could biomarkers direct therapy for the septic...

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Could Biomarkers Direct Therapy for the Septic Patient?

Clark R. Sims, Trung C. Nguyen, and Philip R. Mayeux

Department of Pharmacology and Toxicology, University of Arkansas for Medical

Sciences, Little Rock, Arkansas (C.R.S., P.R.M.), and Department of Pediatrics, Section

of Critical Care Medicine, Baylor College of Medicine/Texas Children’s Hospital,

Houston, Texas (T.C.N)

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 8, 2016 as DOI: 10.1124/jpet.115.230797

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Running Title: (60 characters): Biomarker-Directed Therapy for the Septic Patient?

Corresponding Author:

Philip R. Mayeux, PhD

4301 West Markham Street, #611

Little Rock, AR 72205

501-686-8895

FAX: 501-686-5521

[email protected]

Number of text pages: 23

Number of tables: 0

Number of figures: 2

Number of references: 196

Number of words in Abstract: 190

Number of words in Introduction: 981

Number of words in Discussion: N/A

Section Assignment: Minireviews

Abbreviations: ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin

type 1 motif, member 13; Ang, angiopoietin; CARS, compensatory anti-inflammatory

response syndrome; cfu, colony forming units; CLP, cecal ligation and puncture; CRP,

C-reactive protein; DAMPs, damage-associated molecular patterns; DIC,

disseminated intravascular coagulation; EEG, electroencephalography; GM-CSF,


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granulocyte macrophage colony-stimulating factor; HLA-DR, human leukocyte antigen

- antigen D related; ICAM-1, intercellular adhesion molecule 1; ICU, intensive care

unit; IL, interleukin; KIM-1, kidney injury molecule 1; LPS, lipopolysaccharide; MMP-8,

matrix metalloproteinase-8; MODS, multiple organ dysfunction syndrome; MRI,

magnetic resonance imaging; NAG, N-acetyl-β-(D)-glucosaminidase; NGAL,

neutrophil gelatinase-associated lipocalin; NIRS, near-infrared spectroscopy; PAMPs,

pathogen-associated molecular patterns; PCT, procalcitonin; PD-1, programmed cell

death 1; S1P, sphingosine-1-phosphate; SIRS, systemic inflammatory response

syndrome; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor α; VCAM-1,

vascular cell adhesion molecule 1; VWF, von Willebrand factor


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Abstract

Sepsis is a serious medical condition caused by a severe systemic inflammatory

response to a bacterial, fungal, or viral infection that most commonly affects neonates

and the elderly. Advances in understanding the pathophysiology of sepsis have resulted

in guidelines for care that have helped reduce the risk of dying from sepsis for both

children and older adults. Still, over the past three decades, a large number of clinical

trials have been undertaken to evaluate pharmacological agents for sepsis.

Unfortunately, all of these trials have failed, with the use of some agents even shown to

be harmful. One key issue in these trials was the heterogeneity of the patient population

that participated. What has emerged is the need to target therapeutic interventions to

the specific patient’s underlying pathophysiological processes, rather than looking for a

universal therapy that would be effective in a “typical” septic patient who does not exist.

This review supports the concept that identification of the right biomarkers that can

direct therapy and provide timely feedback on its effectiveness will enable critical care

physicians to decrease mortality of patients with sepsis and improve the quality of life of

survivors.


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Introduction

Sepsis is a serious medical condition caused by a severe systemic inflammatory

response to a bacterial, fungal, or viral infection (Martin, 2012; Dellinger et al., 2013). As

sepsis worsens, organ dysfunction (severe sepsis) or severe sepsis plus hypotension

(septic shock) can develop (Dellinger et al., 2013). Sepsis affects approximately

700,000 people annually in the U.S. alone (Chang et al., 2015). Over 25% of these

patients die before leaving the hospital, making sepsis the 9th leading cause of death

overall (Heron, 2015). In addition, relative to the total number of cases, sepsis is the

most costly medical condition, especially given that over 20% of sepsis survivors are

readmitted within 30 days and nearly 30% of these patients have recurrent sepsis (Torio

and Andrews, 2013; Chang et al., 2015). The estimated annual health care burden is

over $20 billion in the U.S. alone (Torio and Andrews, 2013).

While sepsis affects people of all ages, the most commonly affected populations

are neonates, due to an immature immune system and still developing organs, and the

elderly, due to chronic and comorbid conditions. In the U.S., sepsis is the 6th and 7th

leading cause of death in neonates and infants, respectively, and the 10th leading cause

of death in patients age 65 and older (Heron, 2015). Mortality rates for children with

septic shock range from 9% to 25% (Hartman et al., 2013; Weiss et al., 2015), and for

older adults mortality rates are as high as 45% (Daviaud et al., 2015). Although the

absolute number of deaths from sepsis is increasing due to increasing incidence (Martin,

2012; Gaieski et al., 2013), advances in the understanding of the pathophysiology of

sepsis have reduced the risk of dying from sepsis for both children (Kissoon et al., 2011;

Ruth et al., 2014) and older adults (Stevenson et al., 2014).


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The pathophysiology of sepsis is very complex and still poorly understood.

Initially, local activation of the innate immune response by pathogen-associated

molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)

results in a “cytokine storm” (Wang and Ma, 2008; Gustot, 2011; Laszlo et al., 2015). If

the inflammatory response becomes systemic it manifests as the systemic inflammatory

response syndrome (SIRS, (Conference, 1992)). Patients who survive the initial

hyperinflammatory state may progress to a hypoinflammatory state and develop the

compensatory anti-inflammatory response syndrome (CARS, (Bone, 1996; Ward et al.,

2008)), which increases the susceptibility to recurrence of the primary infection or a

secondary acquired infection. Disruption of the balance between these inflammatory

and anti-inflammatory responses in sepsis leads to multiple organ dysfunction

syndrome (MODS), which significantly increases mortality (Wang and Ma, 2008; Gustot,

2011; Boomer et al., 2014).

Mortality from sepsis has significantly decreased over the years because of early

recognition and resuscitation, as documented in the “Surviving Sepsis Campaign”

(Dellinger et al., 2013). Following recommendations from this document, current

therapies are mostly supportive care. Initial treatment includes broad-spectrum

antibiotics and then fluid resuscitation if necessary. As sepsis severity increases,

therapy may require administration of vasopressors, use of mechanical respiratory

support, and/or renal replacement therapy (Dellinger et al., 2013). Over the past three

decades, there have been over 60 large Phase II and III randomized, controlled clinical

trials of single pharmacological agents for sepsis, all of which have failed, with some

agents even found to be harmful (Fink and Warren, 2014). Reflecting on these failed


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trials, many leaders in the field believe that the heterogeneity of the patient population

that participated in these trials is a key issue (Cohen et al., 2015). To address this issue,

various critical care societies are currently revising the definition of the sepsis syndrome

to refine the inclusion criteria for participants in future sepsis trials. One common theme

is surfacing: infection plus organ dysfunction should be considered as inclusion criteria

rather than just the current two or more SIRS criteria (Vincent et al., 2013; Cohen et al.,

2015; Drewry and Hotchkiss, 2015; Kaukonen et al., 2015). In addition it has been

suggested that there is a need to target therapeutic interventions according to the

specific patient’s underlying pathophysiological processes, rather than looking for a

universal therapy that would be effective in the "typical” patient population with sepsis

(Boomer et al., 2014; Christaki and Giamarellos-Bourboulis, 2014).

The complex pathophysiology of sepsis and the ineffectiveness of current

targeted therapies suggest that treatments guided by biomarkers could provide a new

therapeutic strategy. A biomarker, as defined by an NIH working group, is “a

characteristic that is objectively measured and evaluated as an indicator of normal

biological processes, pathogenic processes, or pharmacologic responses to a

therapeutic intervention” (Group, 2001). There are general and overlapping categories

of biomarkers, which have been reviewed and discussed in detail elsewhere (Marshall

et al., 2009; Kaplan and Wong, 2011; Dupuy et al., 2013; Sandquist and Wong, 2014).

Biomarker categories include 1) screening biomarkers to identify patients at increased

risk and inform on prophylactic interventions, 2) diagnostic biomarkers to establish the

presence or absence of disease and inform treatment decisions, 3) stratification

biomarkers to stage severity and outcome of disease and help identify subgroups of


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patients who may benefit or be harmed by therapy, 4) monitoring biomarkers to inform

on the effectiveness of a therapy, and 5) surrogate biomarkers to inform on disease

progression and potential outcomes of therapy. A number of excellent recent reviews

have been published on the potential diagnostic and prognostic value of biomarkers in

sepsis (Pierrakos and Vincent, 2010; Sandquist and Wong, 2014; Biron et al., 2015;

Cohen et al., 2015). Therefore, this review will not focus on the current status of sepsis

biomarkers. Rather, the goal of this review is to support the concept that the appropriate

biomarkers could be used to direct targeted therapies (Fig. 1). For example, diagnostic

biomarkers could direct therapies such as those targeting immune status, endothelial

injury, coagulation, and microcirculatory failure; and stratification biomarkers could

inform the decision to rapidly employ renal replacement therapy or mechanical

ventilation.

Patient Heterogeneity

Both the incidence and the outcome of sepsis are greatly influenced by patient

heterogeneity including patient-specific factors such as age, comorbid medical

conditions, and genetic predispositions. The very young (<1 year) and the elderly (>65

years) account for 76% of all deaths due to sepsis in the U.S. (Heron, 2015). Premature

and very-low-birth-weight infants suffer the highest mortality (Heron, 2015), which may

be due to increased hemodynamic fragility, low cardiac output, and organ hypoperfusion,

as well as a decreased ability to mount an appropriate immune response (Adkins et al.,

2004; Aneja and Carcillo, 2011; Shah and Padbury, 2014). Likewise, in elderly patients,

debilitated physiological processes, reflected in higher incidences of congestive heart


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failure, coronary artery disease, diabetes, hypertension, and chronic obstructive

pulmonary disease, underlie a 13-fold higher relative risk for the development of sepsis

(Martin et al., 2006) and an increased mortality from sepsis (Boss and Seegmiller, 1981).

These physiological differences in sepsis with respect to age and comorbid medical

conditions suggest that treatments will need to be based on the specific conditions of

the individual patient.

The presence of MODS is an important factor in the treatment of sepsis. While

there is substantial variation in the percentages of patients with any specific organ

involvement, pulmonary, renal, cardiovascular, hepatic, neurological, and hematological

dysfunction may result from primary and secondary causes (Gaieski et al., 2013;

Iskander et al., 2013; Stubbs et al., 2013; Ziaja, 2013; Fiusa et al., 2015). In all

populations, the mortality rate of sepsis greatly increases as multiple organ systems fail

(Gaieski et al., 2013; Iskander et al., 2013; Romanovsky et al., 2013).

Because of the inherent heterogeneity of patients enrolled in clinical trials, a level

of heterogeneity in the therapeutic response should be expected. Most important,

however, while some patients will benefit from the intervention, others could be harmed

(Marshall, 2014). To demonstrate overall efficacy of a treatment, studying larger

numbers of patients to increase statistical power is required in trial design, but this will

increase costs and may still not uncover efficacy in the general population for the

reasons just described. Early-phase sepsis trials should focus on identifying an

appropriate study population and use more specific inclusion criteria to limit trials to the

target populations suggested by preclinical studies. This approach could reduce

heterogeneity and increase the likelihood of demonstrating efficacy.


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Biomarkers

Validation of biomarkers requires an extensive amount of research and

resources. A limitation of biomarker research in human sepsis is that there are rarely

data on preillness levels of a biomarker. Because many septic patients have comorbid

chronic inflammatory conditions such as coronary artery disease and diabetes,

biomarkers alone are unlikely to accurately inform on the severity of sepsis (Ginde et al.,

2014) but may inform on possible outcomes (Kojic et al., 2015). For example, the ideal

biomarker could identify those patients at risk for specific organ dysfunction and thereby

help direct more targeted therapy. In addition, gene expression patterns could lead to

candidate serum protein markers to predict organ-specific injury (Basu et al., 2011).

Given the complex nature of sepsis, achieving this goal will likely require monitoring

multiple biomarkers through the course of the disease and the course of therapy (Wong

et al., 2015a). Biomarkers also depend on the specific pathogen, or often pathogens,

causing sepsis. Consequently, there is a need to accurately and quickly distinguish

sepsis from noninfectious SIRS. These are all areas where proteomics and genomics

(Cao and Robinson, 2014; Cao et al., 2014; Christaki and Giamarellos-Bourboulis,

2014; DeCoux et al., 2015) and rapid identification of positive blood cultures (Bhatti et

al., 2014b; Bhatti et al., 2014a) could become very helpful.

Inflammatory Biomarkers

Should the goal of therapy for septic patients be to decrease inflammation or

boost host immunity? This is an extremely important and difficult question with no


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simple answer. Hotchkiss and Sherwood (Hotchkiss and Sherwood, 2015) have

published a recent commentary on this issue. However, biomarkers have the potential

to help critical care physicians make important clinical decisions regarding

immunotherapy (Hotchkiss et al., 2013b).

In general, blood levels of soluble adhesion molecules such as E-, L- and P-

selectin, ICAM-1, and VCAM-1 correlate with the presence of sepsis in neonates,

children, and adults; however, there are age-related differences in the absolute levels,

and it is still unclear whether changes in levels might correlate positively or negatively

with outcomes (Zonneveld et al., 2014). C-reactive protein (CRP) is an acute-phase

protein released by the liver. It is used as an inflammatory biomarker but cannot

distinguish between infection and noninfectious diseases (Kojic et al., 2015).

Procalcitonin (PCT) is a propeptide of calcitonin released in the blood in response

bacteria-associated proinflammatory mediators and inflammatory cytokines. Its use as a

specific biomarker for sepsis has come under question (Talan, 2015). CRP and PCT

may be more general biomarkers of moderate to severe inflammation but could be

useful for informing on the response to treatment (Wacker et al., 2013; Rule et al., 2015).

However, the levels of PCT and CRP in combination may be able discriminate bacterial

sepsis from other causes of SIRS (Han et al., 2015).

Cytokines including tumor necrosis factor α (TNF-α) and interleukin (IL)-1, IL-6,

IL-8, IL-27, and others have been evaluated individually and in combination as potential

biomarkers for screening and diagnosis (Xiao et al., 2015). Although cytokine levels are

relatively easily and rapidly measured with multiplex assays (Mera et al., 2011), the

cost–benefit of monitoring them has yet to be established. Serial cytokine


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measurements would be required to inform on immune status; however, this would

require a thorough understanding of the complex kinetics of synthesis, release, and

clearance. Moreover, elderly septic patients present with generally higher levels of

inflammatory biomarkers due to comorbid conditions (Ginde et al., 2014), and because

pre-sepsis levels would be unknown, interpreting changes (or not) in cytokine levels

would be extremely difficulty. These issues may explain, at least in part, why clinical

trials targeting cytokines failed to demonstrate efficacy; use of some agents was even

harmful (Fink and Warren, 2014).

Septic patients in the immunosuppressive phase could potentially benefit from

immune-enhancing therapies, but these patients must first be identified. Potential

biomarkers, which have been suggested for this purpose, include decreased monocyte

human leukocyte antigen – antigen D related (HLA-DR) expression for monocyte

unresponsiveness, increased programmed cell death 1 (PD-1) expression for T cell

exhaustion, lymphopenia, and increased percentage of regulatory T cells, among others

(reviewed in (Hotchkiss et al., 2013b) and (Boomer et al., 2014)). The use of specific,

biomarker-directed immunotherapies such as granulocyte macrophage colony

stimulating factor (GM-CSF) to stimulate mature myeloid cell expansion in patients with

granulocytopenia (Mathias et al., 2015), and IL-7 to promote lymphocyte expansion in

patients with lymphopenia (Unsinger et al., 2010; Hutchins et al., 2014) are examples of

new therapeutic strategies with the potential to advance the treatment of the

immunosuppressed septic patient (Hotchkiss et al., 2013a; Boomer et al., 2014).

Endothelial Biomarkers


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Sepsis is associated with severe injury to vascular endothelial cells. Injury to the

endothelium leads to dysregulation of vascular tone, hemostasis, and the permeability

barrier. Microvascular leakage is a hallmark of sepsis and results from damage to the

endothelial cell barrier lining the microvasculature. Identifying possible targeted therapy

to stabilize and even promote repair of the endothelial barrier is a very active area of

research (Darwish and Liles, 2013). Syndecan-1 and heparan sulfate are potential

biomarkers of damage to the endothelial glycocalyx, the antiadhesive and anticoagulant

surface of the endothelial cell (Schmidt et al., 2012) and, if confirmed as such, could be

used as diagnostic and monitoring biomarkers to direct and evaluate anticoagulant

therapy (Ostrowski et al., 2015; Yini et al., 2015).

A number of signaling pathways regulating vascular permeability appear to be

disrupted during sepsis. One such system is the endothelial-specific angiopoietin growth

factor (Ang) and its Tie-2 tyrosine kinase receptor (Maisonpierre et al., 1997; Ziegler et

al., 2013). Plasma levels of Ang-1, Ang-2, and Tie-2 have been show to predict the

onset of acute lung injury (Agrawal et al., 2013), 28-day mortality in septic adults (Fang

et al., 2015), and sepsis severity in children (Wang et al., 2014). If verified, levels of

Ang-1, Ang-2, and Tie-2 in septic patients may inform on the effectiveness and outcome

of therapy affecting the endothelium, such as the use of statins (Ghosh et al., 2015).

Another system is the sphingosine-1-phosphate (S1P) signaling pathway, a key

regulator of endothelial stability and vascular permeability (Lee et al., 1999; Wang and

Dudek, 2009). Serum levels of S1P in septic patients are decreased and inversely

associated with disease severity (Winkler et al., 2015). If S1P levels can be verified as a

biomarker of microvascular injury, S1P could be used to screen, diagnose, and/or


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stratify patients and direct the use of S1P receptor 1 agonists, shown to protect the

endothelial glycocalyx in vitro (Zeng et al., 2014) and to heal the renal microcirculation

in a murine model of sepsis (Wang et al., 2015b).

During sepsis, injured endothelial cells release microparticles containing proteins

and lipids that can augment thrombosis, inflammation, and microvascular injury.

Understanding the role of microparticles in the progression of sepsis and organ injury is

also a very active area of research. Souza and colleagues (Souza et al., 2015) have

published an excellent recent review. As a potential biomarker, microparticles may help

stratify patients and inform on the potential outcomes of therapy targeting the

endothelium.

Coagulation Biomarkers

Sepsis with its associated inflammation induces a state of dysregulated

hemostasis. Up to 30–50% of septic patients have clinical overt disseminated

intravascular coagulation (DIC), which leads to dissemination of fibrin-rich microvascular

thromboses and contributes to MODS (Levi and van der Poll, 2013). Many specific

monotherapeutic agents that interfere with dysregulated hemostasis in septic patients

have been tried without success in large randomized, controlled trials. These agents

include heparin, antithrombin III, recombinant tissue pathway inhibitors, recombinant

activated protein C, and recombinant soluble thrombomodulin (Warren et al., 2001;

Abraham et al., 2003; Nadel et al., 2007; Saito et al., 2007; Jaimes et al., 2009; Ranieri

et al., 2012). These trials, however, enrolled heterogeneous septic patients and did not

use coagulation biomarkers as titratable therapeutic endpoints. On reflection, the failure


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of these trials suggests that, as described earlier, study design and patient selection

rather than the specific agents may be at fault (Cohen et al., 2015). Currently, clinicians

rely on point-of-care global measurement of the hemostatic pathway including

prothrombin time, activated partial thromboplastin time, the fibrin degradation product D-

dimer, and platelet count to provide blood product support for hemostasis (Wada et al.,

2013). For future clinical trials, in addition to the point-of-care assays, measuring,

replacing, and normalizing the activities of heparin, antithrombin III, tissue factor,

activated protein C, and/or thrombomodulin in septic patients with a defined severe

coagulopathy perhaps might lead to better outcomes.

Sepsis-induced thrombotic microangiopathies driven by either von Willebrand

factor (VWF)/platelets and/or complement pathways will lead to dissemination of

VWF/platelet-rich microvascular thromboses and MODS (Nguyen et al., 2014;

Charchaflieh et al., 2015). Measuring VWF, the presence of ultralarge VWF multimers,

and ADAMTS-13 (VWF-cleaving protease) and directing therapies to normalize their

activities have been shown to improve outcome in children with sepsis-induced

thrombocytopenia-associated multiple organ failure (Nguyen et al., 2008; Fortenberry et

al., 2012). Low levels of ADAMTS-13, high VWF activity, and the presence of ultralarge

VWF multimers have been associated with worse outcomes in septic adults (Martin et

al., 2007; Bockmeyer et al., 2008). Sepsis-induced complement pathway dysregulation

leading to DIC and thrombotic microangiopathies has been demonstrated in human

clinical studies and animal models (Charchaflieh et al., 2015; Zhao et al., 2015).

Eculizumab, a monoclonal antibody against complement C5, has been shown to

improve outcome in severe Escherichia coli–induced hemolytic uremic syndrome, which


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leads to thrombotic microangiopathy and MODS (Lapeyraque et al., 2011; Noris et al.,

2012). Research is needed to prove whether other types of infection-induced thrombotic

microangiopathy and MODS can be ameliorated by measuring and normalizing

complement activities.

Physiological Biomarkers

Biomarkers are generally assumed to mean small molecules. However,

physiological biomarkers are the most rapidly determined indices of organ dysfunction

and can often be measured at the bedside. The complex pathophysiology of sepsis can

have a major impact on the translation of experimental therapies to clinical practice.

This is one of the key reasons that animal models of sepsis have had, thus far, a limited

impact on therapy in humans (Dyson and Singer, 2009; Marshall, 2014; Efron et al.,

2015). Changes in cardiac output, organ perfusion, drug delivery to therapeutic target,

and clearance of the drug are dynamic and often unpredictable in septic patients.

Physiological biomarkers could be of great help in interpreting successes, failures, and

undesirable outcomes in sepsis clinical trials.

The application of transthoracic echocardiography with speckle-tracking

technology to detect subclinical myocardial dysfunction may have value in directing

cardioprotective strategies, even if the ejection fraction remains normal (Shahul et al.,

2015). This type of monitoring may also help in establishing prognosis in septic shock

patients because, despite a normal ejection fraction, severe right ventricular wall

longitudinal strain dysfunction is associated with a high rate of mortality in patients with

severe sepsis and septic shock (Orde et al., 2014). There is a pressing need for


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biomarkers to screen for sepsis-induced cardiomyopathy. Extracellular histones may

emerge as such a biomarker (Alhamdi et al., 2015; Kalbitz et al., 2015).

There is also a need for minimally invasive techniques that permit direct

physiological measurements of microvascular flow and organ perfusion in critically ill

patients. One of the most routine measurements made in septic patients is blood lactate

concentration. Lactate production is a protective response to allow cellular energy

production to continue when tissue oxygen supply is inadequate for aerobic metabolism

(Suetrong and Walley, 2015). Serum lactate levels remain a strong, independent

predictor of lung injury (Mikkelsen et al., 2013). Elevated lactate levels are highly

associated with in-hospital mortality and can be used to stratify patient risk (Casserly et

al., 2015). Use of lactate as a biomarker to direct management of the microcirculation

during the later stages of sepsis can be complicated by changes in lactate clearance

(Chertoff et al., 2015). Better understanding of the relationships between lactate levels

and actual status of the microcirculation remains a critical need (De Backer et al., 2013).

Organ-specific microcirculation can be measured in animal models of

experimental sepsis and used to evaluate the therapeutic potential of new approaches

to improve organ perfusion (Dear et al., 2005; Wu et al., 2007; Holthoff et al., 2012; Fink

et al., 2013; Taccone et al., 2014). As therapeutic strategies emerge to target

microvascular dysfunction, physiological biomarkers measureable at the bedside could

assist in directing therapy and confirming efficacy (or revealing harmfulness) (Matejovic

et al., 2015). Cine phase-contrast MRI and contrast-enhanced ultrasound can be

performed on critically ill septic patients to monitor blood flow in the kidney (Schneider

et al., 2013). For example, cine phase-contrast MRI revealed that renal blood flow was


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consistently reduced as a fraction of cardiac output in septic patients with established

acute kidney injury (Prowle et al., 2012). Although these minimally invasive techniques

for monitoring organ perfusion are still in the validation phase, their use could ultimately

guide the choice of therapy and evaluate effectiveness. For example, changes in renal

perfusion paired with renal injury biomarkers such as neutrophil gelatinase-associated

lipocalin (NGAL), kidney injury molecule 1 (KIM-1), N-acetyl-β-(D)-glucosaminidase

(NAG), and matrix metalloproteinase-8 (MMP-8) (Liangos et al., 2007; Basu et al., 2011;

Wang et al., 2015a) could direct the implementation of renal replacement therapies

(hemofiltration and/or dialysis) that could improve outcomes in some patients; however,

this approach is not without risk (Elseviers et al., 2010; Sun et al., 2014; Prowle and

Davenport, 2015; Wald et al., 2015).

Orthogonal polarization spectral imaging and side-stream dark-field imaging

techniques can be used at the bedside to obtain real-time measurements of the

sublingual microcirculation in humans with sepsis (De Backer et al., 2012). Even though

changes in the sublingual microcirculation cannot predict the status of other organ-

specific microvascular beds, there is an excellent correlation between decreases in the

proportion of perfused small sublingual vessels in early sepsis and decreased survival

rates (De Backer et al., 2013; De Backer et al., 2014). These techniques could be used

as diagnostic and monitoring physiological biomarkers to direct therapy targeting the

vasculature, as suggested in animal models of sepsis (Holthoff et al., 2013; Wang et al.,

2015b).

Sepsis-associated encephalopathy with cognitive impairment is a common

complication of sepsis that places a substantial burden on survivors of sepsis, their


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families, and their caregivers (Iwashyna et al., 2010). While the pathophysiology is still

poorly understood, neuroinflammatory and ischemic process are likely contributors to

alterations in the blood–brain barrier, release of neurotoxic mediators, and microglial

activation (Adam et al., 2013). Changes in standard electroencephalogram (EEG) are

associated with increased mortality and delirium in septic ICU patients (Azabou et al.,

2015). Near-infrared spectroscopy (NIRS) is being evaluated in high-risk septic patients

as a method to monitor brain tissue oxygenation to predict the development of acute

neurological dysfunction (Wood et al., (in press)). Interest is growing in identifying

biomarkers for sepsis-induced neurological injury (Bersani et al., 2015), especially

because animal models are revealing long-term brain alterations that affect learning and

memory and persist into adulthood (Comim et al., 2015; Gao et al., 2015). Targeting

neurotoxic inflammatory pathways (Gao et al., 2015) may emerge as an approach that

could be guided by specific small-molecule and physiological biomarkers.

Accumulating data suggest that during sepsis intracellular redox processes result

in increased production of superoxide, hydrogen peroxide, and peroxynitrite, especially

in mitochondria (Mayeux and Macmillan-Crow, 2012; Duran-Bedolla et al., 2014). While

there are studies to suggest that mitochondrial injury occurs in septic patients (Jeger et

al., 2013), the notion that mitochondrial injury is a cause of organ dysfunction is

controversial (Fink, 2015). Nonetheless, if there were rapidly measurable mitochondrial

injury biomarkers, they could be used to direct mitochondria-targeted antioxidants, such

as ubiquinone (MitoQ), vitamin E (MitoVit E), and piperidine nitroxide (MitoTEMPO),

which have been shown in animal models to be protective against sepsis-induced


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cardiomyopathy (Supavekin et al., 2003; Zang et al., 2012; Yao et al., 2015), renal injury

(Patil et al., 2014), and microcirculatory failure (Patil et al., 2014; Sims et al., 2014).

Combinations of Biomarkers

The heterogeneity of septic patients suggests that multiple biomarkers may be

required to effectively guide therapy (Gibot et al., 2012; Liu et al., 2014; Sandquist and

Wong, 2014). Wong and collaborators have begun to use panels of biomarkers to

stratify pediatric septic shock patients to predict risk (Wong et al., 2014) and possibly

inform on therapeutic decisions (Wong et al., 2015a; Wong et al., 2015b). For example,

the PERSEVERE study used a panel consisting of C-C chemokine ligand 3, IL-8, heat

shock protein 70 kDa 1B, granzyme B, and MMP-8 to stratify pediatric sepsis patients

based on mortality probability (Wong et al., 2014). A panel of candidate biomarker

genes has also been used to guide the use of adjuvant corticosteroids (Wong et al.,

2015b). Due to the complexity of sepsis and the diversity of patient populations, it

should expected that combinations of biomarkers (small molecule and physiological) will

be required to best inform on interventional therapies (Fig. 1).

Animal Models

Animal models of sepsis are the subject of a number of excellent reviews that

discuss advantages and disadvantages of the various species studied and the various

methods used to induce sepsis (Zanotti-Cavazzoni and Goldfarb, 2009; Ward, 2012;

Fink, 2013; Efron et al., 2015). It is clear that no animal model of sepsis can fully

replicate the complexities and range of sepsis in humans. However, animal models are


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essential for preclinical testing of new therapies to help suggest dose, potential toxicities,

and efficacy. Animal models are also necessary to identify potential biomarkers and test

new monitoring modalities. From a basic science point of view, in vivo models of sepsis

provide a means to investigate underlying mechanisms of organ injury that are not

possible in very sick septic patients.

Murine and rat models of sepsis are the most frequently used because of their

relatively low cost and their public acceptance as laboratory subjects. However, just

how relevant murine models of inflammation are to inflammatory diseases is an area of

intense debate (Seok et al., 2013; Osuchowski et al., 2014; Efron et al., 2015). Larger

species, such as pigs, sheep, dogs, and subhuman primates, are also used in sepsis

research, and some of these species better replicate more of the “typical”

cardiovascular defects observed in humans with sepsis than do small animals (Zanotti-

Cavazzoni and Goldfarb, 2009; Taylor et al., 2012). Not surprisingly, however, large-

animal models are rarely used in survival studies. This is an important consideration

because increased mortality is a key measure of efficacy in sepsis clinical trials.

Because sepsis is defined as a systemic inflammatory response due to an

infection (Dellinger et al., 2013), the most clinically relevant models of sepsis should

incorporate both a systemic inflammatory response and an active infection. Models of

sepsis that utilize endotoxemia (lipopolysaccharide [LPS] from the outer membrane of

gram-negative bacteria) in rodents and larger species are less clinically relevant

because these models rely solely on rapid activation of the innate immune system and

do not replicate the consequences of active microbial defense and subsequent immune

suppression observed in humans with sepsis (Stearns-Kurosawa et al., 2011).


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In rodents, the cytokine profile and magnitude of cytokine release are very

different between endotoxemia models and active-infection models, such as

polymicrobial peritonitis induced by cecal ligation and puncture (CLP). LPS produces a

very rapid but transient increase in inflammatory cytokines to levels that can be orders

of magnitude higher than with CLP (Kawai et al., 1999; Remick et al., 2000; Miyaji et al.,

2003), where the magnitude and kinetics of cytokine release more closely mirror what

occurs in human sepsis (Rittirsch et al., 2007). Still, there are advantages to studying

endotoxemia. Endotoxemia models are attractive from an experimental point of view

because they are highly reproducible and do produce similar effects on the vasculature

(although perhaps through different mechanisms (Asano et al., 2015) and similar

mortality rates (Remick et al., 2000). A limitation of endotoxemia models is that the

systemic inflammatory effects are mediated through interactions with toll-like receptor-4

(TLR4) (Kawai et al., 1999; Palsson-McDermott and O'Neill, 2004), whereas this is not

the case in polymicrobial sepsis (Dear et al., 2006). These are mechanistically important

differences between endotoxin and polymicrobial models of sepsis that can impact the

translation of experimental therapies to clinical practice (Rittirsch et al., 2007).

Consequently, the CLP model and other models of active infection are generally

considered to be clinically relevant models of sepsis, especially when they incorporate

antibiotic and fluid therapy to more closely mimic the standard care of septic patients

(Miyaji et al., 2003; Rittirsch et al., 2007; Dyson and Singer, 2009; Rittirsch et al., 2009).

However, the CLP model exhibits high inherent variability because it depends on the

resident bacterial flora of the cecal contents, which can vary from animal to animal, and

on the consistency of a technician’s surgical skills.


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Depending on the dose, intravenous infusion of live E. coli in sheep can produce

mild hyperdynamic (increased cardiac output) sepsis or severe hypotensive sepsis

(Ramchandra et al., 2009; Ishikawa et al., 2011) to mimic the range of sepsis severity

and degree of cardiovascular dysfunction observed in humans. Similarly, in adult pigs,

intravenous administration of live bacteria can produce hypotensive sepsis associated

with pulmonary hypertension and a transient increase in systemic vascular resistance

(Garcia-Septien et al., 2010); in addition, when acute kidney injury develops, a

dissociation is produced between systemic hemodynamic changes and changes in the

renal circulation (Benes et al., 2011). In baboons, intravenous challenge with live E. coli

can be titrated to produce mild sepsis or severe sepsis with refractory hypotension and

coagulopathies (Taylor et al., 2012). An advantage of live-bacteria infusion models is

that gram-positive bacteria can also be studied (Soerensen et al., 2012), as well as

controlled combinations of bacteria. Another advantage of these intravenous live culture

models over the CLP model is that sepsis can be induced without subjecting the animal

to a surgical procedure or general anesthesia.

Autologous fecal peritonitis is a variation of the CLP model in which the cecal

contents are collected from an animal and administered at a later time. This model has

been used in sheep (He et al., 2012; Taccone et al., 2014) and pigs (Benes et al., 2011;

Correa et al., 2012; Wepler et al., 2013) to study sepsis-induced cardiovascular injury

and evaluate resuscitation protocols. A variation in which the fecal matter is derived

from multiple animals can also be used in mice too young or too small for the CLP

procedure (Wynn et al., 2007; Wynn et al., 2008). This model offers an additional

advantage in that cecal contents can be harvested from multiple animals, pooled,


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partially purified, stored at –80°C, and then administered to groups of animals (Starr et

al., 2014). The “dose” of fecal contents can be normalized to bacterial colony–forming

units (cfu)/body weight prior to administration, which should reduce interexperimental

variability.

As discussed earlier, sepsis is not identical in neonates, infants, and adults, and

it should not be assumed that results from adult animal models of sepsis are relevant for

translation to neonates or even infants with sepsis. Most preclinical studies are

performed in adult “middle-aged” animals, yet there are clear differences in the

immunological responses (Wynn et al., 2007; Wynn et al., 2008; Turnbull et al., 2009),

degree of organ injury (Miyaji et al., 2003; Starr et al., 2015), and mortality (Turnbull et

al., 2009; Starr et al., 2014) between very young, adult, and old mice made septic by

fecal peritonitis. CLP can be performed in young (weaning age) rat pups as a model of

infant sepsis. These animals develop rapid hemodynamic changes and microvascular

dysfunction that are not the same as in adult rats (Seely et al., 2011; Sims et al., 2014).

Neonatal, 3-day-old piglets subjected to CLP show deterioration of cardiac output,

pulmonary hypertension, and shock (Goto et al., 2010), consistent with cold shock (low

cardiac output with high systemic vascular resistance) frequently observed in newborns

and infants with severe sepsis (Wheeler et al., 2011).

Confounding comorbid or predisposing conditions of most septic patients limit the

translation of findings from most animal models. However, researchers are now

beginning to study the pathophysiology of sepsis in the presence of comorbid conditions,

such as chronic kidney disease (Doi et al., 2008; Leelahavanichkul et al., 2011), type 1

(Osuchowski et al., 2010; Filgueiras et al., 2012) and type 2 (Jacob et al., 2008)


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diabetes, and atherosclerosis (Wepler et al., 2013). Such studies are especially

important because they could uncover more targeted therapy for comorbid conditions.

They could also help explain why a therapy fails in septic patients with preexisting

conditions (Doi et al., 2008; Leelahavanichkul et al., 2011; Filgueiras et al., 2012) or

why survivors of sepsis may have increased susceptibility to subsequent organ injury

(Portella et al., 2013).

Murine models of sepsis are also being used to investigate the development and

potential causes of cognitive impairments following sepsis like those observed in human

survivors of sepsis (Iwashyna et al., 2010). For example, mice surviving severe sepsis

induced by CLP show time-dependent learning and memory impairments (Chavan et al.,

2012; Ji et al., 2015) associated with dendritic degeneration (Chavan et al., 2012) and

oxidative stress in the prefrontal cortex and hippocampus (Ji et al., 2015).

Preclinical research has several important roles to play in the development of

novel therapeutics. However, animal models have limitations that weaken the prediction

of clinical efficacy. Still, animal models are necessary for the discovery of plausible

biological targets and the demonstration that these targets can be manipulated to

improve outcomes, at least in the experimental animal. Preclinical testing seeks to

identify targeted therapy that may be effective. Equally important, however, is the use of

preclinical testing to suggest ineffective or harmful therapies. For example,

administration of TNF-α-neutralizing antibody, while protective in most gram-negative

preclinical models, is actually detrimental in gram-positive bacteria models and some

other types of pathogens, perhaps due to inhibition of innate antimicrobial defenses

(Lorente and Marshall, 2005). Preclinical studies testing efficacy against sepsis-induced


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multiorgan failure are more challenging (Andonegui et al., 2009; Kapoor et al., 2010;

Coldewey et al., 2013) but are necessary to support translational research and clinical

trials.

Personalized Medicine

Septic patients still experience unacceptably high morbidity and mortality, even

with optimal goal-directed supportive therapy that meets approved guidelines. It is

important to consider that the likelihood of survivors of severe sepsis to be readmitted to

an inpatient health care facility is higher than matched nonsepsis survivors (Prescott et

al., 2014). This is most likely related to the significant long-term physical and mental

disabilities that can affect survivors. For example, estimates are that sepsis-associated

alterations in mental status and cognitive deficits occur in up to 87% of septic patients

(Iwashyna et al., 2012). While decreasing mortality is and should be the goal of therapy,

it is becoming increasingly clear that patients who initially survive sepsis experience

functional deficits that can diminish quality of life and even increase long-term mortality.

Consequently, biomarkers that also direct therapies that decrease the morbidity of

survivors would have a major impact on the overall health care burden of sepsis (Fig. 2).

Biomarkers and rapid noninvasive monitoring could offer better and more rapid

stratification of patients to identify those who might benefit (or be harmed) by more

targeted therapies. The advent of high-throughput technologies and access to advanced

bioinformatics have made possible the concept of personalized medicine to direct

therapy in septic patients. A personalized medicine approach may have the best chance

of identifying specific strategies for such a diverse patient population. A systems biology


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approach could complement the use of biomarkers to stratify patients, aid in prognosis,

and help identify subsets of patients who may benefit or be harmed by therapy (Tsalik et

al., 2015). However, the costs are high, and it is too early to know whether knowledge of

the blood metabolome, proteome, and transcriptome could really direct therapy.

Perspectives

In the near future, biomarkers could direct the therapy of septic patients in order

to achieve an optimal outcome. Cohen and colleagues (Cohen et al., 2015) suggested

that the ideal profile of a sepsis biomarker should have these characteristics: fast

kinetics, high sensitivity and specificity, fully automated technology, short turnaround

time, availability as point-of-care tests, and low cost. Many clinicians already use

biomarkers to suggest an active infection (CRP, PCT), estimate the severity of under-

resuscitated septic shock (mixed venous blood oxygen saturation, lactate), and estimate

the severity of organ injury due to sepsis (liver enzymes, troponin, creatinine, ratio of

partial pressure of oxygen to fractional inspired oxygen, platelet count, activated partial

thromboplastin time, fibrinogen and D-dimer). Physiological biomarker monitoring

including echocardiography, pulmonary artery catheter, Doppler ultrasound, and/or

continuous pulse-contour cardiac analysis is already being used to direct sepsis

resuscitation and is recommended in the “Surviving Sepsis Campaign” document

(Dellinger et al., 2013).

After more than three decades of numerous failed large sepsis clinical trials,

sepsis is currently being redefined, which will most likely include “MODS due to non-

resolving inflammatory response to infection.” Future clinical trials of sepsis therapies


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will most likely target an individual patient’s underlying pathophysiological mechanism

rather than a general patient population with sepsis. Some of the current proposed

mechanisms for sepsis-induced MODS include pathological immune activation,

immunoparalysis, mitochondrial dysfunction, disseminated microvascular thromboses,

and others (Carcillo et al., 2011). The genetic makeup of the host and pathological

organism, sex and age of the host, environmental factors, and specific interventions

from different hospitals will all influence the underlying pathophysiological mechanism of

the individual septic patient (Rautanen et al., 2015; Srinivasan et al., 2015). In this

context, future real-time measurements of biomarkers could identify the pathological

mechanism and direct therapeutic strategies in septic patients, including immune

modulation for pathological immune activation, boosting the immune response for

immunoparalysis, optimizing/rescuing mitochondrial function, reducing oxidative stress,

and/or normalizing dysregulated hemostasis. Eventually, more advanced physiological

monitoring, such as assessment of the microcirculation of specific organs, will give

instantaneous feedback of intervention where titration of therapeutic agents could be

optimized.


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Authorship Contributions

Participated in research design: NA.

Conducted experiments: NA.

Contributed new reagents or analytic tools: NA.

Performed data analysis: NA.

Wrote or contributed to the writing of the manuscript: Sims, Nguyen, Mayeux.


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Footnotes

This work was supported by the National Institutes of Health National Institute of

Diabetes and Digestive Kidney Diseases [F31 DK104533]; and National Institutes of

Health Institute of General Medical Sciences [R01 GM106419, GM112806]; the National

Institutes of Health National Center for Advancing Translational Sciences Clinical and

Translational Science Award [Grant UL1-TR000039].


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Figure Legends

Fig. 1. Conceptual framework for the use of discussed biomarkers in combination to

direct the development, validation and use of targeted therapies.

Fig. 2. Age, genetics, environmental factors, and comorbidities contribute to the

diversity of septic patients, with the young and the elderly populations being the most

susceptible to sepsis. As the systemic inflammatory response syndrome (SIRS) and

sepsis progress to severe sepsis with multiple organ dysfunction syndrome (MODS),

biomarkers could be used to guide targeted therapy that would halt the progression of

sepsis to increase survival and decrease morbidity.


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Inflammatory Biomarkers E-, L-, P-selectin ICAM-1, VCAM-1 CRP, PCT TNF-α IL-1, IL-6, IL-8, IL-27 HLA-DR PD-1 Lymphopenia Increased percentage of T

regulatory cells

Endothelial Biomarkers Syndecan-1 Heparan Sulfate Ang-1, Ang-2 Tie-2 S1P Microparticles

Coagulation Biomarkers Prothrombin Time Activated Partial

Thromboplastin Time D-dimer Platelet Count VWF Ultralarge VWF Multimers Fibrinogen ADAMTS-13

Organ-Directed and Physiological Biomarkers Brain:

Changes in EEG Cognitive Impairment Perfusion (NIRS)

Heart: Myocardial Dysfunction

(Echocardiography) Troponin

Kidney:

Perfusion (Ultrasound, MRI) Creatinine Clearance NGAL, KIM-1, NAG,

Liver:

ALT, AST Lung:

Ratio of partial pressure of O2 to fractional inspired O2

Microcirculation:

Blood Lactate Sublingual Perfusion Mixed venous O2 saturation

Tissue Injury: MMP-8, Extracellular Histones

Targeted Therapies Immunomodulation

Endothelium Microvasculature

Coagulation Organ-specific

Figure 1


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Definitions

Systemic Inflammatory Response Syndrome (SIRS): Two or more of the following:

•  fever or hypothermia •  tachycardia •  tachypnea •  leukocytosis or leukopenia

Sepsis: SIRS with a known infection

Severe Sepsis: Sepsis with 1 organ dysfunction or multiple organ dysfunction syndrome (MODS)

Sepsis

Severe Sepsis

SIRS

Death

Decreased Morbidity

Young (< 1 yr) Immature Immune System and Organ Development

Old (> 65 yrs) Weakened Immune System

and Comorbidities

Increased Survival

Biomarker-DirectedTherapy



Figure 2

could biomarkers direct therapy for the septic...

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