamine production and exhibited higher baseline secretion of norepinephrine and epinephrine. The effects of upregulation of phagocyte-derived catecholamines were investigated in two models of acute lung injury (ALI). Increased levels of phagocyte-derived catecholamines were associated with intensification of the acute inflammatory response, as assessed by increased plasma leak of albumin, enhanced myeloperoxidase content in lungs, augmented levels of proinflammatory mediators in bronchoalveolar lavage fluids, and elevated expression of pulmonary ICAM-1 and VCAM-1. In adrenalectomized rats, development of ALI was enhanced and related to alpha(2)-adrenoceptors engagement but not to involvement of mineralocorticoid or glucocorticoid receptors. Collectively, these data demonstrate that catecholamines are potent inflammatory activators of macrophages, upregulating NFkappaB and further downstream cytokine production of these cells. In adrenalectomized animals, which have been used to further assess the role of catecholamines, there appears to be a compensatory increase in catecholamine generating enzymes and catecholamines in macrophages, resulting in amplification of the acute inflammatory response via engagement of alpha(2)-adrenoceptors.

The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis.

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis.

Acute lung injury induced by lipopolysaccharide is independent of complement activation.

Although acute lung injury (ALI) is an important problem in humans, its pathogenesis is poorly understood. Airway instillation of bacterial LPS, a known complement activator, represents a frequently used model of ALI. In the present study, pathways in the immunopathogenesis of ALI were evaluated. ALI was induced in wild-type, C3(-/-), and C5(-/-) mice by airway deposition of LPS. To assess the relevant inflammatory mediators, bronchoalveolar lavage fluids were evaluated by ELISA analyses and various neutralizing Abs and receptor antagonists were administered in vivo. LPS-induced ALI was neutrophil-dependent, but it was not associated with generation of C5a in the lung and was independent of C3, C5, or C5a. Instead, LPS injury was associated with robust generation of macrophage migration inhibitory factor (MIF), leukotriene B(4) (LTB4), and high mobility group box 1 protein (HMGB1) and required engagement of receptors for both MIF and LTB4. Neutralization of MIF or blockade of the MIF receptor and/or LTB4 receptor resulted in protection from LPS-induced ALI. These findings indicate that the MIF and LTB4 mediator pathways are involved in the immunopathogenesis of LPS-induced experimental ALI. Most strikingly, complement activation does not contribute to the development of ALI in the LPS model.

Functional roles for C5a receptors in sepsis.

The function of the C5a receptors, C5ar (encoded by C5ar) and C5l2 (encoded by Gpr77), especially of C5l2, which was originally termed a 'default receptor', remains a controversial topic. Here we investigated the role of each receptor in the setting of cecal ligation and puncture-induced sepsis by using antibody-induced blockade of C5a receptors and knockout mice. In 'mid-grade' sepsis (30-40% survival), blockade or absence of either C5ar or C5l2 greatly improved survival and attenuated the buildup of proinflammatory mediators in plasma. In vivo appearance or in vitro release of high mobility group box 1 protein (HMGB1) required C5l2 but not C5ar. In 'high-grade' sepsis (100% lethality), the only protective condition was the combined blockade of C5l2 and C5ar. These data suggest that C5ar and C5l2 contribute synergistically to the harmful consequences in sepsis and that C5l2 is required for the release of HMGB1. Thus, contrary to earlier speculation, C5l2 is a functional receptor rather than merely a default receptor.