Revisiting Post-ICU Admission Fluid Balance Across Pediatric Sepsis Mortality Risk Strata: A Secondary Analysis of a Prospective Observational Cohort Study.

OBJECTIVES: Post-ICU admission cumulative positive fluid balance (PFB) is associated with increased mortality among critically ill patients. We sought to test whether this risk varied across biomarker-based risk strata upon adjusting for illness severity, presence of severe acute kidney injury (acute kidney injury), and use of continuous renal replacement therapy (CRRT) in pediatric septic shock. DESIGN: Ongoing multicenter prospective observational cohort. SETTING: Thirteen PICUs in the United States (2003-2023). PATIENTS: Six hundred and eighty-one children with septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Cumulative percent PFB between days 1 and 7 (days 1-7 %PFB) was determined. Primary outcome of interest was complicated course defined as death or persistence of greater than or equal to two organ dysfunctions by day 7. Pediatric Sepsis Biomarker Risk Model (PERSEVERE)-II biomarkers were used to assign mortality probability and categorize patients into high mortality (n = 91), intermediate mortality (n = 134), and low mortality (n = 456) risk strata. Cox proportional hazard regression models with adjustment for PERSEVERE-II mortality probability, presence of sepsis-associated acute kidney injury on day 3, and use of CRRT, demonstrated that time-dependent variable days 1-7%PFB was independently associated with an increased hazard of complicated course. Risk-stratified analyses revealed that each 10% increase in days 1-7 %PFB was associated with increased hazard of complicated course only among patients with high mortality risk strata (adjusted hazard ratio 1.24 (95% CI, 1.08-1.43), p = 0.003). However, this association was not causally mediated by PERSEVERE-II biomarkers. CONCLUSIONS: Our data demonstrate the influence of cumulative %PFB on the risk of complicated course in pediatric septic shock. Contrary to our previous report, this risk was largely driven by patients categorized as having a high mortality risk based on PERSEVERE-II biomarkers. Incorporation of such prognostic enrichment tools in randomized trials of restrictive fluid management or early initiation of de-escalation strategies may inform targeted application of such interventions among at-risk patients.

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.

Machine learning-driven identification of the gene-expression signature associated with a persistent multiple organ dysfunction trajectory in critical illness.

BACKGROUND: Multiple organ dysfunction syndrome (MODS) disproportionately drives morbidity and mortality among critically ill patients. However, we lack a comprehensive understanding of its pathobiology. Identification of genes associated with a persistent MODS trajectory may shed light on underlying biology and allow for accurate prediction of those at-risk. METHODS: Secondary analyses of publicly available gene-expression datasets. Supervised machine learning (ML) was used to identify a parsimonious set of genes associated with a persistent MODS trajectory in a training set of pediatric septic shock. We optimized model parameters and tested risk-prediction capabilities in independent validation and test datasets, respectively. We compared model performance relative to an established gene-set predictive of sepsis mortality. FINDINGS: Patients with a persistent MODS trajectory had 568 differentially expressed genes and characterized by a dysregulated innate immune response. Supervised ML identified 111 genes associated with the outcome of interest on repeated cross-validation, with an AUROC of 0.87 (95% CI: 0.85-0.88) in the training set. The optimized model, limited to 20 genes, achieved AUROCs ranging from 0.74 to 0.79 in the validation and test sets to predict those with persistent MODS, regardless of host age and cause of organ dysfunction. Our classifier demonstrated reproducibility in identifying those with persistent MODS in comparison with a published gene-set predictive of sepsis mortality. INTERPRETATION: We demonstrate the utility of supervised ML driven identification of the genes associated with persistent MODS. Pending validation in enriched cohorts with a high burden of organ dysfunction, such an approach may inform targeted delivery of interventions among at-risk patients. FUNDING: H.R.W.'s NIHR35GM126943 award supported the work detailed in this manuscript. Upon his death, the award was transferred to M.N.A. M.R.A., N.S.P, and R.K were supported by NIHR21GM151703. R.K. was supported by R01GM139967.

Obesity Alters cytokine signaling and gut microbiome in septic mice.

Sepsis is a leading cause of mortality. Plasma cytokine levels may identify those at increased risk of mortality from sepsis. Our aim was to understand how obesity alters cytokine levels during early sepsis and its correlation with survival. Six-week-old C57BL/6 male mice were randomized to control (non-obese) or high fat diet (obese) for 5-7 weeks. Sepsis was induced by cecal ligation and perforation (CLP). Cytokine levels were measured from cheek bleeds 8 h after CLP, and mice were monitored for survival. Other cohorts were sacrificed 1 h after CLP for plasma and tissue. Septic obese mice had higher survival. At 8 h after sepsis, obese mice had higher adiponectin, leptin, and resistin but lower TNFα and IL-6 compared to non-obese mice. When stratified by 24-h survival, adipokines were not significantly different in obese and non-obese mice. TNFα and IL-6 were higher in non-obese, compared to obese, mice that died within 24 h of sepsis. Diet and to sepsis significantly impacted the cecal microbiome. IL-6 is a prognostic biomarker during early sepsis in non-obese and obese mice. A plausible mechanism for the survival difference in non-obese and obese mice may be the difference in gut microbiome and its evolution during sepsis.

Prognostic and predictive value of endothelial dysfunction biomarkers in sepsis-associated acute kidney injury: risk-stratified analysis from a prospective observational cohort of pediatric septic shock.

BACKGROUND: Sepsis-associated acute kidney injury (SA-AKI) is associated with high morbidity, with no current therapies available beyond continuous renal replacement therapy (CRRT). Systemic inflammation and endothelial dysfunction are key drivers of SA-AKI. We sought to measure differences between endothelial dysfunction markers among children with and without SA-AKI, test whether this association varied across inflammatory biomarker-based risk strata, and develop prediction models to identify those at highest risk of SA-AKI. METHODS: Secondary analyses of prospective observational cohort of pediatric septic shock. Primary outcome of interest was the presence of ≥ Stage II KDIGO SA-AKI on day 3 based on serum creatinine (D3 SA-AKI SCr). Biomarkers including those prospectively validated to predict pediatric sepsis mortality (PERSEVERE-II) were measured in Day 1 (D1) serum. Multivariable regression was used to test the independent association between endothelial markers and D3 SA-AKI SCr. We conducted risk-stratified analyses and developed prediction models using Classification and Regression Tree (CART), to estimate risk of D3 SA-AKI among prespecified subgroups based on PERSEVERE-II risk. RESULTS: A total of 414 patients were included in the derivation cohort. Patients with D3 SA-AKI SCr had worse clinical outcomes including 28-day mortality and need for CRRT. Serum soluble thrombomodulin (sTM), Angiopoietin-2 (Angpt-2), and Tie-2 were independently associated with D3 SA-AKI SCr. Further, Tie-2 and Angpt-2/Tie-2 ratios were influenced by the interaction between D3 SA-AKI SCr and risk strata. Logistic regression demonstrated models predictive of D3 SA-AKI risk performed optimally among patients with high- or intermediate-PERSEVERE-II risk strata. A 6 terminal node CART model restricted to this subgroup of patients had an area under the receiver operating characteristic curve (AUROC) 0.90 and 0.77 upon tenfold cross-validation in the derivation cohort to distinguish those with and without D3 SA-AKI SCr and high specificity. The newly derived model performed modestly in a unique set of patients (n = 224), 84 of whom were deemed high- or intermediate-PERSEVERE-II risk, to distinguish those patients with high versus low risk of D3 SA-AKI SCr. CONCLUSIONS: Endothelial dysfunction biomarkers are independently associated with risk of severe SA-AKI. Pending validation, incorporation of endothelial biomarkers may facilitate prognostic and predictive enrichment for selection of therapeutics in future clinical trials among critically ill children.

Detrimental effects of PCSK9 loss-of-function in the pediatric host response to sepsis are mediated through independent influence on Angiopoietin-1.

BACKGROUND: Sepsis is associated with significant mortality. Yet, there are no efficacious therapies beyond antibiotics. PCSK9 loss-of-function (LOF) and inhibition, through enhanced low-density lipoprotein receptor (LDLR) mediated endotoxin clearance, holds promise as a potential therapeutic approach among adults. In contrast, we have previously demonstrated higher mortality in the juvenile host. Given the potential pleiotropic effects of PCSK9 on the endothelium, beyond canonical effects on serum lipoproteins, both of which may influence sepsis outcomes, we sought to test the influence of PCSK9 LOF genotype on endothelial dysfunction. METHODS: Secondary analyses of a prospective observational cohort of pediatric septic shock. Genetic variants of PCSK9 and LDLR genes, serum PCSK9, and lipoprotein concentrations were determined previously. Endothelial dysfunction markers were measured in day 1 serum. We conducted multivariable linear regression to test the influence of PCSK9 LOF genotype on endothelial markers, adjusted for age, complicated course, and low- and high-density lipoproteins (LDL and HDL). Causal mediation analyses to test impact of select endothelial markers on the association between PCSK9 LOF genotype and mortality. Juvenile Pcsk9 null and wildtype mice were subject to cecal slurry sepsis and endothelial markers were quantified. RESULTS: A total of 474 patients were included. PCSK9 LOF was associated with several markers of endothelial dysfunction, with strengthening of associations after exclusion of those homozygous for the rs688 LDLR variant that renders it insensitive to PCSK9. Serum PCSK9 was not correlated with endothelial dysfunction. PCSK9 LOF influenced concentrations of Angiopoietin-1 (Angpt-1) upon adjusting for potential confounders including lipoprotein concentrations, with false discovery adjusted p value of 0.042 and 0.013 for models that included LDL and HDL, respectively. Causal mediation analysis demonstrated that the effect of PCSK9 LOF on mortality was mediated by Angpt-1 (p = 0.0008). Murine data corroborated these results with lower Angpt-1 and higher soluble thrombomodulin among knockout mice with sepsis relative to the wildtype. CONCLUSIONS: We present genetic and biomarker association data that suggest a potential direct role of the PCSK9-LDLR pathway on Angpt-1 in the developing host with septic shock and warrant external validation. Further, mechanistic studies on the role of PCSK9-LDLR pathway on vascular homeostasis may lead to the development of pediatric-specific sepsis therapies.

Acute kidney injury biomarker olfactomedin 4 predicts furosemide responsiveness.

BACKGROUND: Acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. Olfactomedin 4 (OLFM4), a secreted glycoprotein expressed in neutrophils and stressed epithelial cells, is upregulated in loop of Henle (LOH) cells following AKI. We hypothesized that urine OLFM4 (uOLFM4) will increase in patients with AKI and may predict furosemide responsiveness. METHODS: Urine from critically ill children was collected prospectively and tested for uOLFM4 concentrations with a Luminex immunoassay. Severe AKI was defined by KDIGO (stage 2/3) serum creatinine criteria. Furosemide responsiveness was defined as > 3 mL/kg/h of urine output in the 4 h after a 1 mg/kg IV furosemide dose administered as part of standard of care. RESULTS: Fifty-seven patients contributed 178 urine samples. Irrespective of sepsis status or AKI cause, uOLFM4 concentrations were higher in patients with AKI (221 ng/mL [IQR 93-425] vs. 36 ng/mL [IQR 15-115], p = 0.007). uOLFM4 concentrations were higher in patients unresponsive to furosemide (230 ng/mL [IQR 102-534] vs. 42 ng/mL [IQR 21-161], p = 0.04). Area under the receiver operating curve for association with furosemide responsiveness was 0.75 (95% CI, 0.60-0.90). CONCLUSIONS: AKI is associated with increased uOLFM4. Higher uOLFM4 is associated with a lack of response to furosemide. Further testing is warranted to determine whether uOLFM4 could identify patients most likely to benefit from earlier escalation from diuretics to kidney replacement therapy to maintain fluid balance. A higher resolution version of the Graphical abstract is available as Supplementary information.

Detrimental effects of PCSK9 loss-of-function in the pediatric host response to sepsis are mediated through independent influence on Angiopoietin-1.

Background: Sepsis is associated with significant mortality, yet there are no efficacious therapies beyond antibiotics and supportive care. In adult sepsis studies, PCSK9 loss-of-function (LOF) and inhibition has shown therapeutic promise, likely through enhanced low-density lipoprotein receptor (LDLR) mediated endotoxin clearance. In contrast, we previously demonstrated higher mortality in septic juvenile hosts with PCSK9 LOF. In addition to direct influence on serum lipoprotein levels, PCSK9 likely exerts pleiotropic effects on vascular endothelium. Both mechanisms may influence sepsis outcomes. We sought to test the influence of PCSK9 LOF genotype on endothelial dysfunction in pediatric sepsis. Methods: Secondary analyses of a prospective observational cohort of pediatric septic shock. Single nucleotide polymorphisms of PCSK9 and LDLR genes were assessed. Serum PCSK9, lipoprotein, and endothelial marker concentrations were measured. Multivariable linear regression tested the influence of PCSK9 LOF genotype on endothelial markers, adjusted for age, complicated course, and low- and high-density lipoproteins (LDL and HDL). Causal mediation analyses assessed impact of select endothelial markers on the association between PCSK9 LOF genotype and mortality. Juvenile Pcsk9 null and wildtype mice were subject to cecal slurry sepsis and endothelial markers were quantified. Results: 474 patients were included. PCSK9 LOF was associated with several markers of endothelial dysfunction, with strengthening of associations after exclusion of patients homozygous for the rs688 LDLR variant that renders it insensitive to PCSK9. Serum PCSK9 levels did not correlate with endothelial dysfunction. PCSK9 LOF significantly influenced concentrations of Angiopoietin-1 (Angpt-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1). However, upon adjusting for LDL and HDL, PCSK9 LOF remained significantly associated with low Angpt-1 alone. Causal Mediation Analysis demonstrated that the effect of PCSK9 LOF on mortality was partially mediated by Angpt-1 (p=0.0008). Murine data corroborated these results with lower Angpt-1 and higher soluble thrombomodulin among knockout mice with sepsis relative to the wildtype. Conclusions: PCSK9 LOF independently influences serum Angpt-1 levels in pediatric septic shock. Angpt-1 likely contributes mechanistically to the effect of PCSK9 LOF on mortality in juvenile hosts. Mechanistic studies on the role of PCSK9-LDLR pathway on vascular homeostasis may lead to the development of novel pediatric-specific sepsis therapies.

OBESE MICE WITH PNEUMONIA HAVE HYPERLEPTINEMIA AND INCREASED PULMONARY SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 ACTIVATION.

ABSTRACT: Obesity is an ongoing epidemic that influences pathobiology in numerous disease states. Obesity is associated with increased plasma leptin levels, a hormone that activates the signal transducer and activator of transcription 3 (STAT3) pathway. Pneumonia is a significant cause of morbidity and mortality. During pneumonia, inflammatory pathways including STAT3 are activated. Outcomes in obese patients with pneumonia are mixed, with some studies showing obesity increases harm and others showing benefit. It is unclear whether obesity alters STAT3 activation during bacterial pneumonia and how this might impact outcomes from pneumonia. We used a murine model of obesity and pneumonia challenge with Pseudomonas aeruginosa in obese and nonobese mice to investigate the effect of obesity on STAT3 activation. We found obese mice with bacterial pneumonia had increased mortality compared with nonobese mice. Inflammatory markers, IL-6 and TNF-α, and lung neutrophil infiltration were elevated at 6 h after pneumonia in both nonobese and obese mice. Obese mice had greater lung injury compared with nonobese mice at 6 h after pneumonia. Leptin and insulin levels were higher in obese mice compared with nonobese mice, and obese mice with pneumonia had higher pulmonary STAT3 activation compared with nonobese mice.

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.

Biomarkers estimating baseline mortality risk for neonatal sepsis: nPERSEVERE: neonate-specific sepsis biomarker risk model.

BACKGROUND: Prognostic biomarker research neonatal sepsis is lacking. We assessed the utility of a validated pediatric prognostic tool called PERSEVERE II that uses decision tree methodology to predict mortality at discharge in neonates who experienced sepsis. METHODS: Prospective study in a dual-center cohort of neonates with sepsis admitted between June 2020 and December 2021. Biomarker analysis was done on serum samples obtained at the time of evaluation for the event. RESULTS: In a cohort of 59 neonates with a mortality rate of 15.3%, PERSEVERE II was 67% sensitive and 59% specific for mortality, p 0.27. Amongst PERSEVERE II biomarkers, IL-8 showed good prognostic performance for mortality prediction with a cutoff of 300 pg/mL (sensitivity 100%, specificity 65%, negative predictive value 100%, AUC 0.87, p 0.0003). We derived a new decision tree that is neonate specific (nPERSEVERE) with improved performance compared to IL-8 (sensitivity 100%, specificity 86%, negative predictive value 100%, AUC 0.95, p < 0.0001). CONCLUSIONS: IL-8 and nPERSEVERE demonstrated good prognostic performance in a small cohort of neonates with sepsis. Moving toward precision medicine in sepsis, our study proposes an important tool for clinical trial prognostic enrichment that needs to be validated in larger studies. IMPACT: Prognostic and predictive biomarker research is lacking in the newborn intensive care unit. Biomarkers can be used at the time of evaluation for neonatal sepsis (blood culture acquisition) to identify neonates with high baseline mortality risk. Stratification is an important step toward precision medicine in neonatal sepsis.

Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia.

INTRODUCTION: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. METHODS: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. RESULTS: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3-76.9%) versus 21.5% (IQR 10.6-34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2-61.5%) compared to those without it at 13.7% (IQR 7.7-26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3-29.0%) versus 32.5% (IQR 18.5-46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. CONCLUSION: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

Olfactomedin 4 as a novel loop of Henle-specific acute kidney injury biomarker.

Acute kidney injury (AKI) is associated with morbidity and mortality. Urinary biomarkers may disentangle its clinical heterogeneity. Olfactomedin 4 (OLFM4) is a secreted glycoprotein expressed in stressed neutrophils and epithelial cells. In septic mice, OLFM4 expression localized to the kidney's loop of Henle (LOH) and was detectable in the urine. We hypothesized that urine OLFM4 (uOLFM4) will be increased in patients with AKI and sepsis. Urine from critically ill pediatric patients was obtained from a prospective study based on AKI and sepsis status. uOLFM4 was quantified with a Luminex immunoassay. AKI was defined by KDIGO severe criteria. Sepsis status was extracted from the medical record based on admission diagnosis. Immunofluorescence on pediatric kidney biopsies was performed with NKCC2, uromodulin and OLFM4 specific antibodies. Eight patients had no sepsis, no AKI; 7 had no sepsis but did have AKI; 10 had sepsis, no AKI; 11 had sepsis and AKI. Patients with AKI had increased uOLFM4 compared to no/stage 1 AKI (p = 0.044). Those with sepsis had increased uOLFM4 compared to no sepsis (p = 0.026). uOLFM4 and NGAL were correlated (r2 0.59, 95% CI 0.304-0.773, p = 0.002), but some patients had high uOLFM4 and low NGAL, and vice versa. Immunofluorescence on kidney biopsies demonstrated OLFM4 colocalization with NKCC2 and uromodulin, suggesting expression in the thick ascending LOH (TALH). We conclude that AKI and sepsis are associated with increased uOLFM4. uOLFM4 and NGAL correlated in many patients, but was poor in others, suggesting these markers may differentiate AKI subgroups. Given OLFM4 colocalization to human TALH, we propose OLFM4 may be a LOH-specific AKI biomarker.

Integrated PERSEVERE and endothelial biomarker risk model predicts death and persistent MODS in pediatric septic shock: a secondary analysis of a prospective observational study.

BACKGROUND: Multiple organ dysfunction syndrome (MODS) is a critical driver of sepsis morbidity and mortality in children. Early identification of those at risk of death and persistent organ dysfunctions is necessary to enrich patients for future trials of sepsis therapeutics. Here, we sought to integrate endothelial and PERSEVERE biomarkers to estimate the composite risk of death or organ dysfunctions on day 7 of septic shock. METHODS: We measured endothelial dysfunction markers from day 1 serum among those with existing PERSEVERE data. TreeNet® classification model was derived incorporating 22 clinical and biological variables to estimate risk. Based on relative variable importance, a simplified 6-biomarker model was developed thereafter. RESULTS: Among 502 patients, 49 patients died before day 7 and 124 patients had persistence of MODS on day 7 of septic shock. Area under the receiver operator characteristic curve (AUROC) for the newly derived PERSEVEREnce model to predict death or day 7 MODS was 0.93 (0.91-0.95) with a summary AUROC of 0.80 (0.76-0.84) upon tenfold cross-validation. The simplified model, based on IL-8, HSP70, ICAM-1, Angpt2/Tie2, Angpt2/Angpt1, and Thrombomodulin, performed similarly. Interaction between variables-ICAM-1 with IL-8 and Thrombomodulin with Angpt2/Angpt1-contributed to the models' predictive capabilities. Model performance varied when estimating risk of individual organ dysfunctions with AUROCS ranging from 0.91 to 0.97 and 0.68 to 0.89 in training and test sets, respectively. CONCLUSIONS: The newly derived PERSEVEREnce biomarker model reliably estimates risk of death or persistent organ dysfunctions on day 7 of septic shock. If validated, this tool can be used for prognostic enrichment in future pediatric trials of sepsis therapeutics.

A neutrophil subset defined by intracellular olfactomedin 4 is associated with mortality in sepsis.

Sepsis is a heterogeneous syndrome clinically and biologically, but biomarkers of distinct host response pathways for early prognostic information and testing targeted treatments are lacking. Olfactomedin 4 (OLFM4), a matrix glycoprotein of neutrophil-specific granules, defines a distinct neutrophil subset that may be an independent risk factor for poor outcomes in sepsis. We hypothesized that increased percentage of OLFM4+ neutrophils on sepsis presentation would be associated with mortality. In a single-center, prospective cohort study, we enrolled adults admitted to an academic medical center from the emergency department (ED) with suspected sepsis [identified by 2 or greater systemic inflammatory response syndrome (SIRS) criteria and antibiotic receipt] from March 2016 through December 2017, followed by sepsis adjudication according to Sepsis-3. We collected 200 µL of whole blood within 24 h of admission and stained for the neutrophil surface marker CD66b followed by intracellular staining for OLFM4 quantitated by flow cytometry. The predictors for 60-day mortality were 1) percentage of OLFM4+ neutrophils and 2) OLFM4+ neutrophils at a cut point of ≥37.6% determined by the Youden Index. Of 120 enrolled patients with suspected sepsis, 97 had sepsis and 23 had nonsepsis SIRS. The mean percentage of OLFM4+ neutrophils was significantly increased in both sepsis and nonsepsis SIRS patients who died (P ≤ 0.01). Among sepsis patients with elevated OLFM4+ (≥37.6%), 56% died, compared with 18% with OLFM4+ <37.6% (P = 0.001). The association between OLFM4+ and mortality withstood adjustment for age, sex, absolute neutrophil count, comorbidities, and standard measures of severity of illness (SOFA score, APACHE III) (P < 0.03). In summary, OLFM4+ neutrophil percentage is independently associated with 60-day mortality in sepsis and may represent a novel measure of the heterogeneity of host response to sepsis.

Olfactomedin 4-Positive Neutrophils Are Upregulated after Hemorrhagic Shock.

Neutrophils are vital to both the inflammatory cascade and tissue repair after an injury. Neutrophil heterogeneity is well established but there is less evidence for significant, different functional roles for neutrophil subsets. OLFM4 (Olfactomedin-4) is expressed by a subset of neutrophils, and high expression of OLFM4 is associated with worse outcomes in patients with sepsis and acute respiratory distress syndrome. We hypothesized that an increased number of OLFM4+ neutrophils would occur in trauma patients with worse clinical outcomes. To test this, we prospectively enrolled patients who suffered a blunt traumatic injury. Blood was collected at the time of admission, Day 3, and Day 7 and analyzed for the percentage of neutrophils expressing OLFM4. We found that a subset of patients who suffered blunt traumatic injury upregulated their percentage of OLFM4+ neutrophils. Those who upregulated their OLFM4 had an increased length of stay, days in the ICU, and ventilator days. A majority of these patients also suffered from hemorrhagic shock. To establish a potential role for OLFM4+ neutrophils, we used a murine model of hemorrhagic shock because mice also express OLFM4 in a subset of neutrophils. These studies demonstrated that wild type mice had higher concentrations of cytokines in the plasma and myeloperoxidase in the lungs compared with OLFM4-null mice. In addition, we used an anti-OLFM4 antibody, which when given to wild type mice led to the reduction of myeloperoxidase in the lungs of mice. These findings suggest that OLFM4+ neutrophils are a unique subset of neutrophils that affect the inflammatory response after tissue injury.

Longitudinal characterization of olfactomedin-4 expressing neutrophils in pediatric patients undergoing bone marrow transplantation.

Sepsis is an important cause of morbidity and mortality in pediatric patients. Increased expression of olfactomedin-4 (OLFM4), a glycoprotein contained within a subpopulation of neutrophils, has been associated with complicated course in sepsis. The factors that regulate OLFM4 expression are unknown. Here, we followed children undergoing bone marrow transplantation (BMT) to document the percentage of neutrophils that express OLFM4 over time. This population was selected because of the ability to observe nascent neutrophils following engraftment, perform frequent blood sampling, and the children are at high risk for clinical complications that may associate with changes in percentage of OLFM4+ neutrophils. We found a surprising degree of variability of OLFM4 expression between patients. In the weeks following initial neutrophil recovery we also saw great variability in OLFM4 expression within individual patients, indicating that multiple external factors may modify OLFM4 expression. We identified decreased expression of CD64 (a marker associated with response to infection), in OLFM4+ neutrophils. This is the first study to demonstrate fluctuation in OLFM4 expression within patients and provides insight into possible mechanisms for OLFM4 regulation in nascent neutrophils.

Pre-operative neutrophil-lymphocyte ratio predicts low cardiac output in children after cardiac surgery.

Neutrophil-lymphocyte ratio has been associated with clinical outcomes in several groups of cardiac patients, including patients with coronary artery disease, cardiac failure, and cardiac transplant recipients. We hypothesised that pre- and/or post-operative haematological cell counts are associated with clinical outcomes in children undergoing cardiac surgery for CHD. We performed a post hoc analysis of data collected as part of a prospective observational cohort study (n = 83, data available n = 47) of children evaluated for glucocorticoid receptor levels after cardiac surgery (July 2015-January 2016). The association of neutrophil-lymphocyte ratio with low cardiac output syndrome, time to inotrope free, and vasoactive-inotropic score was examined using proportional odds analysis, cox regression, and linear regression models, respectively. A majority (80%) of patients were infants (median/interquartile range 4.1/0.2-7.6 months) with conotruncal (36%) and left-sided obstructed lesions (28%). Two patients required mechanical circulatory support and three died. Higher pre-operative neutrophil-lymphocyte ratio was associated with higher cumulative odds of severe/moderate versus mild low cardiac output on post-operative day 1 (odds ratio 2.86; 95% confidence interval 1.18-6.93; p = 0.02). Pre-operative neutrophil-lymphocyte ratio was not significantly associated with time to inotrope free or vasoactive-inotrope score. Post-operative neutrophil-lymphocyte ratio was also not associated with outcomes. In children after congenital heart surgery, higher pre-operative neutrophil-lymphocyte ratio was associated with a higher chance of low cardiac output in the early post-operative period. Pre-operative neutrophil-lymphocyte ratio maybe a useful prognostic marker in children undergoing congenital heart surgery.

Juvenile OLFM4-null mice are protected from sepsis.

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.

The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.

Olfactomedin-4 (OLFM4) identifies a subset of neutrophils conserved in both mouse and man, associated with worse outcomes in several inflammatory conditions. We investigated the role of OLFM4-positive neutrophils in murine intestinal ischemia/reperfusion (IR) injury. Wild-type (WT) C57Bl/6 and OLFM4 null mice were subjected to intestinal IR injury and then monitored for survival or tissues harvested for further analyses. In vivo intestinal barrier function was determined via functional assay of permeability to FITC-dextran. OLFM4 null mice had a significant 7-d survival benefit and less intestinal barrier dysfunction compared with WT. Early after IR, WT mice had worse mucosal damage on histologic examination. Experiments involving adoptive transfer of bone marrow demonstrated that the mortality phenotype associated with OLFM4-positive neutrophils was transferrable to OLFM4 null mice. After IR injury, WT mice also had increased intestinal tissue activation of NFκB and expression of iNOS, 2 signaling pathways previously demonstrated to be involved in intestinal IR injury. In combination, these experiments show that OLFM4-positive neutrophils are centrally involved in the pathologic pathway leading to intestinal damage and mortality after IR injury. This may provide a therapeutic target for mitigation of intestinal IR injury in a variety of common clinical situations.-Levinsky, N. C., Mallela, J., Opoka, A., Harmon, K., Lewis, H. V., Zingarelli, B., Wong, H. R., Alder, M. N. The olfactomedin-4 positive neutrophil has a role in murine intestinal ischemia/reperfusion injury.