Comparison of 16 Pediatric Acute Respiratory Distress Syndrome-Associated Plasma Biomarkers With Changing Lung Injury Severity.

OBJECTIVES: Pediatric acute respiratory distress syndrome (PARDS) is a source of substantial morbidity and mortality in the PICU, and different plasma biomarkers have identified different PARDS and ARDS subgroups. We have a poor understanding of how these biomarkers change over time and with changing lung injuries. We sought to determine how biomarker levels change over PARDS course, whether they are correlated, and whether they are different in critically ill non-PARDS patients. DESIGN: Two-center prospective observational study. SETTING: Two quaternary care academic children's hospitals. PATIENTS: Subjects under 18 years of age admitted to the PICU who were intubated and met the Second Pediatric Acute Lung Injury Consensus Conference-2 PARDS diagnostic criteria and nonintubated critically ill subjects without apparent lung disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples were obtained on study days 1, 3, 7, and 14. The levels of 16 biomarkers were measured using a fluorometric bead-based assay. Compared with non-PARDS subjects, on day 1 PARDS subjects had increased concentrations of tumor necrosis factor-alpha, interleukin (IL)-8, interferon-γ, IL17, granzyme B, soluble intercellular adhesion molecule-1 (sICAM1), surfactant protein D, and IL18 but reduced matrix metalloproteinase 9 (MMP-9) concentrations (all p < 0.05). Day 1 biomarker concentrations and PARDS severity were not correlated. Over PARDS course, changes in 11 of the 16 biomarkers positively correlated with changing lung injury with sICAM1 ( R = 0.69, p = 2.2 × 10 -16 ) having the strongest correlation. By Spearman rank correlation of biomarker concentrations in PARDS subjects, we identified two patterns. One had elevations of plasminogen activator inhibitor-1, MMP-9, and myeloperoxidase, and the other had higher inflammatory cytokines. CONCLUSIONS: sICAM1 had the strongest positive correlation with worsening lung injury across all study time points suggesting that it is perhaps the most biologically relevant of the 16 analytes. There was no correlation between biomarker concentration on day 1 and day 1 PARDS severity; however, changes in most biomarkers over time positively correlated with changing lung injury. Finally, in day 1 samples, 7 of the 16 biomarkers were not significantly different between PARDS and critically ill non-PARDS subjects. These data highlight the difficulty of using plasma biomarkers to identify organ-specific pathology in critically ill patients.

Multi-omic characterization of pediatric ARDS via nasal brushings.

RATIONALE: While nasal brushing transcriptomics can identify disease subtypes in chronic pulmonary diseases, it is unknown whether this is true in pediatric acute respiratory distress syndrome (PARDS). OBJECTIVES: Determine whether nasal transcriptomics and methylomics can identify clinically meaningful PARDS subgroups that reflect important pathobiological processes. METHODS: Nasal brushings and serum were collected on days 1, 3, 7, and 14 from control and PARDS subjects from two centers. PARDS duration was the primary endpoint. MEASUREMENTS AND MAIN RESULTS: Twenty-four control and 39 PARDS subjects were enrolled. Two nasal methylation patterns were identified. Compared to Methyl Subgroup 1, Subgroup 2 had hypomethylation of inflammatory genes and was enriched for immunocompromised subjects. Four transcriptomic patterns were identified with temporal patterns indicating injury, repair, and regeneration. Over time, both inflammatory (Subgroup B) and cell injury (Subgroup D) patterns transitioned to repair (Subgroup A) and eventually homeostasis (Subgroup C). When control specimens were included, they were largely Subgroup C. In comparison with 17 serum biomarkers, the nasal transcriptome was more predictive of prolonged PARDS. Subjects with initial Transcriptomic Subgroup B or D assignment had median PARDS duration of 8 days compared to 2 in A or C (p = 0.02). For predicting PARDS duration ≥ 3 days, nasal transcriptomics was more sensitive and serum biomarkers more specific. CONCLUSIONS: PARDS nasal transcriptome may reflect distal lung injury, repair, and regeneration. A combined nasal PCR and serum biomarker assay could be useful for predictive and diagnostic enrichment. Trial registration Clinicaltrials.gov NCT03539783 May 29, 2018.

The extracellular stress response to pediatric cardiopulmonary bypass.

The heat shock response, also frequently referred to as the stress response, is an ancient, highly conserved, endogenous cellular defense mechanism characterized by the rapid upregulation of a specific class of proteins known collectively as heat shock proteins, or stress proteins. The 70 kDa family of heat shock proteins are highly inducible and have been shown to possess important immunomodulatory effects in both the intracellular and extracellular compartments. In the current prospective translational study, we measured extracellular (i.e. plasma) levels of heat shock protein 72 (Hsp72) in 49 children undergoing cardiopulmonary bypass (CPB) for either palliation or repair of congenital heart disease. There was a significant and transient increase (less than 24 h) in extracellular Hsp72 levels following CPB. Extracellular Hsp72 levels significantly correlated with levels of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8, as well as the anti-inflammatory cytokine, IL-10. In addition, plasma Hsp72 levels correlated with troponin-I levels, a marker of myocardial injury. Increased extracellular Hsp72 levels at 6 h following CPB were independently associated with increased length of stay in the cardiac intensive care unit. Importantly, the source of extracellular Hsp72 does not appear to be cardiomyocytes. However, the mechanism of release and clinical relevance of the increase in extracellular Hsp72 need to be further delineated.

Cerebrospinal fluid levels of extracellular heat shock protein 72: A potential biomarker for bacterial meningitis in children.

Extracellular heat shock protein 72 (Hsp72) is an endogenous danger signal and potential biomarker for critical illness in children. We hypothesized that elevated levels of extracellular Hsp72 in the cerebrospinal fluid (CSF) of children with suspected meningitis could predict bacterial meningitis. We measured extracellular Hsp72 levels in the CSF of 31 critically ill children with suspected meningitis via a commercially available enzyme-linked immunosorbent assay. Fourteen had bacterial meningitis based on CSF pleocytosis and bacterial growth in either blood or CSF culture. Seventeen children with negative cultures comprised the control group. CSF Hsp72 was significantly elevated in children with bacterial meningitis compared to controls. Importantly, CSF Hsp72 levels did not correlate with the CSF white blood cell count. On receiver operator characteristic analysis, using a cut-off of 8.1 ng/mL, CSF Hsp72 has a sensitivity of 79% and a specificity of 94% for predicting bacterial meningitis. We therefore conclude that CSF extracellular Hsp72 levels are elevated in critically ill children with bacterial meningitis versus controls. Hsp72 potentially offers clinicians improved diagnostic information in distinguishing bacterial meningitis from other processes.

The immunomodulatory effects of albumin in vitro and in vivo.

Albumin appears to have proinflammatory effects in vitro. We hypothesized that albumin would induce a state of tolerance to subsequent administration of lipopolysaccharide (LPS) in vitro and in vivo. RAW264.7 and primary peritoneal macrophages were treated with increasing doses of bovine serum albumin (BSA) and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. In separate experiments, RAW264.7 cells were preconditioned with 1 mg/mL BSA for 18 h prior to LPS (10 µg/mL) treatment and harvested for NF-κB luciferase reporter assay or TNF-α ELISA. Finally, C57Bl/6 mice were preconditioned with albumin via intraperitoneal administration 18 h prior to a lethal dose of LPS (60 mg/kg body wt). Blood was collected at 6 h after LPS administration for TNF-α ELISA. Albumin produced a dose-dependent and TLR-4-dependent increase in NF-κB activation and TNF-α gene expression in vitro. Albumin preconditioning abrogated the LPS-mediated increase in NF-κB activation and TNF-α gene expression in vitro and in vivo. The clinical significance of these findings remains to be elucidated.

Pediatric Sepsis - Part V: Extracellular Heat Shock Proteins: Alarmins for the Host Immune System.

Heat shock proteins (HSPs) are molecular chaperones that facilitate the proper folding and assembly of nascent polypeptides and assist in the refolding and stabilization of damaged polypeptides. Through these largely intracellular functions, the HSPs maintain homeostasis and assure cell survival. However, a growing body of literature suggests that HSPs have important effects in the extracellular environment as well. Extracellular HSPs are released from damaged or stressed cells and appear to act as local "danger signals" that activate stress response programs in surrounding cells. Importantly, extracellular HSPs have been shown to activate the host innate and adaptive immune response. With this in mind, extracellular HSPs are commonly included in a growing list of a family of proteins known as danger-associated molecular patterns (DAMPs) or alarmins, which trigger an immune response to tissue injury, such as may occur with trauma, ischemia-reperfusion injury, oxidative stress, etc. Extracellular HSPs, including Hsp72 (HSPA), Hsp27 (HSPB1), Hsp90 (HSPC), Hsp60 (HSPD), and Chaperonin/Hsp10 (HSPE) are especially attractrive candidates for DAMPs or alarmins which may be particularly relevant in the pathophysiology of the sepsis syndrome.

Plasma angiopoietin-2 levels increase in children following cardiopulmonary bypass.

OBJECTIVE: The aim was to investigate the effects of cardiopulmonary bypass (CPB) on plasma levels of the vascular growth factors, angiopoietin (angpt)-1, angpt-2, and vascular endothelial growth factor (VEGF). DESIGN: The design was a prospective, clinical investigation. SETTING: The setting was a 12-bed pediatric cardiac intensive care unit of a tertiary children's medical center. PATIENTS: The patients were 48 children (median age, 5 months) undergoing surgical correction or palliation of congenital heart disease who were prospectively enrolled following informed consent. INTERVENTIONS: There were no interventions in this study. MEASUREMENTS AND RESULTS: Plasma samples were obtained at baseline and at 0, 6, and 24 h following CPB. Angpt-1, angpt-2, and VEGF levels were measured via commercial ELISA. Angpt-2 levels increased by 6 h (0.95, IQR 0.43-2.08 ng mL(-1) vs. 4.62, IQR 1.16-6.93 ng mL(-1), P < 0.05) and remained significantly elevated at 24 h after CPB (1.85, IQR 0.70-2.76 ng mL(-1); P < 0.05). Angpt-1 levels remained unchanged immediately after CPB, but were significantly decreased at 24 h after CPB (0.64, IQR 0.40-1.62 ng mL(-1) vs. 1.99, IQR 1.23-2.63 ng mL(-1), P < 0.05). Angpt-2 levels correlated significantly with cardiac intensive care unit (CICU) length of stay (LOS) and were an independent predictor for CICU LOS on subsequent multivariate analysis. CONCLUSIONS: Angpt-2 appears to be an important biomarker of adverse outcome following CPB in children.

The role of endogenously produced extracellular hsp72 in mononuclear cell reprogramming.

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-kappaB activation. Heat shock conditions consisted of incubation at 43 degrees C for 1 h. Control cells were incubated at 37 degrees C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 mug/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-kappaB luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-kappaB-DNA binding and IkappaBalpha degradation. Heat shock protein 72-mediated inhibition of NF-kappaB activation was further corroborated by a significant decrease in TNF-alpha production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-kappaB was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.

Induction of endotoxin tolerance enhances bacterial clearance and survival in murine polymicrobial sepsis.

The fundamental mechanisms that underlie endotoxin tolerance remain to be elucidated, and the clinical significance of endotoxin tolerance in the context of active systemic infection remains in question. We hypothesized that the endotoxin tolerance phenotype would result in decreased inflammation at the expense of altered bacterial clearance and, thus, higher mortality in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Endotoxin tolerance was induced in C57Bl/6 mice with 5 mg/kg LPS or vehicle 18 h before subsequent CLP. Lung tissue, peritoneal fluid, and blood were collected at 1, 3, 6, and 18 h after surgery for subsequent analysis. Peritoneal macrophages were isolated for ex vivo phagocytosis assay. In separate experiments, mice were allowed to recover, and survival was monitored for 7 days. Endotoxin tolerance attenuated plasma TNF-alpha and IL-6 at 6 h after CLP. Peritoneal fluid cytokines were significantly attenuated as well. Endotoxin tolerance significantly improved bacterial clearance in both blood and peritoneal fluid after CLP. Similarly, ex vivo phagocytosis by primary peritoneal macrophages and RAW264.7 murine peritoneal macrophages was significantly improved after induction of the endotoxin tolerance phenotype. Contrary to our original hypothesis, we conclude that endotoxin tolerance significantly attenuates the host inflammatory response, augments bacterial clearance, and improves survival in this murine model of polymicrobial sepsis.

Admission angiopoietin levels in children with septic shock.

Angiopoietin (angpt) 1 and angpt-2 are circulating proteins first ascribed opposing roles in embryonic angiogenesis. Both bind the tyrosine kinase with immunoglobulin-like loop and epidermal growth factor homology domains (Tie) 2 receptor on endothelial cells, but angpt-1 is a Tie-2 agonist, whereas angpt-2 antagonizes Tie-2 signaling. In the developed vasculature, angpt-1 protects against vascular leak, whereas angpt-2 promotes increased vascular permeability. Because alterations in vascular permeability are common in septic shock, we obtained plasma from critically ill children within 24 h of diagnosis of the systemic inflammatory response syndrome (SIRS, n = 20), sepsis (n = 20), or septic shock (n = 61), as well as 15 healthy controls. Plasma levels of angpt-1 and angpt-2 were measured via a commercially available enzyme-linked immunosorbent assay. Plasma angpt-2 levels were significantly elevated in children with septic shock when compared with healthy children, as well as critically ill children with either SIRS or sepsis, and circulating angpt-2 levels seemed to correlate with disease severity and outcome. In addition, plasma angpt-1 levels were significantly decreased in critically ill children with septic shock compared with critically ill children with either SIRS or sepsis. Given the contrasting effects of angpt-2 and angpt-1 on the vascular endothelium, these two factors may play an important role in the pathophysiology of septic shock in children, and further studies are warranted.

The green tea polyphenol epigallocatechin-3-gallate improves systemic hemodynamics and survival in rodent models of polymicrobial sepsis.

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid found in green tea. Recent in vitro studies have suggested that EGCG inhibits activation of the nuclear factor-kappaB (NF-kappaB) pathway. The NF-kappaB is a transcriptional factor required for gene expression of many inflammatory mediators, including the inducible isoform of nitric oxide synthase (NOS2). Excessive NO production by NOS2 is directly linked to the vasoplegia, shock, and mortality associated with sepsis. Accordingly, we hypothesized that EGCG administration would inhibit NOS2 gene expression and thereby improve survival in a rodent model of polymicrobial sepsis. Polymicrobial sepsis was induced in male Sprague-Dawley rats (hemodynamic study) and C57BL6 mice (mortality study) via cecal ligation and double puncture (CL2P). Rodents were treated with either EGCG (10 mg/kg intraperitoneally) or vehicle at 1 and 6 h after CL2P and every 12 h thereafter. In the hemodynamic study, mean arterial blood pressure was monitored for 18 h, and rats were killed at 3, 6, and 18 h after CL2P. In the mortality study, survival was monitored for 72 h after CL2P in mice. In vehicle-treated rodents, CL2P was associated with profound hypotension and greater than 80% mortality rate. Epigallocatechin-3-gallate treatment significantly improved both the hypotension and survival. In vitro experiments further showed that EGCG inhibited activation of NF-kappaB and subsequent NOS2 gene expression in a primary culture of rat aortic smooth muscle cells. Epigallocatechin-3-gallate may therefore represent a potential nutritional supplement or pharmacologic agent in patients with sepsis.