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.

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.

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.

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.

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.

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.

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.

Antibody responses of variable lymphocyte receptors in the lamprey.

Lamprey and hagfish, the living representatives of jawless vertebrates, use genomic leucine-rich-repeat cassettes for the combinatorial assembly of diverse antigen receptor genes encoding variable lymphocyte receptors of two types: VLRA and VLRB. We describe here the VLRB-bearing lineage of lymphocytes in sea lamprey. These cells responded to repetitive carbohydrate or protein determinants on bacteria or mammalian cells with lymphoblastoid transformation, proliferation and differentiation into plasmacytes that secreted multimeric antigen-specific VLRB antibodies. Lacking a thymus and the ability to respond to soluble protein antigens, lampreys seem to have evolved a B cell-like system for adaptive humoral responses.

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.

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.

Circulating dsDNA, endothelial injury, and complement activation in thrombotic microangiopathy and GVHD.

Transplant-associated thrombotic microangiopathy (TA-TMA) is a common and poorly recognized complication of hematopoietic stem cell transplantation (HSCT) associated with excessive complement activation, likely triggered by endothelial injury. An important missing piece is the link between endothelial injury and complement activation. We hypothesized that neutrophil extracellular traps (NETs) mechanistically link endothelial damage with complement activation and subsequent TA-TMA. Neutrophil activation releases granule proteins together with double-stranded DNA (dsDNA) to form extracellular fibers known as NETs. NETs have been shown to activate complement and can be assessed in humans by quantification of dsDNA in serum. We measured levels of dsDNA, as a surrogate for NETs in 103 consecutive pediatric allogeneic transplant recipients at day 0, +14, +30, +60, and +100. A spike in dsDNA production around day +14 during engraftment was associated with subsequent TA-TMA development. Peak dsDNA production around day +14 was associated with interleukin-8-driven neutrophil recovery. Increased dsDNA levels at days +30, +60, and +100 were also associated with increased mortality and gastrointestinal graft-versus-host disease (GVHD). NETs may serve as a mechanistic link between endothelial injury and complement activation. NET formation may be one mechanism contributing to the clinical overlap between GVHD and TA-TMA.

The glucocorticoid receptor and cortisol levels in pediatric septic shock.

BACKGROUND: There is controversy around the prescription of adjunct corticosteroids in patients with fluid-refractory septic shock, and studies provide mixed results, showing benefit, no benefit, and harm. Traditional means for evaluating whether a patient receives corticosteroids relied on anecdotal experience or measurement of serum cortisol production following stimulation. We set out to measure both serum cortisol and the intracellular signaling receptor for cortisol, the glucocorticoid receptor (GCR), in this group of patients. METHODS: We enrolled pediatric patients admitted to the pediatric intensive care unit with a diagnosis of systemic inflammatory response syndrome (SIRS), sepsis, or septic shock as well as healthy controls. We measured serum cortisol concentration and GCR expression by flow cytometry in peripheral blood leukocytes on the day of admission and day 3. RESULTS: We enrolled 164 patients for analysis. There was no difference between GCR expression comparing SIRS, sepsis, and septic shock. When all patients with septic shock were compared, those patients with a complicated course, defined as two or more organ failures at day 7 or death by day 28, had lower expression of GCR in all peripheral blood leukocytes. Further analysis suggested that patients with the combination of low GCR and high serum cortisol had higher rates of complicated course (75%) compared with the other three possible combinations of GCR and cortisol levels: low GCR and low cortisol (33%), high GCR and high cortisol (33%), and high GCR and low cortisol (13%; P <0.05). CONCLUSIONS: We show that decreased expression of the GCR correlated with poor outcome from septic shock, particularly in those patients with high serum cortisol. This is consistent with findings from transcriptional studies showing that downregulation of GCR signaling genes portends worse outcome.

Characterization of the Glucocorticoid Receptor in Children Undergoing Cardiac Surgery.

OBJECTIVES: Postoperative administration of corticosteroids is common practice for managing catecholamine refractory low cardiac output syndrome. Since corticosteroid activity is dependent on the glucocorticoid receptor, we sought to characterize glucocorticoid receptor levels in children undergoing cardiac surgery and examined the association between glucocorticoid receptor levels and cardiovascular dysfunction. DESIGN: Prospective observational cohort study. SETTING: Large, tertiary pediatric cardiac center. SUBJECTS: Children undergoing corrective or palliative cardiac surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A prospective observational cohort study was conducted in 83 children with congenital heart disease. Total glucocorticoid receptor levels were measured in the peripheral WBCs using flow cytometry. In addition, blood samples were collected for total cortisol levels. The primary outcome studied was the time to being inotrope free. An increase in glucocorticoid receptor level from postoperative day 1 to postoperative day 3 was associated with a longer time to being inotrope free (hazard ratio, 0.49 [0.29-0.81]; p = 0.01) in the univariate analysis. This association remained significant after adjusting for age, weight, cardiopulmonary bypass time, cross clamp time, Risk Adjustment for Congenital Heart Surgery-1 score, and postoperative steroid use (hazard ratio, 0.53 [0.29-0.99]; p = 0.05). Postoperative day 3 glucocorticoid receptor level showed a trend to have longer time to being inotrope free (hazard ratio, 0.66 [0.42-1.02]; p = 0.0.06). The cortisol levels minimally increased during the study duration and did not correlate with glucocorticoid receptor levels. CONCLUSIONS: Increasing glucocorticoid receptor levels in peripheral WBCs of children undergoing cardiac surgery are associated with a longer time to being inotrope free. Cortisol levels minimally increased during the study duration. These results suggest that exposure to high-dose perioperative corticosteroids may suppress the hypothalamic-pituitary-adrenal axis leading to increase in glucocorticoid receptor levels in response to a low cortisol environment. Further studies are required to better delineate the interplay between glucocorticoid receptor levels, cortisol levels, corticosteroid exposure, and postoperative inotropic requirements.

Olfactomedin-4 Is a Candidate Marker for a Pathogenic Neutrophil Subset in Septic Shock.

OBJECTIVES: Heterogeneity in sepsis-related pathobiology presents a significant challenge. Resolving this heterogeneity presents an opportunity to understand pathobiology and improve patient care. Olfactomedin-4 is a neutrophil subset marker and may contribute to sepsis heterogeneity. Our objective was to evaluate the expression of olfactomedin-4 and characterize neutrophil heterogeneity in children with septic shock. DESIGN: Single-center, prospective cohort, as well as secondary analysis of existing transcriptomic and proteomic databases. SETTING: Tertiary care PICU. PATIENTS: Patients from 5 days to 18 years old with septic shock were enrolled. Data collected included the expression of olfactomedin-4 messenger RNA, serum protein concentrations, and percentage of neutrophils that express olfactomedin-4. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Secondary analysis of existing transcriptomic data demonstrated that olfactomedin-4 is the most highly expressed gene in nonsurvivors of pediatric septic shock, compared with survivors. Secondary analysis of an existing proteomic database corroborated these observations. In a prospectively enrolled cohort, we quantified the percentage of olfactomedin-4+ neutrophils in patients with septic shock. Patients with a complicated course, defined as greater than or equal to two organ failures at day 7 of septic shock or 28-day mortality, had a higher percentage of olfactomedin-4+ neutrophils, compared with those without a complicated course. By logistic regression, the percentage of olfactomedin-4+ neutrophils was independently associated with increased risk of a complicated course (odds ratio, 1.09; 95% CI, 1.01-1.17; p = 0.024). CONCLUSIONS: Olfactomedin-4 identifies a subpopulation of neutrophils in patients with septic shock, and those with a high percentage of olfactomedin-4+ neutrophils are at higher risk for greater organ failure burden and death. Olfactomedin-4 might serve as a marker of a pathogenic neutrophil subset in patients with septic shock.

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.

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.

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.

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.