TRIM72 promotes alveolar epithelial cell membrane repair and ameliorates lung fibrosis.

BACKGROUND: Chronic tissue injury was shown to induce progressive scarring in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF), while an array of repair/regeneration and stress responses come to equilibrium to determine the outcome of injury at the organ level. In the lung, type I alveolar epithelial (ATI) cells constitute the epithelial barrier, while type II alveolar epithelial (ATII) cells play a pivotal role in regenerating the injured distal lungs. It had been demonstrated that eukaryotic cells possess repair machinery that can quickly patch the damaged plasma membrane after injury, and our previous studies discovered the membrane-mending role of Tripartite motif containing 72 (TRIM72) that expresses in a limited number of tissues including the lung. Nevertheless, the role of alveolar epithelial cell (AEC) repair in the pathogenesis of IPF has not been examined yet. METHOD: In this study, we tested the specific roles of TRIM72 in the repair of ATII cells and the development of lung fibrosis. The role of membrane repair was accessed by saponin assay on isolated primary ATII cells and rat ATII cell line. The anti-fibrotic potential of TRIM72 was tested with bleomycin-treated transgenic mice. RESULTS: We showed that TRIM72 was upregulated following various injuries and in human IPF lungs. However, TRIM72 expression in ATII cells of the IPF lungs had aberrant subcellular localization. In vitro studies showed that TRIM72 repairs membrane injury of immortalized and primary ATIIs, leading to inhibition of stress-induced p53 activation and reduction in cell apoptosis. In vivo studies demonstrated that TRIM72 protects the integrity of the alveolar epithelial layer and reduces lung fibrosis. CONCLUSION: Our results suggest that TRIM72 protects injured lungs and ameliorates fibrosis through promoting post-injury repair of AECs.

Hypercapnia Alters Alveolar Epithelial Repair by a pH-Dependent and Adenylate Cyclase-Mediated Mechanism.

Lung cell injury and repair is a hallmark of the acute respiratory distress syndrome (ARDS). Lung protective mechanical ventilation strategies in these patients may lead to hypercapnia (HC). Although HC has been explored in the clinical context of ARDS, its effect upon alveolar epithelial cell (AEC) wounding and repair remains poorly understood. We have previously reported that HC alters the likelihood of AEC repair by a pH-sensitive but otherwise unknown mechanism. Adenylate cyclase (AC) is an attractive candidate as a putative AEC CO2 sensor and effector as it is bicarbonate sensitive and controls key mediators of AEC repair. The effect of HC on AC activity and plasma membrane (PM) wound repair was measured in AEC type 1 exposed to normocapnia (NC, 40 Torr) or HC (80 Torr), ± tromethamine (THAM) or sodium bicarbonate (HCO3) ± AC probes in a micropuncture model of AEC injury relevant to ARDS. Intracellular pH and AC activity were measured and correlated with repair. HC decreased intracellular pH 0.56, cAMP by 37%, and absolute PM repair rate by 26%. Buffering or pharmacologic manipulation of AC reduced or reversed the effects of HC on AC activity (THAM 103%, HCO3 113% of NC cAMP, ns; Forskolin 168%, p < 0.05) and PM repair (THAM 87%, HCO3 108% of NC likelihood to repair, ns; Forskolin 160%, p < 0.01). These findings suggest AC to be a putative AEC CO2 sensor and modulator of AEC repair, and may have implications for future pharmacologic targeting of downstream messengers of the AC-cAMP axis in experimental models of ARDS.

Plasma membrane wounding and repair in pulmonary diseases.

Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures, and gastric acid aspiration stress lung cells, and the compromise of plasma membranes occurs as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. Because it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes and discuss relevant genetic, physical, and biological injury mechanisms, as well as mechanisms used by lung cells for cell survival and membrane repair. Finally, we discuss relevant signaling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signaling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells, should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases.

TRIM72 modulates caveolar endocytosis in repair of lung cells.

Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.

Cytokines and clinical predictors in distinguishing pulmonary transfusion reactions.

BACKGROUND: Pulmonary transfusion reactions are important complications of blood transfusion, yet differentiating these clinical syndromes is diagnostically challenging. We hypothesized that biologic markers of inflammation could be used in conjunction with clinical predictors to distinguish transfusion-related acute lung injury (TRALI), transfusion-associated circulatory overload (TACO), and possible TRALI. STUDY DESIGN AND METHODS: In a nested case-control study performed at the University of California at San Francisco and Mayo Clinic, Rochester, we evaluated clinical data and blood samples drawn before and after transfusion in patients with TRALI (n = 70), possible TRALI (n = 48), TACO (n = 29), and controls (n = 147). Cytokines measured included granulocyte-macrophage-colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-α. Logistic regression and receiver operating characteristics curve analyses were used to determine the accuracy of clinical predictors and laboratory markers in differentiating TACO, TRALI, and possible TRALI. RESULTS: Before and after transfusion, IL-6 and IL-8 were elevated in patients with TRALI and possible TRALI relative to controls, and IL-10 was elevated in patients with TACO and possible TRALI relative to that of TRALI and controls. For all pulmonary transfusion reactions, the combination of clinical variables and cytokine measurements displayed optimal diagnostic performance, and the model comparing TACO and TRALI correctly classified 92% of cases relative to expert panel diagnoses. CONCLUSIONS: Before transfusion, there is evidence of systemic inflammation in patients who develop TRALI and possible TRALI but not TACO. A predictive model incorporating readily available clinical and cytokine data effectively differentiated transfusion-related respiratory complications such as TRALI and TACO.

Extravascular lung water and pulmonary vascular permeability index as markers predictive of postoperative acute respiratory distress syndrome: a prospective cohort investigation.

OBJECTIVE: Robust markers of subclinical perioperative lung injury are lacking. Extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index are two promising early markers of lung edema. We aimed to evaluate whether extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index would identify patients at risk for clinically significant postoperative pulmonary edema, particularly resulting from the acute respiratory distress syndrome. DESIGN: Prospective cohort study. SETTING: Tertiary care academic medical center. PATIENTS: Adults undergoing high-risk cardiac or aortic vascular surgery (or both) with risk of acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index measurements were obtained intraoperatively and in the early postoperative period. We assessed the accuracy of peak extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index as predictive markers of clinically significant pulmonary edema (defined as acute respiratory distress syndrome or cardiogenic pulmonary edema) using area under the receiver-operating characteristic curves. Associations between extravascular lung water indexed to predicted body weight and pulmonary vascular permeability patient-important with important outcomes were assessed. Of 150 eligible patients, 132 patients (88%) had extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index measurements. Of these, 13 patients (9.8%) had postoperative acute respiratory distress syndrome and 15 patients (11.4%) had cardiogenic pulmonary edema. Extravascular lung water indexed to predicted body weight effectively predicted development of clinically significant pulmonary edema (area under the receiver-operating characteristic curve, 0.79; 95% CI, 0.70-0.89). Pulmonary vascular permeability index discriminated acute respiratory distress syndrome from cardiogenic pulmonary edema alone or no edema (area under the receiver-operating characteristic curve, 0.77; 95% CI, 0.62-0.93). Extravascular lung water indexed to predicted body weight was associated with the worst postoperative PaO2/FIO2, duration of mechanical ventilation, ICU stay, and hospital stay. Peak values for extravascular lung water indexed to predicted body weight and pulmonary vascular permeability index were obtained within 2 hours of the primary intraoperative insult for the majority of patients (> 80%). CONCLUSIONS: Perioperative extravascular lung water indexed to predicted body weight is an early marker that predicts risk of clinically significant postoperative pulmonary edema in at-risk surgical patients. Pulmonary vascular permeability index effectively discriminated postoperative acute respiratory distress syndrome from cardiogenic pulmonary edema. These measures will aid in the early detection of subclinical lung injury in at-risk surgical populations.

TRIM72 is required for effective repair of alveolar epithelial cell wounding.

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

Dopamine inhibits pulmonary edema through the VEGF-VEGFR2 axis in a murine model of acute lung injury.

The neurotransmitter dopamine and its dopamine receptor D2 (D2DR) agonists are known to inhibit vascular permeability factor/vascular endothelial growth factor (VEGF)-mediated angiogenesis and vascular permeability. Lung injury is a clinical syndrome associated with increased microvascular permeability. However, the effects of dopamine on pulmonary edema, a phenomenon critical to the pathophysiology of both acute and chronic lung injuries, have yet to be established. Therefore, we sought to determine the potential therapeutic effects of dopamine in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Compared with sham-treated controls, pretreatment with dopamine (50 mg/kg body wt) ameliorated LPS-mediated edema formation and lowered myeloperoxidase activity, a measure of neutrophil infiltration. Moreover, dopamine significantly increased survival rates of LPS-treated mice, from 0-75%. Mechanistically, we found that dopamine acts through the VEGF-VEGFR2 axis to reduce pulmonary edema, as dopamine pretreatment in LPS-treated mice resulted in decreased serum VEGF, VEGFR2 phosphorylation, and endothelial nitric oxide synthase phosphorylation. We used D2DR knockout mice to confirm that dopamine acts through D2DR to block vascular permeability in our lung injury model. As expected, a D2DR agonist failed to reduce pulmonary edema in D2DR(-/-) mice. Taken together, our results suggest that dopamine acts through D2DR to inhibit pulmonary edema-associated vascular permeability, which is mediated through VEGF-VEGFR2 signaling and conveys protective effects in an ALI model.

Inhaled carbon monoxide attenuates myocardial inflammatory cytokine expression in a rat model of cardiopulmonary bypass.

Carbon monoxide (CO), a by-product of Heme metabolism, is a potent modulator of inflammation. Low dose inhaled CO has demonstrated reduced lung and kidney injury in animal models of cardiopulmonary bypass (CPB). We evaluated the impact of low dose inhaled CO on systemic, pulmonary, and myocardial inflammatory response to CPB in rats. Sixteen male Sprague-Dawley rats underwent CPB for 1 hour. The CO (n = 8) group received inhaled CO at 250 ppm for 3 hours before CPB. The Air (n = 8) group served as the control. Pulmonary mechanics were assessed pre and post CPB. The animals were recovered for 30 minutes post CPB and subsequently sacrificed. Pre CPB and post CPB serum Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-10 (IL-10) were analyzed by enzyme-linked immunosorbent assay. Gene expression array and real time quantitative polymerase chain reaction (PCR) analysis was performed on the extracted heart tissue. Baseline characteristics were similar between the groups with the expected exception of carboxyhemoglobin levels (p < or = .001) and oxyhemoglobin saturation (p < or = .01) in Air versus CO treated groups, respectively. Serum TNF-alpha (363 +/- 278 vs. 287 +/- 195;p = .13) and IL-10 (237 +/- 26 vs. 302 +/- 137; p = Not Significant) in Air versus CO groups respectively were not statistically different after CPB, despite showing a trend of inflammatory attenuation. Gene expression array of the myocardial tissue suggested a pattern of inflammatory modulation, which was confirmed by real time quantitative PCR demonstrating IL-10 expression 3.13 times higher (p = .02) in the CO treated group compared to the Air group. These data demonstrate that pretreatment with CO at 250 ppm may have a modulatory effect on the inflammatory response to CPB without compromising hemodynamics or oxygen delivery. Further investigation in a survival model of CPB is warranted.

The role of purinergic signaling on deformation induced injury and repair responses of alveolar epithelial cells.

Cell wounding is an important driver of the innate immune response of ventilator-injured lungs. We had previously shown that the majority of wounded alveolus resident cells repair and survive deformation induced insults. This is important insofar as wounded and repaired cells may contribute to injurious deformation responses commonly referred to as biotrauma. The central hypothesis of this communication states that extracellular adenosine-5' triphosphate (ATP) promotes the repair of wounded alveolus resident cells by a P2Y2-Receptor dependent mechanism. Using primary type 1 alveolar epithelial rat cell models subjected to micropuncture injury and/or deforming stress we show that 1) stretch causes a dose dependent increase in cell injury and ATP media concentrations; 2) enzymatic depletion of extracellular ATP reduces the probability of stretch induced wound repair; 3) enriching extracellular ATP concentrations facilitates wound repair; 4) purinergic effects on cell repair are mediated by ATP and not by one of its metabolites; and 5) ATP mediated cell salvage depends at least in part on P2Y2-R activation. While rescuing cells from wounding induced death may seem appealing, it is possible that survivors of membrane wounding become governors of a sustained pro-inflammatory state and thereby perpetuate and worsen organ function in the early stages of lung injury syndromes. Means to uncouple P2Y2-R mediated cytoprotection from P2Y2-R mediated inflammation and to test the preclinical efficacy of such an undertaking deserve to be explored.

Sizing the lung of mechanically ventilated patients.

INTRODUCTION: This small observational study was motivated by our belief that scaling the tidal volume in mechanically ventilated patients to the size of the injured lung is safer and more 'physiologic' than scaling it to predicted body weight, i.e. its size before it was injured. We defined Total Lung Capacity (TLC) as the thoracic gas volume at an airway pressure of 40 cm H2O and tested if TLC could be inferred from the volume of gas that enters the lungs during a brief 'recruitment' maneuver. METHODS: Lung volume at relaxed end expiration (Vrel) as well as inspiratory capacity (IC), defined as the volume of gas that enters the lung during a 5 second inflation to 40 cm H2O, were measured in 14 patients with respiratory failure. TLC was defined as the sum of IC and Vrel. The dependence of IC and Vrel on body mass index (BMI), respiratory system elastance and plateau airway pressure was assessed. RESULTS: TLC was reduced to 59 ± 23% of that predicted. Vrel/TLC, which averaged 0.45 ± 0.11, was no different than the 0.47 ± 0.04 predicted during health in the supine posture. The greater than expected variability in observed Vrel/TLC was largely accounted for by BMI. Vrel and IC were correlated (r = 0.76). Taking BMI into account strengthened the correlation (r = 0.92). CONCLUSIONS: We conclude that body mass is a powerful determinant of lung volume and plateau airway pressure. Effective lung size can be easily estimated from a recruitment maneuver derived inspiratory capacity measurement and body mass index.

Type I alveolar epithelial phenotype in primary culture.

Type I alveolar epithelial cells (ATIs) are very large, thin cells, which extend across several air sacs and cover more than 95% of the alveolar surface area. ATIs are the target of many insults, including ventilator-induced lung injury, and are generally considered terminally differentiated cells arising from type II cell (ATII) lineage. ATIs have proven difficult to harvest and maintain in primary culture, which is why much of ATI biology has been inferred from studies on ex vivo, ATII-derived, so-called ATI-like cells. We report on a modified approach to rat ATI harvest and primary culture, which yielded the following observations: (1) rat ATI can be harvested and maintained with a high degree of purity in primary culture; (2) in vitro growth characteristics of primary ATIs differ from those of ATII-derived ATI-like cells; ATIs, but not ex vivo, ATII-derived ATI-like cells, are capable of cell division; (3) ATIs readily repair plasma membrane wounds without the subsequent loss of their ability to divide; (4) ATI monolayers heal scratch wounds primarily by cell spreading and migration. Although the ability of ATIs to divide may be limited to the in vitro environment, we do believe that their role in alveolar wound repair deserves to be revisited, and the molecular control of ATI-ATII plasticity further explored.

Experimental models to study cell wounding and repair.

Cell wounding, that is a loss of plasma membrane integrity, is a common everyday occurrence in load bearing organs such as muscle, skin, and bone. In general, these injuries trigger adaptive responses to either restore homeostasis or to protect the cells from further damage. The ability to restore plasma membrane integrity after injury is critical for cell survival and all cells possess a means to do so. However, the probability of plasma membrane wound repair depends on the cell type, as well as the size and nature of the lesion. Several in vitro experimental models of cell injury have been developed to simulate specific stresses cells experience in vivo. Motivated by our interest in studying the mechanisms of cell injury and repair relevant to ventilator associated lung injury, we review some of the most frequently used in vitro experimental models of cell wounding and present some new data pertaining to alveolar epithelium.

A prospective trial of elective extubation in brain injured patients meeting extubation criteria for ventilatory support: a feasibility study.

INTRODUCTION: To assess the safety and feasibility of recruiting mechanically ventilated patients with brain injury who are solely intubated for airway protection and randomising them into early or delayed extubation, and to obtain estimates to refine sample-size calculations for a larger study. The design is a single-blinded block randomised controlled trial. A single large academic medical centre is the setting. METHODS: Sixteen neurologically stable but severely brain injured patients with a Glasgow Coma Score (GCS) of 8 or less were randomised to early or delayed extubation until their neurological examination improved. Eligible patients met standard respiratory criteria for extubation and passed a modified Airway Care Score (ACS) to ensure adequate control of respiratory secretions. The primary outcome measured between groups was the functional status of the patient at hospital discharge as measured by a Modified Rankin Score (MRS) and Functional Independence Measure (FIM). Secondary measurements included the number of nosocomial pneumonias and re-intubations, and intensive care unit (ICU) and hospital length of stay. Standard statistical assessments were employed for analysis. RESULTS: Five female and eleven male patients ranging in age from 30 to 93 years were enrolled. Aetiologies responsible for the neurological injury included six head traumas, three brain tumours, two intracerebral haemorrhages, two subarachnoid haemorrhages and three ischaemic strokes. There were no demographic differences between the groups. There were no unexpected deaths and no significant differences in secondary measures. The difference in means between the MRS and FIM were small (0.25 and 5.62, respectively). These results suggest that between 64 and 110 patients are needed in each treatment arm to detect a treatment effect with 80% power. CONCLUSIONS: Recruitment and randomisation of severely brain injured patients appears to be safe and feasible. A large multicentre trial will be needed to determine if stable, severely brain injured patients who meet respiratory and airway control criteria for extubation need to remain intubated.

Cell wounding and repair in ventilator injured lungs.

Acute lung injury (ALI) is a common, frequently hospital-acquired condition with a high morbidity and mortality. The stress associated with invasive mechanical ventilation represents a potentially harmful exposure, and attempts to minimize deforming stress through low tidal ventilation have proven efficacious. Lung cells are both sensors and transducers of deforming stress, and are frequently wounded in the setting of mechanical ventilation. Cell wounding may be one of the drivers of the innate immunologic and systemic inflammatory response associated with mechanical ventilation. These downstream effects of mechanotransduction have been referred to collectively as "Biotrauma". Our review will focus on cellular stress failure, that is cell wounding, and the mechanisms mediating subsequent plasma membrane repair, we hold that a better mechanistic understanding of cell plasticity, deformation associated remodeling and repair will reveal candidate approaches for lung protective interventions in mechanically ventilated patients. We will detail one such intervention, lung conditioning with hypertonic solutions as an example of ongoing research in this arena.

Intraoperative tidal volume as a risk factor for respiratory failure after pneumonectomy.

BACKGROUND: Respiratory failure is a leading cause of postoperative morbidity and mortality in patients undergoing pneumonectomy. The authors hypothesized that intraoperative mechanical ventilation with large tidal volumes (VTs) would be associated with increased risk of postpneumonectomy respiratory failure. METHODS: Patients undergoing elective pneumonectomy at the authors' institution from January 1999 to January 2003 were studied. The authors collected data on demographics, relevant comorbidities, neoadjuvant therapy, pulmonary function tests, site and type of operation, duration of surgery, intraoperative ventilator settings, and intraoperative fluid administration. The primary outcome measure was postoperative respiratory failure, defined as the need for continuation of mechanical ventilation for greater than 48 h postoperatively or the need for reinstitution of mechanical ventilation after extubation. RESULTS: Of 170 pneumonectomy patients who met inclusion criteria, 30 (18%) developed postoperative respiratory failure. Causes of postoperative respiratory failure were acute lung injury in 50% (n = 15), cardiogenic pulmonary edema in 17% (n = 5), pneumonia in 23% (n = 7), bronchopleural fistula in 7% (n = 2), and pulmonary thromboembolism in 3% (n = 1). Patients who developed respiratory failure were ventilated with larger intraoperative VT than those who did not (median, 8.3 vs. 6.7 ml/kg predicted body weight; P < 0.001). In a multivariate regression analysis, larger intraoperative VT (odds ratio, 1.56 for each ml/kg increase; 95% confidence interval, 1.12-2.23) was associated with development of postoperative respiratory failure. The interaction between larger VT and fluid administration was also statistically significant (odds ratio, 1.36; 95% confidence interval, 1.05-1.97). CONCLUSION: Mechanical ventilation with large intraoperative VT is associated with increased risk of postpneumonectomy respiratory failure.

Bronchoscopy in ventilator-associated pneumonia: agreement of calibrated loop and serial dilution.

RATIONALE: Although the serial dilution technique for quantitative culture of bronchoalveolar fluid is considered to be the gold standard for the diagnosis of ventilator-associated pneumonia, it is more labor intensive than the calibrated loop technique. OBJECTIVE: We sought to determine the agreement between the calibrated loop and serial dilution techniques in the diagnosis of ventilator-associated pneumonia. METHODS: We prospectively measured bacterial colony counts by the serial dilution and calibrated loop techniques in 121 bronchoalveolar lavage samples of 104 patients with suspected ventilator-associated pneumonia. MEASUREMENTS AND MAIN RESULTS: At the time of bronchoscopy, patients had received mechanical ventilation for a median of 8 d. Patients were receiving antibiotics when 90 of the 121 (74.4%) bronchoalveolar samples were obtained. The colony counts of 13 bacterial isolates were too numerous to count by the calibrated loop technique; by serial dilution technique, their counts ranged from 4.70 to 6.74 log10 cfu/ml. Fifty other bacteria had paired colony counts measured by each of the two techniques: the bias (95% confidence interval) between the two techniques was -0.380 (-0.665 to -0.095) log10 cfu/ml, with precision of 1.002 log10 cfu/ml and 95% limits of agreement of -2.344 to 1.584 log10 cfu/ml. Using the threshold of 4 log10 cfu/ml as a criterion for the diagnosis of ventilator-associated pneumonia, there was discordance only for one bacterial organism between the two techniques. CONCLUSIONS: The calibrated loop technique can be used for the diagnosis of ventilator-associated pneumonia using bronchoalveolar lavage fluid.

The effects of the alveolar recruitment maneuver and positive end-expiratory pressure on arterial oxygenation during laparoscopic bariatric surgery.

Abnormalities in gas exchange that occur during anesthesia are mostly caused by atelectasis, and these alterations are more pronounced in morbidly obese than in normal weight subjects. Sustained lung insufflation is capable of recruiting the collapsed areas and improving oxygenation in healthy patients of normal weight. We tested the effect of this ventilatory strategy on arterial oxygenation (Pao2) in patients undergoing laparoscopic bariatric surgery. After pneumoperitoneum was accomplished, the recruitment group received up to 4 sustained lung inflations with peak inspiratory pressures up to 50 cm H2O, which was followed by ventilation with 12 cm H2O positive end-expiratory pressure (PEEP). The patient's lungs in the control group were ventilated in a standard fashion with PEEP of 4 cm H2O. Variables related to gas exchange, respiratory mechanics, and hemodynamics were compared between recruitment and control groups. We found that alveolar recruitment effectively increased intraoperative Pao2 and temporarily increased respiratory system dynamic compliance (both P < 0.01). The effects of alveolar recruitment on oxygenation lasted as long as the trachea was intubated, and lungs were ventilated with high PEEP, but soon after tracheal extubation, all the beneficial effects on oxygenation disappeared. The mean number of vasopressor treatments given during surgery was larger in the recruitment group compared with the control group (3.0 versus 0.8; P = 0.04). In conclusion, our data suggest that the use of alveolar recruitment may be an effective mode of improving intraoperative oxygenation in morbidly obese patients. Our results showed the effect to be short lived and associated with more frequent intraoperative use of vasopressors.

Hypercapnic acidosis impairs plasma membrane wound resealing in ventilator-injured lungs.

The objective of this study was to assess the effects of hypercapnic acidosis on lung cell injury and repair by confocal microscopy in a model of ventilator-induced lung injury. Three groups of normocapnic, hypocapnic, and hypercapnic rat lungs were perfused ex vivo, either during or after injurious ventilation, with a solution containing the membrane-impermeant label propidium iodide. In lungs labeled during injurious ventilation, propidium iodide fluorescence identifies all cells with plasma membrane wounds, both permanent and transient, whereas in lungs labeled after injurious ventilation propidium iodide fluorescence identifies only cells with permanent plasma membrane wounds. Hypercapnia minimized the adverse effects of high-volume ventilation on vascular barrier function, whereas hypocapnia had the opposite effect. Despite CO2-dependent differences in lung mechanics and edema the number of injured subpleural cells per alveolus was similar in the three groups (0.48 +/- 0.34 versus 0.51 +/- 0.19 versus 0.43 +/- 0.20 for hypocapnia, normocapnia, and hypercapnia, respectively). However, compared with normocapnia the probability of wound repair was significantly reduced in hypercapnic lungs (63 versus 38%; p < 0.02). This finding was subsequently confirmed in alveolar epithelial cell scratch models. The potential relevance of these observations for lung inflammation and remodeling after mechanical injury is discussed.

Cellular stress failure in ventilator-injured lungs.

The clinical and experimental literature has unequivocally established that mechanical ventilation with large tidal volumes is injurious to the lung. However, uncertainty about the micromechanics of injured lungs and the numerous degrees of freedom in ventilator settings leave many unanswered questions about the biophysical determinants of lung injury. In this review we focus on experimental evidence for lung cells as injury targets and the relevance of these studies for human ventilator-associated lung injury. In vitro, the stress-induced mechanical interactions between matrix and adherent cells are important for cellular remodeling as a means for preventing compromise of cell structure and ultimately cell injury or death. In vivo, these same principles apply. Large tidal volume mechanical ventilation results in physical breaks in alveolar epithelial and endothelial plasma membrane integrity and subsequent triggering of proinflammatory signaling cascades resulting in the cytokine milieu and pathologic and physiologic findings of ventilator-associated lung injury. Importantly, though, alveolar cells possess cellular repair and remodeling mechanisms that in addition to protecting the stressed cell provide potential molecular targets for the prevention and treatment of ventilator-associated lung injury in the future.