Critical role of mitochondrial oxidative stress in acid aspiration induced ALI in mice.

Acute lung injury (ALI) is a pulmonary inflammatory disorder which causes significant mortality in critically ill patients. Intracellular oxidative stress has been considered to be the major component in the pathogenesis of ALI but exact source of intracellular ROS is not clearly known. The present study has been designed to elucidate the role of NADPH oxidase system and/or mitochondrial oxidative stress and its downstream pathway NLRP3 inflammasomes in mouse model of acid aspiration mediated ALI. Our data showed that acid aspiration induced lung inflammation was associated with enhanced oxidative stress as evident by data on MDA levels, nitrite levels and redox imbalance. Further acid aspiration resulted in elevation of expression of NADPH oxidase subunits (gp91 phox/p22 phox/p67 phox) as well as mitochondrial oxidative stress as reflected by aconitase activity, mitochondrial ROS levels. Interestingly, NADPH oxidase inhibitor, apocynin did not alter lung inflammation upon HCl instillation. Conversely, mitochondrial antioxidant mito-tempo resulted in significant amelioration of lung inflammation as indicated by suppression of pulmonary neutrophils and inflammatory cytokines namely IL-1ß, TNF-α, IL-6 in BALF. Analysis of mitochondrial enzymes aconitase/mitochondrial ROS/Mn-SOD confirmed that reduction in lung inflammation by mito-tempo was associated with normalization of oxidative stress in mitochondria. Further, mito-tempo administration blunted phosphorylation of p65- NF-κB at Ser 536. Finally, mito-tempo downregulated HCl-induced NF-κB-dependent pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6) drastically at mRNA levels. Overall, our data support that mitochondrial oxidative stress is crucial in modulating the HCl induced lung inflammation and identifies mitochondrial-targeted antioxidant as a potential therapeutic agent.

Hypoxia-Inducible Factor (HIF)-1α Promotes Inflammation and Injury Following Aspiration-Induced Lung Injury in Mice.

Acid aspiration-induced lung injury is a common disease in the intensive care unit (ICU) and acute respiratory distress syndrome (ARDS). Hypoxia-inducible factor (HIF)-1α is a major transcription factor responsible for regulating the cellular response to changes in oxygen tension. A clear understanding of the function of HIF-1α in lung inflammatory response is currently lacking. Here, we sought to determine the role of HIF-1α in type 2 alveolar epithelial cells (AEC) in the generation of the acute inflammatory response following gastric aspiration (GA). GA led to profound hypoxia at very early time points following GA. This correlated to a robust increase in HIF-1α, tissue albumin and pro-inflammatory mediators following GA in AECs. The extent of lung injury and the release of pro/anti-inflammatory cytokines were significantly reduced in HIF-1α (-/-) mice. Finally, we report that HIF-1α upregulation of the acute inflammatory response is dependent on NF-κB following GA.

Elastin degradation products in acute lung injury induced by gastric contents aspiration.

BACKGROUND: Gastric contents aspiration is a high-risk condition for acute lung injury (ALI). Consequences range from subclinical pneumonitis to respiratory failure, depending on the volume of aspirate. A large increment in inflammatory cells, an important source of elastase, potentially capable of damaging lung tissue, has been described in experimental models of aspiration. We hypothesized that in early stages of aspiration-induced ALI, there is proteolytic degradation of elastin, preceding collagen deposition. Our aim was to evaluate whether after a single orotracheal instillation of gastric fluid, there is evidence of elastin degradation. METHODS: Anesthesized Sprague-Dawley rats received a single orotracheal instillation of gastric fluid and were euthanized 4, 12 and 24 h and at day 4 after instillation (n = 6/group). We used immunodetection of soluble elastin in lung tissue and BALF and correlated BALF levels of elastin degradation products with markers of ALI. We investigated possible factors involved in elastin degradation and evaluated whether a similar pattern of elastin degradation can be found in BALF samples of patients with interstitial lung diseases known to have aspirated. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. RESULTS: We found evidence of early proteolytic degradation of lung elastin. Elastin degradation products are detected both in lung tissue and BALF in the first 24 h and are significantly reduced at day 4. They correlate significantly with ALI markers, particularly PMN cell count, are independent of acidity and have a similar molecular weight as those obtained using pancreatic elastase. Evaluation of BALF from patients revealed the presence of elastin degradation products not present in controls that are similar to those found in BALF of rats treated with gastric fluid. CONCLUSIONS: A single instillation of gastric fluid into the lungs induces early proteolytic degradation of elastin, in relation to the magnitude of alveolar-capillary barrier derangement. PMN-derived proteases released during ALI are mostly responsible for this damage. BALF from patients showed elastin degradation products similar to those found in rats treated with gastric fluid. Long-lasting effects on lung elastic properties could be expected under conditions of repeated instillations of gastric fluid in experimental animals or repeated aspiration events in humans.

Pepsin: biomarker, mediator, and therapeutic target for reflux and aspiration.

Extra-esophageal reflux is suspected to cause a wide range of clinical symptoms in the upper airways. Diagnosis and treatment has focused on acid, but realization of the role of nonacid reflux has resulted in research investigating the use of pepsin as a biomarker for gastric reflux and aspiration. Pepsin analysis can complement the use of questionnaires and office-based diagnosis and lessen the dependency on invasive and expensive diagnostic tests. Furthermore, pepsin as a first-line diagnostic biomarker has been shown to improve the accuracy of reflux diagnosis. In addition to its use as a diagnostic biomarker, pepsin has been shown to cause inflammation independent of the pH of the refluxate and thus despite acid suppression therapy. Research is ongoing to develop new therapies for airway reflux that specifically target pepsin.

Changes in the pattern of fibrosis in the rat lung with repetitive orotracheal instillations of gastric contents: evidence of persistent collagen accumulation.

Recurrent aspiration of gastric contents has been associated with several interstitial lung diseases. Despite this association, the pathogenic role of aspiration in these diseases has been poorly studied and little is known about extracellular matrix (ECM) changes in animal models of repetitive events of aspiration. Our aim was to study the repair phase of lung injury induced by each of several instillations of gastric fluid in Sprague-Dawley rats to evaluate changes in ECM and their reversibility. Anesthetized animals received weekly orotracheal instillations of gastric fluid for 1, 2, 3, and 4 wk and were euthanized at day 7 after last instillation. For reversibility studies, another group received 7 weekly instillations and was euthanized at day 7 or 60 after last instillation. Biochemical and histological measurements were used to evaluate ECM changes. Lung hydroxyproline content increased progressively and hematoxylin and eosin, Masson's trichrome, and alpha-SMA stains showed that after a single instillation, intra-alveolar fibrosis predominated, whereas with repetitive instillations this fibrosis pattern became less prominent and interstitial fibrosis progressively became evident. Both type I and III collagen increased in intra-alveolar and interstitial fibrosis. Imbalance between matrix metalloproteinase-2 (MMP-2) activity and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression was observed, favoring either collagen degradation or accumulation depending on the number of instillations. Caspase-3 activation was also dose dependent. ECM changes were partially reversible at long-term evaluation, since Masson bodies, granulomas, and foreign body giant cells disappeared, whereas interstitial collagen accumulated. In conclusion, repetitive lung instillations of gastric fluid induce progressive fibrotic changes in rat lung ECM that persist at long-term evaluation.

A pilot randomized clinical trial assessing the effect of cricoid pressure on risk of aspiration.

INTRODUCTION: Patients at risk for microaspiration during elective intubation often receive cricoid pressure in the hopes of mitigating such risk. However, there is scarce evidence to either support or reject this practice. The objective of this investigation was to assess the effect of cricoid pressure on microaspiration and to inform the potential feasibility of conducting a larger, more definitive clinical trial. METHODS: This was a pilot randomized clinical trial set in the operating rooms of a tertiary referral hospital between August and October of 2014. Patients with risk factors for microaspiration (obesity, gastroesophageal reflux disease, or diabetes) were enrolled. The patients were randomized to either cricoid pressure or no cricoid pressure during induction of anesthesia with endotracheal intubation. Immediately after intubation, a sample of lower airway secretions was collected and analyzed for pepsin A. MAIN RESULTS: A total of 95 patients were evaluated, randomized and completed the study protocol. 46 were randomized to cricoid pressure and 49 to no cricoid pressure. Seven patients crossed-over treatment arms. A total of 18 (19.6%) patients met the pre-defined criteria for microaspiration. In both the intention-to-treat and per-protocol analyses, there were no statistically significant differences in the rate of microaspiration [OR (95% CI)] = 1.39 (0.49-3.92) and 1.30 (0.44-3.86), respectively. CONCLUSIONS: Utilizing pepsin A as a biomarker of aspiration, this pilot clinical trial did not find evidence for a reduced rate of aspiration or adverse clinical events with the administration of cricoid pressure during elective endotracheal intubation of patients with risk factors for microaspiration.

Synergy between acid and endotoxin in an experimental model of aspiration-related lung injury progression.

Aspiration is a common cause of lung injury, but it is unclear why some cases are self-limited while others progress to acute respiratory distress syndrome (ARDS). Sporadic exposure to more than one insult could account for this variable progression. We investigated whether synergy between airway acid and endotoxin (LPS) amplifies injury severity in mice and whether LPS levels in human patients could corroborate our experimental findings. C57BL/6 mice aspirated acid (pH 1.3) or normal saline (NS), followed by LPS aerosol or nothing. Bronchoalveolar lavage fluid (BALF) was obtained 2 to 49 h later. Mice were injected with FITC-dextran 25 h after aspiration and connected to a ventilator, and lung elastance (H) measured periodically following deep inflation (DI). Endotracheal and gastric aspirates were also collected from patients in the intensive care unit and assayed for pH and LPS. Lung instability (ΔH following DI) and pressure-volume hysteresis in acid- or LPS-exposed mice was greater than in controls but markedly greater in the combined acid/LPS group. BALF neutrophils, cytokines, protein, and FITC-dextran in the acid/LPS mice were geometrically higher than all other groups. BALF from acid-only mice markedly amplified LPS-induced TNF-α production in cultured macrophages. Human subjects had variable endotracheal LPS levels with the highest burden in those at higher risk of aspiration. Acid aspiration amplifies LPS signaling in mice to disrupt barrier function and lung mechanics in synergy. High variation in airway LPS and greater airway LPS burden in patients at higher risk of aspiration could help explain the sporadic progression of aspiration to ARDS.

Anti-infective control in human bronchiolar epithelial cells by mucin phenotypic changes following uptake of N-acetyl-L-cysteine.

PURPOSE: Aspiration pneumonia is infection of the respiratory tract resulting from accumulation of sputum in the larynx. N-acetyl-L-cysteine (NAC) might regulate mucin (MUC) expression and activate inherent anti-infective system in bronchiolar epithelial cells after cellular uptake, and therefore, serve as the preventative agent for chronic lung disease including aspiration pneumonia. The purpose of this in vitro study was to evaluate the effect of uptake of NAC by human bronchiolar epithelial cells on bacterial infection and regulations of mucin expression in association with cellular redox status under co-culture with a representative pathogen for hospital- and community-acquired pneumonia, Streptococcus pneumoniae. MATERIALS AND METHODS: Human bronchiolar epithelial cells preincubated with or without 20 mM NAC for 3 h were co-cultured with or without bacteria for 8 h and evaluated with respect to cellular redox balance, expressions of various types of MUC, proinflammatory cytokines and mediators, and bacterial infection state by biochemical, genetic, and immunofluorescent assays. RESULTS: Markedly increased intracellular reactive oxygen species and oxidized glutathione levels plus increased release and expression of proinflammatory cytokines and mediators were observed in cells co-cultured with bacteria. These bacteria-induced cellular redox disturbance and proinflammatory events were prevented and alleviated by pretreatment with NAC. Cells co-cultured with bacteria did not increase expression of anti-infective membranous MUC4 but exhibited increases in gel-forming MUC5AC expression and bacterial infection. However, NAC-pretreated cells avoided bacterial infection along with enhancement of MUC4, but not MUC5AC, expression. CONCLUSION: Uptake of NAC by human bronchiolar epithelial cells prevented bacterial infection and upregulated membranous, but not gel-forming, MUC expression along with the increase in intracellular antioxidant level under co-culture conditions with S. pneumoniae.

In vivo compartmental analysis of leukocytes in mouse lungs.

The lung has a unique structure consisting of three functionally different compartments (alveolar, interstitial, and vascular) situated in an extreme proximity. Current methods to localize lung leukocytes using bronchoalveolar lavage and/or lung perfusion have significant limitations for determination of location and phenotype of leukocytes. Here we present a novel method using in vivo antibody labeling to enable accurate compartmental localization/quantification and phenotyping of mouse lung leukocytes. Anesthetized C57BL/6 mice received combined in vivo intravenous and intratracheal labeling with fluorophore-conjugated anti-CD45 antibodies, and lung single-cell suspensions were analyzed by flow cytometry. The combined in vivo intravenous and intratracheal CD45 labeling enabled robust separation of the alveolar, interstitial, and vascular compartments of the lung. In naive mice, the alveolar compartment consisted predominantly of resident alveolar macrophages. The interstitial compartment, gated by events negative for both intratracheal and intravenous CD45 staining, showed two conventional dendritic cell populations, as well as a Ly6C(lo) monocyte population. Expression levels of MHCII on these interstitial monocytes were much higher than on the vascular Ly6C(lo) monocyte populations. In mice exposed to acid aspiration-induced lung injury, this protocol also clearly distinguished the three lung compartments showing the dynamic trafficking of neutrophils and exudative monocytes across the lung compartments during inflammation and resolution. This simple in vivo dual-labeling technique substantially increases the accuracy and depth of lung flow cytometric analysis, facilitates a more comprehensive examination of lung leukocyte pools, and enables the investigation of previously poorly defined "interstitial" leukocyte populations during models of inflammatory lung diseases.

Gastric reflux: association with aspiration and oral secretion pH as marker of reflux: a descriptive correlational study.

BACKGROUND: Gastric reflux leading to pulmonary aspiration is a frequent event in mechanically ventilated, gastric-fed patients, which can lead to ventilator-associated complications and pneumonia. OBJECTIVES: The objectives of this study were to determine the association between gastric reflux and aspiration using the presence of pepsin in oral or tracheal secretions as a marker of reflux or aspiration and to determine the association between the pH (range, 0-14) and the presence of pepsin in oral secretions. METHODS: A descriptive correlational study was conducted in mechanically ventilated surgical or medical patients receiving gastric tube feedings. Oral secretions were suctioned hourly and tracheal secretions every 2 to 3 hours for 12-hour periods over 1 to 2 days in 15 patients. RESULTS: There were 142 paired samples of oral tracheal secretions. A majority of samples (60%) had the same results, with 32% both pepsin-positive and 27% both pepsin-negative. The range of pH measurements was 4 to 8, with a mean of 6.3 ± 0.05. Ninety oral specimens had a pH of 4 to 6. Forty-seven of the oral specimens with pH measures between 4 and 6 (52%) were pepsin-positive. The correlation of pH percent pepsin-positive oral secretions was not significant. CONCLUSION: Aspiration events were more frequent than reflux events. Measurement of actual pepsin concentration to detect new reflux and aspiration events is recommended in future studies. Bedside pH measures of oral secretions are not a valid marker of gastric reflux.

Endothelial Semaphorin 7A promotes inflammation in seawater aspiration-induced acute lung injury.

Inflammation is involved in the pathogenesis of seawater aspiration-induced acute lung injury (ALI). Although several studies have shown that Semaphorin 7A (SEMA7A) promotes inflammation, there are limited reports regarding immunological function of SEMA7A in seawater aspiration-induced ALI. Therefore, we investigated the role of SEMA7A during seawater aspiration-induced ALI. Male Sprague-Dawley rats were underwent seawater instillation. Then, lung samples were collected at an indicated time for analysis. In addition, rat pulmonary microvascular endothelial cells (RPMVECs) were cultured and then stimulated with 25% seawater for indicated time point. After these treatments, cells samples were collected for analysis. In vivo, seawater instillation induced lung histopathologic changes, pro-inflammation cytokines release and increased expression of SEMA7A. In vitro, seawater stimulation led to pro-inflammation cytokine release, cytoskeleton remodeling and increased monolayer permeability in pulmonary microvascular endothelial cells. In addition, knockdown of hypoxia-inducible factor (HIF)-1α inhibited the seawater induced increase expression of SEMA7A. Meanwhile, knockdown of SEMA7A by specific siRNA inhibited the seawater induced aberrant inflammation, endothelial cytoskeleton remodeling and endothelial permeability. These results suggest that SEMA7A is critical in the development of lung inflammation and pulmonary edema in seawater aspiration-induced ALI, and may be a therapeutic target for this disease.

TRPV4: an exciting new target to promote alveolocapillary barrier function.

Transient receptor potential (TRP) channels are emerging as important players and drug targets in respiratory disease. Amongst the vanilloid-type TRP channels (which includes the six members of the TRPV family), target diseases include cough, asthma, cancer, and more recently, pulmonary edema associated with acute respiratory distress syndrome. Here, we critically evaluate a recent report that addresses TRPV4 as a candidate target for the management of acute lung injury that develops as a consequence of aspiration of gastric contents, or acute chlorine gas exposure. By use of two new TRPV4 inhibitors (GSK2220691 or GSK2337429A) and a trpv4(-/-) mouse strain, TRPV4 was implicated as a key mediator of pulmonary inflammation after direct chemical insult. Additionally, applied therapeutically, TRPV4 inhibitors exhibited vasculoprotective effects after chlorine gas exposure, inhibiting vascular leakage, and improving blood oxygenation. These observations underscore TRPV4 channels as candidate therapeutic targets in the management of lung injury, with the added need to balance these against the potential drawbacks of TRPV4 inhibition, such as the danger of limiting the immune response in settings of pathogen-provoked injury.

Dietary advanced glycation end-products, its pulmonary receptor, and high mobility group box 1 in aspiration lung injury.

BACKGROUND: Gastric aspiration is a significant cause of acute lung injury and acute respiratory distress syndrome. Environmental risk factors, such as a diet high in proinflammatory advanced glycation end-products (AGEs), may render some patients more susceptible to lung injury after aspiration. We hypothesized that high dietary AGEs increase its pulmonary receptor, RAGE, producing an amplified pulmonary inflammatory response in the presence of high mobility group box 1 (HMGB1), a RAGE ligand and an endogenous signal of epithelial cell injury after aspiration. MATERIALS AND METHODS: CD-1 mice were fed either a low AGE or high AGE diet for 4 wk. After aspiration injury with acidified small gastric particles, bronchoalveolar lavage and whole-lung tissue samples were collected at 5 min, 1 h, 5 h, and 24 h after injury. RAGE, soluble RAGE (sRAGE), HMGB1, cytokine and chemokine concentrations, albumin levels, neutrophil influx, and lung myeloperoxidase activity were measured. RESULTS: We observed that high AGE-fed mice exhibited greater pulmonary RAGE levels before aspiration and increased bronchoalveolar lavage sRAGE levels after aspiration compared with low AGE-fed mice. Lavage HMGB1 levels rose immediately after aspiration, peaking at 1 h, and strongly correlated with sRAGE levels in both dietary groups. High AGE-fed mice demonstrated higher cytokine and chemokine levels with increased pulmonary myeloperoxidase activity over 24 h versus low AGE-fed mice. CONCLUSIONS: This study indicates that high dietary AGEs can increase pulmonary RAGE, augmenting the inflammatory response to aspiration in the presence of endogenous damage signals such as HMGB1.

Comparison of serum amyloid A and C-reactive protein as diagnostic markers of systemic inflammation in dogs.

The diagnostic performance of canine serum amyloid A (SAA) was compared with that of C-reactive protein (CRP) in the detection of systemic inflammation in dogs. Sera from 500 dogs were retrospectively included in the study. C-reactive protein and SAA were measured using validated automated assays. The overlap performance, clinical decision limits, overall diagnostic performance, correlations, and agreement in the clinical classification between these 2 diagnostic markers were compared. Significantly higher concentrations of both proteins were detected in dogs with systemic inflammation (SAA range: 48.75 to > 2700 mg/L; CRP range: 0.4 to 907.4 mg/L) compared to dogs without systemic inflammation (SAA range: 1.06 to 56.4 mg/L; CRP range: 0.07 to 24.7 mg/L). Both proteins were shown to be sensitive and specific markers of systemic inflammation in dogs. Significant correlations and excellent diagnostic agreement were observed between the 2 markers. However, SAA showed a wider range of concentrations and a significantly superior overall diagnostic performance compared with CRP.

Comparaison de la protéine amyloïde sérique A et de la protéine C réactive comme marqueurs diagnostiques de l'inflammation systémique chez les chiens. La performance diagnostique de l'amyloïde sérique canine A (SAA) a été comparée à celle de la protéine C réactive (PCR) dans la détection de l'inflammation systémique chez les chiens. Le sérum de 500 chiens a été inclus rétrospectivement dans l'étude. La protéine C réactive et la SAA ont été mesurées en utilisant des bioanalyses automatisées validées. La performance de chevauchement, les limites de décision cliniques, la performance diagnostique globale, les corrélations et la concordance dans la classification clinique entre ces 2 marqueurs diagnostiques ont été comparés. Des concentrations significativement supérieures des deux protéines ont été détectées chez les chiens avec une inflammation systémique (plage de la SAA : de 48,75 à > 2700 mg/L; plage de la PCR : de 0,4 à 907,4 mg/L) comparativement aux chiens sans inflammation systémique (plage de la SAA : de 1,06 à 56,4 mg/L; plage de la PCR : de 0,07 à 24,7 mg/L). Il a été démontré que les deux protéines étaient sensibles et des marqueurs spécifiques de l'inflammation systémique chez les chiens. Des corrélations significatives et une concordance diagnostique excellente ont été observées entre les deux marqueurs. Cependant, la SAA a indiqué un écart plus vaste pour les concentrations et une performance diagnostique significativement supérieure comparativement à la PCR.(Traduit par Isabelle Vallières).

Pepsin concentrations are elevated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis after lung transplantation.

BACKGROUND: Aspiration of gastroesophageal refluxate has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and the progression of bronchiolitis obliterans syndrome after lung transplantation. The goals of the present study were to identify lung transplant patients at the greatest risk of aspiration and to investigate the causative factors. MATERIALS AND METHODS: From September 2009 to November 2011, 252 bronchoalveolar lavage fluid (BALF) samples were collected from 100 lung transplant patients. The BALF pepsin concentrations and the results of transbronchial biopsy, esophageal function testing, barium swallow, and gastric emptying scan were compared among those with the most common end-stage lung diseases requiring lung transplantation: IPF, chronic obstructive pulmonary disease, cystic fibrosis, and α1-antitrypsin deficiency. RESULTS: Patients with IPF had higher BALF pepsin concentrations and a greater frequency of acute rejection than those with α1-antitrypsin deficiency, cystic fibrosis, or chronic obstructive pulmonary disease (P = 0.037). Moreover, the BALF pepsin concentrations correlated negatively with a lower esophageal sphincter pressure and distal esophageal amplitude; negatively with distal esophageal amplitude and positively with total esophageal acid time, longest reflux episode, and DeMeester score in those with chronic obstructive pulmonary disease; and negatively with the upright acid clearance time in those with IPF. CONCLUSIONS: Our results suggest that patients with IPF after lung transplantation are at increased risk of aspiration and a greater frequency of acute rejection episodes, and that the risk factors for aspiration might be different among those with the most common end-stage lung diseases who have undergone lung transplantation. These results support the role of evaluating the BALF for markers of aspiration in assessing lung transplant patients as candidates for antireflux surgery.

Differential mouse pulmonary dose and time course responses to titanium dioxide nanospheres and nanobelts.

Three anatase titanium dioxide (TiO(2)) nanoparticles (NPs) were prepared; nanospheres (NSs), short nanobelts (NB1), and long nanobelts (NB2). These NPs were used to investigate the effect of NP shape and length on lung toxicity. Mice were exposed (0-30 µg per mouse) by pharyngeal aspiration and pulmonary toxicity was assessed over a 112-day time course. Whole lung lavage data indicated that NB1- and NB2-exposed mice, but not NS-exposed mice, had significant dose- and time-dependent pulmonary inflammation and damage. Histopathological analyses at 112 days postexposure determined no interstitial fibrosis in any NS-exposed mice, an increased incidence in 30 µg NB1-exposed mice, and significant interstitial fibrosis in 30 µg NB2-exposed mice. At 112 days postexposure, lung burden of NS was decreased by 96.4% and NB2 by 80.5% from initial deposition levels. At 112 days postexposure, enhanced dark field microscopy determined that alveolar macro- phages were the dominant deposition site, but a fraction of NB1 and NB2 was observed in the alveolar interstitial spaces. For the 30 µg exposure groups at 112 days postexposure, confocal micro- scopy and immunofluorescent staining demonstrated that retained NB2 but not NS were present in the interstitium subjacent to the terminal bronchiole near the normal location of the smallest lymphatic capillaries in the lung. These lymphatic capillaries play a critical role in particle clearance, and the accumulation of NB2, but not NS, suggests possible impaired lymphatic clearance by the high aspect ratio particles. In summary, our data indicate that TiO(2) NP shape alters pulmonary responses, with severity of responses being ranked as NS < NB1 < NB2.

Effect of high advanced glycation end-product diet on pulmonary inflammatory response and pulmonary function following gastric aspiration.

It is not clear why some patients with aspiration advance to acute lung injury or acute respiratory distress syndrome, whereas others do not. The Western diet is high in advanced glycation end-products (AGEs), which have been found to be proinflammatory. We hypothesize that dietary AGEs exaggerate the pulmonary inflammatory response following gastric aspiration. CD-1 mice were randomized to receive either a low-AGE (LAGE) or a high-AGE (HAGE) diet for 4 weeks. Five hours after intratracheal instillation of acidified small gastric particles, pulmonary function was determined. Polymorphonuclear neutrophil counts, albumin, cytokine/chemokine, and tumor necrosis factor soluble receptor II concentrations in the bronchoalveolar lavage and lung myeloperoxidase activity were measured. Compared with LAGE-fed animals, those fed a HAGE diet had increased lung tissue resistance (P = 0.017), bronchoalveolar lavage albumin concentration (P < 0.05), pulmonary polymorphonuclear neutrophil counts (P = 0.0045), and lung myeloperoxidase activity (P = 0.002) following aspiration. In addition, the plasma levels of tumor necrosis factor soluble receptor II were significantly elevated (P < 0.05), whereas paradoxically levels of keratinocyte chemoattractant and monocyte chemoattractant protein 1 were decreased in mice with HAGE diet. In conclusion, a diet high in AGEs exacerbates acute lung injury following gastric aspiration as evidenced by increases in neutrophil infiltration, airway albumin leakage, and decreased pulmonary compliance. This is the first evidence implicating exacerbation of acute inflammatory lung injury by dietary AGEs. Targeting AGEs in the circulatory system may offer a therapeutic strategy for limiting lung injury following gastric aspiration.

The effect of curcumin on lung injuries in a rat model induced by aspirating gastrointestinal decontamination agents.

BACKGROUND: Aspiration is one of the most feared complications of gastrointestinal decontamination procedures with nonabsorbed polyethylene glycol (PEG) solution and activated charcoal (AC). We aimed to investigate the protective effects of curcumin (CUR) on lung injury in rats induced by aspiration of these agents. METHODS: Experimental rats were divided randomly into 6 groups (n = 7): a saline-aspirated control (group I), sterile saline aspirated with CUR treatment (group II), PEG aspirated (group III), PEG aspirated with CUR treatment (group IV), AC aspirated (group V), and AC aspirated with CUR treatment (group VI). After aspiration, treatment groups II, IV, and VI were given 150 mg/kg CUR intraperitoneally once a day for 7 days. After 7 days, the rats were humanely killed, and both the lungs and serum specimens from all groups were evaluated histopathologically, immunohistochemically, and biochemically. RESULTS: Aspiration of gastrointestinal decontamination agents produced histopathologic changes, elevated levels of malondialdehyde and surfactant protein D, reduced levels of antioxidant enzymes, and increased expression of inflammatory cytokines interleukin-1ß and tumor necrosis factor α. Curcumin treatments effectively attenuated the rats' pulmonary inflammation responses (as shown by reduced alveolar damage), decreased serum malondialdehyde and surfactant protein D levels, and inhibited the expressions of tumor necrosis factor α and interleukin-1ß. CONCLUSIONS: Because of its anti-inflammatory effects, CUR treatment may have preventive effects on lung injuries induced by aspirating gastrointestinal decontamination agents.

Acid-induced acute lung injury in mice is associated with P44/42 and c-Jun N-terminal kinase activation and requires the function of tumor necrosis factor α receptor I.

Aspiration of hydrochloric acid (HCl)-containing gastric juice leads to acute lung injury (ALI) and hypoxemic respiratory failure due to an exuberant inflammatory response associated with pulmonary edema from increased vascular and epithelial permeability. The aim of this study was to determine the role and signaling mechanisms of tumor necrosis factor α (TNF-α) in experimental ALI from HCl aspiration using a combination of genetic animal models and pharmacologic inhibition strategies. To this end, HCl was instilled intratracheally to mice, followed by respiratory system elastance measurement, bronchoalveolar lavage, and lung tissue harvesting 24 h after injection. Hydrochloric acid instillation induced an inflammatory response in the lungs of wild-type mice, evidenced as increased bronchoalveolar lavage total cells, neutrophils, and total protein; histologic lung injury score; and respiratory system elastance, whereas TNF-α receptor I mRNA levels were maintained. These alterations could be prevented by pretreatment with etanercept or genetic deletion of the 55-kd TNF-α receptor I, but not by deletion of the TNF-α gene. Hydrochloric acid induced a 6-fold increase in apoptotic, caspase 3-positive cells in lung sections from wild-type mice, which was abrogated in mice lacking TNF-α receptor I. In immunoblotting and immunohistochemistry studies, HCl stimulated signaling via p44/42 and c-Jun N-terminal kinase, which was blocked in TNF-α receptor I knockout mice. In conclusion, ALI induced by HCl requires TNF-α receptor I function and associates with activation of downstream proinflammatory signaling pathways p44/42 and c-Jun N-terminal kinase.

Acid contact in the rodent pulmonary alveolus causes proinflammatory signaling by membrane pore formation.

Although gastric acid aspiration causes rapid lung inflammation and acute lung injury, the initiating mechanisms are not known. To determine alveolar epithelial responses to acid, we viewed live alveoli of the isolated lung by fluorescence microscopy, then we microinjected the alveoli with HCl at pH of 1.5. The microinjection caused an immediate but transient formation of molecule-scale pores in the apical alveolar membrane, resulting in loss of cytosolic dye. However, the membrane rapidly resealed. There was no cell damage and no further dye loss despite continuous HCl injection. Concomitantly, reactive oxygen species (ROS) increased in the adjacent perialveolar microvascular endothelium in a Ca(2+)-dependent manner. By contrast, ROS did not increase in wild-type mice in which we gave intra-alveolar injections of polyethylene glycol (PEG)-catalase, in mice overexpressing alveolar catalase, or in mice lacking functional NADPH oxidase (Nox2). Together, our findings indicate the presence of an unusual proinflammatory mechanism in which alveolar contact with acid caused membrane pore formation. The effect, although transient, was nevertheless sufficient to induce Ca(2+) entry and Nox2-dependent H(2)O(2) release from the alveolar epithelium. These responses identify alveolar H(2)O(2) release as the signaling mechanism responsible for lung inflammation induced by acid and suggest that intra-alveolar PEG-catalase might be therapeutic in acid-induced lung injury.