The management of respiratory failure often necessitates the use of supplemental oxygen and mechanical ventilation, both crucial interventions in critical care. However, this combination of hyperoxia (defined as exposure to supra-atmospheric levels of oxygen) and mechanical ventilation can lead to significant additional lung injury. The aim of this review is to synthesise current evidence defining how hyperoxia primes the adult lung for increased susceptibility to the development of lung injury during mechanical ventilation and to contextualise these mechanisms within contemporary clinical research and practice. We review experimental and translational studies elucidating the interplay between oxidative stress, cytoskeletal remodelling, inflammation, and alveolar-capillary barrier dysfunction, highlighting how these processes initiate or amplify pre-existing lung injury. We also examine current approaches to oxygen therapy, both as a standalone intervention and in the context of ventilator management. While the injurious role of hyperoxia in animal models is highly consistent and reproducible, clinical trials have been less consistent in directly linking hyperoxia exposure to increased patient mortality. Although clinical trials comparing liberal versus conservative oxygen strategies have yielded conflicting results over decades, emerging mechanistic insights from pre-clinical models may help identify patient subpopulations at greatest risk for hyperoxia-associated lung injury. By integrating evidence from basic science and clinical studies, this review underscores the importance of judicious oxygen titration to mitigate iatrogenic lung injury and optimise outcomes in critically ill patients.

Endothelial glycocalyx (eGCX) shedding contributes to microvascular endotheliopathy in Acute Respiratory Distress Syndrome (ARDS) and is a potential underrecognized source of phenotypic heterogeneity. In pediatric ARDS (PARDS), we examined whether circulating heparan sulfate (HS) signatures, as readouts of eGCX shedding, capture inter-individual variability beyond other eGCX components and protein biomarkers, whether specific HS structural features are enriched, and whether they correlate with heparanase-1 (HPSE) activity. We retrospectively analyzed plasma samples (2018-2020) from children with and without PARDS. Mass spectrometry quantified glycosaminoglycans and sulfation subtypes alongside HPSE activity, while protein biomarkers were measured by multiplex assay. Among 46 children (36 PARDS, 10 no PARDS), principal component analysis identified three components explaining 63% of variance. The primary component (PC1) was driven by 6-O- and N-sulfated HS subtypes, while a secondary component (PC2) reflected inflammatory proteins. In PARDS, higher PC1 scores were associated with worse organ dysfunction and fewer ventilator-free days. Higher total HS levels were associated with enrichment of sulfated HS (including 6-O- and N-sulfated subtypes), whereas the opposite pattern was observed in non-PARDS; higher HPSE activity further correlated with these subtypes. These preliminary findings suggest that variation in circulating HS signatures identifies a distinct endothelial-derived biological axis linked to clinical outcomes.

OBJECTIVES:Molecular subphenotypes, identified through latent class analysis (LCA) of biomarker profiles, have the potential to guide targeted therapeutics in critical care. We have previously published that intensive insulin management has subphenotype-specific beneficial effects among children with hyperglycemia accompanying cardiorespiratory failure. However, the real-time application of subphenotype-based strategies in clinical settings remains challenging due to the operational aspects of biomarker assays. Our study had three objectives: 1) to compare biomarker measurements from rapid immunoassay and conventional multiplex platforms; 2) to evaluate the cross-platform transportability of a conventional assay-based parsimonious classifier for LCA-derived subphenotypes and compare it with a classifier trained directly on rapid immunoassay data; and 3) to assess the prognostic and predictive significance of rapid immunoassay-based subphenotypes. DESIGN:Retrospective cohort study. SETTING:Multicenter PICUs. PATIENTS:Two hundred sixty-nine critically ill children with acute cardiorespiratory failure and hyperglycemia (2012-2016). INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:LCA was previously used to derive hyperinflammatory and hypoinflammatory classes using a conventional multiplex assay of 13 plasma biomarkers. After feature selection, a parsimonious classifier using interleukin (IL)-6, IL-8, and soluble tumor necrosis factor receptor 1 (sTNFR-1) was developed to predict LCA-derived subphenotypes. We applied this classifier to rapid immunoassay biomarker measurements, yielding an area under the receiver operating characteristic curve (AUROC) of 0.90 (95% CI, 0.85-0.95). However, calibration was poor due to systematic underestimation of sTNFR-1 concentrations by the rapid platform. We then derived and internally validated via bootstrapping a de novo classifier using rapid immunoassay data, achieving an AUROC of 0.90 (95% CI, 0.86-0.95) and excellent calibration. Using a probability threshold of ≥ 0.5, the de novo classifier matched LCA-derived classifications in 241 of 269 cases (accuracy 89.6%). Rapid immunoassay-based subphenotypes demonstrated differences in mortality (33.3% in hyperinflammatory vs. 11.8% in hypoinflammatory; p = 0.009) and differential response to intensive insulin management (interaction p = 0.024). CONCLUSIONS:A parsimonious, rapid immunoassay-based classifier can approximate LCA-derived molecular subphenotypes while preserving their prognostic and predictive significance, with the potential to inform future subphenotype-based precision trials.

Low-voltage-activated (LVA, T-type, or CaV3), calcium-selective channels open in response to modest depolarizations, just above the resting membrane potential, supporting neuronal burst-firing patterns and spontaneous firing in cardiac pacemaker cells. How LVA-channels open at low voltages is unclear: traditional gating-current experiments suggest that LVA-channel voltage-sensing domains (VSDs) paradoxically require stronger depolarization to activate than pore opening. Using voltage-clamp fluorometry, we find that the activation of all four VSDs in human CaV3.1-channels precedes opening in voltage, solving the longstanding conundrum. We also uncover confounding effects of La3+ (used for gating-current measurements) on VSD function and clarify the role of distinct LVA-channel structure S6Cyto. CaV3.1-VSDs operate within a narrow voltage-range, resembling the VSDs of related NaV-channels more than those of other CaV-channels. Likely, NaV-like VSDs emerge before sodium selectivity.

Molecular subphenotypes, identified through biomarker profiling independent of clinical diagnosis, have the potential to guide targeted therapeutics in critical care. We have previously published that intensive insulin management has subphenotype-specific beneficial effects among children with hyperglycemia accompanying cardiorespiratory failure. However, due to the operational aspects of biomarker assays, prospective real-time application of subphenotype-based strategies remains daunting. This study compared biomarker values measured via a rapid immunoassay requiring minimal handling to a conventional laboratory-based multiplex assay, assessed its ability to classify subphenotypes with a parsimonious classifier, and compared clinical outcomes between rapid immunoassay-based subphenotypes. This retrospective multicenter study included re-assaying plasma samples from 269 children with acute cardiorespiratory failure and hyperglycemia. Latent class analysis (LCA) was previously used to derive hyper-inflammatory and hypo-inflammatory classes. A parsimonious classifier was fit to LCA-derived subphenotypes and produced a model consisting of IL-6, IL-8, and sTNFR-1. We found that, despite the rapid immunoassay systematically overestimating biomarker values relative to the conventional assay, biomarkers were strongly correlated between platforms (Pearson r = 0.87-0.93). Using the parsimonious classifier, subphenotype classifications matched between platforms in 95% of patients (n = 256/269). When compared to previously derived LCA-derived subphenotypes, rapid immunoassay-based subphenotypes demonstrated an AUC of 0.90 (95% CI 0.85-0.95). The rapid immunoassay-based hyper-inflammatory class was associated with higher mortality (26% vs. 11%; P = 0.01) and heterogeneity of treatment effect to intensive insulin management (interaction P = 0.01). Our findings suggest that subphenotyping using a rapid immunoassay is feasible and accurate, laying the foundation for future precision medicine strategies in pediatric critical care.

Introduction and Rationale: Bacterial pneumonia accounts for one million hospitalizations/year in the US with high mortality rates. Current treatment includes oxygen (hyperoxia; HO) and antibiotic therapy, despite the well-known side effects of antibiotics and lung damaging effects of HO. Thus, new targeted therapeutic approaches are needed. We recently discovered potentially protective effects of TREK-1 potassium channels in “one-hit” lung injury models using HO or influenza-A. This study was designed to determine whether TREK-1 protection also applies to bacterial pneumonia by using a clinically-relevant model of live Pseudomonas aeruginosa (PA) infection, with and without additional HO therapy. Hypothesis: Pharmacological activation of TREK-1 channels protects mice and primary human lung epithelial/endothelial cells against PA- and PA+HO-induced injury via epithelial/endothelial plasma membrane potential (Em) hyperpolarization, inhibition of Em-dependent Ca2+ influx, and reduction of Ca2+-dependent cytokine secretion. Methods: We intratracheally (i.t) infected mice with PA (CFU:20x106) and exposed one subset to HO 4 hours post-infection, while controls remained at room air. Starting at the 4-hour time point, each group was treated once-daily with a TREK-1 activating compound (i.t., ML335, BL1249) or vehicle control. After 72 hours, we quantified lung compliance and analyzed broncho-alveolar lavage (BALF) for: total/differential cell counts, IgM levels (barrier-dysfunction), cell apoptosis, ROS and cytokine secretion. Similarly, we infected primary human lung epithelial and endothelial cells with PA (MOI:5), followed 4 hours later by HO, and quantified alterations in plasma membrane Em, intracellular Ca2+(iCa2+) levels, and cytokine and ROS production. TREK-1 expression in mouse lungs and primary human lung epithelial/endothelial cells was measured by qPCR and ELISA. The role of Ca2+ influx via voltage-gated CaV channels in cytokine/ROS production was investigated with Nifedipine. Results: PA infection, and more so PA+HO, resulted in significant downregulation in TREK-1 gene and protein expression in mouse lungs, and primary human lung epithelial and endothelial cells. Despite this downregulation, a subset of residual TREK-1 channels remained intact and amendable to pharmacological intervention. Indeed, activation of residual TREK-1 channels with 2 structurally different compounds (ML335, BL1249) mitigated PA- and PA+HO -induced BALF macrophage and neutrophil infiltration, ROS and H2O2 production, inflammatory cytokines secretion, barrier dysfunction, and cell apoptosis. Mechanistically, PA and PA+HO exposure resulted in progressive epithelial/endothelial Em depolarization, influx of extracellular iCa2+ via CaV channels, and iCa2+-dependent ROS and cytokine production. These pro-inflammatory effects were counteracted by pharmacological TREK-1 activation. Conclusion: TREK-1 potassium channel activation may represent a novel and targeted therapeutic intervention against bacterial pneumonia.

Rationale: We have previously identified two biomarker-defined inflammatory subphenotypes of pediatric acute respiratory distress syndrome (ARDS) using latent class analysis, which demonstrate differential clinical outcomes in mortality and ICU length of stay. To aid in precision-based target therapy development, further studies are needed to investigate how inflammatory biomarkers interact differently between these two subphenotypes. Network analysis can identify significant protein-protein interactions but is scarcely employed in current ARDS studies. We sought to examine the relationship of previously validated inflammatory biomarkers using a correlation-based protein-protein interaction network depicting changed and unchanged interactions. Methods: We analyzed a panel of circulating inflammatory biomarkers related to inflammation and endothelial function, collected on the first day of diagnosis, from n=248 ARDS patients with pre-assigned subphenotype. Biomarkers were normalized, log-transformed, and retained if missingness was less than 25%. Missing values were imputed using multiple imputation by chained equations. Pairwise Spearman correlation tests were conducted separately for each subphenotype. Significant correlations were identified based on a false discovery rate (FDR) P-value < 0.05 and an absolute Spearman's Rho > 0.4. To construct a protein-protein interaction network (Fig. 1), we calculated the change in correlation between subphenotypes (hyperinflammatory minus hypoinflammatory). Network edges were colored and sized according to the direction and magnitude of significant correlation change. Biomarkers that remained strongly correlated without significant changes between subphenotypes were represented with dashed lines. Node colors indicate community assignments by modularity, while node size reflects eigenvector centrality. Results: We found 24 significant interactions among 41 retained biomarkers. As depicted in Figure 1, the central motif of the resulting network is a decorrelation of Interleukin-10 signaling molecules (green), as well as significant differences among TIMP and MMP markers (pink, yellow, red). Additional markers correspond to leukocyte chemotaxis (blue). Inter-community correlations were largely unchanged (14/20), while intra-community correlations were largely different between subphenotypes (21/28). Conclusion: The hyperinflammatory subphenotype shows significant shifts in correlative structure of inflammatory biomarkers. Our results show that in the hyperinflammatory subphenotype, circulating proteins involved in anti-inflammatory IL-10 signaling exhibit a weakened correlation, which may reduce the inhibition of proinflammatory signals. In contrast, interactions among proinflammatory markers such as MMP1, MMP3, and RAGE are strengthened. The differential correlation of TIMP and MMP markers further suggests a disruption in the TIMP/MMP balance, potentially contributing to excessive tissue remodeling in severe illness. This study expands our understanding of protein interactions contributing to differential outcomes in ARDS subphenotypes.

Acute Lung Injury (ALI) and its most severe form, Acute Respiratory Distress Syndrome (ARDS), are critical pulmonary conditions characterized by life-threatening acute hypoxic respiratory failure, affecting over three million individuals globally each year. ALI involves alveolar inflammation and disruption of the alveolar-capillary barrier, primarily driven by neutrophil infiltration and the release of inflammatory mediators. In our previous study using a lipopolysaccharide (LPS)-induced mouse model of ALI, we demonstrated that C6, a peptide inhibitor of voltage-gated proton channels (Hv1), ameliorates lung injury, identifying Hv1 as a potential therapeutic target. However, (i) whether the anti-inflammatory effects of C6 are translatable to a clinically relevant live bacterial infection model, and (ii) the molecular mechanisms underlying these anti-inflammatory effects, remain unknown, and are a crucial next step towards targeted rational drug development. To induce ALI, we used an intratracheal Pseudomonas aeruginosa infection model, a gram-negative bacterium relevant in ventilated and immunocompromised patients. A separate group of infected mice also received intravenous treatment with C6 (4 mg/kg). Lung injury severity was evaluated using histopathological analysis. Bronchoalveolar lavage (BAL) fluid was collected to quantify neutrophil infiltration and proinflammatory cytokines concentrations. In addition, reactive oxygen species (ROS) production and intracellular calcium levels in BAL neutrophils were measured. RNA sequencing of BAL neutrophils was conducted to assess C6-induced transcriptional changes. Key findings were validated in vitro using human neutrophils. C6 mitigates P. aeruginosa-induced ALI in mice by reducing neutrophil infiltration into the alveolar space by 86

Rationale: Red blood cell (RBC) transfusions can trigger inflammation which can lead to or worsen multi-organ dysfunction (MODS). Inflammatory biomarkers clustered through latent profile analysis are associated with differential mortality at baseline in acute respiratory distress and sepsis. However, shifts in inflammation-related biomarkers post-transfusion have not yet been studied in a sufficient number of critically ill children with MODS. Methods: We analyzed a broad panel of 250 inflammation related markers in plasma from 238 critically ill children with MODS undergoing transfusions. Biomarkers were assayed before and within 24 hours post-transfusion. The raw values were normalized, log-transformed and pre-post differences computed. Using latent profile analysis, patients were grouped both on pre-transfusion biomarker levels and post-transfusion changes. Cross-tabulation of these changes formed three distinct composite clusters that were analyzed for 7-day pediatric logistic organ dysfunction (PELOD-2) score trajectories. Biomarkers were clustered by silhouette and tested for functional enrichment against multiple databases, controlling for baseline enrichment of all markers at adjusted Fisher's Exact P < 0.05. Results: Pre-transfusion analysis revealed two distinct profiles: hypoinflamed (n=136) and hyperinflamed (n=96). Post-transfusion changes identified two key patterns, steady or reactive. Cross-tabulation yielded three composite groups: The steady hypoinflamed (n=52) group maintained low biomarker levels with minimal change post-transfusion. The reactive hypoinflamed (n=79) group, with somewhat elevated baseline levels, exhibited pronounced bidirectional post-transfusion changes. The steady hyperinflamed (n=88) group displayed consistently high inflammation markers at baseline with moderate post-transfusion shifts. Analysis of 7-day PELOD-2 score trajectories showed significant differences among the composite groups (Kruskal-Wallis P<0.001). All groups had decreasing PELOD-2 scores, indicating clinical improvement; however, steady hyperinflamed patients consistently had the highest scores. Reactive hypoinflamed patients showed a more substantial post-transfusion improvement in scores compared to steady hypoinflamed patients despite equal baselines (Figure 1). Functional enrichment produced three groups using the silhouette method. Steady patients saw increased enrichments for B cell mediated immunity, T cell activation, and the adaptive immune response (Cluster 1) as well as MAP kinase activity, PI3K/AKT signaling, and tyrosine kinase activity (Cluster 2), while reactive patients decreased in related marker levels. Steady patients had decreasing enrichments associated with neutrophils and T-cells, acute-phase immune response, and oxidative pathways (Cluster 3), whereas reactive patients saw increased levels. Conclusion: Inflammatory response to RBC transfusion varies by baseline inflammation and is associated with clinical outcomes. Pre-transfusion profiles may eventually guide personalized treatment in pediatric MODS and improve outcomes by anticipating post-transfusion inflammation and associated clinical trajectories.

RATIONALE: Degradation of the endothelial glycocalyx, an endothelial surface layer rich in sulfated glycosaminoglycans (sGAGs), contributes to microvascular barrier dysfunction in ARDS. Elevated levels of circulating sGAG fragments, measured using gold-standard mass spectrometry (LC-MS/MS), correlate with poor clinical outcomes in pediatric ARDS. However, LC-MS/MS is impractical for bedside use. The dimethyl-methylene blue (DMMB) assay provides a simple alternative for measuring sGAGs in biological matrices. The feasibility and validity of this approach in plasma from children with acute respiratory failure (ARF) have not been studied. METHODS: We measured plasma concentrations of sGAGs (ng/ml) using LC-MS/MS from a cohort of invasively mechanically ventilated children (ages ≥1 month to ≤17 years) with ARF (2018-2020). We compared these results to the DMMB assay (Chondrex, Inc.), a cationic dye that binds polyanionic sGAGs, causing a metachromatic absorbance shift measured at 530 nm via spectrophotometry. We analyzed both standard (chondroitin-6-sulfate) and patient samples in duplicates, calculating standard linearity (R²) and intra-assay coefficient of variation (CV). We assessed systematic bias by examining the relationship between inter-assay concentration differences (DMMB – LC-MS/MS) and mean sGAG levels ([DMMB + LC-MS/MS]/2) and evaluated for inter-assay heteroskedasticity by modeling residuals against mean sGAG levels. Inter-assay agreement was represented with Bland-Altman plots. sGAG levels were log-transformed to correct for skewness. RESULTS: We analyzed plasma from 11 children with ARF. Median age was 3.3 years (interquartile range [IQR] 1.9-14.2) and pneumonia/sepsis caused 54.5% of ARF. Standard curve R² was 0.99, and the intra-assay CV was 8.8%. sGAGs positively correlated with DMMB (Pearson r=0.58; 95% confidence interval [CI] -0.04, 0.87; p=0.06); in contrast, hyaluronan, an unsulfated GAG measured by LC-MS/MS, weakly correlated with DMMB (r=0.15; 95% CI -0.50, 0.67; p=0.66). There was a systematic linear increase in inter-assay differences with higher mean sGAG levels (p<0.01). Following regression adjustment, we derived a corrected DMMB equation: (DMMB + [12.28 − 1.32 x mean sGAG levels]). After correction, we found no linear association between inter-assay differences and mean sGAG levels (coefficient 0.01; 95% CI -1.44, 1.44; p=1.00), nor did we detect inter-assay heteroskedasticity (coefficient -0.22; 95% CI -0.95, 0.52; p=0.52). Mean inter-assay difference was 0.001 (95% limit of agreement -0.79, 0.79; Figure 1). CONCLUSIONS: The DMMB assay moderately correlated with LC-MS/MS, demonstrating a correctable linear bias likely due to matrix effects from plasma constituents. Our findings suggest that the DMMB assay could serve as an accurate and rapid tool for detecting circulating sGAGs in children with ARF.

Rationale: Bacterial pneumonia is a life-threatening disease accounting for 1.4 million deaths annually worldwide. Pathophysiologically, it is characterized by severe alveolar inflammation that compromises the alveolar-capillary barrier, which is a key contributor to the observed high morbidity and mortality rates. Besides antibiotics, current treatments are mostly supportive, and targeted therapies aimed at antibiotic-resistant strains are lacking. In the inpatient setting, Gram-negative bacteria pose particular challenges, with Pseudomonas aeruginosa (P. aeruginosa) being one of the leading causes of inpatient mortality. At cellular level, pulmonary endothelial cells are key sentinel cells that respond to infection by releasing proinflammatory mediators that promote lung injury and facilitating neutrophil transmigration. In previous studies, we found that pharmacological activation of large conductance Ca2+-activated K+(BK) channels protects mice and pulmonary endothelial cells against lipopolysaccharide (LPS)-induced inflammation. However, whether BK channel-mediated protection can be translated into a clinically more relevant system is unknown. Therefore, this study focuses on exploring the potentially protective effects of endothelial BK channel activation in a live bacterial P. aeruginosa pneumonia model. Methods: To investigate BK-mediated protection in vivo, we intra-tracheally infected wild-type and endothelial cell-specific BK knockout (endo BK-KO) mice with P. aeruginosa (20 x 10⁶ CFUs) and treated them once-daily intra-tracheally with the BK activator NS1619 (0.66 mg/kg) or a vehicle control starting 24 hours post infection. Physiological parameters, cellular integrity, barrier function, and bronchoalveolar (BAL) fluid and plasma biochemical markers of lung injury were assessed at 72 hours. Endpoints included measurements of inflammatory cytokines in mouse BAL fluid (BALF) and plasma, and BALF reactive oxygen species (ROS), total protein and IgM levels using DCFDA-imaging and ELISA assays, respectively. Results:P. aeruginosa infection (i) decreased peripheral oxygen saturations, quasi-static lung compliance, body temperature, and body weight, and (ii) increased BALF and plasma cytokines (IL-6, TNF-α, IL-1β, and RANTES), and BALF ROS, total protein and IgM levels, and total cell and neutrophil counts. Once-daily intra-tracheal treatment of mice with the BK activator NS1619 reversed these effects, except ROS production, suggesting a BK-independent mechanism for oxidative stress regulation. In endo BK-KO mice, NS1619 had no impact on these inflammatory processes during P. aeruginosa infection, validating the key contribution of endothelial BK channels in the NS1619-mediated protective effects and underscoring its anti-inflammatory potential. Conclusions: Endothelial BK channels may represent promising therapeutic targets for the development of new treatment strategies against bacterial pneumonia, potentially decreasing the frequency and duration of antibiotic use.

Bacterial pneumonia causes 1.4 million deaths annually worldwide. Besides antibiotics, current treatments are mostly supportive, and no other targeted therapies exist that improve patient outcomes. Key features of bacterial pneumonia include alveolar inflammation, including inflammatory cell infiltration, mediator release, and alveolar-capillary barrier dysfunction. We previously demonstrated that plasma membrane hyperpolarization via large conductance K+ (BK) channels reduces pro-inflammatory mediator release from TNF-α- or lipopolysaccharide (LPS)-treated pulmonary endothelial cells. Building on those findings, this study evaluates pharmacological BK channel activation as a potential treatment for LPS-induced pneumonia in a mouse model and explores its molecular mechanisms. We found that BK channel activation with NS1619 in LPS-infected mice reduced broncho-alveolar lavage fluid total cell and neutrophil counts, CCL-2 concentrations, and ROS and H2O2 production, and increased antioxidant superoxide dismutase and catalase levels. These effects were not linked to glutathione, neutrophil myeloperoxidase, elastase, or extracellular traps. These protective effects were replicated with a structurally different BK channel activator, NS19504. At the cellular level, both NS1619 and NS19504 reduced LPS-induced ROS production in primary human alveolar epithelial cells, whereas LPS had no effect on endothelial ROS production. Our findings suggest that pharmacological BK channel activation could serve as a new therapeutic target against bacterial pneumonia.

OBJECTIVES:Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on immune, endothelial, and epithelial cells. In the setting of inflammation, it becomes upregulated and spliced into a soluble form (soluble ICAM-1 [sICAM-1]). This study examined the association of sICAM-1 with clinical outcomes in two large pediatric cohorts with acute respiratory distress syndrome (ARDS) and acute respiratory failure (ARF) and examined the relationships between sICAM-1 and other protein biomarkers utilizing network analysis to contextualize its role in ARDS pathophysiology. DESIGN:Secondary analysis of prospective cohort studies. SETTING:Multicenter PICUs. PATIENTS OR SUBJECTS:Critically ill children with ARDS (Pediatric Acute Lung Injury [PALI], 2008-2014) and ARF (Coagulation and Fibrinolysis in Pediatric Insulin Titration Trial [CAF-PINT], 2012-2016). INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:sICAM-1 levels were measured from plasma collected within 72 hours of diagnosis. The primary outcome was in-hospital mortality, and secondary outcomes included multiple organ dysfunction and ventilator-free days. We constructed a biomarker correlation-based network that included sICAM-1 and 32 plasma biomarkers reflective of inflammation, endothelial and epithelial injury, and extracellular matrix degradation. Key biomarkers with centrality metrics in the top 10% (≥ 90th percentile) were defined as critical hubs within the network. The study included 214 children from PALI and 251 from CAF-PINT. In-hospital mortality was 18% and 14%, respectively. Baseline median oxygenation index ratios were 10 (interquartile range [IQR], 5.6-19.7) and 8.5 (IQR, 3.5-17.7). Higher plasma sICAM-1 was associated with in-hospital mortality, multiple organ dysfunction, and fewer ventilator-free days in each of the two cohorts (all p < 0.05). Tissue inhibitor of metalloproteinase-1 (composite centrality, 0.99), tumor necrosis factor receptor-1 (0.83), sICAM-1 (0.74), and interleukin-8 (0.74) were identified as network hubs. CONCLUSIONS:Elevated sICAM-1 levels were associated with poor outcomes in two separate cohorts of ARDS and ARF patients. Network analysis revealed sICAM-1 as a central hub, characterized by high centrality metrics. These findings underscore the multifaceted role of sICAM-1 in leukocyte transmigration, inflammation, and endothelial dysfunction and highlight its critical role in ARDS pathophysiology.

No targeted interventions exist that improve the outcomes of patients with acute lung injury/ARDS. A few studies investigated Na + and Ca 2+ channels/transporters for potential therapeutic intervention but with limited translational success. This study highlights the regulatory role of TREK-1 K + channels during HO+stretch/mechanical ventilation-induced lung injury in ROS production, caspase activation, cytokine secretion, and explores the underlying TREK-1-mediated signaling mechanisms. These preclinical findings lay the groundwork for future rational drug design targeting TREK-1 channels.

For more than two centuries, digoxin has been used to treat heart failure by increasing the strength of cardiac contraction and, more recently, is used for heart rate control. The proposed, yet unproven, mechanism underlying digoxin’s positive inotropic effect is as follows: By inhibiting the Na + -K + ATPase (NKA), digoxin partially dissipates the transmembrane Na + gradient, which is used by the Na + -Ca 2+ exchanger (NCX1) to extrude Ca 2+ from myocytes, thus causing accumulation of cytosolic Ca 2+ and therefore increased cardiac contractility. Here, we demonstrate that digoxin critically relies on a specific allosteric regulation of NCX1, known as Na + -dependent inactivation, to exert its positive inotropic effect, establishing the precise mechanism of action of this historic drug. These findings identify a distinct molecular target for the development of positive inotropes that avoid the undesirable effects associated with the blockade of NKA. As the structural information for the region involved with NCX1 Na + -dependent inhibition is well resolved, we provide the mechanistic foundation for drug development.