Evaluation of the amelioration effect of Ganoderma formosanum extract on delaying PM2.5 damage to lung macrophages.

SCOPE: Particulate matter (PM) contains toxic organic matter and heavy metals that enter the entire body through blood flow and may cause mortality. Ganoderma formosanum mycelium, a valuable traditional Chinese medicine that has been used since ancient times, contains various active ingredients that can effectively impede inflammatory responses on murine alveolar macrophages induced by PM particles. METHODS AND RESULTS: An experimental study assessing the effect of G. formosanum mycelium extract's water fraction (WA) on PM-exposed murine alveolar macrophages using ROS measurement shows that WA reduces intracellular ROS by 12% and increases cell viability by 16% when induced by PM particles. According to RNA-Sequencing, western blotting, and real-time qPCR are conducted to analyze the metabolic pathway. The WA reduces the protein ratio in p-NF-κB/NF-κB by 18% and decreases the expression of inflammatory genes, including IL-1ß by 38%, IL-6 by 29%, and TNF-α by 19%. Finally, the identification of seven types of anti-inflammatory compounds in the WA fraction is achieved through UHPLC-ESI-Orbitrap-Elite-MS/MS analysis. These compounds include anti-inflammatory compounds, namely thiamine, adenosine 5'-monophosphate, pipecolic acid, L-pyroglutamic acid, acetyl-L-carnitine, D-mannitol, and L-malic acid. CONCLUSIONS: The study suggests that the WA has the potential to alleviate the PM -induced damage in alveolar macrophages, demonstrating its anti-inflammatory properties.

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PURPOSE: Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. METHODS: Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. RESULTS: Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. CONCLUSIONS: A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Effects of Euterpe oleracea Mart. (AÇAÍ) extract in acute lung inflammation induced by cigarette smoke in the mouse.

Short term inhalation of cigarette smoke (CS) induces significant lung inflammation due to an imbalance of oxidant/antioxidant mechanisms. Açai fruit (Euterpe oleracea) has significant antioxidant and anti-inflammatory actions. The present study aimed to determine whether oral administration of an açai stone extract (ASE) could reduce lung inflammation induced by CS. Thirty C57BL/6 mice were assigned to three groups (n=10 each): the Control+A group was exposed to ambient air and treated orally with ASE 300 mg/kg/day; the CS group was exposed to smoke from 6 cigarettes per day for 5 days; and the CS+A group was exposed to smoke from 6 cigarettes per day for 5 days and treated orally with ASE (300 mg/kg/day). On day 6, all mice were sacrificed. After bronchoalveolar lavage, the lungs were removed for histological and biochemical analyses. The CS group exhibited increases in alveolar macrophage (AMs) and neutrophil numbers (PMNs), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase activities (GPx), TNF-α expression, and nitrites levels in lung tissue when compared with the control ones (p<0.001 for all parameters). The AMs, PMNs, MPO, SOD, CAT, GPx and nitrite were significantly reduced by oral administration of ASE when compared with CS group (p<0.001 for all parameters, with exception of AMs p<0.01). The present results suggested that systemic administration of an ASE extract could reduce the inflammatory and oxidant actions of CS. Thus, the results of this study in mice should stimulate future studies on ASE as a potential agent to protect against CS-induced inflammation in humans.

Effects of decreased calmodulin protein on the survival mechanisms of alveolar macrophages during Pneumocystis pneumonia.

Pneumocystis infection causes increased intracellular levels of reactive oxygen species (ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of key prosurvival molecules in Amø and bronchoalveolar lavage fluids from infected rats and mice showed low levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and reduced activation of phosphoinositide-3 kinase (PI-3K). Ubiquitous calcium-sensing protein calmodulin protein and mRNA levels were also reduced in Amø during Pneumocystis pneumonia (Pcp). Calmodulin has been implicated in control of GM-CSF production and PI-3K activation in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from infected animals. Supplementation of Amø with exogenous calmodulin increased survival signaling via GM-CSF and PI-3K and reduced ROS and apoptosis. These data support the hypotheses that calmodulin levels at least partially control survival signaling in Amø and that restoration of GM-CSF or PI-3K signaling will improve host response to the organism.

Cellular interaction of different forms of aluminum nanoparticles in rat alveolar macrophages.

Nanomaterials, with dimensions in the 1-100 nm range, possess numerous potential benefits to society. However, there is little characterization of their effects on biological systems, either within the environment or on human health. The present study examines cellular interaction of aluminum oxide and aluminum nanomaterials, including their effect on cell viability and cell phagocytosis, with reference to particle size and the particle's chemical composition. Experiments were performed to characterize initial in vitro cellular effects of rat alveolar macrophages (NR8383) after exposure to aluminum oxide nanoparticles (Al2O3-NP at 30 and 40 nm) and aluminum metal nanoparticles containing a 2-3 nm oxide coat (Al-NP at 50, 80, and 120 nm). Characterization of the nanomaterials, both as received and in situ, was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), and/or CytoViva150 Ultra Resolution Imaging (URI)). Particles showed significant agglomeration in cell exposure media using DLS and the URI as compared to primary particle size in TEM. Cell viability assay results indicate a marginal effect on macrophage viability after exposure to Al2O3-NP at doses of 100 microg/mL for 24 h continuous exposure. Al-NP produced significantly reduced viability after 24 h of continuous exposure with doses from 100 to 250 microg/mL. Cell phagocytotic ability was significantly hindered by exposure to 50, 80, or 120 nm Al-NP at 25 microg/mL for 24 h, but the same concentration (25 microg/mL) had no significant effect on the cellular viability. However, no significant effect on phagocytosis was observed with Al2O3-NP. In summary, these results show that Al-NP exhibit greater toxicity and more significantly diminish the phagocytotic ability of macrophages after 24 h of exposure when compared to Al2O3-NP.

Oxidant stress modulates murine allergic airway responses.

The allergic inflammation occurring in asthma is believed to be accompanied by the production of free radicals. To investigate the role of free radicals and the cells affected we turned to a murine model of allergic inflammation produced by sensitization to ovalbumin with subsequent aerosol challenge. We examined oxidant stress by measuring and localizing the sensitive and specific marker of lipid peroxidation, the F2-isoprostanes. F2-isoprostanes in whole lung increased from 0.30 +/- 0.08 ng/lung at baseline to a peak of 0.061 +/- 0.09 ng/lung on the ninth day of daily aerosol allergen challenge. Increased immunoreactivity to 15-F2t-IsoP (8-iso-PGF2alpha) or to isoketal protein adducts was found in epithelial cells 24 h after the first aerosol challenge and at 5 days in macrophages. Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased immunoreactivity after ovalbumin challenge. Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes while supplemental vitamin E suppressed their formation. Similar changes in immunoreactivity to F2-isoprostanes were seen. Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant. Our findings indicate that allergic airway inflammation in mice is associated with an increase in oxidant stress, which is most striking in airway epithelial cells and macrophages. Oxidant stress plays a role in the production of airway responsiveness.

Influence of supplementation of all-rac-alpha-tocopheryl acetate preweaning and vitamin C postweaning on alpha-tocopherol and immune responses of piglets.

This study was designed to test whether dietary maternal supplementation of all-rac-alpha-tocopheryl acetate during lactation and dietary vitamin C supplementation after weaning could increase the alpha-tocopherol status pre- and postweaning and the immune responses of piglets postweaning. The experiment involved 12 crossbred sows that were fed increasing levels of all-rac-alpha-tocopheryl (70, 150, and 250 IU/kg, as-fed basis) during lactation. After weaning (d 28 of age), litters were divided into two groups that were supplemented with or without vitamin C (500 mg/kg of feed, as-fed basis). Milk and blood samples were obtained from the sows during lactation. Pigs were bled at 4, 16, 28, 35, 42, and 49 d of age. Liver, heart, muscle, and s.c. adipose tissues were collected (on 28, 35, 42, and 49 d of age) and analyzed for alpha-tocopherol. On the same days, alveolar macrophages of the lungs were collected, and analyzed for the concentration of alpha-tocopherol and its stereoisomer composition, fatty acid composition, and release of prostaglandin E2, leukotriene B4, and thromboxane B2. Increasing dietary all-rac-alpha-tocopheryl acetate concentration increased the concentration of alpha-tocopherol in plasma (P = 0.02) and milk (P = 0.007) of sows, and the sow milk concentrations of alpha-tocopherol and vitamin A were greater on d 2 of lactation than later on during lactation. The plasma concentration of alpha-tocopherol in piglets decreased from d 4 to later on during suckling (P < 0.001) and again as the postweaning period progressed (P < 0.001). When lipid-standardized, plasma alpha-tocopherol was increased in piglets of sows fed 250 IU of all-rac-alpha-tocopheryl acetate compared with other sow-groups (P = 0.005). At 28 d of age, alpha-tocopherol concentrations in tissues were increased with supplementation of the high dietary all-rac-alpha-tocopheryl acetate levels to the sows; however, after weaning, a decrease in alpha-tocopherol concentration in most tissues (except liver) was observed, but the decrease tended to be less in the muscle (P = 0.099) and adipose tissue (P = 0.11) of piglets suckling sows fed 150 and 250 IU of all-rac-alpha-tocopheryl acetate. Vitamin C supplementation after weaning increased liver alpha-tocopherol (P = 0.01) and serum immunoglobulin M concentration (P = 0.04), and vitamin C supplementation increased the proportion of the RRR-alpha-tocopherol (P = 0.03) at the expense of the RRS-stereoisomer form (P = 0.05) of alpha-tocopherol in alveolar macrophages of the piglets. In conclusion, this study on maternal all-rac-alpha-tocopheryl acetate and postweaning vitamin C supplementation suggests a nutritional strategy for increasing alpha-tocopherol status and immune responses of weaned piglets.

Acute pulmonary inflammation induced by lung overloading with selenium particles: leukocyte response and in situ detection of selenium at high resolution.

The kinetics of the acute inflammatory response of the lung was triggered in CD-1 mice by a single intratracheal instillation of a large amount of Se (10 mg); it was studied by quantitative cytology of bronchoalveolar lavage samples, light microscopy, and scanning electron microscopy coupled with x-ray elemental microanalysis. Bronchoalveolar lavage leukocytes were mostly neutrophils and increased from 12 to 24 h of Se treatment and decreased at 72 h. Only less than half of the granulocytes showed ingested Se particles; in contrast, virtually all BAL macrophages contained Se particles. Scanning electron microscopy coupled with X-ray elemental microanalysis revealed that the intracellular Se particles were heterogeneous in size (diameters from 0.4 and up to 14 microm), and that Se inclusions were sometimes accumulated at a pole of the cell. At 72 h after instillation of the particles, Se-loaded alveolar macrophages were migrated in the interstitial space of the alveoli. Se-positive regions had a focal distribution in the lung; accumulation of inflammatory cells erased the alveolar architecture of these areas of the deep lung. Our data indicates that Se overloading of the lung results in: (1) an acute inflammatory response that is dominated by neutrophils; (2) early removal of Se done mostly by alveolar macrophages, and (3) formation of focal areas of invasion of the lung parenchyma by inflammatory infiltrates.

Prevention of gram-negative translocation reduces the severity of hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is characterized by intrapulmonary vascular dilatations and an increased alveoloarterial oxygen difference (AaPO(2)). These abnormalities are related to augmented pulmonary nitric oxide (NO) production, dependent primarily on increases in the expression and activity of inducible NO-synthase (iNOS) within pulmonary intravascular macrophages and, to a lesser extent, of endothelial NOS (eNOS). Production of iNOS by pulmonary intravascular macrophages might be related to translocated gut bacteria present in the pulmonary circulation. To test this hypothesis, we determined whether macrophage sequestration, lung iNOS expression and activity, and HPS severity were decreased after norfloxacin was given for 5 weeks to prevent Gram-negative bacterial translocation in rats with common bile duct ligation-induced cirrhosis. Norfloxacin decreased the incidence of Gram-negative translocation from 70 to 0% and the percentage of pulmonary microvessels containing more than 10 macrophages from 52 +/- 7 to 21 +/- 8% (p < 0.01). AaPO(2) and cerebral uptake of intravenous (99m)Tc-labeled albumin macroaggregates (reflecting intrapulmonary vascular dilatations) were intermediate to those of untreated cirrhotic and sham-operated rats. The activity and expression of lung iNOS, but not eNOS, were reduced to normal. Norfloxacin may reduce HPS severity by inhibiting Gram-negative bacterial translocation, thereby decreasing NO production by pulmonary intravascular macrophages. Bacterial translocation may be the key to the pathogenesis of HPS.

The role of oxidative stress in indium phosphide-induced lung carcinogenesis in rats.

Indium phosphide (IP), widely used in the microelectronics industry, was tested for potential carcinogenicity. Sixty male and 60 female Fischer 344 rats were exposed by aerosol for 6 h/day, 5 days/week, for 21 weeks (0.1 or 0.3 mg/m(3); stop exposure groups) or 105 weeks (0 or 0.03 mg/m(3) groups) with interim groups (10 animals/group/sex) evaluated at 3 months. After 3-month exposure, severe pulmonary inflammation with numerous infiltrating macrophages and alveolar proteinosis appeared. After 2 years, dose-dependent high incidences of alveolar/bronchiolar adenomas and carcinomas occurred in both sexes; four cases of squamous cell carcinomas appeared in males (0.3 mg/m(3)), and a variety of non-neoplastic lung lesions, including simple and atypical hyperplasia, chronic active inflammation, and squamous cyst, occurred in both sexes. To investigate whether inflammation-related oxidative stress functioned in the pathogenesis of IP-related pulmonary lesions, we stained lungs of control and high-dose animals immunohistochemically for four markers indicative of oxidative stress: inducible nitric oxide synthase (i-NOS), cyclooxygenase-2 (COX-2), glutathione-S-transferase Pi (GST-Pi), and 8-hydroxydeoxyguanosine (8-OHdG). Paraffin-embedded samples from the 3-month and 2-year control and treated females were used. i-NOS and COX-2 were highly expressed in inflammatory foci after 3 months; at 2 years, all four markers were expressed in non-neoplastic and neoplastic lesions. Most i-NOS staining, mainly in macrophages, occurred in chronic inflammatory and atypical hyperplastic lesions. GST-Pi and 8-OHdG expression occurred in cells of carcinoma epithelium, atypical hyperplasia, and squamous cysts. These findings suggest that IP inhalation causes pulmonary inflammation associated with oxidative stress, resulting in progression to atypical hyperplasia and neoplasia.

Effect of amiodarone on the lung shown by polarized light microscopy.

OBJECTIVE: To show lipid deposits in the lung of patients treated with amiodarone. MATERIALS AND METHODS: Formalin-fixed, unstained frozen sections were used to reveal lipid deposits by means of polarized light. Lung tissues obtained at the postmortem examination of one patient and the surgical biopsy of another were available. RESULTS: Birefringent lipid deposits were seen in the alveolar lining cells and macrophages, as well as in the walls of arterioles and venules. CONCLUSIONS: To assess the effect of amiodarone on the lung, polarized light microscopy of unstained, frozen sections is a simple and rapid method.

Localization of surfactant protein A (SP-A) in alveolar macrophage subpopulations of normal and fibrotic rat lung.

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1+/SP-A+ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.

Fatty acid composition of lung, macrophage and surfactant phospholipids after short-term enteral feeding with n-3 lipids.

Utilization of enteral feeding modalities may prove clinically relevant for rapid modulation of lung phospholipid polyunsaturated fatty acids (PUFA) that serve as substrates for the formation of vasoactive dienoic eicosanoids. We compared the effects of short-term enteral feeding with formulations enriched with either fish (n-3) or corn (n-6) oil PUFA on the fatty acid composition of rat lung, alveolar macrophage and surfactant phospholipids. The diets were infused continuously for 72 h through a surgically placed gastroduodenal feeding catheter by a syringe pump. The n-3 PUFA derived from the fish oil enriched diet were readily incorporated into the phospholipid membranes of the alveolar macrophages, lung tissue and pulmonary surfactant. The relative percentages of the n-3 PUFA were significantly higher and individual and total n-6 PUFA significantly lower in the macrophage, lung and surfactant phospholipids from the n-3-supplemented rats in comparison with those present in the rats infused enterally with the n-6 diet or untreated, chow-fed rats (baseline). In contrast, there was a significant increase in linoleic acid (18:2n-6) without modification of arachidonic acid (20:4n-6) in the alveolar macrophages, lung tissue and surfactant from rats enterally receiving the n-6 diet relative to levels measured in the rats at baseline. The results suggest that short-term continuous delivery of n-3-enriched enteral preparations can foster rapid modification of membrane phospholipid PUFA composition of lung tissue, alveolar macrophages and lung surfactant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pentoxifylline on in vivo leukocyte function and clearance of group B streptococci from preterm rabbit lungs.

OBJECTIVES: Pentoxifylline was evaluated for its ability to enhance inactivation of group B streptococci in lungs of prematurely born rabbits. Mechanisms associated with intrapulmonary streptococcal clearance and the pharmacodynamics of pentoxifylline were also investigated. DESIGN: Randomized, controlled animal trial. SETTING: University research laboratory. SUBJECTS: A total of 123 New Zealand rabbits were delivered prematurely by cesarean section and were used for clearance studies. Twenty-three preterm pups were additionally utilized to study the pharmacodynamics of pentoxifylline. INTERVENTIONS: Preterm rabbits were infected with group B streptococcal aerosols and given intraperitoneal injections of either pentoxifylline (25, 12.5, and 12.5 mg/kg) or placebo at 0, 6, and 12 hrs after infection. MEASUREMENTS AND MAIN RESULTS: At 0, 4, and 24 hrs, the numbers of streptococci were determined in the left lung, while the right lung underwent bronchoalveolar lavage to quantify intra-alveolar leukocytes, phagocytosis of inhaled bacteria, and concentrations of lysozyme and tumor necrosis factor. In a separate experiment, blood and bronchoalveolar fluid from infected animals were analyzed for pentoxifylline content. Streptococcal proliferation was less in pentoxifylline-treated animals than in controls at 24 hrs (p < .01). Pulmonary macrophages and polymorphonuclear leukocytes recovered from bronchoalveolar lavage fluid did not differ in numbers or phagocytic activity. Pentoxifylline-treated animals had lower levels of lysozyme (p < .02) and tumor necrosis factor (p < .005) in bronchoalveolar lavage fluid compared with placebo-treated pups. Therapeutic levels of pentoxifylline were achieved in blood and bronchoalveolar lavage fluid. CONCLUSIONS: Despite lowering lysozyme and tumor necrosis factor content in epithelial lining fluid, pentoxifylline improves the inactivation of group B streptococci in preterm rabbit lungs. These findings suggest that increased group B streptococcal clearance was coincident with an anti-inflammatory effect due to pentoxifylline. We conclude pentoxifylline may be clinically useful as an adjunctive therapy for group B streptococcal pneumonia in newborns.