Insanity is doing the same thing over and over again and expecting different results.
—Albert Einstein (1879–1955)
Severe sepsis and septic shock is responsible for significant morbidity, mortality, and health care resource consumption worldwide. Although mortality has decreased over the last decade largely through early diagnosis (1), interventions, and supportive care, it continues to be unacceptable. Sepsis is the most expensive diagnosis necessitating hospitalization, with hospital costs exceeding $60 billion per year in the United States (2).
Sepsis is initiated by an infectious agent stimulating the expression of a wide and complex array of proinflammatory, anti-inflammatory, and apoptotic biomarkers (3). Elevated circulatory concentrations of these biomarkers have been implicated in the pathogenesis of organ dysfunction and mortality. Logically, these observations have given rise to numerous trials based on the hypothesis that antagonizing these biomarkers will improve outcomes. Although showing great promise in animal models, these therapies have been either equivalent or inferior to placebo in humans. After many decades and billions of dollars invested in animal models and clinical immunotherapy therapeutic trials (4); a product does not exist for clinical use. In the last year alone, three large multicenter randomized control trials have resulted in no outcome benefit. These trials include human recombinant activated protein C (5), eritoran tetrasodium (E5564, a Toll-like receptor 4 [TLR-4] antagonist) (6), and talactoferrin α (recombinant human lactoferrin) (7).
Improving the time to diagnosis and intervention has been shown to have a significant impact on outcomes in acute myocardial infarction, stroke, and trauma. This was made possible through a better understanding of the early pathogenesis of these diseases, leading to more specific targeting for adjunctive therapies. Over the last decade, a similar paradigm shift in clinical management has occurred for sepsis and has robustly led to significant mortality reduction (8). Although the causes for previously failed immunotherapy trials are numerous; a prolonged and inconsistent time from disease onset to therapeutic intervention is a common feature.
To maximize the use of targeted adjunctive immunotherapy, a comprehensive examination of early circulatory biomarker activity is needed to realize the optimal outcome benefit. The purpose of this article was to present new patient-level data on the early natural history of circulatory biomarker activity in the most proximal phases of severe sepsis and septic shock presentation. These findings will then be superimposed upon previous human outcome trials identified in the current literature targeting these specific biomarkers. It is quite possible that the timing of enrollment and intervention may be a significant underlying cause for repeated failures of these immunotherapy trials.
MATERIALS AND METHODS
Description of patient cohort
Plasma samples were obtained from a serum repository including patients enrolled in the study examining early goal-directed therapy (EGDT) in severe sepsis and septic shock (8, 9). To understand the natural pattern of biomarker activity and limit the influence of hemodynamic optimization on inflammation, only control patients (not receiving EGDT) were examined (9). Patients were excluded if they were on immunosuppressive therapy or replacement corticosteroid therapy or had care limitations provided on admission. No patients received recombinant activated protein C.
Timing of data collection
Demographics, baseline characteristics, and organ dysfunction scores were obtained at enrollment. Plasma samples and laboratory data were collected at 0, 3, 6, 12, 24, 48, 60, and 72 h after patient enrollment. Patients were followed up until hospital discharge and at 28 and 60 days.
The following biomarkers were assayed (Table 1): interleukin 1β (IL-1β), IL-1ra, IL-6, IL-8, IL-10, intercellular adhesion molecule (ICAM), tumor necrosis factor-α (TNF-α), caspase 3, D-dimer, high-mobility group protein 1 (HMGB1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP), and myeloperoxidase (MPO). Assay results were sent to the Henry Ford Hospital Department of Public Health Sciences, which maintained the database and performed the statistical analyses independent of the study investigators. The demographics, baseline clinical data, organ dysfunction scores, and mortality were examined by descriptive statistics.
We reviewed the literature for randomized immunotherapy trials to examine the timing of targeted immunomodulatory interventions in each study. We searched PubMed, MEDLINE, and Google Scholar for adult trials published in the last 25 years. Search terms used alone and in combination were “sepsis,” “severe sepsis,” “septic shock,” “biomarker activity,” “animal model of sepsis,” “human model of sepsis,” “immunotherapy trials,” “randomized trial,” “interleukins,” “timing of biomarker activity,” “immune-modulation,” “recombinant human activated protein C (rh-APC or drotrecogin alfa activated),” “IL-1ra,” “TNF-α,” “IL-6,” “IL-1β,” “IL-8,” “D-dimer,” “HMGB1,” “MMP-9,” “VEGF,” “ICAM-1,” “MPO,” “caspase 3,” and “IL-10.” We focused on these previously mentioned biomarkers during our search because of the available data from our own patient cohort. In addition, some of the biomarkers have been prominent targets in clinical trials examining immunotherapy. A secondary search focused on animal models of sepsis and sepsis simulations to determine the timing of targeted biomarker in the randomized immunotherapy sepsis trials. The findings of these two searches were then superimposed upon published and unpublished early biomarker activity of human severe sepsis and septic shock patients (8, 9). We excluded review articles, pediatric studies, studies enrolling healthy volunteers, studies without specified time frame of enrollment, and studies examining statin or dietary supplements. We identified a total of 39 studies for our analysis (Fig. 1).
Complete biomarker data were available in 104 patients from the control group in the EDGT study. The mean age was 63.0 ± 17.1 years, and there was an enrollment time of 1.7 ± 2.2 h after hospital arrival (Table 2). Organ dysfunction scores, including Acute Physiology and Chronic Health Evaluation II score, Simplified Acute Physiology Score II, Multiple Organ Dysfunction Score, Sequential Organ Failure Assessment, were commensurate with previous sepsis immunotherapy outcome trials. The majority of patients met criteria for septic shock at enrollment, with overall in-hospital mortality of 39.2%.
The peak and nadir of the biomarkers examined over the 72-h study period are illustrated in Table 3, relative to the time frames of immunotherapeutic interventions identified in our literature review. Interleukin 6, IL-1β, and IL-10 were highest at presentation. At 3 h after patient enrollment, we observed a peak in IL-1ra, TNF-α, and VEGF levels. Matrix metalloproteinase 9 peaked at 6 h, and IL-8 at 12 h. Within 24 to 48 h, peak levels of D-dimer, HMGB1, ICAM-1, MPO, and caspase 3 were reached. Several markers had a bimodal pattern in their peak levels, with all markers decreasing noticeably to their nadir within 48 h of patient enrollment.
The history of biomarker activity
The immunological pathogenesis of sepsis was eloquently characterized by Bone (3) in two phases—an early proinflammatory phase shifts into a later anti-inflammatory phase. An ideal response occurs when these two phases are balanced. The proinflammatory phase lasts the initial 24 to 72 h following the insult. The anti-inflammatory phase begins after 72 h and can last 2 to 8 weeks or even longer. When there is an exaggerated early response, early mortality associated with shock may be the result. With an excessive later response, immune suppression may occur with later-onset infections and multiorgan failure. In some patients, this late anti-inflammatory response can predominate and lead to “immune-paralysis,” requiring immunomodulation therapy to restore necessary immune integrity. Based on this observed early biomarker activity in animal models and our presented data; the phases of inflammation described by Bone are not distinct and essentially overlap. This observation questions the appropriateness of single treatment target for immunomodulation in sepsis. Furthermore, the greatest biomarker variability is measured in hours, not days, and can be bimodal following the initial presentation. The bimodal peaks of some biomarkers indicate that additional stimuli or interactions may contribute to the natural history of circulatory biomarker concentrations. For example, high levels of TNF-α, IL-6, IL-1ra, and IL-8 are associated with early hemodynamic deterioration and mortality in intensive care unit (ICU)–based studies (9, 10). This hemodynamic deterioration leading to tissue hypoxia can itself be a stimulus for inflammation in the progression septic shock (9, 11). This “second hit,” which is multifactorial, has been previously described in the development of multiorgan failure and, particularly, the delayed need for mechanical ventilation (12) and renal replacement therapy (13). Outcome differences have been observed for early-versus late-onset septic shock (10).
The animal and human models of biomarker activity
In animal models, the sepsis insult is distinct (i.e., cecal perforation), and the elevation of biomarkers is seen within hours. In well-controlled human studies simulating sepsis, the response to an insult (endotoxin) begins with symptoms of the systemic inflammatory response syndrome within 1 h, with biomarker elevation within 3 h after endotoxin introduction (14, 15). Changes in organ function are seen within 3 to 6 h, with many peaking by 3 to 5 h (14, 16). The consistently reproducible insults of these human and animal experimental sepsis models, lack of comorbidities, well-controlled standards of care, and the timing of insult to immunotherapy intervention create a high level of homogeneity. Although this concept is debatable (17), this level of homogeneity is difficult to reproduce in human outcome trials (17). A step toward understanding why successful human and animal models in sepsis have yet to yield success begins with addressing and understanding the variables contributing to this heterogeneity (Table 4).
The clinical reality of sepsis management and its outcome implications
Based on a review of temporal biomarker activity in animal and human models of sepsis and our own observations in patients presenting with community-acquired sepsis; the most effective time to intervention with immunotherapy is immediate or within the first 12 h of onset of severe sepsis and septic shock. This timing is either to directly antagonize or to prevent the peak levels of biomarkers that occur within this period.
However, the reality is that there are significant delays in both diagnosis and therapeutic interventions, with outcome and pathogenic implications when patients are enrolled in clinical trials. The emergency department (ED) is the portal of entry for more than 52.4%, general practice unit (GPU) for 34.8%, and the ICU for 12.8% of all sepsis patients presenting to the hospital (18). The mortality rates are 27.6%, 41.3%, and 46.8% for each of these respective locations. Thus, it is critically important that patients are diagnosed as soon as possible because mortality can almost be 20% higher if a patient is admitted to the GPU instead of the ICU from the ED. Patients with a sepsis diagnosis can frequently have symptoms for up to 24 h before hospital arrival. When this is combined with an ED waiting time from 5 to 24 h, there is significant “incubation” time before definitive care and study enrollment (19).
After hospital presentation, the disease severity can be underestimated, and these patients are not uncommonly admitted to the GPU to later decompensate requiring ICU transfer (20). Carr et al. (21) showed that up to 10% of in-hospital cardiac arrests carry an admission diagnosis of pneumonia. This median time to the cardiac arrest associated with septic shock was 18.9 h after ICU and 24.8 h after GPU admission. Thus, observational studies reveal that 87.2% of severe sepsis and septic shock patients originate outside the ICU (18). This source of heterogeneity in illness severity and location (community- vs. hospital-acquired sepsis) upon enrollment may provide insight into the failure of previous human trials of immunotherapy.
The influence of hemodynamic status and optimization on biomarker activity
The degree of resuscitation impacts biomarker activity before enrollment. The temporal patterns of IL-1ra, ICAM-1, TNF-α, caspase 3, and IL-8 from admission to the first 72 h of hospitalization are significantly associated with the severity of global tissue hypoxia, organ dysfunction, and mortality. These findings identify global tissue hypoxia as an important contributor to the early inflammatory response and support the role of hemodynamic optimization in supplementing other established therapies during this diagnostic and therapeutic window of opportunity (9, 10).
Sevransky et al. (22) noted in a review that clinical trials inconsistently specify hemodynamic goals. The wide range of hemodynamic treatment targets suggests a lack of agreement for management of patients with sepsis. The integral components to hemodynamic optimization such as fluid, vasoactive therapy, transfusion, mechanical ventilation, and sedation may all impact biomarker activity. This lack of consistency in hemodynamic goals may contribute to heterogeneity in treatment effects for clinical trials of novel sepsis therapies (22).
The influence of antibiotic therapy on inflammation
Antibiotic-induced endotoxin release has been observed in both animal and human models of sepsis after antibiotic administration (23). The antibiotic ceftazidime exerts an antioxidant effect in vitro. It inhibits perferryl-mediated lipoperoxidation iron scavenging, neutralizes hydrogen peroxide chloride, and quenches singlet oxygen (24). Multiple animal studies have shown a reduction in levels of IL-1 and TNF-α and mortality, when administering tetracycline (a proposed inhibitor of MMP-9) 20 min before i.v. lipopolysaccharide (LPS) with repeat half dose at 6 and 24 h, compared with controls (25). These effects were no longer statistically significant when tetracycline administration was delayed as little as 1 h following LPS (40%–60% survival), and at 4 h after LPS, tetracycline offered no benefit over controls (10%–20% survival). Similar findings have been shown up to 12 h in the cecal perforation model (26). Experimental studies of gram-negative sepsis have shown considerable attenuation of the systemic inflammatory response following i.v. administration of macrolides such as clarithromycin (27). Thus, the influence of antibiotic administration refers not only to its outcome implications (28) but also to its contribution to the heterogeneity in early biomarker levels (18).
Generalized immune modulation
Toll-like receptors bind to pathogen-associated molecular patterns and initiate intracellular signaling cascades that result in the synthesis and release of proinflammatory biomarkers such as TNF-α, IL-1, IL-6, and IL-8. Toll-like receptor 4, in particular, binds LPS and other ligands, forming a TLR-4–CD14 complex, which plays a prominent role in the inflammatory reaction to infection (29). TAK-242, a TLR-4–mediated signaling inhibitor, interferes with signal transduction mediated through the CD14–TLR-4 complex without directly inhibiting the binding of LPS to TLR-4 and has been shown to provide a survival advantage in vitro (29). Despite promising phase II trials, the most recent phase III trial of eritoran tetrasodium treatment blocking TLR-4 has failed to reduce mortality (6, 30). The mean time from shock or respiratory failure was approximately 19 h with initiation of the study drug within 36 h of onset of shock or respiratory failure (31).
Glucocorticoids have a range of anti-inflammatory actions leading to general suppression of inflammation. Glucocorticoids can inhibit the intracellular upregulation of necrosis factor κB, which stimulates the production of key proinflammatory biomarkers TNF-α and IL-1. They also inhibit production of the inducible nitric oxide synthase and decrease the release of other biomarkers including platelet activating factor (32). High-dose corticosteroids in sepsis have not consistently resulted in outcome benefit (33). The two most recent multicenter trials examining low-dose corticosteroids in septic shock show conflicting results in regard to outcome (34, 35). The time of intervention, 8 h in the first trial (positive benefit) versus up to 72 h in the second trial (no benefit), may hold an explanation for these divergent findings.
Outcome studies examining ibuprofen (36) administered up to 24 h after organ failure and cyclooxygenase inhibitor (lornoxicam), which allowed for enrollment up to 8 h following ICU admission, have shown no differences in mortality between treatment and control arm (37). Despite a recent trial showing no outcome benefit (5), it is plausible that the beneficial effects may be realized if drotrecogin alfa activated is administered at an even earlier period.
Talactoferrin, an iron-binding protein found not only in mucosal secretions but also in secondary granules of neutrophils, has been shown to have anti-infective and anti-inflammatory properties. Among various immunomodulatory properties of recombinant human lactoferrin is the ability to compete with E-selectin for LPS binding. This binding has been shown to downregulate to production of reactive oxygen species in neutrophils and has been associated with the prevention of further tissue damage following infection if administered early. The phase 2 trial of oral talactoferrin in patients with severe sepsis and septic shock achieved an absolute 28-day all-cause mortality reduction of 12.5% (placebo group mortality 26.9%, talactoferrin group 14.4%, P = 0.05) and in patients without cardiovascular dysfunction (23.3% placebo vs. 2.6% talactoferrin group) (30). The most recent phase II/III trial of oral talactoferrin in sepsis (OASIS trial) was discontinued because of no observed mortality benefit (38). Randomization had to occur within 24 h of severe sepsis or septic shock onset with an additional 4 h to initiation of treatment representing a significant lead time based on the dynamic nature of early biomarker activity.
Other approaches of generalized immunomodulation have yet to show that they are feasible for the general sepsis population. Smaller trials of hemofiltration show promising results in a well-defined surgical population (39). Trials with granulocyte-macrophage colony-stimulating factor have been shown to be safe and effective in restoring the monocytic immunocompetence (40). The treatment, however, was not able to demonstrate significant mortality reductions.
LIMITATIONS AND FUTURE DIRECTIONS
Biomarkers may have significant effects at the receptor or tissue level, which suggest that detectable circulatory levels may not accurately reflect disease activity within the cell. The development of diagnostic tools that allow real-time and accurate determination of biomarker concentrations may significantly contribute to improved characterization of the individual inflammatory state of the patient. Some of the larger failed trials did not publish their data, and others did not detail the time to treatment from patient presentation. These limitations hinder investigators who seek methodological information to avoid continued failure in the design of future sepsis clinical trials.
At the most proximal point of hospital presentation, patients presenting with community-acquired severe sepsis and septic shock already have elevated circulatory biomarker levels. These circulatory biomarkers overlap, have bimodal patterns, and generally peak between 3 and 36 h while diminishing over the subsequent 72 h of observation. When this early natural history and the clinical realities of sepsis care are taken into account, prior sepsis immunotherapy trial enrollment is generally delayed after the window of peak circulatory biomarker concentrations. Thus, there is a need to recalibrate the timing of enrollment and intervention in future immunotherapy trials that still may hold great promise for this deadly disease.
The authors thank the EGDT Collaborative Group for this study composed of the following: Quanniece Rivers, BS; Katie Floyd, MA; Shajuana Rivers; Stacie Young; Ruben Flores, PhD; Scott Rongey, PhD; Scok-Won Lee, PhD; H. Matilda Horst, MD, Kandis K. Rivers, MD; Khalil K. Rivers, Arturo Suarez, MD, Damon Goldsmith, Peter Nwoke, MD, Bernhard Knoblich, MD; the nursing, technical, administrative, and support staff in the ED and ICUs. They also thank the patients and their families; the Department of Emergency Medicine nurses, residents, senior staff attending physicians, pharmacists, patient advocates, technicians, billing and administration personnel, and medical and surgical ICU nurses and technicians; and the Departments of Respiratory Therapy, Pathology, Medical Records, and Admitting and Discharge for their patience and cooperation in making this study possible.
ALI — acute lung injury
ARDS — acute respiratory distress syndrome
CD14 — cluster of differentiation 14
ED — emergency department
EGDT — early goal-directed therapy
GPU — general practice unit
HMGB1 — high-mobility group protein 1
ICAM-1 — intercellular adhesion molecule 1
ICU — intensive care unit
IL-1ra — interleukin 1 receptor antagonist
IL-1β — interleukin 1beta
IL-6 — interleukin 6
IL-8 — interleukin 8
IL-10 — interleukin 10
LPS — lipopolysaccharide
MMP — matrix metalloproteinase
MPO — myeloperoxidase
NO — nitric oxide
TLR-4 — Toll-like receptor 4
TNF-α — tumor necrosis factor alpha
VEGF — vascular endothelial growth factor
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