Distinct patterns of SARS-CoV-2 BA.2.87.1 and JN.1 variants in immune evasion, antigenicity, and cell-cell fusion.

The rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly, BA.2.87.1 is more resistant to neutralization by XBB.1.5-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines. IMPORTANCE: This study investigates the recently emerged SARS-CoV-2 variants, BA.2.87.1 and JN.1, in comparison to earlier variants and the parental D614G. Varied infectivity and cell-cell fusion activity among these variants suggest potential disparities in their ability to infect target cells and possibly pathogenesis. BA.2.87.1 exhibits lower nAb escape from bivalent mRNA vaccinee and BA.2.86/JN.1-infected sera than JN.1 but is relatively resistance to XBB.1.5-vaccinated hamster sera, revealing distinct properties in immune reason and underscoring the significance of continuing surveillance of variants and reformulation of vaccines. Antigenic differences between BA.2.87.1 and other earlier variants yield critical information not only for antibody evasion but also for viral evolution. In conclusion, this study furnishes timely insights into the spike biology and immune escape of the emerging variants BA.2.87.1 and JN.1, thus guiding effective vaccine development and informing public health interventions.

Distinct Patterns of SARS-CoV-2 BA.2.87.1 and JN.1 Variants in Immune Evasion, Antigenicity and Cell-Cell Fusion.

The rapid evolution of SARS-CoV-2 variants presents a constant challenge to the global vaccination effort. In this study, we conducted a comprehensive investigation into two newly emerged variants, BA.2.87.1 and JN.1, focusing on their neutralization resistance, infectivity, antigenicity, cell-cell fusion, and spike processing. Neutralizing antibody (nAb) titers were assessed in diverse cohorts, including individuals who received a bivalent mRNA vaccine booster, patients infected during the BA.2.86/JN.1-wave, and hamsters vaccinated with XBB.1.5-monovalent vaccine. We found that BA.2.87.1 shows much less nAb escape from WT-BA.4/5 bivalent mRNA vaccination and JN.1-wave breakthrough infection sera compared to JN.1 and XBB.1.5. Interestingly. BA.2.87.1 is more resistant to neutralization by XBB.15-monovalent-vaccinated hamster sera than BA.2.86/JN.1 and XBB.1.5, but efficiently neutralized by a class III monoclonal antibody S309, which largely fails to neutralize BA.2.86/JN.1. Importantly, BA.2.87.1 exhibits higher levels of infectivity, cell-cell fusion activity, and furin cleavage efficiency than BA.2.86/JN.1. Antigenically, we found that BA.2.87.1 is closer to the ancestral BA.2 compared to other recently emerged Omicron subvariants including BA.2.86/JN.1 and XBB.1.5. Altogether, these results highlight immune escape properties as well as biology of new variants and underscore the importance of continuous surveillance and informed decision-making in the development of effective vaccines.

Corrigendum: Brief research report: impact of vaccination on antibody responses and mortality from severe COVID-19.

[This corrects the article DOI: 10.3389/fimmu.2024.1325243.].

Brief research report: impact of vaccination on antibody responses and mortality from severe COVID-19.

Introduction: While it is established that vaccination reduces risk of hospitalization, there is conflicting data on whether it improves outcome among hospitalized COVID-19 patients. This study evaluated clinical outcomes and antibody (Ab) responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection/vaccines in patients with acute respiratory failure (ARF) and various comorbidities. Methods: In this single-center study, 152 adult patients were admitted to Ohio State University hospital with ARF (05/2020 - 11/2022) including 112 COVID-19-positive and 40 COVID-19-negative patients. Of the COVID-19 positive patients, 23 were vaccinated for SARS-CoV-2 (Vax), and 89 were not (NVax). Of the NVax COVID-19 patients, 46 were admitted before and 43 after SARS-CoV-2 vaccines were approved. SARS-CoV-2 Ab levels were measured/analyzed based on various demographic and clinical parameters of COVID-19 patients. Additionally, total IgG4 Ab concentrations were compared between the Vax and NVax patients. Results: While mortality rates were 36% (n=25) and 27% (n=15) for non-COVID-19 NVax and Vax patients, respectively, in COVID-19 patients mortality rates were 37% (NVax, n=89) and 70% (Vax, n=23). Among COVID-19 patients, mortality rate was significantly higher among Vax vs. NVax patients (p=0.002). The Charlson's Comorbidity Index score (CCI) was also significantly higher among Vax vs. NVax COVID-19 patients. However, the mortality risk remained significantly higher (p=0.02) when we compared COVID-19 Vax vs. NVax patients with similar CCI score, suggesting that additional factors may increase risk of mortality. Higher levels of SARS-CoV-2 Abs were noted among survivors, suggestive of their protective role. We observed a trend for increased total IgG4 Ab, which promotes immune tolerance, in the Vax vs. NVax patients in week 3. Conclusion: Although our cohort size is small, our results suggest that vaccination status of hospital-admitted COVID-19 patients may not be instructive in determining mortality risk. This may reflect that within the general population, those individuals at highest risk for COVID-19 mortality/immune failure are likely to be vaccinated. Importantly, the value of vaccination may be in preventing hospitalization as opposed to stratifying outcome among hospitalized patients, although our data do not address this possibility. Additional research to identify factors predictive of aberrant immunogenic responses to vaccination is warranted.

Immune evasion, infectivity, and fusogenicity of SARS-CoV-2 BA.2.86 and FLip variants.

Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and XBB-derived variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose-vaccinated and bivalent-vaccinated healthcare workers, XBB.1.5-wave-infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially different conformational stability of BA.2.86 spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

Synergistic Role of NK Cells and Monocytes in Promoting Atherogenesis in Severe COVID-19 Patients.

Clinical data demonstrate an increased predisposition to cardiovascular disease (CVD) following severe COVID-19 infection. This may be driven by a dysregulated immune response associated with severe disease. Monocytes and vascular tissue resident macrophages play a critical role in atherosclerosis, the main pathology leading to ischemic CVD. Natural killer (NK) cells are a heterogenous group of cells that are critical during viral pathogenesis and are known to be dysregulated during severe COVID-19 infection. Their role in atherosclerotic cardiovascular disease has recently been described. However, the contribution of their altered phenotypes to atherogenesis following severe COVID-19 infection is unknown. We demonstrate for the first time that during and after severe COVID-19, circulating proinflammatory monocytes and activated NK cells act synergistically to increase uptake of oxidized low-density lipoprotein (Ox-LDL) into vascular tissue with subsequent foam cell generation leading to atherogenesis despite recovery from acute infection. Our data provide new insights, revealing the roles of monocytes/macrophages, and NK cells in COVID-19-related atherogenesis.

Immune Evasion, Infectivity, and Fusogenicity of SARS-CoV-2 Omicron BA.2.86 and FLip Variants.

Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and the XBB-lineage variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose vaccinated and bivalent vaccinated healthcare workers, XBB.1.5-wave infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant Spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially differences conformational stability of BA.2.86 Spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.

Persistent hypoxia promotes myofibroblast differentiation via GPR-81 and differential regulation of LDH isoenzymes in normal and idiopathic pulmonary fibrosis fibroblasts.

Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1-dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α-smooth muscle actin (α-SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF-ß under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR-81, a G-protein-coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate-mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR-81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen-deficient microenvironments.

The aged extracellular matrix and the profibrotic role of senescence-associated secretory phenotype.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal lung disease that is primarily found in the elderly population, and several studies have demonstrated that aging is the major risk factor for IPF. IPF is characterized by the presence of apoptosis-resistant, senescent fibroblasts that generate an excessively stiff extracellular matrix (ECM). The ECM profoundly affects cellular functions and tissue homeostasis, and an aberrant ECM is closely associated with the development of lung fibrosis. Aging progressively alters ECM components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction through the expression of factors linked to a senescence-associated secretary phenotype (SASP). There is growing evidence that SASP factors affect various cell behaviors and influence ECM turnover in lung tissue through autocrine and/or paracrine signaling mechanisms. Since life expectancy is increasing worldwide, it is important to elucidate how aging affects ECM dynamics and turnover via SASP and thereby promotes lung fibrosis. In this review, we will focus on the molecular properties of SASP and its regulatory mechanisms. Furthermore, the pathophysiological process of ECM remodeling by SASP factors and the influence of an altered ECM from aged lungs on the development of lung fibrosis will be highlighted. Finally, recent attempts to target ECM alteration and senescent cells to modulate fibrosis will be introduced.NEW & NOTEWORTHY Aging is the most prominent nonmodifiable risk factor for various human diseases including Idiopathic pulmonary fibrosis. Aging progressively alters extracellular matrix components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction. In this review, we will discuss the pathological impact of aging and senescence on lung fibrosis via senescence-associated secretary phenotype factors and potential therapeutic approaches to limit the progression of lung fibrosis.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

A role for Toll-like receptor 3 in lung vascular remodeling associated with SARS-CoV-2 infection.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs). In vitro , SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

Nedd4L suppression in lung fibroblasts facilitates pathogenesis of lung fibrosis.

Ubiquitination-mediated protein degradation is associated with the development of pulmonary fibrosis. We and others have shown that Nedd4L plays anti-inflammatory and anti-fibrotic roles by targeting lysophosphatidic acid receptor 1 (LPAR1), p-Smad2/3, and ß-catenin, and other molecules for their degradation in lung epithelial cells and fibroblasts. However, the molecular regulation of Nedd4L expression in lung fibroblasts has not been studied. In this study, we find that Nedd4L levels are significantly suppressed in lung myofibroblasts in IPF patients and in experimental pulmonary fibrosis, and in TGF-ß1-treated lung fibroblasts. Nedd4L knockdown promotes TGF-ß1-mediated phosphorylation of Smad2/3 and lung myofibroblast differentiation. Mechanistically, Nedd4L targets TGF-ß receptor II (TßRII), the first key enzyme of TGF-ß1-mediated signaling, for its ubiquitination and degradation. Further, we show that inhibition of transcriptional factor E2F rescues Nedd4L levels and mitigates experimental pulmonary fibrosis. Together, our data reveal insight into mechanisms by which E2F-mediated Nedd4L suppression contributes to the pathogenesis of lung fibrosis. This study provides evidence showing that upregulation of Nedd4L is a potential therapeutic strategy to treat fibrotic disorders including lung fibrosis.

Serum Ferritin as a Predictor of Outcomes in Hospitalized Patients with Covid-19 Pneumonia.

Purpose: Elevated ferritin levels are associated with poor outcomes in Covid-19 patients. Optimal timing of ferritin assessment and the merit of longitudinal values remains unclear. Methods: Patients admitted to Henry Ford Hospital with confirmed SARS-CoV-2 were studied. Regression models were used to determine the relation between ferritin and mortality, need for mechanical ventilation, ICU admission, and days on the ventilator. Results: 2265 patients were evaluated. Patients with an initial ferritin of > 490 ng/mL had an increased risk of death (OR 3.4, P < .001), admission to the ICU (OR 2.78, P < .001) and need for mechanical ventilation (OR 3.9, P < .001). There was no difference between admission and Day 1 ICU ferritin levels (611.5 ng/mL vs. 649 ng/mL respectively; P = .07). The decline in ferritin over ICU days 1-4 was similar between survivors and non-survivors. A change in ferritin levels from admission to ICU Day 1 (P = .330), or from ICU Day 1 to 2 (P = .788), did not predict days on the ventilator. Conclusions: Initial Ferritin levels were highly predictive of ICU admission, the need for mechanical ventilation and in-hospital mortality. However, longitudinal measures of ferritin throughout the hospital stay did not provide additional predictive value.

Rationale and design of a randomized controlled clinical trial; Titration of Oxygen Levels (TOOL) during mechanical ventilation.

BACKGROUND: Both hyperoxemia and hypoxemia are deleterious in critically ill patients. Targeted oxygenation is recommended to prevent both of these extremes, however this has not translated to the bedside. Hyperoxemia likely persists more than hypoxemia due to absence of immediate discernible adverse effects, cognitive biases and delay in prioritization of titration. METHODS: We present the methodology for the Titration Of Oxygen Levels (TOOL) trial, an open label, randomized controlled trial of an algorithm-based FiO2 titration with electronic medical record-based automated alerts. We hypothesize that the study intervention will achieve targeted oxygenation by curbing episodes of hyperoxemia while preventing hypoxemia. In the intervention arm, electronic alerts will be used to titrate FiO2 if SpO2 is ≥94% with FiO2 levels ≥0.4 over 45 min. FiO2 will be titrated per standard practice in the control arm. This study is being carried out with deferred consent. The sample size to determine efficacy is 316 subjects, randomized in a 1:1 ratio to the intervention vs. control arm. The primary outcome is proportion of time during mechanical ventilation spent with FiO2 ≥ 0.4 and SpO2 ≥ 94%. We will also assess proportion of time during mechanical ventilation spent with SpO2 < 88%, duration of mechanical ventilation, length of ICU and hospital stay, hospital mortality, and adherence to electronic alerts as secondary outcomes. CONCLUSION: This study is designed to evaluate the efficacy of a high fidelity, bioinformatics-based, electronic medical record derived electronic alert system to improve targeted oxygenation in mechanically ventilated patients by reducing excessive FiO2 exposure.

Pathogenesis of pneumonia and acute lung injury.

Pneumonia and its sequelae, acute lung injury, present unique challenges for pulmonary and critical care healthcare professionals, and these challenges have recently garnered global attention due to the ongoing Sars-CoV-2 pandemic. One limitation to translational investigation of acute lung injury, including its most severe manifestation (acute respiratory distress syndrome, ARDS) has been heterogeneity resulting from the clinical and physiologic diagnosis that represents a wide variety of etiologies. Recent efforts have improved our understanding and approach to heterogeneity by defining sub-phenotypes of ARDS although significant gaps in knowledge remain. Improving our mechanistic understanding of acute lung injury and its most common cause, infectious pneumonia, can advance our approach to precision targeted clinical interventions. Here, we review the pathogenesis of pneumonia and acute lung injury, including how respiratory infections and lung injury disrupt lung homoeostasis, and provide an overview of respiratory microbial pathogenesis, the lung microbiome, and interventions that have been demonstrated to improve outcomes-or not-in human clinical trials.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.