Bronchoalveolar Lavage Total Protein Research Articles

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Local nebulization of 1\u03b1,25(OH)2D3 attenuates LPS-induced acute lung inflammation

BackgroundEvidence supports a critical role of vitamin D status on exacerbation in chronic obstructive pulmonary disease, indicating the need to avoid vitamin D deficiency in these patients. However, oral vitamin D supplementation is limited by the potential risk for hypercalcemia. In this study, we investigated if local delivery of vitamin D to the lungs improves vitamin D-mediated anti-inflammatory action in response to acute inflammation without inducing hypercalcemia.MethodsWe studied vitamin D sufficient (VDS) or deficient (VDD) mice in whom 1α,25(OH)2D3 (0.2 μg/kg) or a vehicle followed by lipopolysaccharide (LPS 25 µg) were delivered to the lung as a micro-spray.ResultsLocal 1α,25(OH)2D3 reduced LPS-induced inflammatory cells in bronchoalveolar lavage (BAL) in VDS (absolute number of cells: − 57% and neutrophils − 51% p < 0.01) and tended to diminish LPS-increased CXCL5 BAL levels in VDS (− 40%, p = 0.05) while it had no effect on CXCL1 and CXCL2 in BAL and mRNA in lung of VDS and VDD. It also significantly attenuated the increased IL-13 in BAL and lung, especially in VDD mice (− 41 and − 75%, respectively). mRNA expression of Claudin-18 in lung was significantly lower in VDS mice with local 1α,25(OH)2D3 while Claudin-3, -5 and -8 mRNA levels remained unchanged. Finally, in VDD mice only, LPS reduced lung mRNA expression of adhesion junction Zona-occludens-1, in addition to increasing uric acid and total protein in BAL, which both were prevented by local 1α,25(OH)2D3.ConclusionUnder normal levels of vitamin D, local 1α,25(OH)2D3 nebulization into the lung efficiently reduced LPS induction of inflammatory cells in BAL and slightly attenuated LPS-increase in CXCL5. In case of severe vitamin D deficiency, although local 1α,25(OH)2D3 nebulization failed to significantly minimize cellular inflammation in BAL at this dose, it prevented epithelial barrier leakage and damage in lung. Additional research is needed to determine the potential long-term beneficial effects of local 1α,25(OH)2D3 nebulization on lung inflammation.

A two-hit model of sepsis plus hyperoxia causes lung permeability and inflammation.

Mouse models of acute lung injury (ALI) have been instrumental for studies of the biological underpinnings of lung inflammation and permeability, but murine models of sepsis generate minimal lung injury. Our goal was to create a murine sepsis model of ALI that reflects the inflammation, lung edema, histological abnormalities, and physiological dysfunction that characterize ALI. Using a cecal slurry (CS) model of polymicrobial abdominal sepsis and exposure to hyperoxia (95%), we systematically varied the timing and dose of the CS injection, fluids and antibiotics, and dose of hyperoxia. We found that CS alone had a high mortality rate that was improved with the addition of antibiotics and fluids. Despite this, we did not see evidence of ALI as measured by bronchoalveolar lavage (BAL) cell count, total protein, C-X-C motif chemokine ligand 1 (CXCL-1) or by lung wet:dry weight ratio. Addition of hyperoxia [95% fraction of inspired oxygen ([Formula: see text])] to CS immediately after CS injection increased BAL cell counts, CXCL-1, and lung wet:dry weight ratio but was associated with 40% mortality. Splitting the hyperoxia treatment into two 12-h exposures (0-12 h and 24-36 h) after CS injection increased survival to 75% and caused significant lung injury compared with CS alone as measured by increased BAL total cell count (92,500 vs. 240,000, P = 0.0004), BAL protein (71 vs. 103 µg/mL, P = 0.0030), and lung wet:dry weight ratio (4.5 vs. 5.5, P = 0.0005), and compared with sham as measured by increased BAL CXCL-1 (20 vs. 2,372 pg/mL, P < 0.0001) and histological lung injury score (1.9 vs. 4.2, P = 0.0077). In addition, our final model showed evidence of lung epithelial [increased BAL and plasma receptor for advanced glycation end products (RAGE)] and endothelial (increased Syndecan-1 and sulfated glycosaminoglycans) injury. In conclusion, we have developed a clinically relevant mouse model of sepsis-induced ALI using intraperitoneal injection of CS, antibiotics and fluids, and hyperoxia. This clinically relevant model can be used for future studies of sepsis-induced ALI.

Bolus intravenous 0.9% saline leads to interstitial permeability pulmonary edema in healthy volunteers

Bolus intravenous administration of 0.9% saline has been associated with the development of pulmonary edema, and increased mortality. An animal model has previously demonstrated that rapid intravenous administration of 0.9% saline was associated with non-hydrostatic lung injury with increased lung lavage protein. We hypothesized that this non-hydrostatic effect would also occur in human volunteers. In a randomized, cross-over study of 14 healthy male subjects, the lung lavage protein concentration and cardiorespiratory effects of an intervention with rapid intravenous administration of 30mL/kg of 0.9% saline were compared with sham intervention. Bronchoalveolar lavage (BAL) was performed after fluid administration. Doppler echocardiography, lung ultrasound, pulmonary function tests, and blood sampling were performed before and after each intervention. The BAL total protein concentration was greater after 0.9% saline administration than with sham (196.1µg/mL (SD 87.6) versus 129.8µg/mL (SD 55.4), respectively (p = 0.020). Plasma angiopoietin-2 concentration was also increased to 2.26ng/mL (SD 0.87) after 0.9% saline administration compared with sham 1.53ng/mL (SD 0.69) (p < 0.001). There were small increases in stroke volume (from 58mL (IQR 51-74) to 66mL (IQR 58-74), p = 0.045) and Doppler echocardiography left ventricle E/e' ratio (from 5.0 (IQR 4.5-6.0) to 5.7 (IQR 5.3-6.3), p = 0.007), but no changes to right ventricular function. Rapid intravenous administration of 0.9% saline leads to interstitial permeability pulmonary edema in healthy human volunteers. Further research is now warranted to understand these effects in critically ill patients.

A study of the diagnostic utility of the complement activation product C4d in bronchoalveolar lavage fluid of patients with bronchogenic carcinoma

Background Molecular markers may improve the diagnosis of lung cancer. In the past few years, multiple biomarkers have been detected in bronchial lavage fluids and tissues of patients with lung cancer. Aim The present prospective case–control study aimed to evaluate the role of complement activation product (C4d) as a diagnostic aid for bronchogenic carcinoma and to predict its diagnostic levels in bronchoalveolar lavage fluid (BALF) and in sputum supernatant of patients diagnosed with lung cancer. Patients and methods The study included 40 patients. They were classified into two groups: group 1 represented lung cancer group (which was diagnosed in this study by histopathological examination), which consisted of 25 patients and group 2 represented control group with nonmalignant chest diseases, which consisted of 15 patients. Clinical assessment, bronchoscopy, measurement of C4d in BALF and in sputum supernatant, and measurement of total protein in BALF were performed for all studied participants. Results C4d in bronchoalveolar lavage (BAL), C4d in sputum supernatant, and total protein in BAL were significantly higher in the lung cancer group compared with the control group. Conclusion C4d in BAL and sputum as well as total protein in BAL can be used as a diagnostic aid in patients with bronchogenic carcinoma compared with other lung diseases with moderate sensitivity and specificity.

Analysis of Acrolein Exposure Induced Pulmonary Response in Seven Inbred Mouse Strains and Human Primary Bronchial Epithelial Cells Cultured at Air-Liquid Interface

Background Acrolein is a major component of environmental pollutants, cigarette smoke, and is also formed by heating cooking oil. We evaluated the interstrain variability of response to subchronic inhalation exposure to acrolein among inbred mouse strains for inflammation, oxidative stress, and tissue injury responses. Furthermore, we studied the response to acrolein vapor in the lung mucosa model using human primary bronchial epithelial cells (PBEC) cultured at an air-liquid interface (ALI) to evaluate the findings of mouse studies. Methods Female 129S1/SvlmJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ mice were exposed to 1 part per million (ppm) acrolein or filtered air for 11 weeks. Total cell counts and protein concentrations were measured in bronchoalveolar lavage (BAL) fluid to assess airway inflammation and membrane integrity. PBEC-ALI models were exposed to acrolein vapor (0.1 and 0.2 ppm) for 30 minutes. Gene expression of proinflammatory, oxidative stress, and tissue injury-repair markers was assessed (cut off: ≥2 folds; p < 0.05) in the lung models. Results Total BAL cell numbers and protein concentrations remained unchanged following acrolein exposure in all mouse strains. BALB/cByJ, C57BL/6J, and 129S1/SvlmJ strains were the most affected with an increased expression of proinflammatory, oxidative stress, and/or tissue injury markers. DBA/2J, C3H/HeJ, A/J, and FVB/NJ were affected to a lesser extent. Both matrix metalloproteinase 9 (Mmp9) and tissue inhibitor of metalloproteinase 1 (Timp1) were upregulated in the strains DBA/2J, C3H/HeJ, and FVB/NJ indicating altered protease/antiprotease balance. Upregulation of lung interleukin- (IL-) 17b transcript in the susceptible strains led us to investigate the IL-17 pathway genes in the PBEC-ALI model. Acrolein exposure resulted in an increased expression of IL-17A, C, and D; IL-1B; IL-22; and RAR-related orphan receptor A in the PBEC-ALI model. Conclusion The interstrain differences in response to subchronic acrolein exposure in mouse suggest a genetic predisposition. Altered expression of IL-17 pathway genes following acrolein exposure in the PBEC-ALI models indicates that it has a central role in chemical irritant toxicity. The findings also indicate that genetically determined differences in IL-17 signaling pathway genes in the different mouse strains may explain their susceptibility to different chemical irritants.

Investigating the effect of TRPV4 inhibition on pulmonary-vascular barrier permeability following segmental endotoxin challenge

BackgroundAcute Respiratory Distress Syndrome (ARDS) is associated with increased pulmonary-vascular permeability. In the lung, transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel, is a regulator of endothelial permeability and pulmonary edema. We performed a Phase I, placebo-controlled, double-blind, randomized, parallel group, proof-of-mechanism study to investigate the effects of TRPV4 channel blocker, GSK2798745, on pulmonary-vascular barrier permeability using a model of lipopolysaccharide (LPS)-induced lung inflammation. MethodsHealthy participants were randomized 1:1 to receive 2 single doses of GSK2798745 or placebo, 12 h apart. Two hours after the first dose, participants underwent bronchoscopy and segmental LPS instillation. Total protein concentration and neutrophil counts were measured in bronchoalveolar lavage (BAL) samples collected before and 24 h after LPS challenge, as markers of barrier permeability and inflammation, respectively. The primary endpoint was baseline adjusted total protein concentration in BAL at 24 h after LPS challenge. A Bayesian framework was used to estimate the posterior probability of any percentage reduction (GSK2798745 relative to placebo). Safety endpoints included the incidence of adverse events (AEs), vital signs, 12-lead electrocardiogram, clinical laboratory and haematological evaluations, and spirometry. ResultsForty-seven participants were dosed and 45 completed the study (22 on GSK2798745 and 23 on placebo). Overall, GSK2798745 was well tolerated. Small reductions in mean baseline adjusted BAL total protein (~9%) and neutrophils (~7%) in the LPS-challenged segment were observed in the GSK2798745 group compared with the placebo group; however, the reductions did not meet pre-specified success criteria of at least a 95% posterior probability that the percentage reduction in the mean 24-h post LPS BAL total protein level (GSK2798745 relative to placebo) exceeded zero. Median plasma concentrations of GSK2798745 were predicted to inhibit TRPV4 on lung vascular endothelial cells by ~70–85% during the 24 h after LPS challenge; median urea-corrected BAL concentrations of GSK2798745 were 3.0- to 8.7-fold higher than those in plasma. ConclusionsGSK2798745 did not affect segmental LPS-induced elevation of BAL total protein or neutrophils, despite blood and lung exposures that were predicted to be efficacious. Clinicaltrials.gov identifierNCT03511105.

Myeloid Cell Recruitment and Activation Following Ozone Exposure in a Murine Model of Mutant Surfactant Protein‐C Pulmonary Dysfunction

The missense isoleucine to threonine substitution at position 73 [I73T] in the Surfactant Protein‐C [SP‐C] gene [SFTPC], an alveolar type‐2 cell [AT2] restricted protein, has been linked to sporadic and familial cases of idiopathic pulmonary fibrosis (IPF). We have developed a hypomorphic SP‐CI73T mouse model which expresses 20% of wild‐type SP‐C output. In this setting, AT2 cells mistraffick pro‐SP‐C on the plasma membrane and temporally acquire a late block in AT2 cell macroautophagy. This is associated with age‐dependent enlargement of alveolar septae, AT2 cell hyperplasia and minor inflammatory cell accumulation, beginning at 16 wk of age. Ozone is a ubiquitous air pollutant that causes parenchymal injury and monocyte/macrophage dominant inflammation. Clinically, acute exposure to ozone is known to exacerbate respiratory injury in susceptible population (i.e. asthma). Therefore, we tested whether SP‐CI73T dysfunction is associated with susceptibility to ozone induced injury (0.8 ppm, 3 h) in young (8–12wk) and aging (52 wk) mouse cohorts. We found that young WT mice exposed to ozone did not cause significant alterations in bronchoalveolar lavage (BAL) fluid immune cell composition or cell numbers at 24 h. Conversely, differential analysis of cytospins and flow cytometry analysis of tissue digest showed increased cell counts with significant neutrophilia and lymphocyte accumulation in SP‐CI73T mice. Analysis of aging cohorts confirmed significant deterioration of lung architecture in air‐exposed 52 wk SP‐CI73T mice. Ozone exposure was associated with histological evidence of perivascular edema and immune cells influx. BAL total protein analysis also showed increased epithelial‐vascular barrier disruption at 24 h and 48 h. Taken together, our findings support the concept that epithelial dysfunction driven by mutant SP‐CI73T expression is associated with increased susceptibility to environmental toxicant and that these effects are heightened with aging.Support or Funding InformationGrant support: P30ES013508 (MFB), VA 1I01BX001176 (MFB), ES004738 (DL), ES005022 (DL).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Effects of Human SP-A1 and SP-A2 on the BAL Proteome and Signaling Pathways in Response to Klebsiella pneumoniae and Ozone Exposure

Surfactant protein A (SP-A) plays critical roles in host defense, regulation of inflammation and surfactant metabolism in the lung. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2 encoding surfactant proteins SP-A1 and SP-A2, respectively. Structural and functional differences exist between SP-A1 and SP-A2 in vitro and in vivo. Ozone is a major air pollutant with a negative impact on many biological processes. In this study we used humanized transgenic (hTG) SP-A1 and SP-A2 mice, and SP-A KO mice to study in vivo effects of SP-A1 and SP-A2 on the bronchoalveolar lavage (BAL) proteomic profile and associated signaling pathways in response to ozone or filtered air (FA) exposure and Klebsiella pneumoniae infection. The BAL samples were harvested 24 h after ozone (2 ppm for 3 h) or FA exposure and infection and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-ToF/ToF. We found: that (1) Ozone exposure, but not infection, is a major factor for increases in total BAL protein content. (2) A total of 36 proteins were identified, accounting for 89.62% of the BAL proteins resolved by the 2D-DIGE system. (3) The number of proteins in which levels were altered more than 25% following infection and FA exposure was: SP-A2 > SP-A1 > KO for male mice, and SP-A2 ≈ SP-A1 > KO for female mice. (4) The number of proteins with more than 25% increase/decrease after ozone exposure and infection was: SP-A2 > SP-A1 ≈ KO, with the majority being increases in male mice and decreases in female mice. (5) Eleven out of the 36 proteins, including annexin A5, glutathione S-transferase A4, SP-A1/SP-A2, and 14-3-3 zeta protein, exhibited significant differences among SP-A genotypes. The acute phase response (APR) that includes the NF-kB signaling pathway plays a critical role, followed by Nrf2-mediated oxidative response, and others. These associated with SP-A genotype, sex, and ozone-induced oxidative stress in response to infection. We concluded that human SP-A2 and SP-A1 exhibit differential genotype-and sex-dependent innate immune responses to microbial pathogens and/or ozone-induced oxidative stress by modulating proteomic patterns and signaling pathways in the lung.

Aquaporin 5 -1364A/C Promoter Polymorphism Is Associated with Pulmonary Inflammation and Survival in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome is defined according to clinical criteria, but lack of precise characterization may contribute to negative trials and impede personalized care. Polymorphisms of aquaporin-5, a key mediator of inflammation, may impact outcome. In acute respiratory distress syndrome attributable to bacterial pneumonia, the C-allele of the aquaporin-5 -1364A/C promoter polymorphism is associated with less pulmonary inflammation and greater survival. This may improve characterization of acute respiratory distress syndrome and ultimately facilitate individualized care. The aquaporin-5 (AQP5) -1364A/C promoter single-nucleotide polymorphism is associated with an altered AQP5 expression and mortality in sepsis. Because AQP5 expression alters neutrophil cell migration, it could affect pulmonary inflammation and survival in bacterially evoked acute respiratory distress syndrome. Accordingly, the authors tested the hypotheses that the AC/CC genotype in patients with bacterially evoked pneumonia resulting in acute respiratory distress syndrome is associated with (1) attenuated pulmonary inflammation and (2) higher 30-day survival. In this prospective, observational study, bronchoalveolar lavage and blood sampling were performed within 24 h of intensive care unit admission. In 136 Caucasian patients with bacterially evoked acute respiratory distress syndrome, genotype of the AQP5 -1364A/C promoter polymorphism, bronchoalveolar lavage total protein, albumin, white cell concentrations, and lactate dehydrogenase activity were measured to evaluate the relationship between genotypes and survival. AC/CC patients as well as survivors showed lower bronchoalveolar lavage protein (0.9 mg/ml vs. 2.3 mg/ml, P < 0.001 and 1.6 mg/ml vs. 2.6 mg/ml, P = 0.035), albumin (0.2 mg/ml vs. 0.6 mg/ml, P = 0.019 and 0.3 mg/ml vs. 0.6 mg/ml, P = 0.028), leukocytes (424 /ml vs. 1,430/ml; P = 0.016 and 768 /ml vs. 1,826/ml; P = 0.025), and lactate dehydrogenase activity (82 U/l vs. 232 U/l; P = 0.006 and 123 U/l vs. 303 U/l; P = 0.020). Thirty-day survival was associated with AQP5 -1364A/C genotypes (P = 0.005), with survival of 62% for AA genotypes (58 of 93) but 86% for C-allele carriers (37 of 43). Furthermore, multiple proportional hazard analysis revealed the AA genotype was at high risk for death within 30 days (hazard ratio, 3.53; 95% CI, 1.38 to 9.07; P = 0.009). In acute respiratory distress syndrome attributable to bacterial pneumonia, the C-allele of the AQP5 -1364A/C promoter polymorphism is associated with an attenuated pulmonary inflammation and higher 30-day survival. Thus, the AQP5 genotype impacts on inflammation and prognosis in acute respiratory distress syndrome.

Remote ischemic preconditioning attenuates cardiopulmonary bypass-induced lung injury.

The use of cardiopulmonary bypass (CPB) in cardiac surgeries is known to induce pathological changes in vital organs such as lungs. Remote ischemic preconditioning (RIPC) is a protective strategy that has shown to be able to reduce tissue damage related to ischemia-reperfusion injury (IRI). The current study seeks to evaluate the beneficial effects of limb RIPC on lung tissues and function in a rat CPB model. RIPC, which consisted of three cycles of 5-min ischemia and subsequently 5-min reperfusion, was induced in the hind limbs of the animals via a tourniquet. Bronchoalveolar lavage (BAL) fluid analysis and hematoxylin and eosin staining revealed that limb RIPC could significantly attenuate CPB-induced pulmonary injury, as evidenced by a combination of lower total BAL protein content, less severe alveolar wall thickening and reduced intra-alveolar neutrophil infiltration. Consistently, RIPC was also found to improve the proliferation capacity of the bronchioalveolar stem cells isolated from the lung tissues in rats subjected to surgical procedure with CPB. These beneficial effects translated into significantly improved lung function. Further investigation suggested that RIPC could up-regulate the serum levels of several anti-inflammatory cytokines such as interleukin (IL)-4 and 10, which might play a role in its pulmonoprotective effects. Taken together, the current study provided convincing evidence that limb RIPC could be a useful strategy for minimizing CPB-induced organ injuries in patients undergoing CPB surgery.

The role of Src & ERK1/2 kinases in inspiratory resistive breathing induced acute lung injury and inflammation

BackgroundInspiratory resistive breathing (IRB), a hallmark of obstructive airway diseases, is associated with large negative intrathoracic pressures, due to strenuous contractions of the inspiratory muscles. IRB is shown to induce lung injury in previously healthy animals. Src is a multifunctional kinase that is activated in the lung by mechanical stress. ERK1/2 kinase is a downstream target of Src. We hypothesized that Src is activated in the lung during IRB, mediates ERK1/2 activation and IRB-induced lung injury.MethodsAnaesthetized, tracheostomized adult rats breathed spontaneously through a 2-way non-rebreathing valve. Resistance was added to the inspiratory port to provide a peak tidal inspiratory pressure of 50% of maximum (inspiratory resistive breathing). Activation of Src and ERK1/2 in the lung was estimated during IRB. Following 6 h of IRB, respiratory system mechanics were measured by the forced oscillation technique and bronchoalveolar lavage (BAL) was performed to measure total and differential cell count and total protein levels. IL-1b and MIP-2a protein levels were measured in lung tissue samples. Wet lung weight to total body weight was measured and Evans blue dye extravasation was estimated to measure lung permeability. Lung injury was evaluated by histology. The Src inhibitor, PP-2 or the inhibitor of ERK1/2 activation, PD98059 was administrated 30 min prior to IRB.ResultsSrc kinase was activated 30 min after the initiation of IRB. Src inhibition ameliorated the increase in BAL cellularity after 6 h IRB, but not the increase of IL-1β and MIP-2a in the lung. The increase in BAL total protein and lung injury score were not affected. The increase in tissue elasticity was partly inhibited. Src inhibition blocked ERK1/2 activation at 3 but not at 6 h of IRB. ERK1/2 inhibition ameliorated the increase in BAL cellularity after 6 h of IRB, blocked the increase of IL-1β and returned Evans blue extravasation and wet lung weight to control values. BAL total protein and the increase in elasticity were partially affected. ERK1/2 inhibition did not significantly change total lung injury score compared to 6 h IRB.ConclusionsSrc and ERK1/2 are activated in the lung following IRB and participate in IRB-induced lung injury.

High circulating angiopoietin-2 levels exacerbate pulmonary inflammation but not vascular leak or mortality in endotoxin-induced lung injury in mice

BackgroundElevated plasma levels of angiopoietin-2 (ANGPT2) have been reported in patients with acute lung injury (ALI); however, it remains unclear whether this increase contributes to, or just marks, the underlying...

Tiotropium bromide exerts anti-inflammatory effects during resistive breathing, an experimental model of severe airway obstruction.

IntroductionResistive breathing (RB), a hallmark of obstructive airway diseases, is characterized by strenuous contractions of the inspiratory muscles that impose increased mechanical stress on the lung. RB is shown to induce pulmonary inflammation in previous healthy animals. Tiotropium bromide, an anticholinergic bronchodilator, is also shown to exert anti-inflammatory effects. The effect of tiotropium on RB-induced pulmonary inflammation is unknown.MethodsAdult rats were anesthetized, tracheostomized and breathed spontaneously through a two-way non-rebreathing valve. Resistances were connected to the inspiratory and/or expiratory port, to produce inspiratory resistive breathing (IRB) of 40% or 50% Pi/Pi,max (40% and 50% IRB), expiratory resistive breathing (ERB) of 60% Pe/Pe,max (60% ERB) or combined resistive breathing (CRB) of both 40% Pi/Pi,max and 60% Pe/Pe,max (40%/60% CRB). Tiotropium aerosol was inhaled prior to RB. After 6 h of RB, mechanical parameters of the respiratory system were measured and bronchoalveolar lavage (BAL) was performed. IL-1β and IL-6 protein levels were measured in lung tissue. Lung injury was estimated histologically.ResultsIn all, 40% and 50% IRB increased macrophage and neutrophil counts in BAL and raised IL-1β and IL-6 lung levels, tissue elasticity, BAL total protein levels and lung injury score. Tiotropium attenuated BAL neutrophil number, IL-1β, IL-6 levels and lung injury score increase at both 40% and 50% IRB. The increase in macrophage count and protein in BAL was only reversed at 40% IRB, while tissue elasticity was not affected. In all, 60% ERB raised BAL neutrophil count and total protein and reduced macrophage count. IL-1β and IL-6 levels and lung injury score were increased. Tiotropium attenuated these alterations, except for the decrease in macrophage count and the increase in total protein level. In all, 40%/60% CRB increased macrophage and neutrophil count in BAL, IL-1β and IL-6 levels, tissue elasticity, total protein in BAL and histological injury score. Tiotropium attenuated the aforementioned alterations.ConclusionTiotropium inhalation attenuates RB-induced pulmonary inflammation.

Interleukin-17A Is Associated With Alveolar Inflammation and Poor Outcomes in Acute Respiratory Distress Syndrome

Interleukin-17A is a proinflammatory cytokine known to play a role in host defense and pathologic inflammation in murine models of lung injury. The relationship between interleukin-17A and inflammation in human lung injury is unknown. Our primary objective was to determine whether interleukin-17A levels are associated with alveolar measures of inflammation and injury in patients with acute respiratory distress syndrome. Our secondary objective was to test whether interleukin-17A levels are associated with acute respiratory distress syndrome-related outcomes. Observational study. Six North American medical centers. We studied two groups of patients with acute respiratory distress syndrome: 1) patients previously enrolled in a placebo-controlled clinical trial of omega-3 fatty acids performed at five North American medical centers (n = 86, acute respiratory distress syndrome 1), and 2) patients with systemic inflammatory response syndrome admitted to an ICU who developed acute respiratory distress syndrome (n = 140, acute respiratory distress syndrome 2). In acute respiratory distress syndrome 1, we used paired serum and bronchoalveolar lavage fluid samples obtained within 48 hours of acute respiratory distress syndrome onset, whereas in acute respiratory distress syndrome 2, we used plasma obtained within the first 24 hours of ICU admission. None. We measured circulating interleukin-17A in acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2. We also measured interleukin-17A, neutrophil counts, and total protein in bronchoalveolar lavage fluid from acute respiratory distress syndrome 1. We found that bronchoalveolar lavage interleukin-17A was strongly associated with higher bronchoalveolar lavage percent neutrophils (p < 0.001) and bronchoalveolar lavage total protein (p < 0.01) in acute respiratory distress syndrome1. In both acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2, elevated interleukin-17A was associated with higher Sequential Organ Failure Assessment scores (p < 0.05). Elevated circulating and alveolar levels of interleukin-17A are associated with increased percentage of alveolar neutrophils, alveolar permeability, and organ dysfunction in acute respiratory distress syndrome.

Cigarette smokers have exaggerated alveolar barrier disruption in response to lipopolysaccharide inhalation.

Cigarette smoke exposure is associated with an increased risk of the acute respiratory distress syndrome (ARDS); however, the mechanisms underlying this relationship remain largely unknown. To assess pathways of lung injury and inflammation in smokers and non-smokers with and without lipopolysaccharide (LPS) inhalation using established biomarkers. We measured plasma and bronchoalveolar lavage (BAL) biomarkers of inflammation and lung injury in smokers and non-smokers in two distinct cohorts of healthy volunteers, one unstimulated (n=20) and one undergoing 50 μg LPS inhalation (n=30). After LPS inhalation, cigarette smokers had increased alveolar-capillary membrane permeability as measured by BAL total protein, compared with non-smokers (median 274 vs 208 μg/mL, p=0.04). Smokers had exaggerated inflammation compared with non-smokers, with increased BAL interleukin-1β (p=0.002), neutrophils (p=0.02), plasma interleukin-8 (p=0.003), and plasma matrix metalloproteinase-8 (p=0.006). Alveolar epithelial injury after LPS was more severe in smokers than non-smokers, with increased plasma (p=0.04) and decreased BAL (p=0.02) surfactant protein D. Finally, smokers had decreased BAL vascular endothelial growth factor (VEGF) (p<0.0001) with increased soluble VEGF receptor-1 (p=0.0001). Cigarette smoke exposure may predispose to ARDS through an abnormal response to a 'second hit,' with increased alveolar-capillary membrane permeability, exaggerated inflammation, increased epithelial injury and endothelial dysfunction. LPS inhalation may serve as a useful experimental model for evaluation of the acute pulmonary effects of existing and new tobacco products.

Amine modification of nonporous silica nanoparticles reduces inflammatory response following intratracheal instillation in murine lungs.

Amorphous silica nanoparticles (NPs) possess unique material properties that make them ideal for many different applications. However, the impact of these materials on human and environmental health needs to be established. We investigated nonporous silica NPs both bare and modified with amine functional groups (3-aminopropyltriethoxysilane (APTES)) in order to evaluate the effect of surface chemistry on biocompatibility. In vitro data showed there to be little to no cytotoxicity in a human lung cancer epithelial cell line (A549) for bare silica NPs and amine-functionalized NPs using doses based on both mass concentration (below 200μg/mL) and exposed total surface area (below 14m(2)/L). To assess lung inflammation, C57BL/6 mice were administered bare or amine-functionalized silica NPs via intra-tracheal instillation. Two doses (0.1 and 0.5mg NPs/mouse) were tested using the in vivo model. At the higher dose used, bare silica NPs elicited a significantly higher inflammatory response, as evidence by increased neutrophils and total protein in bronchoalveolar lavage (BAL) fluid compared to amine-functionalized NPs. From this study, we conclude that functionalization of nonporous silica NPs with APTES molecules reduces murine lung inflammation and improves the overall biocompatibility of the nanomaterial.

Inhalation toxicity of soman vapor in non-anesthetized rats: A preliminary assessment of inhaled bronchodilator or steroid therapy

Respiratory toxicity, injury and treatment following vapor inhalational exposure to the chemical warfare nerve agent (CWNA) soman (GD) were examined in non-anesthetized rats. This study exposed male Sprague–Dawley rats (250–300g) to 520, 560, 600, 825 or 1410mg×min/m3 of soman in a customized head-out inhalation system. Signs of CWNA-induced cholinergic crises were observed in all soman-exposed animals. The LCt50 of vaporized soman as determined by probit analysis was 593.1mg×min/m3. All animals exposed to 825 and 1410mg×min/m3 developed severe convulsions and died within 4–8min post-exposure. Edema measured by wet/dry weight ratio of the left lung lobe increased in a dose-dependent manner in all soman-exposed animals. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase (AChE) activities were inhibited dose-dependently in soman-exposed groups at 24h. A significant increase in total BAL protein was observed in soman-exposed animals at all doses. AChE activity was inhibited in lung and whole brain tissues in all soman-exposed animals. Histopathological analysis of the lungs of animals exposed to 600mg×min/m3 of soman revealed prominent morphological changes including alveolar histiocytosis, hemorrhage and inflammation consisting of neutrophilic exudate. Exposure of animals to 600mg×min/m3 of soman followed by treatment with two actuations for 10s of Combivent (21μg of ipratropium bromide and 120μg of albuterol sulfate) and Symbicort (80μg budesonide and 4.5μg formoterol) by inhalation into a modified metered dose inhaler (MDI) 10min post-exposure resulted in increased minute volume, but did not decrease mortality. These results indicate that inhalation exposure to soman vapor causes acute respiratory toxicity and injury in untreated, un-anesthetized rats and that inhalation treatment with Combivent or Symbicort did improve the respiratory outcomes, but did not influence lethality.

Chemokine Localization in Bronchial Angiogenesis

Angiogenesis in the lung involves the systemic bronchial vasculature and becomes prominent when chronic inflammation prevails. Mechanisms for neovascularization following pulmonary ischemia include growth factor transit from ischemic parenchyma to upstream bronchial arteries, inflammatory cell migration/recruitment through the perfusing artery, and paracrine effects of lung cells within the left bronchus, the niche where arteriogenesis takes place. We analyzed left lung bronchoalveolar lavage (BAL) fluid and left bronchus homogenates after left pulmonary artery ligation (LPAL) in rats, immediately after the onset of ischemia (0 h), 6 h and 24 h later. Additionally, we tested the effectiveness of dexamethasone on decreasing inflammation (0–24 h LPAL) and angiogenesis at early (3 d LPAL; bronchial endothelial proliferation) and late (14 d LPAL; blood flow) stages. After LPAL (6 h), BAL protein, total inflammatory cells, macrophages, and polymorphonuclear cells increased significantly. In parallel, pro-angiogenic CXC chemokines increased in BAL and the left main-stem bronchus (CXCL1) or only within the bronchus (CXCL2). Dexamethasone treatment reduced total BAL protein, inflammatory cells (total and polymorphonuclear cells), and CXCL1 but not CXCL2 in BAL. By contrast, no decrease was seen in either chemokine within the bronchial tissue, in proliferating bronchial endothelial cells, or in systemic perfusion of the left lung. Our results confirm the presence of CXC chemokines within BAL fluid as well as within the left mainstem bronchus. Despite significant reduction in lung injury and inflammation with dexamethasone treatment, chemokine expression within the bronchial tissue as well as angiogenesis were not affected. Our results suggest that early changes within the bronchial niche contribute to subsequent neovascularization during pulmonary ischemia.

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis

Autotaxin Emerges as a Therapeutic Target for Idiopathic Pulmonary Fibrosis