Identification of molecular subphenotypes in two cohorts of paediatric ARDS.

BACKGROUND: Two subphenotypes of acute respiratory distress syndrome (ARDS), hypoinflammatory and hyperinflammatory, have been reported in adults and in a single paediatric cohort. The relevance of these subphenotypes in paediatrics requires further investigation. We aimed to identify subphenotypes in two large observational cohorts of paediatric ARDS and assess their congruence with prior descriptions. METHODS: We performed latent class analysis (LCA) separately on two cohorts using biomarkers as inputs. Subphenotypes were compared on clinical characteristics and outcomes. Finally, we assessed overlap with adult cohorts using parsimonious classifiers. FINDINGS: In two cohorts from the Children's Hospital of Philadelphia (n=333) and from a multicentre study based at the University of California San Francisco (n=293), LCA identified two subphenotypes defined by differential elevation of biomarkers reflecting inflammation and endotheliopathy. In both cohorts, hyperinflammatory subjects had greater illness severity, more sepsis and higher mortality (41% and 28% in hyperinflammatory vs 11% and 7% in hypoinflammatory). Both cohorts demonstrated overlap with adult subphenotypes when assessed using parsimonious classifiers. INTERPRETATION: We identified hypoinflammatory and hyperinflammatory subphenotypes of paediatric ARDS from two separate cohorts with utility for prognostic and potentially predictive, enrichment. Future paediatric ARDS trials should identify and leverage biomarker-defined subphenotypes in their analysis.

Multi-omic factors associated with future wheezing in infants.

BACKGROUND: The pathophysiology of wheezing is multifactorial, impacted by medical, demographic, environmental, and immunologic factors. We hypothesized that multi-omic analyses of host and microbial factors in saliva would enhance the ability to identify infants at risk for wheezing. METHODS: This longitudinal cohort study included 161 term infants. Infants who developed wheezing (n = 27) within 24 months of delivery were identified using the International Study of Asthma and Allergies in Childhood Written Questionnaire and review of the medical record. Standardized surveys were used to assess infant traits and environmental exposures. Saliva was collected for multi-omic assessment of cytokines, microRNAs, mRNAs, and microbiome/virome RNAs. RESULTS: Two infant factors (daycare attendance, family history of asthma) and three salivary "omic" features (miR-26a-5p, Elusimicrobia, Streptococcus phage phiARI0131-1) differed between the two groups (adjusted p < 0.05). miR-26a-5p levels were correlated with Elusimicrobia (R = -0.87, p = 3.7 × 10-31). A model employing the three omic features plus daycare attendance and family asthma history yielded the highest predictive accuracy for future wheezing episodes (AUC = 0.74, 95% CI: 0.703-0.772, 77% sensitivity, 62% specificity). CONCLUSIONS: Host-microbiome interactions in saliva may yield pathophysiologic clues about the origins of wheezing and aid identification of infants at risk of future wheezing episodes. IMPACT: Wheezing is multi-factorial, but the relative contributions of infant traits, environment, and underlying biology are poorly understood. This multi-omic study identifies three molecular factors, including salivary microRNAs, microbes, and viral phages associated with increased risk of infant wheezing. Measurement of these molecular factors enhanced predictive accuracy for future wheezing when combined with family asthma history and daycare attendance. Validation of this approach could be used to identify infants at risk for wheezing and guide personalized medical management.

Multi-Omic Factors Associated with Frequency of Upper Respiratory Infections in Developing Infants.

Susceptibility to upper respiratory infections (URIs) may be influenced by host, microbial, and environmental factors. We hypothesized that multi-omic analyses of molecular factors in infant saliva would identify complex host-environment interactions associated with URI frequency. A cohort study involving 146 infants was used to assess URI frequency in the first year of life. Saliva was collected at 6 months for high-throughput multi-omic measurement of cytokines, microRNAs, transcripts, and microbial RNA. Regression analysis identified environmental (daycare attendance, atmospheric pollution, breastfeeding duration), microbial (Verrucomicrobia, Streptococcus phage), and host factors (miR-22-5p) associated with URI frequency (p < 0.05). These results provide pathophysiologic clues about molecular factors that influence URI susceptibility. Validation of these findings in a larger cohort could one day yield novel approaches to detecting and managing URI susceptibility in infants.

Lower respiratory tract delivery, airway clearance, and preclinical efficacy of inhaled GM-CSF in a postinfluenza pneumococcal pneumonia model.

Inhaled granulocyte/macrophage colony-stimulating factor (GM-CSF) shows promise as a therapeutic to treat viral and bacterial pneumonia, but no mouse model of inhaled GM-CSF has been described. We sought to 1) develop a mouse model of aerosolized recombinant mouse GM-CSF administration and 2) investigate the protection conferred by inhaled GM-CSF during influenza A virus (IAV) infection against secondary bacterial infection with pneumococcus. To assess lower respiratory tract delivery of aerosolized therapeutics, mice were exposed to aerosolized fluorescein (FITC)-labeled dextran noninvasively via an aerosolization tower or invasively using a rodent ventilator. The efficiency of delivery to the lower respiratory tracts of mice was 0.01% noninvasively compared with 0.3% invasively. The airway pharmacokinetics of inhaled GM-CSF fit a two-compartment model with a terminal phase half-life of 1.3 h. To test if lower respiratory tract levels were sufficient for biological effect, mice were infected intranasally with IAV, treated with aerosolized recombinant mouse GM-CSF, and then secondarily infected with Streptococcus pneumoniae. Inhaled GM-CSF conferred a significant survival benefit to mice against secondary challenge with S. pneumoniae (P < 0.05). Inhaled GM-CSF did not reduce airway or lung parenchymal bacterial growth but significantly reduced the incidence of S. pneumoniae bacteremia (P < 0.01). However, GM-CSF overexpression during influenza virus infection did not affect lung epithelial permeability to FITC-dextran ingress into the bloodstream. Therefore, the mechanism of protection conferred by inhaled GM-CSF appears to be locally mediated improved lung antibacterial resistance to systemic bacteremia during IAV infection.

Higher serum PD-L1 level predicts increased overall survival with lapatinib versus trastuzumab in the CCTG MA.31 phase 3 trial.

BACKGROUND: The purpose of this retrospective biomarker study of the Canadian Cancer Trials Group (CCTG) MA.31 randomized phase 3 trial (lapatinib vs trastuzumab) of HER2-positive metastatic breast cancer (MBC) was to evaluate the prognostic and predictive biomarker utility of pretreatment serum programmed death ligand 1 (PD-L1) levels. METHODS: CCTG MA.31 accrued 652 HER2-positive patients; 387 had serum available (185 in the trastuzumab arm and 202 in the lapatinib arm). The Ella immunoassay platform (ProteinSimple, San Jose, California) was used to quantitate serum PD-L1 levels. Stepwise forward Cox multivariable analyses were performed for progression-free survival and overall survival (OS). RESULTS: In the whole trial population, continuous pretreatment serum PD-L1 levels were not associated with OS. However, within the trastuzumab arm, a higher continuous pretreatment serum PD-L1 level was significant for shorter OS (hazard ratio [HR], 3.85; P = .04), but within the lapatinib arm, pretreatment serum PD-L1 was not associated with OS (P = .37). In the whole trial, in a multivariable analysis for OS, serum PD-L1 (median cut point) remained a significant independent covariate (HR, 2.38; P = .001). There was a significant interaction between treatment arm and continuous serum PD-L1 (bootstrap method; P = .0025): at or above 214.2 pg/mL (the 89th percentile), serum PD-L1 was associated with significantly shorter OS with trastuzumab treatment versus lapatinib treatment. CONCLUSIONS: In the CCTG MA.31 trial, serum PD-L1 was a significant predictive factor: a higher pretreatment serum PD-L1 level was associated with shorter OS with trastuzumab treatment but with longer OS with lapatinib treatment. Immune evasion may decrease the effectiveness of trastuzumab therapy. Further evaluation of elevated serum PD-L1 in advanced breast cancer is warranted to identify patients with HER2-positive MBC who may benefit from novel immune-targeted therapies in addition to trastuzumab.

Cytokine Panels and Pediatric Acute Respiratory Distress Syndrome: A Translational Investigation.

OBJECTIVES: To identify and compare serum and lower respiratory tract fluid biomarkers of lung injury using well-characterized mouse models of lung injury. To explore the relationship between these preclinical biomarkers and clinical outcomes in a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. DESIGN: Prospective, observational cohort study. SETTING: A basic science laboratory and the PICU of a tertiary-care children's hospital. PATIENTS: PICU patients intubated for respiratory failure from a suspected respiratory infection. INTERVENTIONS: Prospective enrollment and collection of lower respiratory tract fluid samples. MEASUREMENTS AND MAIN RESULTS: C57BL6/J mice were intranasally inoculated with escalating doses of influenza A virus or toll-like receptor agonists to simulate varying degrees of lung injury. Serum and bronchoalveolar lavage fluid were measured for the presence of cytokines using commercially available multiplex cytokine assays. Elevated levels of C-C motif chemokine ligand 7 at the peak of inflammation in both bronchoalveolar lavage fluid and serum correlated with lethality, with the bronchoalveolar lavage fluid ratio of C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 providing the best prediction in the mouse models. These preclinical biomarkers were examined in the plasma and lower respiratory tract fluid of a discovery cohort of pediatric patients with acute respiratory failure from pneumonia. The primary clinical outcome measure was ventilator-free days, with secondary outcomes of pediatric acute respiratory distress syndrome severity and mortality. Elevation in peak lower respiratory tract fluid C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratios demonstrated a significant negative correlation with ventilator-free days (r = -0.805; p < 0.02). CONCLUSIONS: This study provides evidence that lung immune profiling via lower respiratory tract fluid cytokine analysis is feasible and may provide insight into clinical outcomes. Further validation of markers, including the C-C motif chemokine ligand 7:C-C motif chemokine ligand 22 ratio in this limited study, in a larger cohort of patients is necessary.

DNA Viremia Is Associated with Hyperferritinemia in Pediatric Sepsis.

OBJECTIVE: To evaluate the relationship between detection of DNA viruses, ferritin, and outcomes in children with severe sepsis. STUDY DESIGN: We enrolled 75 pediatric patients with severe sepsis admitted to a tertiary care children's hospital. Plasma ferritin was measured within 48 hours of diagnosis and subsequently twice weekly. Herpes simplex type 1, human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, and adenovirus DNAemia were assessed by polymerase chain reaction. RESULTS: The incidence of DNAemia was increased significantly in patients with ferritin ≥1000 ng/mL (78% vs 28%; P < .05). Patients with ferritin ≥1000 ng/mL were more likely to have multiple DNA viruses detected in plasma (39% vs 4%; P < .001). The number of viruses detected in plasma directly correlated with the degree of hyperferritinemia and development of combined hepatobiliary and hematologic dysfunction after we controlled for bacterial and fungal coinfections (P < .05) as well as increased mortality after we controlled for severity of illness and cancer diagnosis (OR 2.6, 95% CI 1.1-6.3, P < .05). CONCLUSIONS: Viral DNAemia was associated with hyperferritinemia and adverse outcome in pediatric severe sepsis. Prospective studies are needed to determine whether hyperferritinemia may be used to identify patients at risk of occult DNAemia.

GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/macrophage polarization.

BACKGROUND: Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. METHODS: Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. RESULTS: Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an "M1-like" to a more "M2-like" activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). CONCLUSIONS: Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic "M1-like" macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

No Requirement for Targeted Theophylline Levels for Diuretic Effect of Aminophylline in Critically Ill Children.

OBJECTIVES: To determine the relationship between theophylline trough levels and urine output in critically ill children administered aminophylline as adjunctive diuretic therapy. DESIGN: Retrospective cohort study. SETTING: The PICU of a tertiary care children's hospital. PATIENTS: A mixed population of medical/surgical including postoperative cardiothoracic surgery patients less than 18 years old. INTERVENTIONS: Electronic medical records of all PICU patients admitted from July 2010 to June 2015 were reviewed, and patients who received aminophylline as diuretic therapy were identified. MEASUREMENTS AND MAIN RESULTS: Patient cohort data including demographics, daily aminophylline, furosemide and chlorothiazide dosing, theophylline trough levels, fluid intake, urine output and total fluid balance, blood urea nitrogen, and creatinine levels were abstracted. Multivariate analysis based on a generalized estimating equations approach demonstrated that aminophylline administration, when analyzed as a categorical variable, was associated with an increase in urine output and decreased fluid balance. However, aminophylline dosing, when analyzed as a continuous variable, was associated with neither an increase in urine output nor decreased fluid balance. Theophylline trough levels were not correlated with urine output at 24 hours (p = 0.78) and were negatively correlated with urine output at 48 hours (r = 0.078; p < 0.005). CONCLUSIONS: Aminophylline administration provided a measure of increased diuresis, regardless of dosage, and theophylline trough levels. Therefore, achieving a prescribed therapeutic trough level may not be necessary for full diuretic effect. Because, as opposed to the diuretic effect, the side effect profile of aminophylline is dose-dependent, low maintenance dosing may optimize the balance between providing adjunctive diuretic effect while minimizing the risk of toxicity.

Viral DNAemia and Immune Suppression in Pediatric Sepsis.

OBJECTIVES: Demonstrate that DNA viremia is common in pediatric sepsis and quantitate its associations with host immune function and secondary infection risk. DESIGN: Retrospective analysis of a prospective cohort study. PATIENTS: Seventy-three children admitted with sepsis-induced organ failure. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: This study was performed as an ancillary investigation to a single-center prospective study of children with severe sepsis. Longitudinally collected, batched, frozen plasma was examined using real time-polymerase chain reaction for the presence of cytomegalovirus, Epstein-Barr virus, herpes simplex virus, human herpes virus-6, torque teno virus, and adenovirus DNA. Innate immune function was also measured longitudinally via quantification of ex vivo lipopolysaccharide -induced tumor necrosis factor-α production capacity. Viral DNAemia with a virus other than torque teno virus was detected in 28 of 73 subjects (38%) and included cytomegalovirus 5%, Epstein-Barr virus 11%, herpes simplex virus 4%, human herpes virus-6 8%, and adenovirus 26%. In addition, torque teno virus was detected in 89%. Epstein-Barr virus DNAemia was associated with preexisting immune suppression (p = 0.007) Viral DNAemia was associated with preexisting immune suppression and high risk for the subsequent development of secondary infection (p < 0.05 for both). Subjects with viral DNAemia had lower innate immune function over time compared with those who were virus negative (p < 0.05). CONCLUSIONS: DNAemia from multiple viruses can be detected in septic children and is strongly associated with preexisting immune suppression and secondary infection risk. The role of DNA viruses in the perpetuation of impaired host defense in this setting should be the subject of prospective study.

A Systemic Inflammation Mortality Risk Assessment Contingency Table for Severe Sepsis.

OBJECTIVES: We tested the hypothesis that a C-reactive protein and ferritin-based systemic inflammation contingency table can track mortality risk in pediatric severe sepsis. DESIGN: Prospective cohort study. SETTING: Tertiary PICU. PATIENTS: Children with 100 separate admission episodes of severe sepsis were enrolled. INTERVENTIONS: Blood samples were attained on day 2 of sepsis and bi-weekly for biomarker batch analysis. A 2 × 2 contingency table using C-reactive protein and ferritin thresholds was developed. MEASUREMENTS AND MAIN RESULTS: A C-reactive protein of 4.08 mg/dL and a ferritin of 1,980 ng/mL were found to be optimal cutoffs for outcome prediction at first sampling (n = 100) using the Youden index. PICU mortality was increased in the "high-risk" C-reactive protein greater than or equal to 4.08 mg/dL and ferritin greater than or equal to 1,980 ng/mL category (6/13 [46.15%]) compared with the "intermediate-risk" C-reactive protein greater than or equal to 4.08 mg/dL and ferritin less than 1,980 ng/mL or C-reactive protein less than 4.08 mg/dL and ferritin greater than or equal to 1,980 ng/mL categories (2/43 [4.65%]), and the "low-risk" C-reactive protein less than 4.08 mg/dL and ferritin less than 1,980 ng/mL category (0/44 [0%]) (odds ratio, 36.43 [95% CI, 6.16-215.21]). The high-risk category was also associated with the development of immunoparalysis (odds ratio, 4.47 [95% CI, 1.34-14.96]) and macrophage activation syndrome (odds ratio, 24.20 [95% CI, 5.50-106.54]). Sixty-three children underwent sequential blood sampling; those who were initially in the low-risk category (n = 24) and those who subsequently migrated (n = 19) to the low-risk category all survived, whereas those who remained in the "at-risk" categories had increased mortality (7/20 [35%]; p < 0.05). CONCLUSIONS: A C-reactive protein- and ferritin-based contingency table effectively assessed mortality risk. Reduction in systemic inflammation below a combined threshold C-reactive protein of 4.08 mg/dL and ferritin of 1,980 ng/mL appeared to be a desired response in children with severe sepsis.

Three Hypothetical Inflammation Pathobiology Phenotypes and Pediatric Sepsis-Induced Multiple Organ Failure Outcome.

OBJECTIVES: We hypothesize that three inflammation pathobiology phenotypes are associated with increased inflammation, proclivity to develop features of macrophage activation syndrome, and multiple organ failure-related death in pediatric severe sepsis. DESIGN: Prospective cohort study comparing children with severe sepsis and any of three phenotypes: 1) immunoparalysis-associated multiple organ failure (whole blood ex vivo tumor necrosis factor response to endotoxin < 200 pg/mL), 2) thrombocytopenia-associated multiple organ failure (new onset thrombocytopenia with acute kidney injury and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 activity < 57%), and/or 3) sequential multiple organ failure with hepatobiliary dysfunction (respiratory distress followed by liver dysfunction with soluble Fas ligand > 200 pg/mL), to those without any of these phenotypes. SETTING: Tertiary children's hospital PICU. PATIENTS: One hundred consecutive severe sepsis admissions. INTERVENTIONS: Clinical data were recorded daily, and blood was collected twice weekly. MEASUREMENTS AND MAIN RESULTS: Multiple organ failure developed in 75 cases and eight died. Multiple organ failure cases with any of the three inflammation phenotypes (n = 37) had higher inflammation (C-reactive protein, p = 0.009 and ferritin, p < 0.001) than multiple organ failure cases without any of these phenotypes (n = 38) or cases with only single organ failure (n = 25). Development of features of macrophage activation syndrome and death were more common among multiple organ failure cases with any of the phenotypes (macrophage activation syndrome: 10/37, 27%; death: 8/37, 22%) compared to multiple organ failure cases without any phenotype (macrophage activation syndrome: 1/38, 3%; p = 0.003 and death: 0/38, 0%; p = 0.002). CONCLUSIONS: Our approach to phenotype categorization remains hypothetical, and the phenotypes identified need to be confirmed in multicenter studies of pediatric multiple organ dysfunction syndrome.

Predicting Organ Dysfunction in Septic and Critically Ill Patients: A Prospective Cohort Study Using Rapid Ex Vivo Immune Profiling.

OBJECTIVES: While cytokine response patterns are pivotal in mediating immune responses, they are also often dysregulated in sepsis and critical illness. We hypothesized that these immunological deficits, quantifiable through ex vivo whole blood stimulation assays, may be indicative of subsequent organ dysfunction. DESIGN: In a prospective observational study, adult septic patients and critically ill but nonseptic controls were identified within 48 hours of critical illness onset. Using a rapid, ex vivo assay based on responses to lipopolysaccharide (LPS), anti-CD3/anti-CD28 antibodies, and phorbol 12-myristate 13-acetate with ionomycin, cytokine responses to immune stimulants were quantified. The primary outcome was the relationship between early cytokine production and subsequent organ dysfunction, as measured by the Sequential Organ Failure Assessment score on day 3 of illness (SOFAd3). SETTING: Patients were recruited in an academic medical center and data processing and analysis were done in an academic laboratory setting. PATIENTS: Ninety-six adult septic and critically ill nonseptic patients were enrolled. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Elevated levels of tumor necrosis factor and interleukin-6 post-endotoxin challenge were inversely correlated with SOFAd3. Interferon-gamma production per lymphocyte was inversely related to organ dysfunction at day 3 and differed between septic and nonseptic patients. Clustering analysis revealed two distinct immune phenotypes, represented by differential responses to 18 hours of LPS stimulation and 4 hours of anti-CD3/anti-CD28 stimulation. CONCLUSIONS: Our rapid immune profiling technique offers a promising tool for early prediction and management of organ dysfunction in critically ill patients. This information could be pivotal for early intervention and for preventing irreversible organ damage during the acute phase of critical illness.

SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages.

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210L and a short (S) SP-R210S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210L antagonizes type I interferon ß (IFNß), as depletion of SP-R210L potentiates IFNß secretion. SP-R210 antibodies enhance and attenuate IFNß secretion in SP-R210L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210L, however, results in macropinocytic retention of the virus that depends on both SP-R210S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

Inflammatory and tissue injury marker dynamics in pediatric acute respiratory distress syndrome.

BACKGROUNDThe molecular signature of pediatric acute respiratory distress syndrome (ARDS) is poorly described, and the degree to which hyperinflammation or specific tissue injury contributes to outcomes is unknown. Therefore, we profiled inflammation and tissue injury dynamics over the first 7 days of ARDS, and associated specific biomarkers with mortality, persistent ARDS, and persistent multiple organ dysfunction syndrome (MODS).METHODSIn a single-center prospective cohort of intubated pediatric patients with ARDS, we collected plasma on days 0, 3, and 7. Nineteen biomarkers reflecting inflammation, tissue injury, and damage-associated molecular patterns (DAMPs) were measured. We assessed the relationship between biomarkers and trajectories with mortality, persistent ARDS, or persistent MODS using multivariable mixed effect models.RESULTSIn 279 patients (64 [23%] nonsurvivors), hyperinflammatory cytokines, tissue injury markers, and DAMPs were higher in nonsurvivors. Survivors and nonsurvivors showed different biomarker trajectories. IL-1α, soluble tumor necrosis factor receptor 1, angiopoietin 2 (ANG2), and surfactant protein D increased in nonsurvivors, while DAMPs remained persistently elevated. ANG2 and procollagen type III N-terminal peptide were associated with persistent ARDS, whereas multiple cytokines, tissue injury markers, and DAMPs were associated with persistent MODS. Corticosteroid use did not impact the association of biomarker levels or trajectory with mortality.CONCLUSIONSPediatric ARDS survivors and nonsurvivors had distinct biomarker trajectories, with cytokines, endothelial and alveolar epithelial injury, and DAMPs elevated in nonsurvivors. Mortality markers overlapped with markers associated with persistent MODS, rather than persistent ARDS.FUNDINGNIH (K23HL-136688, R01-HL148054).

Reduced frequency of CD56 dim CD16 pos natural killer cells in pediatric systemic inflammatory response syndrome/sepsis patients.

BACKGROUND: Sepsis continues to be a leading cause of death in infants and children. Natural killer (NK) cells serve as a bridge between innate and adaptive immunity, yet their role in pediatric sepsis has not been well characterized. METHODS: We tested the hypothesis that decreased NK cell cytotoxicity is a common feature of pediatric systemic inflammatory response syndrome (SIRS)/sepsis patients by measuring, using flow cytometry, NK cell cytotoxicity and cell surface phenotype in the peripheral blood of 38 pediatric intensive care patients who demonstrated signs and symptoms of SIRS and/or sepsis. RESULTS: NK cell cytotoxicity was significantly reduced in peripheral blood mononuclear cells (PBMCs) of pediatric SIRS/sepsis patients as compared with healthy controls, and the percentage of CD56(dim) CD16(+) cytotoxic NK cells in PBMCs was lower in patients with SIRS/sepsis than in normal donors. However, on a per cell basis, CD56(dim) CD16(+) NK cells in patients mediated cytotoxicity as well as those in normal donors. CONCLUSION: The NK cell dysfunction in pediatric SIRS/sepsis patients reflects a quantitative rather than a qualitative difference from healthy controls.

Ex Vivo Endotoxin Stimulation of Blood for Predicting Survival in Patients With Sepsis: A Systematic Review.

BACKGROUND: Sepsis is a syndrome characterized by host immune dysfunction, with the extent of immunoparalysis differing among patients. Lipopolysaccharide (LPS) is used commonly to assess the immune function of critically ill patients with sepsis. However, the reliability of this ex vivo diagnostic test in predicting clinical outcomes remains uncertain. RESEARCH QUESTION: Does LPS-induced tumor necrosis factor (TNF) production from the blood of patients with sepsis predict mortality? Secondary outcomes included ICU and hospital stay durations, nosocomial infection rate, and organ recovery rate. STUDY DESIGN AND METHODS: Human sepsis studies from various databases through April 2023 were evaluated. Inclusion criteria encompassed LPS-stimulated blood assays, English language, and reported clinical outcomes. Bias risk was evaluated using the Newcastle-Ottawa scale (NOS). Relationships between TNF production and mortality were analyzed at sepsis onset and during established sepsis, alongside secondary outcomes. RESULTS: Of 11,580 studies, 17 studies (14 adult and three pediatric) were selected for analysis. Although 15 studies were evaluated as moderate to high quality using the NOS, it is important to note that some of these studies also had identifiable biases, such as unclear methods of participant recruitment. Nine studies detailed survival outcomes associated with LPS-induced TNF production at sepsis onset, whereas five studies explored TNF production's relationship with mortality during established sepsis. Trends suggested that lower LPS-induced TNF production correlated with higher mortality. However, heterogeneity in methodologies, especially the LPS assay protocol, hindered definitive conclusions. Publication bias was highlighted using funnel plot analysis. Concerning secondary outcomes, diminished TNF production might signify worsening organ dysfunction, although the link between cytokine production and nosocomial infection varied among studies. INTERPRETATION: For functional immune profiling in sepsis, streamlined research methodologies are essential. This entails organizing cohorts based on microbial sources of sepsis, establishing standardized definitions of immunoparalysis, using consistent types and dosages of immune stimulants, adhering to uniform blood incubation conditions, and adopting consistent clinical outcomes.

Case Report: Successful avoidance of etoposide for primary hemophagocytic lymphohistiocytosis-induced multiple organ dysfunction syndrome using emapalumab.

We describe the case of an infant who presented with simple rhinovirus/enterovirus bronchiolitis whose condition worsened with rapid progression to multiple organ dysfunction syndrome (MODS). The patient was presumed to have either primary or secondary hemophagocytic lymphohistiocytosis (HLH), and treatment was initiated using dexamethasone, anakinra, and intravenous immunoglobulin to modulate the immune system. Due to the organ dysfunction, the use of etoposide was avoided and instead, emapalumab, an interferon gamma antagonist, was administered at a dose of 6 mg/kg. The patient's organ failure improved, and the levels of inflammatory markers decreased. The flow cytometry analysis revealed that cytotoxic cells lacked perforin expression, and subsequent genetic analysis confirmed homozygous pathogenic mutations in the perforin gene. This case highlights the potential avoidance of etoposide in cases of primary HLH, the possible benefit of an elevated initial dose of emapalumab, and the contribution offered by a multi-specialty team approach to complex diagnosis.

Comparison of whole blood cytokine immunoassays for rapid, functional immune phenotyping in critically ill patients with sepsis.

BACKGROUND: Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7-10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality. RESULTS: Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation. CONCLUSIONS: Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.

Immunocompromised Children With Acute Respiratory Distress Syndrome Possess a Distinct Circulating Inflammatory Profile.

Immunocompromised status, with and without stem cell transplant, confers a worse prognosis in pediatric acute respiratory distress syndrome. An improved understanding of the biochemical profile of immunocompromised children with acute respiratory distress syndrome would inform whether specific pathways are targetable, or merely bystanders, in order to improve outcomes in this high-risk subgroup. OBJECTIVES: We aimed to identify a biomarker profile of immunocompromised children, with and without stem cell transplant, independent of illness severity. DESIGN SETTINGS AND PARTICIPANTS: This was a secondary analysis of a prospective cohort study of intubated children with Berlin-defined acute respiratory distress syndrome with existing biomarker measurements conducted in a large academic PICU between 2014 and 2019. MAIN OUTCOMES AND MEASURES: Biomarker levels were compared between immunocompetent and immunocompromised children, with and without stem cell transplant, both prior to and after adjusting for severity of illness. RESULTS: In 333 children with acute respiratory distress syndrome, 84 were immunocompromised, of whom 39 had a stem cell transplant. Circulating neutrophil levels were strongly correlated with biomarkers, with 14 of 18 measured proteins differentially expressed in patients with versus without neutropenia. In order to identify biomarker levels independent of severity of illness, acute respiratory distress syndrome etiology, and neutrophil levels, we computed predicted (log-transformed) biomarker levels after adjusting for confounders using linear regression and then compared these severity-adjusted levels between immunocompetent and immunocompromised (with and without stem cell transplant) subjects using analyses of variance and post hoc Bonferroni. After multivariable adjustment, 11 biomarkers were higher in immunocompromised subjects without stem cell transplant, relative to immunocompetent, implicating endotheliopathy (angiopoietin-2), tissue damage (procollagen type III N-terminal peptide), and innate immunity. A single biomarker, C-C motif chemokine ligand 22, was lower in immunocompromised subjects with and without stem cell transplant. CONCLUSIONS AND RELEVANCE: Immunocompromised children with acute respiratory distress syndrome were characterized by elevations in pro-inflammatory and endothelial damage biomarkers. Our study provides insight into mechanisms underlying the molecular heterogeneity of this population and potentially identifies targetable pathways to mitigate their increased mortality risk.