Total Protein In Bronchoalveolar Lavage Fluid Research Articles

Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials

BackgroundA standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide.MethodsRats were exposed to test material aerosols (ranging from 0.5 to 50 mg/m3) for five consecutive days with 14- or 21-day post-exposure observation. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined. Pulmonary deposition and clearance and test material translocation into extra-pulmonary organs were assessed.ResultsInhaled nanomaterials were found in the lung, in alveolar macrophages, and in the draining lymph nodes. Polyacrylate-coated silica was also found in the spleen, and both zinc oxides elicited olfactory epithelium necrosis. None of the other nanomaterials was recorded in extra-pulmonary organs. Eight nanomaterials did not elicit pulmonary effects, and their no observed adverse effect concentrations (NOAECs) were at least 10 mg/m3. Five materials (coated nano-TiO2, both ZnO, both CeO2) evoked concentration-dependent transient pulmonary inflammation. Most effects were at least partially reversible during the post-exposure period.Based on the NOAECs that were derived from quantitative parameters, with BALF polymorphonuclear (PMN) neutrophil counts and total protein concentration being most sensitive, or from the severity of histopathological findings, the materials were ranked by increasing toxic potency into 3 grades: lower toxic potency: BaSO4; SiO2.acrylate (by local NOAEC); SiO2.PEG; SiO2.phosphate; SiO2.amino; nano-ZrO2; ZrO2.TODA; ZrO2.acrylate; medium toxic potency: SiO2.naked; higher toxic potency: coated nano-TiO2; nano-CeO2; Al-doped nano-CeO2; micron-scale ZnO; coated nano-ZnO (and SiO2.acrylate by systemic no observed effect concentration (NOEC)).ConclusionThe STIS revealed the type of effects of 13 nanomaterials, and micron-scale ZnO, information on their toxic potency, and the location and reversibility of effects. Assessment of lung burden and material translocation provided preliminary biokinetic information. Based upon the study results, the STIS protocol was re-assessed and preliminary suggestions regarding the grouping of nanomaterials for safety assessment were spelled out.

Airway Pressure Release Ventilation Prevents Ventilator-Induced Lung Injury in Normal Lungs

Up to 25% of patients with normal lungs develop acute lung injury (ALI) secondary to mechanical ventilation, with 60% to 80% progressing to acute respiratory distress syndrome (ARDS). Once established, ARDS is treated with mechanical ventilation that can paradoxically elevate mortality. A ventilation strategy that reduces the incidence of ARDS could change the clinical paradigm from treatment to prevention. To demonstrate that (1) mechanical ventilation with tidal volume (VT) and positive end-expiratory pressure (PEEP) settings used routinely on surgery patients causes ALI/ARDS in normal rats and (2) preemptive application of airway pressure release ventilation (APRV) blocks drivers of lung injury (ie, surfactant deactivation and alveolar edema) and prevents ARDS. Rats were anesthetized and tracheostomy was performed at State University of New York Upstate Medical University. Arterial and venous lines, a peritoneal catheter, and a rectal temperature probe were inserted. Animals were randomized into 3 groups and followed up for 6 hours: spontaneous breathing ventilation (SBV, n = 5), continuous mandatory ventilation (CMV, n = 6), and APRV (n = 5). Rats in the CMV group were ventilated with Vt of 10 cc/kg and PEEP of 0.5 cm H2O. Airway pressure release ventilation was set with a P(High) of 15 to 20 cm H2O; P(Low) was set at 0 cm H2O. Time at P(High) (T(High)) was 1.3 to 1.5 seconds and a T(Low) was set to terminate at 75% of the peak expiratory flow rate (0.11-0.14 seconds), creating a minimum 90% cycle time spent at P(High). Bronchoalveolar lavage fluid and lungs were harvested for histopathologic analysis at necropsy. Acute lung injury/ARDS developed in the CMV group (mean [SE] PaO2/FiO2 ratio, 242.96 [24.82]) and was prevented with preemptive APRV (mean [SE] PaO2/FIO2 ratio, 478.00 [41.38]; P < .05). Airway pressure release ventilation also significantly reduced histopathologic changes and bronchoalveolar lavage fluid total protein (endothelial permeability) and preserved surfactant proteins A and B concentrations as compared with the CMV group. Continuous mandatory ventilation in normal rats for 6 hours with Vt and PEEP settings similar to those of surgery patients caused ALI. Preemptive application of APRV blocked early drivers of lung injury, preventing ARDS. Our data suggest that APRV applied early could reduce the incidence of ARDS in patients at risk.

Preemptive Application of Airway Pressure Release Ventilation Prevents Development of Acute Respiratory Distress Syndrome in a Rat Traumatic Hemorrhagic Shock Model

Once established, the acute respiratory distress syndrome (ARDS) is highly resistant to treatment and retains a high mortality. We hypothesized that preemptive application of airway pressure release ventilation (APRV) in a rat model of trauma/hemorrhagic shock (T/HS) would prevent ARDS. Rats were anesthetized, instrumented for hemodynamic monitoring, subjected to T/HS, and randomized into two groups: (a) volume cycled ventilation (VC) (n = 5, tidal volume 10 mL/kg; positive end-expiratory pressure 0.5 cmH(2)O) or (b) APRV (n = 4, P(high) = 15-20 cmH(2)O; T(high) = 1.3-1.5 s to achieve 90% of the total cycle time; T(low) = 0.11-0.14 s, which was set to 75% of the peak expiratory flow rate; P(low) = 0 cmH(2)O). Study duration was 6 h. Airway pressure release ventilation prevented lung injury as measured by PaO(2)/FIO(2) (VC 143.3 ± 42.4 vs. APRV 426.8 ± 26.9, P < 0.05), which correlated with a significant decrease in histopathology as compared with the VC group. In addition, APRV resulted in a significant decrease in bronchoalveolar lavage fluid total protein, increased surfactant protein B concentration, and an increase in epithelial cadherin tissue expression. In vivo microscopy demonstrated that APRV significantly improved alveolar patency and stability as compared with the VC group. Our findings demonstrate that preemptive mechanical ventilation with APRV attenuates the clinical and histologic lung injury associated with T/HS. The mechanism of injury prevention is related to preservation of alveolar epithelial and endothelial integrity. These data support our hypothesis that preemptive APRV, applied using published guidelines, can prevent the development of ARDS.

Transcriptional Events during the Recovery from MRSA Lung Infection: A Mouse Pneumonia Model

Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging threat to human health throughout the world. Rodent MRSA pneumonia models mainly focus on the early innate immune responses to MRSA lung infection. However, the molecular pattern and mechanisms of recovery from MRSA lung infection are largely unknown. In this study, a sublethal mouse MRSA pneumonia model was employed to investigate late events during the recovery from MRSA lung infection. We compared lung bacterial clearance, bronchoalveolar lavage fluid (BALF) characterization, lung histology, lung cell proliferation, lung vascular permeability and lung gene expression profiling between days 1 and 3 post MRSA lung infection. Compared to day 1 post infection, bacterial colony counts, BALF total cell number and BALF protein concentration significantly decreased at day 3 post infection. Lung cDNA microarray analysis identified 47 significantly up-regulated and 35 down-regulated genes (p<0.01, 1.5 fold change [up and down]). The pattern of gene expression suggests that lung recovery is characterized by enhanced cell division, vascularization, wound healing and adjustment of host adaptive immune responses. Proliferation assay by PCNA staining further confirmed that at day 3 lungs have significantly higher cell proliferation than at day 1. Furthermore, at day 3 lungs displayed significantly lower levels of vascular permeability to albumin, compared to day 1. Collectively, this data helps us elucidate the molecular mechanisms of the recovery after MRSA lung infection.

Cardiopulmonary toxicity of pulmonary exposure to occupationally relevant zinc oxide nanoparticles

Exposure to zinc oxide (ZnO) metal fumes is linked to adverse human health effects; however, the hazards of ZnO nanoparticles (ZnONPs) remain unclear. To determine pulmonary exposure to occupationally relevant ZnONPs cause cardiopulmonary injury, Sprague-Dawley rats were exposed to ZnONPs via intratracheal (IT) instillation and inhalation. The relationship between intrapulmonary zinc levels and pulmonary oxidative-inflammatory responses 72 h after ZnONP instillation was determined in bronchoalveolar lavage fluid (BALF). Instilled ZnONPs altered zinc balance and increased the levels of total cells, neutrophils, lactate dehydrogenase (LDH) and total protein in BALF and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in blood after 72 h. The ZnONPs accumulated predominantly in the lungs over 24 h, and trivial amounts of zinc were determined in the heart, liver, kidneys and blood. Furthermore, the inflammatory-oxidative responses induced by occupationally relevant levels of 1.1 and 4.9 mg/m3 of ZnONP inhalation for 2 weeks were determined in BALF and blood at 1, 7 and 30 days post-exposure. Histopathological examinations of the rat lungs and hearts were performed. Inhalation of ZnONP caused an inflammatory cytological profile. The total cell, neutrophil, LDH and total protein levels were acutely increased in the BALF, and there was an inflammatory pathology in the lungs. There were subchronic levels of white blood cells, granulocytes and 8-OHdG in the blood. Cardiac inflammation and the development of fibrosis were detected 7 days after exposure. Degeneration and necrosis of the myocardium were detected 30 days after exposure. The results demonstrate that ZnONPs cause cardiopulmonary impairments. These findings highlight the occupational health effects for ZnONP-exposed workers.

The Triterpenoid CDDO-Me Inhibits Bleomycin-Induced Lung Inflammation and Fibrosis

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p≤0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p≤0.001), and excess production of the pro-fibrotic cytokine TGFβ by 50%. CDDO-Me also inhibited α-smooth muscle actin and fibronectin mRNA by 50% (p≤0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and αSMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.

Effects of COX-2 inhibitor on ventilator-induced lung injury in rats

Mechanical ventilation especially with large tidal volume has been demonstrated to activate inflammatory response inducing lung injury, which could be attenuated by cyclooxygenase (COX)-2 inhibitors. As the main small integral membrane proteins that selectively conduct water molecules' transportation, aquaporin (AQP)-1 downregulation significantly related to lung edema and inflammation. This study aims to investigate the role of AQP1 in ventilator-induced lung injury in rats and evaluates the effects of COX-2 inhibition. Forty rats were allocated into four groups, where rats in Groups LD (low volume+DMSO) and LN (low volume+NS-398) were given intravenously 2ml DMSO and 8mg/kg NS-398 (a specific COX-2 inhibitor, dissolved in 2ml DMSO) before 4-hour lower tidal volume ventilation (8ml/kg), respectively, while DMSO and NS-398 were administrated in the same manner before 4-hour injurious ventilation (40ml/kg) in Groups HD (high volume+DMSO) and HN (high volume+NS-398). The arachidonic acid metabolites (6-keto prostaglandin F1α, thromboxane B2), inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, 6, 8) and total protein levels in bronchoalveolar lavage (BAL) fluid and COX-2 mRNA and AQP1 protein expression in lung tissue were detected; water content and lung morphology were also evaluated. Compared to Groups LD and LN, the rats in Groups HD and HN suffered obvious lung morphological changes with higher wet-to-dry weight ratio and lung injury score, and the levels of arachidonic acid metabolites, inflammatory cytokines and total protein in BAL fluid were increased, the expression of COX-2 mRNA was significantly upregulated and AQP1 protein was downregulated in lung tissue (p<0.05). The changes in BAL fluid and the severity of lung injury were attenuated, and AQP1 expression was upregulated in Group HN as compared to HD (p<0.05). Ventilation with large tidal volume causes inflammatory mediator production and AQP1 downregulation, which could be attenuated by COX-2 inhibition.

Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.

TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALBLps-d) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-d over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-d BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALBLps-d BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening.

Pulmonary toxicity screening of triclosan in rats after intratracheal instillation

Triclosan (TCS) is a chemical compound used in household products as biocide. However, their pulmonary toxicity has been unclear. Thus, the purpose of this study was to investigate the possibility of injury to the lung by inhalation of TCS. Rats were exposed to TCS by single intratracheal instillation of 10 µg/B.W. kg for the low-dose group and 1,000 µg/B.W. kg for the high-dose group, respectively. TCS induced increase in the level of total cell (TC) count, polymorphonuclear leukocytes (PMNs), total protein (TP), lactate acid dehydrogenase (LDH), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF) at 1 day after instillation. However, most pulmonary toxicity marker levels except TP in BALF were restored 14 days after instillation. In addition, TCS led to reduction of cell viability with morphological change in lung eptiehelial cells (L2 cell). Therefore, TCS may affect responses of acute inflammation and permeability in the lung.

Tissue plasminogen activator attenuates ventilator-induced lung injury in rats

To test the hypothesis that the tissue plasminogen activator (tPA) may counteract the inhibitory effect of plasminogen activator inhibitors (PAI) and attenuate lung injury in a rat model of ventilator-induced lung injury (VILI). Adult male Sprague-Dawley rats were ventilated with a HVZP (high-volume zero PEEP) protocol for 2 h at a tidal volume of 30 mL/kg, a respiratory rate of 25 breaths/min, and an inspired oxygen fraction of 21%. The rats were divided into 3 groups (n=7 for each): HVZP+tPA group receiving tPA (1.25 mg/kg, iv) 15 min before ventilation, HVZP group receiving HVZP+vehicle injection, and a control group receiving no ventilation. After 2 h of ventilation, the rats were killed; blood and lungs were collected for biochemical and histological analyses. HVZP ventilation significantly increased total protein content and the concentration of macrophage inflammatory protein-2 (MIP-2) in the bronchoalveolar lavage fluid (BALF) as well as the lung injury score. Rats that received HVZP ventilation had significantly higher lung PAI-1 mRNA expression, plasma PAI-1 and plasma D-dimer levels than the control animals. tPA treatment significantly reduced the BALF total protein and the lung injury score as compared to the HVZP group. tPA treatment also significantly decreased the plasma D-dimer levels and the HVZP ventilation-induced lung vascular fibrin thrombi. tPA treatment showed no effect on MIP-2 level in BALF. These results demonstrate that VILI increases lung PAI-1 mRNA expression, plasma levels of PAI-1 and D-dimers, lung injury score and vascular fibrin deposition. tPA can attenuate VILI by decreasing capillary-alveolar protein leakage as well as local and systemic coagulation as shown by decreased lung vascular fibrin deposition and plasma D-dimers.

Ubiquitin and Stromal Cell-Derived Factor-1α in Bronchoalveolar Lavage Fluid After Burn and Inhalation Injury

The objective of the study was to determine whether the CXC chemokine receptor (CXCR) 4 ligands ubiquitin and stromal cell-derived factor (SDF)-1α are detectable in bronchoalveolar lavage fluid (BALF) after burn and inhalation injury and whether their concentrations in BALF are associated with injury severity, physiological variables, or clinical outcomes. BALF was obtained on hospital admission from 51 patients (48 ± 18 years) with burn (TBSA: 23 ± 24%) and inhalation injury (controls: 10 healthy volunteers, 42 ± 8 years). BALF was analyzed for total protein and for ubiquitin and SDF-1α by enzyme-linked immunosorbent assay. Ubiquitin/SDF-1α levels were normalized to total BALF protein content. The extent of inhalation injury was determined during bronchoscopy using a standardized scoring system. Percent TBSA, Baux scores, revised Baux scores, and clinical variables were documented. Ubiquitin and SDF-1α were detectable in 40% of normal BALF specimens. After injury, ubiquitin was detectable in 90% (P < .01 vs control) and SDF-1α in 10% of the specimens (P < .05 vs control). While SDF-1α levels were reduced in patients (P < .01), ubiquitin levels were increased (P < .01). Ubiquitin concentrations correlated inversely with grade of inhalation injury, revised Baux scores, and resuscitation fluid requirements (Spearman correlation coefficients [r]: -.3, -.33, and -.45, respectively). Ubiquitin levels correlated positively with arterial oxygenation at the time of bronchoscopy (r: .35). BALF levels of CXCR4 agonists are differentially regulated after burn and inhalation injury. Increases in BALF ubiquitin after inhalation injury may maintain CXCR4-mediated lung protection and repair processes. The finding that BALF ubiquitin decreased with higher grades of inhalation injury may provide a biological correlate for an insufficient local inflammatory response after severe inhalation injury.

The role of the extracellular matrix protein mindin in airway response to environmental airways injury.

Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored.Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone.Methods: We exposed C57BL/6J and mindin-deficient (–/–) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies.Results: After inhalation of LPS, mindin–/– mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin–/– mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin–/– mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin–/– mice demonstrated reduced constriction in response to carbachol.Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.

Leukotriene biosynthesis inhibition ameliorates acute lung injury following hemorrhagic shock in rats

BackgroundHemorrhagic shock followed by resuscitation is conceived as an insult frequently induces a systemic inflammatory response syndrome and oxidative stress that results in multiple-organ dysfunction syndrome including acute lung injury. MK-886 is a leukotriene biosynthesis inhibitor exerts an anti inflammatory and antioxidant activity.ObjectivesThe objective of present study was to assess the possible protective effect of MK-886 against hemorrhagic shock-induced acute lung injury via interfering with inflammatory and oxidative pathways.Materials and methodsEighteen adult Albino rats were assigned to three groups each containing six rats: group I, sham group, rats underwent all surgical instrumentation but neither hemorrhagic shock nor resuscitation was done; group II, Rats underwent hemorrhagic shock (HS) for 1 hr then resuscitated with Ringer's lactate (1 hr) (induced untreated group, HS); group III, HS + MK-886 (0.6 mg/kg i.p. injection 30 min before the induction of HS, and the same dose was repeated just before reperfusion period). At the end of experiment (2 hr after completion of resuscitation), blood samples were collected for measurement of serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The trachea was then isolated and bronchoalveolar lavage fluid (BALF) was carried out for measurement of leukotriene B4 (LTB4), leukotriene C4 (LTC4) and total protein. The lungs were harvested, excised and the left lung was homogenized for measurement of malondialdehyde (MDA) and reduced glutathione (GSH) and the right lung was fixed in 10% formalin for histological examination.ResultsMK-886 treatment significantly reduced the total lung injury score compared with the HS group (P < 0.05). MK-886 also significantly decreased serum TNF-α & IL-6; lung MDA; BALF LTB4, LTC4 & total protein compared with the HS group (P < 0.05). MK-886 treatment significantly prevented the decrease in the lung GSH levels compared with the HS group (P < 0.05).ConclusionsThe results of the present study reveal that MK-886 may ameliorate lung injury in shocked rats via interfering with inflammatory and oxidative pathways implicating the role of leukotrienes in the pathogenesis of hemorrhagic shock-induced lung inflammation.

Effect of maternal exposure to ozone on reproductive outcome and immune, inflammatory, and allergic responses in the offspring

There is growing concern that exposure to air pollutants during pregnancy affects health outcomes in the offspring due to alterations in the development of immune and other homeostatic processes. To assess the risks of maternal inhalation exposure to ozone (O3), timed pregnant BALB/c mice were exposed to different concentrations of O3 (0, 0.4, 0.8, and 1.2 ppm) for 4 h/day for 10 days during gestation (GD9–GD18), and pulmonary inflammation and immune responses were assessed in the offspring at 6 weeks-of-age. Maternal O3 exposure reduced the number of productive dams by 25% at the highest O3 concentration (1.2 ppm) and decreased the rate of weight gain in the offspring. Delayed-type hypersensitivity responses to bovine serum albumin were suppressed in the female offspring by maternal exposure to the two highest concentrations of O3, whereas humoral immune responses to sheep red blood cells were not altered in either sex. Maternal exposure to 1.2 ppm O3 increased lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluid (BALF) of the offspring but did not affect the number of inflammatory cells or levels of total protein, IFN-γ, IL-17, and IL-4 cytokines in BALF, or CD4+, CD8+, CD25+, and TCRβ+CD1d+ T-cells in the spleen. Offspring born from air-exposed dams sensitized early in life (postnatal day [PND] 3) to ovalbumin (OVA) antigen and then challenged as adults developed eosinophilia, elevated levels of LDH activity and total protein in BALF, and increased pulmonary responsiveness to methacholine, compared with animals sensitized at PND42. Maternal O3 exposure in the 1.2 ppm O3 group decreased BALF eosinophilia and serum OVA-specific IgE in the female offspring sensitized early in life but did not affect development of allergic airway inflammation by offspring sensitized late in life. In summary, maternal exposure to O3 affected reproductive outcome and produced modest decreases in immune function and indicators of allergic lung disease in surviving offspring.

Acute and Subchronic Airway Inflammation after Intratracheal Instillation of Quartz and Titanium Dioxide Agglomerates in Mice

This study investigated the acute and subchronic inflammatory effects of micrometer-size (micro-size) and nanometer-size (nano-size) particles after intratracheal (i.t.) installation in mice. The role of the type of compound, polymorphism, and size of the particles was investigated. Studied compounds were the two micro-size reference quartzes, SRM1878a and DQ12, a micro- and nano-size rutile titanium dioxide (TiO2), a nano-size anatase, and an amorphous TiO2. Particles were administered by a single i.t. instillation in mice at a fixed dose of 5, 50, and 500 μg, respectively. Inflammation was evaluated from the bronchoalveolar lavage fluid (BALF) content of inflammatory cells, the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), as well as from lung histology. Evaluations were at 24 h (acute effects) and 3 months (subchronic effects) after instillations. Both types of quartz induced a dose-dependent acute increase of neutrophils, IL-6, and total protein in BALF. Limited subchronic inflammation was observed. All types of TiO2 induced a dose-dependent acute increase of neutrophils in BALF. In the acute phase, micro- and nano-size rutile and nano-size amorphous TiO2 induced elevated levels of IL-6 and total protein in BALF at the highest dose. At the nano-size rutile and amorphous TiO2, subchronic lung inflammation was apparent from a dose-dependent increase in BALF macrophages. Histology showed little inflammation overall. The two types of quartz showed virtually similar inflammatory effects. Nearly similar effects were observed for two sizes of rutile TiO2. Differences were seen between the different polymorphs of nano-size TiO2, with rutile being the most inflammogenic and amorphous being the most potent in regard to acute tissue damage.

CCR2-Antagonist Prophylaxis Reduces Pulmonary Immune Pathology and Markedly Improves Survival during Influenza Infection

Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.

Ventilator-Associated Lung Injury Superposed to Oleic Acid Infusion or Surfactant Depletion: Histopathological Characteristics of Two Porcine Models of Acute Lung Injury

Background: The pathophysiological concept of acute lung injury (ALI) in combination with ventilator-associated lung injury (VALI) is still unclear. We characterized the histopathological features of intravenous injection of oleic acid (OAI) and lung lavage (LAV) combined with VALI. Methods: Pigs were randomized to the control, LAV or OAI group and ventilated by pressure-controlled ventilation. Measurements included: haemodynamics, spirometry, blood gas analysis, lung wet-to-dry weight ratio (W/D), total protein content in broncho-alveolar lavage fluid (BALF), and lung pathological description and scoring. Results: Five hours after lung injury induction, gas exchange was significantly impaired in both the OAI and the LAV groups. Compared to controls, we found an increase in W/D and histopathological total injury scores in both the LAV and OAI groups and an increase in BALF total protein content in the OAI group. In contrast to the LAV group, the OAI group showed septal necrosis and alveolar oedema. Both groups exhibited dorsal and caudal atelectasis and interstitial oedema. In addition, the OAI group demonstrated a propensity to dorsal necrosis and congestion whereas the LAV group tended to develop ventral overdistension and barotrauma. Conclusions: This study presents a comparison of porcine OAI and LAV models combined with VALI, providing information for study design in research on ALI.

Propofol Prevents Lung Injury Following Intestinal Ischemia-Reperfusion

The antioxidant properties of propofol have been shown to improve ischemia/reperfusion injury. We investigated whether anesthesia with propofol can ameliorate remote lung injury induced by intestinal ischemia-reperfusion (IIR). Thirty male Wistar rats were randomly allocated in three groups (n = 10 each): animals in group Sham were anesthetized with ketamine and xylazine and then laparotomy and sham IIR followed. Animals in group IIR received ketamine and xylazine and were then subjected to clamping of the superior mesenteric artery for 45 min and reperfusion for 4 h. Group IIR+P received anesthesia with propofol and then IIR was induced, as in group IIR. Blood samples for blood gases and malondialdehyde measurements were drawn at the end of reperfusion. Bronchoalveolar lavage fluid (BALF) was obtained to measure cell counts, total protein, and phospholipids levels. Induction of IIR resulted in deteriorated oxygenation, acidemia, and inflammatory cells sequestration, along with increased BALF protein content and increased proportions of small surfactant aggregates. Anesthesia with propofol alleviated intestinal injury and efficiently prevented lipid oxidation. In group IIR+P inflammatory cell infiltration and pulmonary histologic changes were significantly limited. The increase in BALF total protein and the changes in surfactant aggregates were prevented, leading to normal systemic oxygenation. Using propofol to induce and maintain anesthesia efficiently prevented IIR-induced lung injury. Systemic antioxidant protection, improvement of intestinal injury, inhibition of the inflammatory response, and preservation of the alveolar-capillary permeability seem to be crucial mediating mechanisms for this simple and clinically relevant intervention.

A study of the role of heme oxygenase-1 expression in ventilator induced lung injury and its mechanism in rats

To observe the changes in heme oxygenase-1 (HO-1) expression in lung tissue with ventilator induced lung injury (VILI) in rats, and to explore the mechanism of preventive effect of HO-1 inducer hemin on VILI. Fifty-six male Sprague-Dawley (SD) rats were randomly divided into control group (group C), VILI model group (group M), hemin group 1, 2, 3, 4 (group H1, H2, H3, H4, with intraperitoneal injection of hemin 40, 80, 120, 160 micromol/kg, respectively, 24 hours before model was reproduced), and suppressor Z group (intraperitoneal injection of ZnPP 10 micromol/kg 24 hours before reproduction of the model). After 4 hours of ventilation, all rats, except those of group C, were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected. Total protein, the contents of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) in BALF were measured. The lung tissue specimen was collected, the wet-to-dry weight ratio (W/D), the level of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA) and HO-1 protein expression were determined. Lung pathological changes were observed with microscope. In group M, rat lung tissue was seriously damaged, and total protein in BALF, TNF-alpha, IL-10, W/D of lung, MDA and LDH activity, as well as HO-1 protein expression were markedly higher than those in group C, denoting that VILI model was successfully reproduced. Compared with group M, the total protein in BALF (g/L) in group H1, H2, H3 was gradually reduced (0.74+/-0.06, 0.73+/-0.07, 0.70+/-0.07 vs. 0.84+/-0.08, all P<0.01) with an increase in hemin dose. W/D of lung was reduced (4.93+/-0.27, 4.91+/-0.24, 4.87+/-0.23 vs. 5.53+/-0.48, all P<0.01). The activity of SOD (U/mg) was higher (85+/-9, 82+/-15, 93+/-11 vs. 55+/-12, all P<0.01), and the content of MDA (nmol/mg) was decreased (15+/-3, 15+/-3, 13+/-2 vs. 18+/-4, P<0.05 or P<0.01). The content of IL-10 (pg/L) in BALF was higher (0.42+/-0.06, 0.46+/-0.06, 0.47+/-0.05 vs. 0.36+/-0.07), and the content of TNF-alpha (pg/L) was decreased (0.18+/-0.07, 0.14+/-0.03, 0.10+/-0.07 vs. 0.23+/-0.06), but only the difference between group H2, H3 and group M was statistically significant (all P<0.01). The activity of LDH (U/g) was decreased (11 353+/-1 317, 11 516+/-1 613, 9 631+/-1 520 vs. 12 361+/-1 841), but only the difference between group H3 and group M was statistically significant (P<0.01). HO-1 expression [absorbance (A) value] was increased compared with that of group M (0.164+/-0.010, 0.190+/-0.149, 0.205+/- 0.018 vs. 0.122+/-0.016, all P<0.01), and the degree of lung injury was reduced with increase in dosage. With the further increase of hemin dose, lung injury in group H4 was more serious than that of group H1, H2 and H3. With ZnPP to inhibit HO-1 expression, the protective effect of HO-1 disappeared. A moderate expression of HO-1 as induced by hemin can alleviate VILI, and the best dose of hemin is 120 micromol/kg. Its protective effect on lung tissue may possibly be attributed by its anti-inflammatory effect and anti-oxidative stress.

Acute respiratory toxicity following inhalation exposure to soman in guinea pigs

Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m 3 concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m 3 soman survived, while animals exposed to 841, and 1121 mg/m 3 resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt 50 for soman determined by probit analysis was 827.2 mg/m 3. A majority of the animals that died at 1121 mg/m 3 developed seizures and died within 15–30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5–6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24 h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs.