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Objectives: We previously reported the expression of the two-pore-domain K channel TREK-1 in lung epithelial cells and proposed a role for this channel in the regulation of alveolar epithelial cytokine secretion. In this study, we focused on investigating the role of TREK-1 in vivo in the development of hyperoxia-induced lung injury.Design: Laboratory animal experiments.Setting: University research laboratory.Subjects: Wild-type and TREK-1-deficient mice.Interventions: Mice were anesthetized and exposed to 1) room air, no mechanical ventilation, 2) 95% hyperoxia for 24 hours, and 3) 95% hyperoxia for 24 hours followed by mechanical ventilation for 4 hours.Measurements and Main Results: Hyperoxia exposure accentuated lung injury in TREK-1-deficient mice but not controls, resulting in increase in lung injury scores, bronchoalveolar lavage fluid cell numbers, and cellular apoptosis and a decrease in quasi-static lung compliance. Exposure to a combination of hyperoxia and injurious mechanical ventilation resulted in further morphological lung damage and increased lung injury scores and bronchoalveolar lavage fluid cell numbers in control but not TREK-1-deficient mice. At baseline and after hyperoxia exposure, bronchoalveolar lavage cytokine levels were unchanged in TREK-1-deficient mice compared with controls. Exposure to hyperoxia and mechanical ventilation resulted in an increase in bronchoalveolar lavage interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α levels in both mouse types, but the increase in interleukin-6 and monocyte chemotactic protein-1 levels was less prominent in TREK-1-deficient mice than in controls. Lung tissue macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin-1β gene expression was not altered by hyperoxia in TREK-1-deficient mice compared with controls. Furthermore, we show for the first time TREK-1 expression on alveolar macrophages and unimpaired tumor necrosis factor-α secretion from TREK-1-deficient macrophages.Conclusions: TREK-1 deficiency resulted in increased sensitivity of lungs to hyperoxia, but this effect is less prominent if overwhelming injury is induced by the combination of hyperoxia and injurious mechanical ventilation. TREK-1 may constitute a new potential target for the development of novel treatment strategies against hyperoxia-induced lung injury. [ABSTRACT FROM AUTHOR]

Background: Across Canada, Child Protection Services (CPS) disrupt Indigenous families by apprehending their children at alarmingly high rates. The harms borne by children in out-of-home care (OoHC) have been extensively documented. We examined the impact of OoHC on Manitoba children's health and legal system outcomes to provide rigorous evidence on how discretionary decision-making by CPS agencies can affect these outcomes., Methods: In partnership with First Nations researchers, we used linked administrative data to identify Manitoba children (born 2007-2018) served by First Nations and other Manitoba CPS agencies. We compared those taken into OoHC (n = 19,324) with those never in care but with an open CPS file due to child protection concerns (n = 27,290). We used instrumental variable analysis (CPS agency rates of OoHC as the instrument) to obtain odds ratios (aOR) and 95% confidence intervals adjusted for child, maternal, and family factors., Findings: Mean age (yrs ± standard deviation) at first CPS contact for children taken into OoHC was 2.8 ± 3.7 (First Nations) and 3.0 ± 3.8 (other), and for children never in care was 4.5 ± 4.5 (First Nations) and 5.1 ± 4.7 (other). Among children served by a First Nations agency, males made up 50.6% (n = 5496) in OoHC and 51.0% (n = 6579) never in care. Among children served by other agencies, males made up 51.0% (n = 4324) in OoHC and 51.0% (n = 7428) never in care. Odds of teen pregnancy (First Nations aOR 3.69, 1.40-9.77; other aOR 5.10, 1.83-14.25), teen birth (First Nations aOR 3.23, 1.10-9.49; other aOR 5.06, 1.70-15.03), and sexually transmitted infections (other aOR 7.21, 3.63-14.32) were higher for children in care than children never in care, as were odds of being accused (other aOR 2.71, 1.27-5.75), a victim (other aOR 1.68, 1.10-2.56), charged with a crime (other aOR 2.68, 1.21-5.96), or incarcerated (First Nations aOR 3.64, 1.95-6.80; other aOR 1.19, 1.19-8.04)., Interpretation: Being in OoHC worsened children's health and legal system outcomes. The importance of reducing the number of children taken into care was emphasized in briefings to provincial and First Nations governments. The government response will be monitored., Funding: Social Sciences and Humanities Research Council (no. 890-2018-0029)., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The phytohormone auxin is the major coordinative signal in plant development 1 , mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination 2,3 . Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: Cherry juice (CJ) and cold water immersion (CWI) are both effective recovery strategies following strenuous endurance exercise. However, athletes routinely combine recovery interventions and less is known about the impact of a combined CJ and CWI protocol. Therefore, this study investigated the effects of combining CWI and CJ (a "cocktail" (CT)) on inflammation and muscle damage following a marathon., Methods: A total 39 endurance trained males were randomly assigned to a placebo (PL), CWI, CJ, or CT group before completing a trail marathon run. Muscle damage (creatine kinase (CK)), muscle function (maximal voluntary isometric contraction (MVIC)), and inflammation (interleukin-6 (IL-6); C-reactive protein (CRP)) were measured at baseline, immediately after marathon (only IL-6), 24 h, and 48 h after marathon., Results: There were no statistically significant differences between groups and no group × time interaction effects for any of the dependent variables. Confidence intervals (CI) illustrated that CT had unclear effects on inflammation (IL-6; CRP) and MVIC, but may have increased CK to a greater extent than PL and CJ conditions., Conclusion: There is no evidence of an additive effect of CJ and CWI when the treatments are used in conjunction with each other. On the contrary, combining CJ and CWI may result in slightly increased circulating CK., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Difranco, Cockburn, Dimitriou, Paice, Sinclair, Faki, Hills, Gondek, Wood and Wilson.)

Decades of sustainable dam planning efforts have focused on containing dam impacts in regime conditions, when the dam is fully filled and operational, overlooking potential disputes raised by the filling phase. Here, we argue that filling timing and operations can catalyze most of the conflicts associated with a dam's lifetime, which can be mitigated by adaptive solutions that respond to medium-to-long term hydroclimatic fluctuations. Our retrospective analysis of the contested recent filling of Gibe III in the Omo-Turkana basin provides quantitative evidence of the benefits generated by adaptive filling strategies, attaining levels of hydropower production comparable with the historical ones while curtailing the negative impacts to downstream users. Our results can inform a more sustainable filling of the new megadam currently under construction downstream of Gibe III, and are generalizable to the almost 500 planned dams worldwide in regions influenced by climate feedbacks, thus representing a significant scope to reduce the societal and environmental impacts of a large number of new hydropower reservoirs.

Airway narrowing due to hyperresponsiveness severely limits gas exchange in patients with asthma. Imaging studies in humans and animals have shown that bronchoconstriction causes patchy patterns of ventilation defects throughout the lungs, and several computational models have predicted that these regions are due to constriction of smaller airways. However, these imaging approaches are often limited in their ability to capture dynamic changes in small airways, and the patterns of constriction are heterogeneous. To directly investigate regional variations in airway narrowing and the response to deep inspirations (DIs), we utilized tantalum dust and microfocal X-ray imaging of rat lungs to obtain dynamic images of airways in an intact animal model. Airway resistance was simultaneously measured using the flexiVent system. Custom-developed software was used to track changes in airway diameters up to generation 19 (~0.3-3 mm). Changes in diameter during bronchoconstriction were then measured in response to methacholine (MCh) challenge. In contrast with the model predictions, we observed significantly greater percent constriction in larger airways in response to MCh challenge. Although there was a dose-dependent increase in total respiratory resistance with MCh, the percent change in airway diameters was similar for increasing doses. A single DI following MCh caused a significant reduction in resistance but did not cause a significant increase in airway diameters. Multiple DIs did, however, cause significant increases in airway diameters. These measurements allowed us to directly quantify dynamic changes in airways during bronchoconstriction and demonstrated greater constriction in larger airways.

Objective: Vancomycin empirical dosing studies in thermally injured patients have netted low successful target attainment and most excluded renal dysfunction, limiting applicability. In a previous study, the authors performed a retrospective analysis of 124 patients' measured pharmacokinetic parameters to calculate optimal dose and interval for intermittent infusion regimens and find predictors of clearance and total daily dose. The objective of this study was to improve the accuracy of attaining goal therapeutic targets with initial vancomycin regimens in patients with thermal injury through retrospective modeling., Methods: In this phase 2 study, variables collected and calculated regimens in phase 1 were utilized to try and create an improved empiric vancomycin dosing algorithm in patients with thermal injury. Logistic regression was utilized to determine best predictors of dosing vancomycin every 6 and 8h. The strongest models were built as individual algorithms and tested for accuracy of target attainment. Each algorithm produced a regimen for each patient that was then tested utilizing each patient's actual measured pharmacokinetic parameters., Results: Univariable logistic regression of 41 variables identified 27 and 23 to be predictive of dosing every 8 or 6h, respectively. The most predictive multivariable model for dosing every 8h consisted of creatinine clearance (CrCl)≥80ml/min, Acute Kidney Injury Network classification <1, and total body surface area burned≥10 percent. For dosing every 6h, CrCl≥80ml/min, age≤40years old, days since injury≤6, and serum creatinine (SCr)≤0.8 were most predictive. Based on the top 5 multivariable models for each dosing interval, 7 algorithms were built to produce recommended regimens. The highest performing algorithm resulted in trough concentrations of <10mg/L (23%), 10-20mg/L (65%), 15-20mg/L (26%), and >20mg/L (11%); area under the concentration curve (AUC)>400mghr/L (83%); and AUC >400mghr/L without having a trough >20mg/L (72%)., Conclusions: The algorithm that resulted in the highest target attainment without overdosing recommended 15mg/kg dosed every 24h for CrCl≥30, every 12h for CrCl 31-79, every 8h for patients with CrCl≥80ml/min, and every 6h only if the patient with a CrCl≥80ml/min is also≤40 years old and has a SCr≤0.8. Caution is warranted for groups underrepresented in this study, such as those with very low CrCl, a low BMI, or receiving renal replacement therapy. This algorithm should be validated in other centers for patients with thermal injuries., (Copyright © 2018 Elsevier Ltd and ISBI. All rights reserved.)

Sepsis is the largest cause of mortality in thermally injured patients. Traditional systemic inflammatory response syndrome (SIRS) criteria do not aid diagnosis of sepsis in burn centers. Studies have attempted identification of the best indicators of sepsis in the thermal injured patient, but predictive variables are inconsistent across the various studies. Currently, consensus guidelines lack evidential support as to which patients will benefit most from prompt antimicrobial therapy. The purpose of our study was to evaluate novel diagnostic parameters for thermal injured patients with known sepsis and compare these parameters with existing diagnostic criteria. This study was a retrospective, electronic medical record review. Baseline demographics were analyzed utilizing chi-square, Mann-Whitney U test, or t test. Each patient served as their own control. Generalized linear mixed modeling was utilized for univariable and multivariable analysis. Several models with ≤6 variables each were built with the top performing variables. Performance was analyzed using area under receiver operating curves, sensitivity, specificity, positive predictive value, and negative predictive value. Three hundred and ninety-nine patients were screened. Twenty-nine patients remained after exclusions, leaving 198 blood culture results (62 positive) for analysis. Forty variables were statistically significant during univariable analysis. From multivariable analysis, the best performing model was: Temperature > 39°C or < 36°C, heart rate > 130 beats/min, 10% decrease in mean arterial pressure, and gastric residual volumes twice the feeding rate. Meeting at least one variable of the presented model best identified incidence of sepsis with positive bloodstream infections and outperformed current models in our patients.

Low bioavailable concentrations of the micronutrient zinc (Zn) limit agricultural production on 40% of cultivated land. Here, we demonstrate that plant acclimation to Zn deficiency involves systemic regulation. Physiological Zn deficiency of Arabidopsis thaliana shoots results in increased root transcript levels of the membrane transport protein-encoding genes METAL TRANSPORT PROTEIN2 ( MTP2 ) and HEAVY METAL ATPASE2 ( HMA2 ), which are unresponsive to the local Zn status of roots. MTP2 and HMA2 act additively in the partitioning of Zn from roots to shoots. Chimeric GFP fusion proteins of MTP2 complement an mtp2 mutant and localize in the endoplasmic reticulum (ER) membrane of the outer cell layers from elongation to root hair zone of lateral roots. MTP2 restores Zn tolerance in a hypersensitive yeast mutant. These results are consistent with cell-to-cell movement of Zn toward the root vasculature inside the ER-luminal continuum through the desmotubules of plasmodesmata, under Zn deficiency. The previously described Zn deficiency response comprises transcriptional activation of target genes, including ZINC-REGULATED TRANSPORTER IRON-REGULATED TRANSPORTER PROTEIN genes ZIP4 and ZIP9, by the F-group bZIP transcription factors bZIP19 and bZIP23. We show that ZIP4 and ZIP9 respond to the local Zn status in both roots and shoots, in contrast to the systemic regulation identified here. Our findings are relevant for crop management and improvement toward combating human nutritional Zn deficiency that affects 30 to 50% of the world's population., (© 2018 American Society of Plant Biologists. All rights reserved.)

Purpose: Cryotherapy is an increasingly popular recovery strategy used in an attempt to attenuate the negative impact of strenuous physical activity on subsequent exercise. Therefore, this study aimed to assess the effects of whole body cryotherapy (WBC) and cold water immersion (CWI) on markers of recovery following a marathon., Methods: Thirty-one endurance trained males completed a marathon. Participants were randomly assigned to a CWI, WBC or placebo group. Perceptions of muscle soreness, training stress and markers of muscle function were recorded before the marathon and at 24 and 48 h post exercise. Blood samples were taken at baseline, post intervention and 24 and 48 h post intervention to assess inflammation and muscle damage., Results: WBC had a harmful effect on muscle function compared to CWI post marathon. WBC positively influenced perceptions of training stress compared to CWI. With the exception of C-reactive protein (CRP) at 24 and 48 h, neither cryotherapy intervention positively influenced blood borne markers of inflammation or structural damage compared to placebo., Conclusion: The findings show WBC has a negative impact on muscle function, perceptions of soreness and a number of blood parameters compared to CWI, contradicting the suggestion that WBC may be a superior recovery strategy. Further, cryotherapy is no more effective than a placebo intervention at improving functional recovery or perceptions of training stress following a marathon. These findings lend further evidence to suggest that treatment belief and the placebo effect may be largely responsible for the beneficial effects of cryotherapy on recovery following a marathon.

Cannabinoids, the bioactive components of marijuana, have adverse cardiovascular consequences, including symptomatic sinus bradycardia, sinus arrest and ventricular asystole. Physicians should be aware of these deleterious consequences which can appear in otherwise healthy persons who are chronic marijuana users., (Copyright © 2018 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Etiolated growth in darkness or the irreversible transition to photomorphogenesis in the light engages alternative developmental programs operating across all organs of a plant seedling. Dark-grown Arabidopsis de-etiolated by zinc (dez) mutants exhibit morphological, cellular, metabolic, and transcriptional characteristics of light-grown seedlings. We identify the causal mutation in TRICHOME BIREFRINGENCE encoding a putative acyl transferase. Pectin acetylation is decreased in dez, as previously found in the reduced wall acetylation2-3 mutant, shown here to phenocopy dez. Moreover, pectin of dez is excessively methylesterified. The addition of very short fragments of homogalacturonan, tri-galacturonate, and tetra-galacturonate, restores skotomorphogenesis in dark-grown dez and similar mutants, suggesting that the mutants are unable to generate these de-methylesterified pectin fragments. In combination with genetic data, we propose a model of spatiotemporally separated photoreceptive and signal-responsive cell types, which contain overlapping subsets of the regulatory network of light-dependent seedling development and communicate via a pectin-derived dark signal., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Case Presentation: An African American man in his late 30s was referred to the pulmonary clinic for evaluation of a persistent cough of several weeks' duration. Cough was productive of mucopurulent sputum mixed with blood. He also noted generalized weakness and dyspnea with minimal exertion., (Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Caring for patients with burn injuries is challenging secondary to the acute disease process, chronic comorbidities, and underrepresentation in evidence-based literature. Much current practice relies on extrapolation of guidance from different patient populations and wide variations in universal practices. Identifying infections or sepsis in this hypermetabolic population is imperfect and often leads to overprescribing of antimicrobials, suboptimal dosing, and multidrug resistance. An understanding of pharmacokinetics and pharmacodynamics may aid optimization of dosing regimens to better attain treatment targets. This article provides an overview of the current status of burn infection and attempts recommendations for consideration to improve universally accepted care., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and β-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.

The use of the essential element zinc (Zn) in the biochemistry of land plants is widespread, and thus comparable to that in other eukaryotes. Plants have evolved the ability to adjust to vast fluctuations in external Zn supply, and they can store considerable amounts of Zn inside cell vacuoles. Moreover, among plants there is overwhelming, but yet little explored, natural genetic diversity that phenotypically affects Zn homeostasis. This results in the ability of specific races or species to thrive in different soils ranging from extremely Zn-deficient to highly Zn-polluted. Zn homeostasis is maintained by a tightly regulated network of low-molecular-weight ligands, membrane transport and Zn-binding proteins, as well as regulators. Here we review Zn homeostasis of land plants largely based on the model plant Arabidopsis thaliana, for which our molecular understanding is most developed at present. There is some evidence for substantial conservation of Zn homeostasis networks among land pants, and this review can serve as a reference for future comparisons. Major progress has recently been made in our understanding of the regulation of transcriptional Zn deficiency responses and the role of the low-molecular-weight chelator nicotianamine in plant Zn homeostasis. Moreover, we have begun to understand how iron (Fe) and Zn homeostasis interact as a consequence of the chemical similarity between their divalent cations and the lack of specificity of the major root iron uptake transporter IRT1. The molecular analysis of Zn-hyperaccumulating plants reveals how metal homeostasis networks can be effectively modified. These insights are important for sustainable bio-fortification approaches. This article is part of a Special Issue entitled: Cell Biology of Metals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.

Both hyperoxia and mechanical ventilation can independently cause lung injury. In combination, these insults produce accelerated and severe lung injury. We recently reported that pre-exposure to hyperoxia for 12 hours, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone. We also reported that such injury and apoptosis are inhibited by antioxidant treatment. In this study, we hypothesized that apoptosis signal-regulating kinase-1 (ASK-1), a redox-sensitive, mitogen-activated protein kinase kinase kinase, plays a role in lung injury and apoptosis in this model. To determine the role of ASK-1 in lung injury, the release of inflammatory mediators and apoptosis, attributable to 12 hours of hyperoxia, were followed by large tidal volume mechanical ventilation with hyperoxia. Wild-type and ASK-1 knockout mice were subjected to hyperoxia (Fi(O(2)) = 0.9) for 12 hours before 4 hours of large tidal mechanical ventilation (tidal volume = 25 μl/g) with hyperoxia, and were compared with nonventilated control mice. Lung injury, apoptosis, and cytokine release were measured. The deletion of ASK-1 significantly inhibited lung injury and apoptosis, but did not affect the release of inflammatory mediators, compared with the wild-type mice. ASK-1 is an important regulator of lung injury and apoptosis in this model. Further study is needed to determine the mechanism of lung injury and apoptosis by ASK-1 and its downstream mediators in the lung.

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.

Based on molecular mechanisms and physiologic data, a strong association has been established between dysregulated systemic inflammation and progression of ARDS. In ARDS patients, glucocorticoid receptor-mediated down-regulation of systemic inflammation is essential to restore homeostasis, decrease morbidity and improve survival and can be significantly enhanced with prolonged low-to-moderate dose glucocorticoid treatment. A large body of evidence supports a strong association between prolonged glucocorticoid treatment-induced down-regulation of the inflammatory response and improvement in pulmonary and extrapulmonary physiology. The balance of the available data from controlled trials provides consistent strong level of evidence (grade 1B) for improving patient-centered outcomes. The sizable increase in mechanical ventilation-free days (weighted mean difference, 6.58 days; 95% CI, 2.93 -10.23; P<0.001) and ICU-free days (weighted mean difference, 7.02 days; 95% CI, 3.20-10.85; P<0.001) by day 28 is superior to any investigated intervention in ARDS. The largest meta-analysis on the subject concluded that treatment was associated with a significant risk reduction (RR=0.62, 95% CI: 0.43-0.91; P=0.01) in mortality and that the in-hospital number needed to treat to save one life was 4 (95% CI 2.4-10). The balance of the available data, however, originates from small controlled trials with a moderate degree of heterogeneity and provides weak evidence (grade 2B) for a survival benefit. Treatment decisions involve a tradeoff between benefits and risks, as well as costs. This low cost highly effective therapy is familiar to every physician and has a low risk profile when secondary prevention measures are implemented., (Copyright © 2011. Published by Elsevier Masson SAS.)

Both prolonged exposure to hyperoxia and large tidal volume mechanical ventilation can each independently cause lung injury. However, the combined impact of these insults is poorly understood. We recently reported that preexposure to hyperoxia for 12 h, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone (Makena et al. Am J Physiol Lung Cell Mol Physiol 299: L711-L719, 2010). The upstream mechanisms of this lung injury and apoptosis have not been clearly elucidated. We hypothesized that lung injury in this model was dependent on oxidative signaling via the c-Jun NH(2)-terminal kinases (JNK). We, therefore, evaluated lung injury and apoptosis in the presence of N-acetyl-cysteine (NAC) in both mouse and cell culture models, and we provide evidence that NAC significantly inhibited lung injury and apoptosis by reducing the production of ROS, activation of JNK, and apoptosis. To confirm JNK involvement in apoptosis, cells treated with a specific JNK inhibitor, SP600125, and subjected to preexposure to hyperoxia, followed by mechanical stretch, exhibited significantly reduced evidence of apoptosis. In conclusion, lung injury and apoptosis caused by preexposure to hyperoxia, followed by high tidal volume mechanical ventilation, induces ROS-mediated activation of JNK and mitochondrial-mediated apoptosis. NAC protects lung injury and apoptosis by inhibiting ROS-mediated activation of JNK and downstream proapoptotic signaling.

After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.

Both high tidal volume mechanical ventilation (HV) and hyperoxia (HO) have been implicated in ventilator-induced lung injury. However, patients with acute lung injury are often exposed to HO before the application of mechanical ventilation. The potential priming of the lungs for subsequent injury by exposure to HO has not been extensively studied. We provide evidence that HO (90%) for 12 h followed by HV (25 μl/g) combined with HO for 2 or 4 h (HO-12h+HVHO-2h or -4h) induced severe lung injury in mice. Analysis of lung homogenates showed that lung injury was associated with cleavage of executioner caspases, caspases-3 and -7, and their downstream substrate poly(ADP-ribose) polymerase-1 (PARP-1). No significant lung injury or caspase cleavage was seen with either HO for 16 h or HV for up to 4 h. Ventilation for 4 h with HO (HVHO) did not cause significant lung injury without preexposure to HO. Twelve-hour HO followed by lower tidal volume (6 μl/g) mechanical ventilation failed to produce significant injury or caspase cleavage. We also evaluated the initiator caspases, caspases-8 and -9, to determine whether the death receptor or mitochondrial-mediated pathways were involved. Caspase-9 cleavage was observed in HO-12h+HVHO-2h and -4h as well as HO for 16 h. Caspase-8 activation was observed only in HO-12h+HVHO-4h, indicating the involvement of both pathways. Immunohistochemistry and in vitro stretch studies showed caspase cleavage in alveolar epithelial cells. In conclusion, preexposure to HO followed by HV produced severe lung injury associated with alveolar epithelial cell apoptosis.

Experimental and clinical evidence has demonstrated a strong cause-and-effect relationship between persistence vs reduction in systemic inflammation and progression (unresolving) vs resolution (resolving) of ARDS. In this review, the cellular mechanisms involved in activating and regulating inflammation are contrasted between patients with resolving and unresolving ARDS. At the cellular level, patients with unresolving ARDS have deficient glucocorticoid (GC)-mediated down-regulation of inflammatory cytokine and chemokine transcription despite elevated levels of circulating cortisol, a condition defined as systemic inflammation-associated acquired GC resistance. These patients, contrary to those with resolving ARDS, have persistent elevation in levels of both systemic and BAL fluid inflammatory cytokines and chemokines, markers of alveolar-capillary membrane permeability and fibrogenesis. At the tissue level, the continued production of inflammatory mediators leads to tissue injury, intravascular and extravascular coagulation, and the proliferation of mesenchymal cells, all resulting in maladaptive lung repair and progression of extrapulmonary organ dysfunction. In ARDS, down-regulation of systemic inflammation is essential to restoring homeostasis, decreasing morbidity, and improving survival. Prolonged low-to-moderate dose GC therapy promotes the down-regulation of inflammatory cytokine transcription at the cellular level. Eight controlled studies have consistently reported a significant reduction in markers of systemic inflammation, pulmonary and extrapulmonary organ dysfunction scores, duration of mechanical ventilation, and ICU length of stay. In the aggregate (n = 628), reduction in mortality was substantial for all patients (relative risk [RR], 0.75; 95% CI, 0.63 to 0.89; p < 0.001; I(2), 43%) and for those treated before day 14 (RR, 0.71; 95% CI, 0.59 to 0.85; p < 0.001; I(2), 40%).

Purpose: Ventilator-induced lung injury (VILI) is a recognized complication of mechanical ventilation. Although the specific mechanism by which mechanical ventilation causes lung injury remains an active area of study, both alveolar overdistension and cyclical airway collapse and recruitment have been suggested as contributing causes. We hypothesized that mechanical ventilation in the absence of positive end-expiratory pressure (PEEP) causes VILI to be more severe and regionally variable as compared with PEEP = 8 cm H(2)O., Materials and Methods: To test this hypothesis, anesthetized, supine rabbits were mechanically ventilated with an end-inspiratory pressure of 28 cm H(2)O and either 0 or 8 cm H(2)O PEEP for 4 hours. Regional lung injury was determined by histologic scoring., Results: In the absence of PEEP, lung injury was regionally variable and greatest in the dorsal-caudal lung. This regional injury heterogeneity was abolished by the addition of PEEP = 8 cm H(2)O., Conclusions: These results suggest that VILI is regionally heterogeneous and spatially correlates with regions in which cyclical airway collapse and recruitment is most likely to occur.

Rationale: Mechanical ventilation with large tidal volumes causes ventilator-induced lung injury in animal models. Little direct evidence exists regarding the deformation of airways in vivo during mechanical ventilation, or in the presence of positive end-expiratory pressure (PEEP)., Objectives: To measure airway strain and to estimate airway wall tension during mechanical ventilation in an intact animal model., Methods: Sprague-Dawley rats were anesthetized and mechanically ventilated with tidal volumes of 6, 12, and 25 cm(3)/kg with and without 10-cm H(2)O PEEP. Real-time tantalum bronchograms were obtained for each condition, using microfocal X-ray imaging. Images were used to calculate circumferential and longitudinal airway strains, and on the basis of a simplified mathematical model we estimated airway wall tensions., Measurements and Main Results: Circumferential and longitudinal airway strains increased with increasing tidal volume. Levels of mechanical strain were heterogeneous throughout the bronchial tree. Circumferential strains were higher in smaller airways (less than 800 mum). Airway size did not influence longitudinal strain. When PEEP was applied, wall tensions increased more rapidly than did strain levels, suggesting that a "strain limit" had been reached. Airway collapse was not observed under any experimental condition., Conclusions: Mechanical ventilation results in significant airway mechanical strain that is heterogeneously distributed in the uninjured lung. The magnitude of circumferential but not axial strain varies with airway diameter. Airways exhibit a "strain limit" above which an abrupt dramatic rise in wall tension is observed.

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O(2)) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130(Cas). RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.

Study Objectives: Hypercapnic acidosis has antiinflammatory effects in animal models of acute lung injury (ALI) and improves ventilation-perfusion (V/Q) matching in normal lungs. The effect of hypercapnia on V/Q matching in ALI is conflicting. Hypercapnic acidosis produced by reduced tidal volumes (Vts) was associated with an increased shunt fraction (QS/QT) in patients with ALI compared with control subjects. Vt differences between groups make the assessment of hypercapnic acidosis on V/Q matching difficult. Adding CO2 to the inhaled gas allows the comparison of gas exchange under identical Vt conditions. We hypothesized the presence of hypercapnic acidosis from inspired carbon dioxide (ICD) would improve gas exchange in ALI and would be superior to that of low minute ventilation (LVe) produced by reduced respiratory rate, rather than Vt., Design: University laboratory study of anesthetized New Zealand White rabbits., Interventions: Assessment of V/Q relationships using the multiple inert gas elimination technique was performed in 10 saline solution-lavaged animals, which were ventilated with 6 mL/kg Vts and a positive end-expiratory pressure of 8 cm H2O. Each rabbit was studied while it was in eucapnia, followed by hypercapnia (Pa(CO2), 95 to 100 mm Hg) induced by LVe from decreased respiratory rate and by 10% ICD, in random order., Measurements and Results: The Pa(O2) was greater in ICD and LVe compared to eucapnia, but no significant differences in alveolar-arterial oxygen pressure difference or Pa(O2)/fraction of inspired oxygen ratio occurred. LVe statistically reduced the mean V/Q distributions compared with ICD and eucapnia. Log SDs of ventilation and combined retention and excretion curves of the dispersion index were both increased during LVe, indicating the presence of unfavorable changes in ventilation distribution. Neither LVe nor ICD altered the QS/QT., Conclusions: LVe slightly impairs overall gas exchange and ventilation distribution, but does not increase QS/QT compared with eucapnia and ICD. While ICD does not significantly improve gas exchange, it may be superior to LVe in achieving the antiinflammatory effects of "therapeutic" hypercapnia, since it does not adversely alter gas exchange and has the potential to make the lung more uniformly acidotic.

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.

To investigate whether hypercapnic acidosis protects against ventilator-induced lung injury (VILI) in vivo, we subjected 12 anesthetized, paralyzed rabbits to high tidal volume ventilation (25 cc/kg) at 32 breaths per minute and zero positive end-expiratory pressure for 4 hours. Each rabbit was randomized to receive either an FI(CO(2)) to achieve eucapnia (Pa(CO(2)) approximately 40 mm Hg; n = 6) or hypercapnic acidosis (Pa(CO(2)) 80-100 mm Hg; n = 6). Injury was assessed by measuring differences between the two groups' respiratory mechanics, gas exchange, wet:dry weight, bronchoalveolar lavage fluid protein concentration and cell count, and injury score. The eucapnic group showed significantly higher plateau pressures (27.0 +/- 2.5 versus 20.9 +/- 3.0; p = 0.016), change in Pa(O(2)) (165.2 +/- 19.4 versus 77.3 +/- 87.9 mm Hg; p = 0.02), wet:dry weight (9.7 +/- 2.3 versus 6.6 +/- 1.8; p = 0.04), bronchoalveolar lavage protein concentration (1,350 +/- 228 versus 656 +/- 511 micro g/ml; p = 0.03), cell count (6.86 x 10(5) +/- 0.18 x 10(5) versus 2.84 x 10(5) +/- 0.28 x 10(5) nucleated cells/ml; p = 0.021), and injury score (7.0 +/- 3.3 versus 0.7 +/- 0.9; p < 0.0001). We conclude that hypercapnic acidosis is protective against VILI in this model.