Biomarkers in ALI and ARDS

Mohammed Attia, MD, FRCPCH (UK)

Foretelling using dreams y Dreaming of«.
y

a deep well prison a mirror a second wife a shining moon forgiveness a large cat a large crop

If someone sees himself ««
dead
long life

eating crocodile flesh
he will become a village official

with his face in a mirror
a new life

bringing in cattle
will evangelize the spirit of the community

If someone sees himself ««
uncovering his own backside
he will become an orphan

putting one¶s face to the ground
the dead want something

plunging into cold waters
absolution of all ills

making love to his wife in daylight
god will discover his misdeeds

Introduction
y

ALI occurs
In 1 ± 4% of all PICU cases 10% of all children receiving mechanical ventilation on PICU

y

ALI is associated with a mortality of 25% to 75% depending on
Diagnostic criteria used to identify cases Coexisting risk factors Presence of nonpulmonary organ failure

The Ideal Biomarker
Increases pathologically in the presence of the disease (high sensitivity) y Does not increase in the absence of the disease (high specificity) y Relates to the disease burden and extent y Changes in accordance with the clinical evolution, reflecting the current status of disease
y

Anticipates clinical changes before it happens y Shows no major fluctuation in serum level y Adds independent information about the risk or prognosis y Reproducible y Easy and cheap determination
y

Markers in ARDS and ALI
y

The main characteristics of the syndrome are
Diffuse inflammation Increased microvascular permeability

y

Although a variety of insults may lead to ARDS, a common pathway may probably result in the lung damage

A clinically useful biomarker for ALI might add information regarding the development of ARDS in at-risk patients. y The clinically useful marker will help the intensivist to monitor the disease and evaluate or modulate treatments.
y

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Biomarkers of ARDS and ALI
y

Cytokines
IL-1 IL-2 IL-6 IL-8 IL-15 TNFIL-10 IL-1 ra

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Biomarkers of ARDS and ALI
y

Markers of endothelium activation
Adhesion molecules
x E, L-selectin x I-CAM-1 x V-CAM-1

VWF

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Biomarkers of ARDS and ALI
y

Markers of neutrophil activation
Matrix metalloproteinase-9 (MMP-9) LTB4

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Epithelial damage Neutrophil activation Inflammatory mediators release

Endothelial activation

Biomarkers of ARDS and ALI
y

Lung epithelium specific proteins
Surfactant-associated proteins
x SP-A x SP-B x SP-D

Mucin-associated antigens
x Krebs von den Lungen-6 (KL-6/MUC1)

Cytokines
Cytokines IL-1 IL-2 IL-6 IL-8 IL-15 TNFIL-10 IL-1 ra

Pro-inflammatory cytokines TNFIL-1F IL-6 IL-8 IL-4

Anti-inflammatory cytokines IL-10 IL-1ra sTNFR1 sTNFR2 IL-6

Cytokines
y

Cytokines are produced either by
local resident cells
x x x x Alveolar macrophages Pneumocytes Endothelial cells Fibroblasts

cells arriving to the lung in response to local or systemic injury
x Neutrophils x Lymphocytes x Platelets

TNF- and IL-1 serum levels are related to ARDS disease severity and (1,2) mortality . y Serum levels of IL-6, IL-8 and CD 11b could identify patients with septic shock at risk for organ failure, (3,4) including respiratory failure .
y
1- Marks JD et al., Am Rev Respir Dis 1990, 141:94-7. 2- Damas P et al., Crit Care Med 1989, 17:975-8. 3- Pinsky MR et al., Chest 1993,103:565-75. 4- Takala A et al., Clin Sci (Lond) 1999, 97:529-38.

y

Some researchers found that serum cytokines could not be used as a routine laboratory test to predict the outcome in septic-shock patients.

Calandra T et al., Am J Med 1991, 91:23-9.

y

With multiple study limitations, it was found that plasma TNF- , IL-6, IL-8 fail to associate with severity and course of ARDS in 19 leukocytopenic patients. Meanwhile, BALF levels appeared to differentiate between responders and non-responders to treatment before clinical differences become apparent.

y

Kiehl MG et al., Crit Care Med 1998,9-26:1194

Meduri et al. studied plasma levels of various cytokines in 27 ARDS patients. y Consistent, efficient and independent predictive value for IL-1 and IL-6 serum concentrations over time in severe ARDS. y Superiority of IL-1 and IL-6 plasma levels monitoring over commonly applied clinico physiologic parameters.
y
Meduri GU et al., Chest 1995, 107:1062-73

y

Agouridakis et al. found an excellent predictive value of plasma IL-2 and IL15 levels compared to those observed in BALF. A remarkable finding was the emergence of the discriminative usefulness of elevated IL-2 and IL-15 serum levels in patients with ARDS or at risk for ARDS.

y

Agouridakis P, et al., Eur J Clin Invest 2002, 32:862-7.

In contrast Lesur et al. found lower blood IL-2 levels in patients with ARDS compared to those that never developed ARDS. y There was a strong association of early low serum IL-2 levels with the patients¶ survival y A major criticism to this study is opposite and disproportional fluctuations of IL-2 content in serum and BALF in patients Lesur O et al., Crit Care Med 2000, 28:3814-22. with or without ARDS
y

AntiAnti-inflammatory cytokines
y

A large prospective (77 patients at risk of ARDS) found that
IL-1ra and IL-10 were elevated in patients at risk for ARDS IL-1ra and IL-10 exhibited a remarkable association with the disease outcome, but could not predict the development of the syndrome

Parsons PE et al., . Am J Respir Crit Care Med 1997, 155:1469-73.

y

The inability of serum cytokine levels to predict the development of ARDS in patients with ALI was reproduced by (1,2) other researchers .

1- Takala A et al., Shock.7-17:252 ,2002 2- Bouros D et al., BMC Pulm Med 2004, 4:6.

There is a remarkable consistently elevated serum levels of IL-6 and IL-8 in ARDS and/or severe pneumonia, differentiating these entities from cardiogenic pulmonary oedema. y Serum IL-6 and IL-8 were unable to differentiate ARDS from severe pneumonia
y

Schutte H, et al., Eur Respir J.67-9:1858 ,1996

Limitations in cytokine researches
Number of patients is small in most studies y Including heterogeneous groups of patients in the same study y Blood versus BALF levels of cytokines y Studying limited number of molecules
y

Limitations in cytokine researches
Not looking at the anti-inflammatory limb of the cytokine cascade y Lack of serial measurements y Statistical analysis not including ROC and not reporting cut-off values
y

Markers of endothelium activation
Adhesion molecules E, L-selectin I-CAM-1 V-CAM-1 VWF

The patho-physiologic sequence characterizing ALI involves a cascade of leukocyte-endothelium interactions and adhesions. y The adhesion is followed by transendothelial migration of neutrophils and release of their cytotoxic products resulting in microvascular and tissue injury.
y

y

Adhesion of neutrophils to the endothelium is regulated by
selectins (E, L and P) Integrins immunoglobulin superfamily
x ICAM-1 x VCAM-1

y

Interactions of leukocytes and the endothelium
An initial ³loose´ contact (rolling): selectin Firm adhesion: integrin ( 2) and ICAM-1

Adhesion Molecules
y

Soluble isoforms have been detected in the blood under various inflammatory conditions
s-E-selectin sICAM-1 sVCAM-1

y

Mechanisms to explain an increase in circulating adhesion molecules:
overexpression by the endothelial cells induced by cytokines (IL-1, TNF- ) increased proteolytic cleavage of endothelialbound adhesion molecules secondary to endothelial damage

Adhesion Molecules
y

The expression is almost restricted to stimulated endothelial cells Their presence in serum should potentially reflect the state of endothelium in disease and subsequently the disease severity in ALI

y

vonvon-Willebrand factor antigen
y

A macromolecular antigen that is produced by
endothelial cells platelets and megakaryocytes

y

Endothelial injury (as in ARDS) results in the release of VWF from preformed stores into the circulation.

y

Donnelly et al. demonstrated in a large cohort of patients at risk for ARDS that
Mean circulating levels of sE-selectin were not correlated with subsequent ARDS development and patients' mortality. Low values of sL-selectin exhibited a significant prognostic value

Donnelly SC et al. , Lancet 1994,9-215:(344)23

y

Cowley et al. showed a superiority plasma levels of sE-selectin in predicting organ dysfunction and death in a group of patients with SIRS compared to sICAM-1 peripheral concentrations.

Cowley HC et al., Crit Care Med 1994,.7-22:651

y

sICAM-1 sequential plasma levels, were suggestive of a strong association between the severity of shock
the presence of hypotension the requirement of vasoactive drugs

and the circulating concentrations of the sICAM-1.
Sessler CN et al., Am J Respir Crit Care Med 1995, 151:1420-7. Kayal S et al., Am J Respir Crit Care Med 1998, 157:776-84.

ICAMICAM-1 and VCAM-1 VCAMWere not found to be independent factor for ARDS development, y Exhibited a considerable negative predictive value for ARDS development. y ROC curve analysis showed a clear superiority of plasma parameters in correlating with the disease outcome compared with BALF molecules
y
Agouridakis P et al., Respir Res 2002, 3:25.

VWF
y

Marked and independent association of circulating VWF with the disease severity as assessed by other commonly applied clinical variables. Serum VWF levels were an independent predictor of hospital mortality and were associated with longer duration of MV

y

Kayal S et al., Am J Respir Crit Care Med 1998, 157:776-84. Ware LB et al., Crit Care Med 2001, 29:2325-31.

VWF
In a multicenter study of 559 patients with ALI and ARDS y A significant correlation of elevated VWF plasma levels with adverse outcomes
Mortality duration of unassisted ventilation organ failure

An association between vWF and presence or absence of sepsis, supporting the hypothesis that ALI might be an independent cause of systemic endothelial activation and injury. y No modulation of plasma VWF concentrations by protective mechanical Ware LB et al., Crit Care Med 2001, 29:2325-31. ventilation.
y

Markers of neutrophil activation
MMP-9 LTB4

There is an increasing evidence implicating neutrophils in most cases of ARDS. y Neutrophils have been reported by several studies to exert an important role in the early phase of ALI characterized by
y

architecture remodeling Surfactant and epithelial toxicity

y

y

y

Neutrophils use a wide array of enzymes during the process of transmigration through alveolar-capillary barrier. These enzymes include, metalloproteinases (MMPs) such as MMP-9 also called gelatinase B which is secreted from preformed neutrophil granules in response to proinflammatory cytokines (IL-8, TNF- ). MMP-9 is secreted as a zymogen, and then activated by other proteases such as elastase, and plays a crucial role in digesting basement membranes.

Pulmonary edema fluid levels of IL-8, MMP-2, MMP-9 could differentiate ARDS and cardiogenic pulmonary edema whereas plasma levels proved to be of poor discriminative value. y These findings raise the issue that the inflammatory response of ARDS patients is well compartmentalized, with little spillover into the circulation.
y
Pugin J et al., CritCare Med 1999, 27:304-12.

Leukotrienes
y

y y

LTs (B4, C4, D4, E4) exert a synergistic role with IL-8 in the neutrophil influx and activation leading to a massive recruitment of neutrophils and a massive inflammatory response. Their BALF levels have been found elevated in patients with ARDS. Contribute to alterations of microvascular permeability and accumulation of pulmonary edema.

LTB4 plasma levels could serve as a valuable predictive marker of ARDS in terms of specificity and sensitivity. y There is a strong association of both LTB4 and IL-8 peripheral concentrations with the patients' survival.
y

Amat M et al., Crit Care Med 2000, 28:57-62.

Lung epithelium-specific epitheliumproteins
Surfactant-associated proteins SP-A SP-B SP-D Mucin-associated antigens Krebs von den Lungen-6 (KL6/MUC1)

y

The lung epithelium produces mucus blanket and surfactant proteins Study of BALF is the classical means of studying the proteins secreted by the lung epithelium and investigating their alterations in lung disorders.

y

y

The presence of these proteins in the bloodstream can be explained by several mechanisms including
leakage from the lung into the bloodstream Increased production by the alveolar type II cells diminished clearance rates from the circulation

SurfactantSurfactant-associated Proteins
Pulmonary surfactant is a complex and highly surface active material covering the alveolar space of the lung. y Structure
y

Mainly phospholipids Surfactant proteins

SurfactantSurfactant-associated Proteins
y

Functions of the alveolar surfactant system:
Reduce surface tensions of alveoli Prevention of alveolar edema Pronounced influence, especially of the collectins (SP-A and SP-D) in the innate immune system of the lung

y

Serum levels reflect the epithelial damage and turnover.

SurfactantSurfactant-associated Proteins
y

Four surfactant-specific proteins
SP-A SP-B SP-C SP-D

SP-B and SP-C are low MW hydrophobic y SP-A and SP-D are high MW hydrophilic
y

SP-A and SP-D belong to the collectin subgroup of the C-type lectin superfamily. y SP-A and SP-D are produced by two types of epithelial cells in the peripheral airway
y

Clara cells Alveolar type II cells.

SPSP-A
Acute indicator of lung function and alveolocapillary membrane injury (1) y SP-A BALF levels are strongly related to outcome and likelihood of disease (2) progression y Plasma levels are associated with severity of clinical lung injury and with (3) disease outcome
y
1- Doyle IR et al., Am J Respir Crit Care Med 1995, 152:307-17. 2- Greene KE et al., Am J Respir Crit Care Med 1999, 160:1843-50. 3- Cheng IW et al., Crit Care Med 2003, 31:20-7.

SPSP-A
There is a potential value of SP-A plasma levels in discriminating at risk patients who developed ARDS from (1) those with sepsis and aspiration. y discriminating patients with ALI of various etiologic factors
y

severe pneumonia (2) cardiogenic lung edema.
1- Greene KE et al., Chest 1999, 116:90S-91S. 2- Gunther A et al., Am J Respir Crit Care Med 1996, 153:176-84

SPSP-B
y

Superior to SP-A plasma levels as a marker of
lung function alveolocapillary membrane injury.(1)

SP-B cut-off plasma levels predict ARDS development in at-risk patients suffering a direct lung injury y An increase of blood SP-B levels was observed before other changes of ALI become apparent.(2)
y
1- Doyle IR et al., Am J Respir Crit Care Med 1997, 156:1217-29. 2- Bersten AD et al., Am J Respir Crit Care Med 2001, 164:648-52.

SPSP-D
y

y

SP-A BALF levels are strongly related to outcome and likelihood of disease (1) progression. In a large RCT
Elevated SPD levels were associated with worse clinical outcomes
x Greater risk of death x Fewer ventilator- free days x Fewer organ failure-free days

Attenuation of SP-D plasma levels by lower (2) volume ventilation strategies .
1- Greene KE et al., Am J Respir Crit Care Med 1999, 160:1843-50. 2- Eisner MD et al., Thorax 2003, 58:983-8.

MucinMucin-associated Antigens
Mucins are major components of the mucus layer covering the airway epithelium. y Mucins are either
y

associated with cell membranes or secreted at the surface of the respiratory tract

KLKL-6
Krebs von den Lungen-(KL)-6 is a mucin-associated antigen mainly associated with cellular membranes. y KL-6 is mainly detected in
y

alveolar type II cells epithelial cells of the respiratory bronchioles.
y

It is also present on other somatic cells
pancreatic cells eosophageal cells fundic cells of the stomach

KLKL-6
KL-6 is a sensitive indicator of damage to alveolar type II cells y Its raise would theoretically represent
y

the destruction of the normal lung parenchyma and architecture the increased permeability of the air-blood barrier

KLKL-6
Association of optimal cut-off values of KL-6 serum levels with patients' mortality with high sensitivity (87%) and specificity (100%) y This finding supports the theory that disruption of the alveolar barrier represents a major determinant of prognosis of ALI
y

Ishizaka A et al., Am J Physiol Lung Cell Mol Physiol 2004, 286:L1088-94.

KLKL-6
y

Association of KL-6 serum levels with variables of lung injury severity and with mortality rates. No statistically significant correlation between blood levels and ventilation strategies.

y

Sato H et al., Eur Respir J 2004,.5-23:142

Summary
The ideal biomarker y Cytokines y Products of endothelial activation y Products of neutrophil activation y Lung epithelium-specific proteins
y

Future directions
y

Finding and applying the ideal biomarker in the clinical setting would be very helpful in terms of
Defining categories of patients for different therapies or prognosis. For the purpose of counseling families and patients regarding high/low risk of complications. Possibly identifying novel therapeutic targets.

Future directions
Development of a prognostic index that combines clinical and biological determinants may be useful to ameliorate the limitations encountered using only biomarkers. y A combination index could be crucial for the selection of more homogeneous groups of patients with ALI/ARDS for further studies.
y

Future directions
y

Crossing the boundary from research to clinical application requires
Validation in multiple settings Experimental evidence supporting a pathophysiologic role Ideally intervention trials showing that modification improves the outcome

Conclusion

Conclusion
Currently, the application status in routine clinical practice for most of these biologic markers of ALI is still in its infancy and remains exploratory. y It is important to note that the greatest promise is in only few (VWF, IL-1 , IL6, ICAM-1, VCAM-I, E-selectin) which exhibited independent discriminatory power.
y

Conclusion
y

There is a necessity for further investigations in the context of large prospective studies analyzing homogeneous and well defined group of ARDS or at risk patients and the assessment of novel molecules to serve as diagnostic and prognostic tools, as well as markers of the disease activity and severity.

The question remains «.

How much have we moved from here?

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