Donor smoking is associated with pulmonary edema, inflammation and epithelial dysfunction in ex vivo human donor lungs.

Although recipients of donor lungs from smokers have worse clinical outcomes, the underlying mechanisms are unknown. We tested the association between donor smoking and the degree of pulmonary edema (as estimated by lung weight), the rate of alveolar fluid clearance (AFC; measured by airspace instillation of 5% albumin) and biomarkers of lung epithelial injury and inflammation (bronchoalveolar lavage [BAL] surfactant protein-D (SP-D) and IL-8) in ex vivo lungs recovered from 298 organ donors. The extent of pulmonary edema was higher in current smokers (n = 127) compared to nonsmokers (median 408 g, interquartile range [IQR] 364-500 vs. 385 g, IQR 340-460, p = 0.009). Oxygenation at study enrollment was worse in current smokers versus nonsmokers (median PaO2 /FiO2 214 mm Hg, IQR 126-323 vs. 266 mm Hg, IQR 154-370, p = 0.02). Current smokers with the highest exposure (≥20 pack years) had significantly lower rates of AFC, suggesting that the effects of cigarette smoke on alveolar epithelial fluid transport function may be dose related. BAL IL-8 was significantly higher in smokers while SP-D was lower. These findings indicate that chronic exposure to cigarette smoke has important effects on inflammation, gas exchange, lung epithelial function and lung fluid balance in the organ donor that could influence lung function in the lung transplant recipient.

Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples.

BACKGROUND: Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance. METHODS: In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples. RESULTS: We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations. CONCLUSIONS: In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.

Preoperative plasma club (clara) cell secretory protein levels are associated with primary graft dysfunction after lung transplantation.

Inherent recipient factors, including pretransplant diagnosis, obesity and elevated pulmonary pressures, are established primary graft dysfunction (PGD) risks. We evaluated the relationship between preoperative lung injury biomarkers and PGD to gain further mechanistic insight in recipients. We performed a prospective cohort study of recipients in the Lung Transplant Outcomes Group enrolled between 2002 and 2010. Our primary outcome was Grade 3 PGD on Day 2 or 3. We measured preoperative plasma levels of five biomarkers (CC-16, sRAGE, ICAM-1, IL-8 and Protein C) that were previously associated with PGD when measured at the postoperative time point. We used multivariable logistic regression to adjust for potential confounders. Of 714 subjects, 130 (18%) developed PGD. Median CC-16 levels were elevated in subjects with PGD (10.1 vs. 6.0, p<0.001). CC-16 was associated with PGD in nonidiopathic pulmonary fibrosis (non-IPF) subjects (OR for highest quartile of CC-16: 2.87, 95% CI: 1.37, 6.00, p=0.005) but not in subjects with IPF (OR 1.38, 95% CI: 0.43, 4.45, p=0.59). After adjustment, preoperative CC-16 levels remained associated with PGD (OR: 3.03, 95% CI: 1.26, 7.30, p=0.013) in non-IPF subjects. Our study suggests the importance of preexisting airway epithelial injury in PGD. Markers of airway epithelial injury may be helpful in pretransplant risk stratification in specific recipients.

Plasma receptor for advanced glycation end products and clinical outcomes in acute lung injury.

OBJECTIVES: To determine whether baseline plasma levels of the receptor for advanced glycation end products (RAGE), a novel marker of alveolar type I cell injury, are associated with the severity and outcomes of acute lung injury, and whether plasma RAGE levels are affected by lower tidal volume ventilation. DESIGN, SETTING AND PARTICIPANTS: Measurement of plasma RAGE levels from 676 subjects enrolled in a large randomised controlled trial of lower tidal volume ventilation in acute lung injury. MEASUREMENTS AND MAIN RESULTS: Higher baseline plasma RAGE was associated with increased severity of lung injury. In addition, higher baseline RAGE was associated with increased mortality (OR for death 1.38 (95% CI 1.13 to 1.68) per 1 log increment in RAGE; p = 0.002) and fewer ventilator free and organ failure free days in patients randomised to higher tidal volumes. These associations persisted in multivariable models that adjusted for age, gender, severity of illness and the presence of sepsis or trauma. Plasma RAGE was not associated with outcomes in the lower tidal volume group (p = 0.09 for interaction in unadjusted analysis). In both tidal volume groups, plasma RAGE levels declined over the first 3 days; however, the decline was 15% greater in the lower tidal volume group (p = 0.02; 95% CI 2.4% to 25.0%). CONCLUSIONS: Baseline plasma RAGE levels are strongly associated with clinical outcomes in patients with acute lung injury ventilated with higher tidal volumes. Lower tidal volume ventilation may be beneficial in part by decreasing injury to the alveolar epithelium.

Acute inflammation in a sheep model of unilateral lung ischemia: the role of interleukin-8 recruitment of polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMN) contribute to post-ischemic injury in many organs and in a variety of clinical situations. PMN accumulate in both lungs during unilateral lung ischemia in sheep, but the mechanism has not been defined. In this study, we tested the hypothesis that PMN accumulation is a response to chemotactic signals generated during lung ischemia. Chemotactic activity was measured in a modified Boyden chamber using normal sheep PMN as the responding cells. Increased chemotactic activity was observed in both plasma and lung lymph in a time-dependent manner after ischemia. These data indicate that a chemotactic substance immunoreactive to interleukin-8 antibody is formed as a result of unilateral lung ischemia in sheep in vivo and is a possible mediator of PMN inflammation in this model.

Postischemic hypoperfusion during unilateral lung reperfusion in vivo.

The incomplete restoration of blood flow during reperfusion may amplify injury by prolonging ischemia; this "no-reflow" has been studied extensively in systemic organs. Our goal was to examine lung blood flow and microvascular function, specifically to determine whether blood flow is altered during lung reperfusion injury in vivo. In a unilateral lung model of ischemia-reperfusion in awake sheep, we measured pulmonary vascular resistance in each lung by radiolabeled microspheres. Measurements were made before 14 h of ischemia and again 4 h after reperfusion. Vascular resistance in the reperfused lung increased 3-fold (9.64 +/- 0.85 to 27.04 +/- 4.73 cm H2O/L/min) during reperfusion. The increase in vascular resistance in the reperfused lung fully accounted for the small increase in overall pulmonary vascular resistance (4.04 +/- 0.26 to 5.52 +/- 0.70 cm H2O/L/min). Microvascular permeability in the reperfused lung increased 52% more than in the contralateral lung, measured by an improved indicator dilution method with additional markers sensitive to surface area (butanediol). We conclude that changes in vascular resistance and microvascular function occur during lung reperfusion injury in vivo. The demonstration that postischemic hypoperfusion occurs during lung reperfusion in vivo suggests possible new avenues of approach to related clinical disorders.

Lung ischemia-reperfusion injury in awake sheep: protection with verapamil.

We caused unilateral lung ischemia-reperfusion injury in awake sheep by simultaneously occluding the left pulmonary artery and left main stem bronchus for 12 h. The occluded left lung was inflated with nitrogen. Reperfusion resulted in an elevation of lung lymph flow from 1.3 to 5.0 ml/15 min and an increase in lymph-to-plsma protein concentration ratios. Reperfusion, but not ischemia alone, caused an increase in wet-to-dry weight ratios in both the reperfused left lung and the contralateral right lung. Granulocytes increased in both lungs during the ischemic period and after reperfusion, and hypoxemia developed after reperfusion. The calcium channel antagonist, verapamil, given just before reperfusion, caused a marked attenuation in the reperfusion-induced changes in the lung lymph variables and wet-to-dry weight ratio. However, verapamil did not affect the hypoxemia or granulocyte sequestration seen after reperfusion. We conclude that reperfusion of ischemic sheep lung results in increased microvascular permeability that can be partially prevented by verapamil.

Acute endotoxin-induced lymphocyte subset sequestration in sheep lungs.

Endotoxemia is associated with an early phase of pulmonary hypertension and a later increase in microvascular permeability. These physiologic changes are attended by peripheral blood and lung lymph leukopenia and a rapid accumulation of both granulocytes and lymphocytes in the peripheral lung. In the present study, the numbers of lymphocytes in blood, lung lymph, and lung tissue after infusion of endotoxin were determined by fluorescent labeling of lymphocyte populations with monoclonal antibodies to sheep T1, T4, T8, or leukocyte common antigen and with rabbit anti-sheep immunoglobulins (B cells). Peripheral blood, lung lymph, and lung tissue samples were collected at baseline and 15, 30, 60, 120, 180, and 240 minutes after the start of intravenous infusion of E. coli endotoxin (1.25 micrograms/kg, N = 6) or saline (N = 4) from open-chest anesthetized sheep. Pulmonary artery pressure and lung lymph flow were also monitored at these times. Endotoxin caused marked reductions in the number of T and B lymphocytes in blood and lung lymph. As compared with baseline, total blood leukocytes and granulocytes were significantly reduced below control levels from 30 minutes of endotoxin, and lymphocyte numbers were reduced from 60 minutes. T1, T4, T8 and B lymphocyte subsets contributed to the fall in blood lymphocytes. Endotoxin caused a significant fall in number of lung lymph leukocytes (T1, T4 and T8 cells) from 120 minutes; numbers of B lymphocytes were also reduced. Counts of the number of lymphocytes in the biopsy tissue revealed a significant rise in T lymphocytes sequestered in the lung. We conclude endotoxemia in sheep causes a reduction in lymphocytes in blood and lung lymph and an increase in these cell types in peripheral lung tissue. These findings suggest that lymphocyte subpopulations accumulate in the lungs during periods of pulmonary hypertension and increased permeability and may participate in endotoxin-induced lung injury.

Allergen-stimulated release of mediators into sheep bronchoalveolar lavage fluid. Effect of cyclooxygenase inhibition.

Products of arachidonic acid have been implicated as potential mediators of allergic airway responses in sheep and in humans. We therefore measured the release of arachidonate metabolites into sheep bronchoalveolar lavage fluid (BALF) after local instillation of Ascaris antigen, using a highly specific and sensitive approach, gas chromatography-negative ion-chemical ionization mass spectrometry. Allergen instillation caused increases in the levels of the potent bronchoconstrictors prostaglandin (PG) D2 and thromboxane (TX) A2 (as reflected by levels of TXB2) and of their enzymatic metabolites, 9 alpha, 11 beta-PGF2 and 11-dehydro-TXB2, respectively. Potential bronchodilators, PGI2 and PGE2, were also formed in increased amounts. Levels of the 5-lipoxygenase products, leukotriene (LT) B4 and C4, were not significantly increased. Pretreatment of sheep with cyclooxygenase inhibitors was associated with a decrease in the release of cyclooxygenase products and a concomitant increase in the allergen-stimulated release of LTB4 and LTC4. Eicosanoids are formed promptly in vivo in sheep after allergen instillation: inhibition of cyclooxygenase results in augmented generation of leukotrienes in the airways of sensitive sheep in response to antigen challenge.

Corticosteroids prevent acute lung dysfunction caused by thoracic irradiation in unanesthetized sheep.

We sought to determine the effect of corticosteroid therapy in a new acute model of oxidant lung injury, thoracic irradiation in awake sheep. Sheep were irradiated with 1,500 rads to the whole chest except for blocking the heart and adjacent ventral lung. Seven experimental sheep were given methylprednisolone (1 g intravenously every 6 h for four doses) and thoracic irradiation; control sheep received only irradiation. In irradiated control sheep, lung lymph flow increased from baseline (7.6 ml/h) to peak at 3 h (13.2), and lung lymph protein clearance increased from 5.1 to 9.7 ml/h. Mean pulmonary artery pressure increased in the irradiated control sheep from 19 to 32.4 cm H2O, whereas the lung lymph thromboxane concentration increased from 0.09 to 6.51 ng/ml at 3 h. Arterial oxygen tension in irradiated control sheep fell gradually from 86 mm Hg at baseline to 65 mm Hg at 8 h. Methylprednisolone administration significantly prevented the increase in lung lymph protein clearance, mean pulmonary artery pressure, and lung lymph thromboxane concentration. Methylprednisolone also prevented the fall in arterial oxygen tension after thoracic irradiation, but did not prevent a further decrease in lymphocytes in blood or lung lymph after radiation. We conclude that corticosteroid therapy prevents most of the acute physiologic changes caused by thoracic irradiation in awake sheep.

Effects of hypoproteinemia on lung microvascular protein sieving and lung lymph flow.

Experiments were conducted on five chronically instrumented unanesthetized sheep to determine the effects of sustained hypoproteinemia on lung fluid balance. Plasma total protein concentration was decreased from a control value of 6.17 +/- 0.019 to 3.97 +/- 0.17 g/dl (mean +/- SE) by acute plasmapheresis and maintained at this level by chronic thoracic lymph duct drainage. We measured pulmonary arterial pressure, left atrial pressure, aortic pressure, central venous pressure, cardiac output, oncotic pressures of both plasma and lung lymph, lung lymph flow rate, and lung lymph-to-plasma ratio of total proteins and six protein fractions for both control base-line conditions and hypoproteinemia base-line conditions. Moreover, we estimated the average osmotic reflection coefficient for total proteins and the solvent drag reflection coefficients for the six protein fractions during hypoproteinemia. Hypoproteinemia caused significant decreases in lung lymph total protein concentration, lung lymph-to-plasma total protein concentration ratio, and oncotic pressures of plasma and lung lymph. There were no significant alterations in the vascular pressures, lung lymph flow rate, cardiac output, or oncotic pressure gradient. The osmotic reflection coefficient for total proteins was found to be 0.900 +/- 0.004 for hypoproteinemia conditions, which is equal to that found in a previous investigation for sheep with a normal plasma protein concentration. Our results suggest that hypoproteinemia does not alter the lung filtration coefficient nor the reflection coefficients for plasma proteins. Possible explanations for the reported increase in the lung filtration coefficient during hypoproteinemia by other investigators are also made.

Relation of blood-free to blood-inclusive postmortem lung water measurements in sheep.

Our purpose was to see if the postmortem weight ratio of extravascular lung water to blood-free dry lung (blood-free ratio) was related to similar ratios in blood-inclusive lung and in blood. We developed linear regressions of blood-free ratio on ratios for blood-inclusive lung and blood together and for blood-inclusive lung alone for 73 sheep studied under 11 different protocols and for two subgroups of sheep, one with plasma space expansion and the other without expansion. The relation of ratios of blood-free to blood-inclusive lungs was different between the two subgroups. Although all regressions were highly correlated, the fits of the blood-free ratio on ratios for blood-inclusive lung and blood together were better than for blood-inclusive lung alone. The mean error of prediction of extravascular lung water for all sheep was significantly less for the regression of blood-free ratio on ratios for blood and blood-inclusive lung together (11 g) than for blood-inclusive lung alone (18 g). This study shows that weights of lung homogenate and blood samples before and after simple oven drying can be used to provide accurate inexpensive estimates of postmortem extravascular lung water.