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.

PyCoCa:A quantifying tool of carbon content in airway macrophage for assessment the internal dose of particles.

Given the lack of a comprehensive understanding of the complex metabolism and variable exposure environment, carbon particles in macrophages have become a potentially valuable biomarker to assess the exposure level of atmospheric particles, such as black carbon. However, the tedious and subjective quantification method limits the application of carbon particles as a valid biomarker. Aiming to obtain an accurate carbon particles quantification method, the deep learning and binarization algorithm were implemented to develop a quantitative tool for carbon content in airway macrophage (CCAM), named PyCoCa. Two types of macrophages, normal and foamy appearance, were applied for the development of PyCoCa. In comparison with the traditional methods, PyCoCa significantly improves the identification efficiency for over 100 times. Consistency assessment with the gold standard revealed that PyCoCa exhibits outstanding prediction ability with the Interclass Correlation Coefficient (ICC) values of over 0.80. And a proper fresh dye will enhance the performance of PyCoCa (ICC = 0.89). Subsequent sensitivity analysis confirmed an excellent performance regarding accuracy and robustness of PyCoCa under high/low exposure environments (sensitivity > 0.80). Furthermore, a successful application of our quantitative tool in cohort studies indicates that carbon particles induce macrophage foaming and the foaming decrease the carbon particles internalization in reverse. Our present study provides a robust and efficient tool to accurately quantify the carbon particles loading in macrophage for exposure assessment.

The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

Proteome Analysis in PAM Cells Reveals That African Swine Fever Virus Can Regulate the Level of Intracellular Polyamines to Facilitate Its Own Replication through ARG1.

In 2018, African swine fever broke out in China, and the death rate after infection was close to 100%. There is no effective and safe vaccine in the world. In order to better characterize and understand the virus-host-cell interaction, quantitative proteomics was performed on porcine alveolar macrophages (PAM) infected with ASFV through tandem mass spectrometry (TMT) technology, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The proteome difference between the simulated group and the ASFV-infected group was found at 24 h. A total of 4218 proteins were identified, including 306 up-regulated differentially expressed proteins and 238 down-regulated differentially expressed proteins. Western blot analysis confirmed changes in the expression level of the selected protein. Pathway analysis is used to reveal the regulation of protein and interaction pathways after ASFV infection. Functional network and pathway analysis can provide an insight into the complexity and dynamics of virus-host cell interactions. Further study combined with proteomics data found that ARG1 has a very important effect on ASFV replication. It should be noted that the host metabolic pathway of ARG1-polyamine is important for virus replication, revealing that the virus may facilitate its own replication by regulating the level of small molecules in the host cell.

Critical Choices in Predicting Stone Wool Biodurability: Lysosomal Fluid Compositions and Binder Effects.

The hazard potential, including carcinogenicity, of inhaled man-made vitreous fibers (MMVFs) is correlated with their biodurability in the lung, as prerequisite for biopersistence. Abiotic dissolution testing serves to predict biodurability. We re-analyzed the International Agency for Research on Cancer Monograph on MMVFs and found that the correlation between in vivo biopersistence and abiotic dissolution presented therein confounded different simulant fluids and further confounded evaluation of leaching vs structural elements. These are critical choices for abiotic dissolution testing, as are binder removal and the rate of the flow that removes ions during testing. Therefore, we experimentally demonstrated how fluid composition and binder affect abiotic dissolution of a representative stone wool MMVF. We compared six simulant fluids (all pH 4.5, reflecting the environment of alveolar macrophage lysosomes) that differed in organic acids, which have a critical role in their ability to modulate the formation of Si-rich gels on the fiber surfaces. Removing the binder accelerates the average dissolution rate by +104% (max. + 273%) across the fluids by suppression of gel formation. Apart from the high-citrate fluid that predicted a 10-fold faster dissolution than is observed in vivo, none of the five other fluids resulted in dissolution rates above 400 ng/cm2/h, the limit associated with the exoneration from classification for carcinogenicity in the literature. These findings were confirmed with and without binder. For corroboration, five more stone wool MMVFs were assessed with and without binder in one specific fluid. Again, the presence of the binder caused gel formation and reduced dissolution rates. To enhance the reliability and robustness of abiotic predictions of biodurability, we recommend replacing the critically influential citric acid in pH 4.5 fluids with other organic acids. Also, future studies should consider structural transformations of the fibers, including changes in fiber length, fiber composition, and reprecipitation of gel layers.

Prolonged distribution of aerosolized PEGylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic lung disease characterized by abnormal remodeling of the lung parenchyma with subsequent scarring of the alveolar structure. In this study, we examined the distribution characteristics of aerosolized polyethylene glycol (PEG)ylated liposomes in the lungs of mice with bleomycin-induced pulmonary fibrosis. SIGNIFICANCE: The present study details the utility of aerosolized PEGylated liposomes for improving intrapulmonary pharmacokinetics in fibrotic lungs. METHODS: Aerosolized PEGylated liposomes were administered to fibrotic mouse lungs using a MicroSprayer. Intrapulmonary pharmacokinetics was evaluated via in vivo imaging, measurement of liposome concentrations in bronchoalveolar lavage fluid (BALF) and alveolar macrophages (AMs), and observation of lung tissue sections. In addition, in vitro accumulation experiments using WI-38, A549, and RAW264.7 cells were performed. RESULTS: The decrease of the fluorescence intensity of the PEGylated liposomes was slower than that of the non-modified liposomes. Compared with the non-modified liposomes, the PEGylated liposomes were determined higher in BALF, whereas those in the AMs were lower. Both PEGylated and non-modified liposomes were widely dispersed in fibrotic regions in tissue sections. No difference in accumulation in WI-38 and A549 cells was noted between PEGylated and non-modified liposomes, whereas the PEGylated liposomes exhibited lower intracellular accumulation than non-modified liposomes in RAW264.7 cells. CONCLUSION: Aerosolized drug delivery systems using PEGylated liposomes exhibited prolonged distribution in both healthy and fibrotic mouse lungs. PEGylated liposomes were determined to be efficient drug delivery systems for anti-fibrotic agents targeting lung fibroblasts and alveolar epithelial cells for optimizing the treatment of IPF.

Human cystic fibrosis monocyte derived macrophages display no defect in acidification of phagolysosomes when measured by optical nanosensors.

BACKGROUND: Defective macrophage phagolysosomal acidification is implicated in numerous lung diseases including Cystic Fibrosis (CF) and may contribute to defective pathogen killing. Conflicting reports relating to phagolysosomal pH in CF macrophages have been published, in part related to the use of pH-sensitive fluorescent probes where potential inadequacies in experimental design can be a contributing factor (e.g. employing probes with incorrect pKa for the cellular compartment of interest). We developed a reliable method to quantify macrophage phagolysosomal pH using surface-enhanced Raman spectroscopy-based nanosensors. METHODS: Monocyte-derived macrophages from CF and healthy control participants were incubated with nanosensors. Live cell imaging identified phagocytosed nanosensors, and surface-enhanced Raman spectroscopy was performed using para-mercaptobenzoic acid functionalised gold nanoparticles which produce Raman spectra that change predictably with their environmental pH. Conventional fluorescence spectroscopy was carried out in comparison. Nanosensor localisation to phagolysosomes was confirmed by transmission electron microscopy. RESULTS: Nanosensors were actively phagocytosed by macrophages into phagolysosomes and acidification occurred rapidly and remained stable for at least 60 min. There was no difference in phagolysosomal pH between healthy control and CF macrophages (5.41 ±â€¯0.11 vs. 5.41 ±â€¯0.20, p > .9999), further confirmed by inhibiting Cystic Fibrosis Transmembrane Conductance Regulator in healthy control monocyte-derived macrophages. CONCLUSIONS: Optical nanosensors accurately measure macrophage phagolysosomal pH and demonstrate no phagolysosomal acidification defect in human CF monocyte-derived macrophages. Further studies using alveolar macrophages could extend the impact of our findings. Nanosensors represent a novel and precise means to measure organelle functions with widespread potential for the study and monitoring of several lung diseases.

LOC152742 as a biomarker in the diagnosis of pulmonary tuberculosis infection.

AIM: Long intergenic noncoding RNAs are long noncoding transcripts from the intergenic regions of annotated protein-coding genes. The elevated expression of long noncoding RNA (lnRNA) LOC152742 has been found in tuberculosis infection yet its roles in antimycobacterial responses remain to be elucidated. PATIENTS AND METHODS: In this study, the expression levels of LOC152742 in sputum, plasma of normal individuals, active tuberculosis patients, obsolete tuberculosis patients, and individuals affected with bacillus Calmette-Guerin (BCG) were analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and the sensitivity and specificity of the candidate biomarker LOC152742 were obtained. At the same time, the expression levels of LOC152742 in pulmonary epithelial and macrophages cells infected with H37Ra or H37Rv were detected by qRT-PCR. RESULTS: LOC152742 in sputum and plasma had a higher specificity in active tuberculosis compared with that in obsolete tuberculosis and BCG patients. Additionally, LOC152742 in pulmonary epithelial cells macrophages infected with H37Ra or H37Rv was increased significantly compared with uninfected groups indicating that LOC152742 may potentially act as a novel biomarker for diagnosis and therapy of active tuberculosis.

PP2ACα of Alveolar Macrophages Is a Novel Protective Factor for LPS-Induced Acute Respiratory Distress Syndrome.

Protein phosphatase 2A (PP2A) is one main serine/threonine phosphatase in eukaryotes, and its activation changes have been linked to modulation of numerous pathological processes, such as cancer, inflammation, fibrosis, and neurodegenerative diseases. Acute respiratory distress syndrome (ARDS), the major cause of respiratory failure, remains with limited therapies available up to now. Alveolar macrophages (AMs) are essential to innate immunity and host defense, participating in the pathogenesis of ARDS. As a result, AMs are considered as a potential therapeutic target for ARDS. In our study, we firstly found that PP2A activity was significantly decreased in the lipopolysaccharide (LPS)-stimulated AMs. Furthermore, adoptive transfer of AMs with enhanced PP2A enzyme activity that was improved by C2-ceramide prior to LPS exposure alleviated acute lung inflammation. Conversely, AM-specific ablation of PP2ACα exacerbated inflammatory responses to LPS. Mechanistically, PP2ACα negatively regulates LPS-induced cytokine secretion of AMs by NF-κB and MAPK pathways. Together, these findings provide the evidence to guide the development of novel therapeutic options targeting PP2ACα for ARDS/acute lung injury.

R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells.

The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ±â€¯42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14+ macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.

Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

Studies on the Tissue Localization of HKDC1, a Putative Novel Fifth Hexokinase, in Humans.

Hexokinase domain component 1 (HKDC1) is a recently discovered novel protein, which is being promoted as a putative fifth hexokinase. Although the exact role HKDC1 plays in physiology is still unclear, it has been shown to be important during pregnancy in the regulation of glucose homeostasis. In this study, we have comprehensively studied the expression pattern of HKDC1 in the human body. Using human tissue sample, immunohistochemistry imaging was performed. Our studies indicate that the tissues with highest HKDC1 expression were the brush border epithelium of the intestines, parts of the pancreas, and lung alveolar macrophages. Future directions will be to understand the role of this fifth hexokinase in these tissues, with a focus on its relative function as compared with other endogenously expressed hexokinases.

Intrapulmonary Pharmacokinetics of Levonadifloxacin following Oral Administration of Alalevonadifloxacin to Healthy Adult Subjects.

Alalevonadifloxacin (WCK 2349) is a novel l-alanine ester prodrug of levonadifloxacin that is being developed as an oral fluoroquinolone antibiotic. The primary objective of this study was to determine and compare plasma, epithelial lining fluid (ELF), and alveolar macrophage (AM) concentrations of levonadifloxacin following oral administration of alalevonadifloxacin to healthy adult subjects. Levonadifloxacin concentrations in plasma, ELF, and AM samples from 30 healthy subjects were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following oral dosing of alalevonadifloxacin (1,000 mg twice daily for 5 days). Six subjects were assigned to each bronchoalveolar lavage (BAL) fluid sampling time, i.e., 2, 4, 6, 8, or 12 h after the ninth oral dose. Noncompartmental pharmacokinetic (PK) parameters were determined from serial total plasma concentrations collected over a 12-h interval following the first and ninth oral doses. Penetration ratios were calculated from the areas under the concentration-time curves from 0 to 12 h (AUC0-12) for plasma, ELF, and AM by using mean (and median) concentrations at each BAL sampling time. Unbound plasma concentrations (∼85% plasma protein binding) were used to determine site-to-plasma penetration ratios. Plasma PK parameter values for levonadifloxacin were similar after the first and ninth doses. The respective AUC0-12 values based on mean ELF and AM concentrations were 172.6 and 35.3 mg · h/liter, respectively. The penetration ratios for ELF and AM levonadifloxacin concentrations to unbound plasma levonadifloxacin concentrations were 7.66 and 1.58, respectively. Similar penetration ratios were observed with median concentrations. The observed plasma, ELF, and AM concentrations of levonadifloxacin support further studies of alalevonadifloxacin for treatment of lower respiratory tract bacterial infections caused by susceptible pathogens. (This study has been registered at ClinicalTrials.gov under identifier NCT02253342.).

Coal Mine Dust Desquamative Chronic Interstitial Pneumonia: A Precursor of Dust-Related Diffuse Fibrosis and of Emphysema.

BACKGROUND: Diseases associated with coal mine dust continue to affect coal miners. Elucidation of initial pathological changes as a precursor of coal dust-related diffuse fibrosis and emphysema, may have a role in treatment and prevention. OBJECTIVE: To identify the precursor of dust-related diffuse fibrosis and emphysema. METHODS: Birefringent silica/silicate particles were counted by standard microscope under polarized light in the alveolar macrophages and fibrous tissue in 25 consecutive autopsy cases of complicated coal worker's pneumoconiosis and in 21 patients with tobacco-related respiratory bronchiolitis. RESULTS: Coal miners had 331 birefringent particles/high power field while smokers had 4 (p<0.001). Every coal miner had intra-alveolar macrophages with silica/silicate particles and interstitial fibrosis ranging from minimal to extreme. All coal miners, including those who never smoked, had emphysema. Fibrotic septa of centrilobular emphysema contained numerous silica/silicate particles while only a few were present in adjacent normal lung tissue. In coal miners who smoked, tobacco-associated interstitial fibrosis was replaced by fibrosis caused by silica/silicate particles. CONCLUSION: The presence of silica/silicate particles and anthracotic pigment-laden macrophages inside the alveoli with various degrees of interstitial fibrosis indicated a new disease: coal mine dust desquamative chronic interstitial pneumonia, a precursor of both dust-related diffuse fibrosis and emphysema. In studied coal miners, fibrosis caused by smoking is insignificant in comparison with fibrosis caused by silica/silicate particles. Counting birefringent particles in the macrophages from bronchioalveolar lavage may help detect coal mine dust desquamative chronic interstitial pneumonia, and may initiate early therapy and preventive measures.

Genome-wide analysis of long noncoding RNA profiling in PRRSV-infected PAM cells by RNA sequencing.

Porcine reproductive and respiratory syndrome (PRRS) is a major threat to the global swine industry and causes tremendous economic losses. Its causative agent, porcine reproductive and respiratory syndrome virus (PRRSV), primarily infects immune cells, such as porcine alveolar macrophages and dendritic cells. PRRSV infection results in immune suppression, antibody-dependent enhancement, and persistent infection. Highly pathogenic strains in China cause high fever and severe inflammatory responses in the lungs. However, the pathogenesis of PRRSV is still not fully understood. In this study, we analysed the long noncoding RNA (lncRNA) and mRNA expression profiles of the HP-PRRSV GSWW15 and the North American strain FL-12 in infected porcine alveolar macrophages (PAMs) at 12 and 24 hours post-infection. We predicted 12,867 novel lncRNAs, 299 of which were differentially expressed after viral infection. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses of the genes adjacent to lncRNAs showed that they were enriched in pathways related to viral infection and immune response, indicating that lncRNAs might play regulatory roles in virus-host interactions. Our study provided information about lncRNAs in the porcine immune system and offers new insights into the pathogenic mechanism of PRRSV infection and novel antiviral therapy development.

Pulmonary microRNA profiling: implications in upper lobe predominant lung disease.

BACKGROUND: Numerous pulmonary diseases manifest with upper lobe predominance including cystic fibrosis, smoking-related chronic obstructive pulmonary disease, and tuberculosis. Zonal hypoxia, characteristic of these pulmonary maladies, and oxygen stress in general is known to exert profound effects on various important aspects of cell biology. Lung macrophages are major participants in the pulmonary innate immune response and regional differences in macrophage responsiveness to hypoxia may contribute in the development of lung disease. MicroRNAs are ubiquitous regulators of human biology and emerging evidence indicates altered microRNA expression modulates respiratory disease processes. The objective of this study is to gain insight into the epigenetic and cellular mechanisms influencing regional differences in lung disease by investigating effect of hypoxia on regional microRNA expression in the lung. All studies were performed using primary alveolar macrophages (n = 10) or bronchoalveolar lavage fluid (n = 16) isolated from human subjects. MicroRNA was assayed via the NanoString nCounter microRNA assay. RESULTS: Divergent molecular patterns of microRNA expression were observed in alternate lung lobes, specifically noted was disparate expression of miR-93 and miR-4454 in alveolar macrophages along with altered expression of miR-451a and miR-663a in bronchoalveolar lavage fluid. Gene ontology was used to identify potential downstream targets of divergent microRNAs. Targets include cytokines and matrix metalloproteinases, molecules that could have a significant impact on pulmonary inflammation and fibrosis. CONCLUSIONS: Our findings show variant regional microRNA expression associated with hypoxia in alveolar macrophages and BAL fluid in the lung-upper vs lower lobe. Future studies should address whether these specific microRNAs may act intracellularly, in a paracrine/endocrine manner to direct the innate immune response or may ultimately be involved in pulmonary host-to-pathogen trans-kingdom cross-talk.

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.

Hemosiderin in sputum macrophages may predict infective exacerbations of chronic obstructive pulmonary disease: a retrospective observational study.

BACKGROUND: Infective exacerbations of COPD are common and are accompanied by neutrophilic bronchitis in sputum. Increased respiratory iron content has been associated with respiratory tract infection, though it is unclear if this represents a predisposing factor for infection or the sequelae of inflammation. Iron overload, as assessed in the airways, may be an important biomarker for recurrent infective exacerbations of COPD. The purpose of our study was to determine if hemosiderin in sputum macrophages is related to infective exacerbations of COPD. METHODS: We undertook a retrospective observational study of 54 consecutive patients who presented with an exacerbation of COPD and had sputum examined including assessment for hemosiderin in alveolar macrophages. The relation between infective exacerbations in the previous two years and the percent of hemosiderin-positive macrophages was analyzed with linear regression. To account for the non-parametric distribution of infective exacerbations, negative binomial regression modelling was used to account for other covariates. RESULTS: The percent of hemosiderin positive alveolar macrophages (hemosiderin index), analyzed parametrically and non-parametrically, demonstrated a significant correlation with increasing numbers of infective exacerbations in the previous two years. In a multivariate regression analysis, hemosiderin index was an independent predictor of infective exacerbations. COPD patients with raised hemosiderin index (≥20%) had higher levels of sputum IL-6 compared to patients with lower levels (<20%). CONCLUSIONS: High hemosiderin index in sputum alveolar macrophages measured at the time of AECOPD may be related to the frequency of infective exacerbations of COPD.