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1.
Life Sci Alliance ; 6(8)2023 08.
Article in English | MEDLINE | ID: mdl-37311583

ABSTRACT

Immunological targeting of pathological cells has been successful in oncology and is expanding to other pathobiological contexts. Here, we present a flexible platform that allows labeling cells of interest with the surface-expressed model antigen ovalbumin (OVA), which can be eliminated via either antigen-specific T cells or newly developed OVA antibodies. We demonstrate that hepatocytes can be effectively targeted by either modality. In contrast, pro-fibrotic fibroblasts associated with pulmonary fibrosis are only eliminated by T cells in initial experiments, which reduced collagen deposition in a fibrosis model. This new experimental platform will facilitate development of immune-based approaches to clear potential pathological cell types in vivo.


Subject(s)
Antibodies , Pulmonary Fibrosis , Humans , Fibroblasts , Hepatocytes , Kinetics
2.
Am J Respir Cell Mol Biol ; 55(6): 869-877, 2016 12.
Article in English | MEDLINE | ID: mdl-27486964

ABSTRACT

Protein kinase Cζ (PKCζ) is highly expressed in the lung, where it plays several regulating roles in the pathogenesis of acute lung injury (ALI). Proliferation and differentiation of integrin ß4+ distal lung epithelial progenitor cells seem to play a key role in proper lung regeneration. We investigated the effects of a myristoylated PKCζ inhibitor (PKCζi) in a murine model of bleomycin-induced ALI. After intratracheal injury, we treated mice three times a week with PKCζi or its vehicle, DMSO. We found that mice injured with bleomycin and then treated with PKCζi for one week showed decreased activation of PKCζ, improved lung compliance, and decreased lung protein permeability compared to injured mice treated with DMSO. Mice treated continuously with PKCζi for 6 weeks showed reduced evidence of lung fibrosis by computed tomographic images, decreased lung collagen deposition, and decreased active transforming growth factor-ß in the bronchoalveolar lavage fluid. In addition, we found an increased number of lung ß4+ cells compared to DMSO at Week 6. Therefore, we grew isolated integrin ß4+ lung progenitor cells in the presence of PKCζi or DMSO and found that ß4+ cells treated with PKCζi proliferated more in vitro compared to DMSO. We conclude that the use of a PKCζi promotes resolution of lung fibrosis in a bleomycin ALI model and increases the number of ß4+ progenitor cells with regenerative potential in the lung.


Subject(s)
Acute Lung Injury/drug therapy , Acute Lung Injury/enzymology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Acute Lung Injury/pathology , Animals , Bleomycin , Bronchoalveolar Lavage Fluid/cytology , Cell Death/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Separation , Compliance , Disease Models, Animal , Enzyme Activation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Mice, Inbred C57BL , Models, Biological , Neutrophils/cytology , Neutrophils/drug effects , Permeability , Protein Kinase C/metabolism , Protein Kinase Inhibitors/pharmacology , Pulmonary Fibrosis/complications , Pulmonary Fibrosis/enzymology , Pulmonary Fibrosis/pathology , Reactive Oxygen Species/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Transforming Growth Factor beta/metabolism
3.
Environ Monit Assess ; 187(6): 348, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25968554

ABSTRACT

It is common for hydrology researchers to collect data using in situ sensors at high frequencies, for extended durations, and with spatial distributions that produce data volumes requiring infrastructure for data storage, management, and sharing. The availability and utility of these data in addressing scientific questions related to water availability, water quality, and natural disasters relies on effective cyberinfrastructure that facilitates transformation of raw sensor data into usable data products. It also depends on the ability of researchers to share and access the data in useable formats. In this paper, we describe a data management and publication workflow and software tools for research groups and sites conducting long-term monitoring using in situ sensors. Functionality includes the ability to track monitoring equipment inventory and events related to field maintenance. Linking this information to the observational data is imperative in ensuring the quality of sensor-based data products. We present these tools in the context of a case study for the innovative Urban Transitions and Aridregion Hydrosustainability (iUTAH) sensor network. The iUTAH monitoring network includes sensors at aquatic and terrestrial sites for continuous monitoring of common meteorological variables, snow accumulation and melt, soil moisture, surface water flow, and surface water quality. We present the overall workflow we have developed for effectively transferring data from field monitoring sites to ultimate end-users and describe the software tools we have deployed for storing, managing, and sharing the sensor data. These tools are all open source and available for others to use.


Subject(s)
Environmental Monitoring/instrumentation , Information Storage and Retrieval , Water Pollution/analysis , Publications , Software , Water Pollution/statistics & numerical data , Workflow
4.
Environ Sci Nano ; 1(2): 123-132, 2014 Apr.
Article in English | MEDLINE | ID: mdl-25221673

ABSTRACT

Given the increased use of iron-containing nanoparticles in a number of applications, it is important to understand any effects that iron-containing nanoparticles can have on the environment and human health. Since iron concentrations are extremely low in body fluids, there is potential that iron-containing nanoparticles may influence the ability of bacteria to scavenge iron for growth, affect virulence and inhibit antimicrobial peptide (AMP) function. In this study, Pseudomonas aeruginosa (PA01) and AMPs were exposed to iron oxide nanoparticles, hematite (α-Fe2O3), of different sizes ranging from 2 to 540 nm (2 ± 1, 43 ± 6, 85 ± 25 and 540 ± 90 nm) in diameter. Here we show that the greatest effect on bacterial growth, biofilm formation, and AMP function impairment is found when exposed to the smallest particles. These results are attributed in large part to enhanced dissolution observed for the smallest particles and an increase in the amount of bioavailable iron. Furthermore, AMP function can be additionally impaired by adsorption onto nanoparticle surfaces. In particular, lysozyme readily adsorbs onto the nanoparticle surface which can lead to loss of peptide activity. Thus, this current study shows that co-exposure of nanoparticles and known pathogens can impact host innate immunity. Therefore, it is important that future studies be designed to further understand these types of impacts.

5.
Oncotarget ; 5(13): 4694-708, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-25051361

ABSTRACT

Myc (c-Myc) counteracts p27 effects, and low p27 usually correlates with high Myc expression in human cancer. However there is no information on the co-expression of both genes in chronic lymphocytic leukemia (CLL). We found a lack of correlation between RNA and protein levels of p27 and Myc in CLL cells, so we determined the protein levels by immunoblot in 107 cases of CLL. We observed a high p27 protein expression in CLL compared to normal B cells. Ectopic p27 expression in a CLL-derived cell line resulted in cell death resistance. Surprisingly, Myc expression was very low or undetectable in most CLL cases analyzed, with a clear correlation between high p27 and low Myc protein levels. This was associated with low Skp2 expression, which is consistent with the Skp2 role in p27 degradation and with SKP2 being a Myc target gene. High Myc expression did not correlate with leukemia progression, despite that cell cycle-related Myc target genes were upregulated. However, biochemical analysis showed that the high p27 levels inhibited cyclin-Cdk complexes even in Myc expressing CLL cells. Our data suggest that the combination of high p27 and low Myc is a marker of CLL cells which is mediated by Skp2.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Proto-Oncogene Proteins c-myb/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Cycle/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27/genetics , Cyclins/genetics , Cyclins/metabolism , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Leukemic , Humans , Immunoblotting , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Microscopy, Fluorescence , Middle Aged , Proto-Oncogene Proteins c-myb/genetics , Reverse Transcriptase Polymerase Chain Reaction , S-Phase Kinase-Associated Proteins/genetics
6.
PLoS One ; 9(1): e84434, 2014.
Article in English | MEDLINE | ID: mdl-24400090

ABSTRACT

BACKGROUND: Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury. METHODS: Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded. RESULTS: Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection. CONCLUSION: Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection.


Subject(s)
Aryldialkylphosphatase/genetics , Hypoxia/genetics , Oxidative Stress/genetics , Animals , Animals, Genetically Modified , Aryldialkylphosphatase/metabolism , Central Nervous System/metabolism , Diet , Disease Models, Animal , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression , Hypoxia/metabolism , Hypoxia/mortality , Male , Polymorphism, Genetic , Reactive Oxygen Species/metabolism
7.
PLoS One ; 8(2): e57673, 2013.
Article in English | MEDLINE | ID: mdl-23469047

ABSTRACT

Air pollution is a risk factor for respiratory infections, and one of its main components is particulate matter (PM), which is comprised of a number of particles that contain iron, such as coal fly ash (CFA). Since free iron concentrations are extremely low in airway surface liquid (ASL), we hypothesize that CFA impairs antimicrobial peptides (AMP) function and can be a source of iron to bacteria. We tested this hypothesis in vivo by instilling mice with Pseudomonas aeruginosa (PA01) and CFA and determine the percentage of bacterial clearance. In addition, we tested bacterial clearance in cell culture by exposing primary human airway epithelial cells to PA01 and CFA and determining the AMP activity and bacterial growth in vitro. We report that CFA is a bioavailable source of iron for bacteria. We show that CFA interferes with bacterial clearance in vivo and in primary human airway epithelial cultures. Also, we demonstrate that CFA inhibits AMP activity in vitro, which we propose as a mechanism of our cell culture and in vivo results. Furthermore, PA01 uses CFA as an iron source with a direct correlation between CFA iron dissolution and bacterial growth. CFA concentrations used are very relevant to human daily exposures, thus posing a potential public health risk for susceptible subjects. Although CFA provides a source of bioavailable iron for bacteria, not all CFA particles have the same biological effects, and their propensity for iron dissolution is an important factor. CFA impairs lung innate immune mechanisms of bacterial clearance, specifically AMP activity. We expect that identifying the PM mechanisms of respiratory infections will translate into public health policies aimed at controlling, not only concentration of PM exposure, but physicochemical characteristics that will potentially cause respiratory infections in susceptible individuals and populations.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Coal Ash/pharmacology , Coal , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Respiratory System/drug effects , Respiratory System/microbiology , Animals , Biological Availability , Coal Ash/chemistry , Epithelium/drug effects , Epithelium/metabolism , Epithelium/microbiology , Humans , Iron/analysis , Iron/pharmacokinetics , Male , Mice , Mice, Inbred C57BL , Pseudomonas aeruginosa/metabolism , Respiratory System/metabolism
8.
Environ Health Perspect ; 121(6): 691-8, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23478268

ABSTRACT

BACKGROUND: On 20 March 2010, the Icelandic volcano Eyjafjallajökull erupted for the first time in 190 years. Despite many epidemiological reports showing effects of volcanic ash on the respiratory system, there are limited data evaluating cellular mechanisms involved in the response to ash. Epidemiological studies have observed an increase in respiratory infections in subjects and populations exposed to volcanic eruptions. METHODS: We physicochemically characterized volcanic ash, finding various sizes of particles, as well as the presence of several transition metals, including iron. We examined the effect of Eyjafjallajökull ash on primary rat alveolar epithelial cells and human airway epithelial cells (20-100 µg/cm(2)), primary rat and human alveolar macrophages (5-20 µg/cm(2)), and Pseudomonas aeruginosa (PAO1) growth (3 µg/104 bacteria). RESULTS: Volcanic ash had minimal effect on alveolar and airway epithelial cell integrity. In alveolar macrophages, volcanic ash disrupted pathogen-killing and inflammatory responses. In in vitro bacterial growth models, volcanic ash increased bacterial replication and decreased bacterial killing by antimicrobial peptides. CONCLUSIONS: These results provide potential biological plausibility for epidemiological data that show an association between air pollution exposure and the development of respiratory infections. These data suggest that volcanic ash exposure, while not seriously compromising lung cell function, may be able to impair innate immunity responses in exposed individuals.


Subject(s)
Bacteria/growth & development , Immunity, Innate , Volcanic Eruptions/adverse effects , Adult , Animals , Autophagy , Bacteria/drug effects , Cells, Cultured , Female , Humans , Iceland , MAP Kinase Signaling System , Macrophages/immunology , Male , Microbial Viability/drug effects , Rats , Rats, Sprague-Dawley , beta-Defensins/pharmacology
9.
Am J Respir Cell Mol Biol ; 48(3): 306-13, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23221045

ABSTRACT

Recently, we reported that diesel exhaust particles (DEPs) disrupt tight junctions (TJs) in alveolar epithelial cells (AECs) via an increase in reactive oxygen species (ROS). In this study, we investigated the role of protein kinase C (PKC)-ζ activation in DEP-induced lung injury. C57/bl6 mice were instilled intratracheally with 50 µl of saline containing 100 µg of DEPs or titanium dioxide (TiO2). Twenty-four hours later, bronchoalveolar lavage was performed to assess neutrophil counts and protein concentrations. In addition, in vitro experiments were performed in primary rat and human AECs exposed to DEPs (50 µg/cm(2)) for 3 hours. Transepithelial electrical conductance was measured, and TJ protein association was analyzed by immunoprecipitation. To determine whether the overexpression of antioxidants prevented DEP-induced lung injury, AECs and mice were infected with adenoviruses containing catalase and manganese superoxide dismutase (MnSOD) plasmids. In vivo, the overexpression of catalase and MnSOD prevented DEP-induced neutrophil recruitment. The inhibition of PKC-ζ activation also prevented DEP-induced neutrophil recruitment in vivo. In vitro, DEPs activated PKC-ζ in AECs, but not in alveolar macrophages. Using a specific myristolated PKC-ζ pseudosubstrate pepetide (PKC-ζ ps), we showed that PKC-ζ mediated the DEP-induced dissociation of occludin and zonula occludin-1 (ZO1) in rat and human AECs. In addition, the overexpression of constitutively active PKC-ζ induced the dissociation of occludin and ZO1 in AECs. DEP-induced TJ disruption occurs via PKC-ζ. TJ disruption seems to be in part responsible for DEP-induced lung injury.


Subject(s)
Lung Injury/chemically induced , Lung Injury/enzymology , Protein Kinase C/metabolism , Vehicle Emissions/toxicity , Animals , Bronchoalveolar Lavage , Catalase/metabolism , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Lung Injury/metabolism , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Male , Mice , Mice, Inbred C57BL , Neutrophils/drug effects , Neutrophils/metabolism , Occludin/metabolism , Protein Kinase C/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism , Titanium/toxicity , Zonula Occludens-1 Protein/metabolism
10.
PLoS One ; 7(5): e37759, 2012.
Article in English | MEDLINE | ID: mdl-22662213

ABSTRACT

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.


Subject(s)
Co-Repressor Proteins/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Mitosis/genetics , S Phase/genetics , Transcription, Genetic , Cell Line , Cluster Analysis , Computational Biology/methods , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/chemistry , Cyclins/genetics , Gene Expression Profiling , Gene Expression Regulation , Humans , K562 Cells , Keratinocytes/metabolism , Promoter Regions, Genetic , Protein Binding
11.
Endocrinology ; 153(1): 512-21, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22109890

ABSTRACT

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular (125)iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.


Subject(s)
Chloride-Bicarbonate Antiporters/metabolism , Iodides/metabolism , Thyroid Gland/drug effects , Thyroid Gland/metabolism , Thyrotropin/pharmacology , Animals , Cell Line , Cell Membrane/metabolism , Chloride-Bicarbonate Antiporters/genetics , Colforsin/pharmacology , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Goiter, Nodular/genetics , Goiter, Nodular/metabolism , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/metabolism , Humans , Ion Transport/drug effects , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutagenesis, Site-Directed , Phosphorylation , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Sulfate Transporters , Thyrotropin/metabolism
12.
J Biol Chem ; 286(11): 9815-25, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21245140

ABSTRACT

SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.


Subject(s)
Down-Regulation , Gene Expression Regulation, Leukemic , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia/metabolism , Proto-Oncogene Proteins c-myc/metabolism , S-Phase Kinase-Associated Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cyclin-Dependent Kinase Inhibitor p27 , Female , G1 Phase/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , K562 Cells , Leukemia/genetics , Leukemia/pathology , Lymphoma/genetics , Lymphoma/metabolism , Lymphoma/pathology , Mice , Mice, Nude , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Response Elements/genetics , S Phase/genetics , S-Phase Kinase-Associated Proteins/genetics , Ubiquitin-Protein Ligases/genetics
13.
Am J Physiol Lung Cell Mol Physiol ; 300(4): L569-78, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21257729

ABSTRACT

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC.


Subject(s)
Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/enzymology , Cell Membrane/metabolism , Electric Conductivity , Membrane Proteins/metabolism , Protein Kinase C/metabolism , Protein Phosphatase 2/metabolism , Animals , Catalase/metabolism , Cell Hypoxia , Hydrogen-Ion Concentration , Lanthanum/metabolism , Male , Occludin , Partial Pressure , Protein Kinase C/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Signal Transduction , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Superoxides/metabolism
14.
Respirology ; 16(2): 340-9, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21122029

ABSTRACT

BACKGROUND AND OBJECTIVE: Inhaled particulate matter (PM) causes lung inflammation and epithelial dysfunction. However, the direct effect of PM on alveolar epithelial barrier integrity is not well understood. Our aim is to determine whether PM exposure affects the alveolar epithelial cells (AEC) transepithelial electrical conductance (Gt) and tight junction (TJ) proteins. METHODS: Human AEC (A549) and primary rat AEC were exposed to PM of <10 µm in size (PM(10) ) and diesel exhaust particles (DEP), using titanium dioxide (TiO(2) ) as a control for particle size effects. Gt and permeability to fluorescein isothiocyanate-dextran (FITC-Dextran) were measured to assess barrier integrity. TJ integrity was evaluated by analysing penetration of Lanthanum nitrate (La(3+) ) under transmission electron microscopy. Surface proteins were labelled with biotin and analysed by western blot. Immunofluorescence was performed to assess colocalization of TJ proteins including occludin and zonula occludens-1 (ZO-1). PM induced dissociation of occludin-ZO-1 was evaluated by co-immunoprecipitation. RESULTS: PM(10) and DEP increased Gt and disrupted TJ after 3 h of treatment. PM(10) and DEP induced occludin internalization from the plasma membrane into endosomal compartments and dissociation of occludin from ZO-1. Overexpression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and catalase, prevented PM-induced Gt increase, occludin reduction from the plasma membrane and its dissociation from ZO-1. CONCLUSIONS: PM induces alveolar epithelial dysfunction in part via occludin reduction at the plasma membrane and ZO-1 dissociation in AEC. Furthermore, these effects are prevented by overexpression of two different antioxidant enzymes.


Subject(s)
Electric Conductivity , Membrane Proteins/physiology , Particulate Matter/pharmacology , Pulmonary Alveoli/physiology , Animals , Catalase/metabolism , Cell Line , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cells, Cultured , Dextrans , Fluorescein-5-isothiocyanate/analogs & derivatives , Humans , Lanthanum , Male , Membrane Proteins/drug effects , Occludin , Phosphoproteins/drug effects , Phosphoproteins/physiology , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/enzymology , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolism , Tight Junctions/drug effects , Tight Junctions/physiology , Titanium/metabolism , Titanium/physiology , Vehicle Emissions/analysis , Zonula Occludens-1 Protein
15.
Invest Clin ; 51(1): 53-63, 2010 Mar.
Article in Spanish | MEDLINE | ID: mdl-20815156

ABSTRACT

Ischemia-reperfusion (IR) lung injury is a significant cause of morbidity and mortality in certain clinical scenarios that include transplantation, thromboendarterectomy and reexpansion injury of the lung. Edema of the contralateral lung after IR injury of one lung has been reported and this study was aimed to clarify the pathophysiology of this phenomenon. One-lung ischemia/hypoxia followed by reperfusion with either blood or an acellular plasma substitute was achieved in an isolated rabbit lung model by hilum clamping. After reperfusion, we studied the isolated effects of vasoconstriction and inflammation on contralateral lung injury by using papaverine or hydrocortisone as vasodilator and anti-inflammatory, respectively. We observed that IR of one lung induces edema of the contralateral lung. Absence of leukocytes and platelets in the perfusate or use of hydrocortisone completely inhibits IR injury. Moreover, papaverine suppresses edema of the contralateral, but not that of the reperfused lung. We concluded that IR of one lung produces edema in the contralateral lung that requires vasoconstriction of the latter.


Subject(s)
Lung/blood supply , Pulmonary Edema/etiology , Reperfusion Injury/complications , Reperfusion Injury/physiopathology , Vasoconstriction , Animals , Female , In Vitro Techniques , Rabbits
16.
Invest. clín ; 51(1): 53-63, Mar. 2010. ilus, tab
Article in Spanish | LILACS | ID: lil-574083

ABSTRACT

El daño pulmonar por isquemia-reperfusión (IR) es una importante causa de morbilidad y mortalidad en ciertas condiciones clínicas que incluyen trasplantes, tromboendarterectomía y daño pulmonar por reexpansión. El edema pulmonar contralateral posterior al daño por IR de un pulmón ha sido reportado y esta investigación tiene como objetivo esclarecer la fisiopatología de dicho fenómeno. En un modelo de pulmones aislados y perfundidos de conejo, fue ocluido el hilio pulmonar de forma unilateral induciendo isquemia/hipoxia de dicho órgano, seguido de reperfusiones con sangre o con un substituto plasmático acelular. Los efectos aislados de vasoconstricción e inflamación en el daño pulmonar contralateral fueron estudiados posterior a la reperfusión, usando papaverina e hidrocortisona como agente vasodilatador y antiinflamatorio, respectivamente. En esta investigación se observó que la IR de un pulmón induce edema en el pulmón contralateral. La ausencia de leucocitos y plaquetas en la perfusión y el uso de hidrocortisona inhibió por completo el daño por IR. La papaverina suprimió el edema en el pulmón contralateral mas no en el reperfundido. Se concluye que la IR de un pulmón produce edema en el pulmón contralateral, para lo cual se requiere la presencia de vasoconstricción.


Ischemia-reperfusion (IR) lung injury is a significant cause of morbidity and mortality in certain clinical scenarios that include transplantation, thromboendarterectomy and reexpansion injury of the lung. Edema of the contralateral lung after IR injury of one lung has been reported and this study was aimed to clarify the pathophysiology of this phenomenon. One-lung ischemia/hypoxia followed by reperfusion with either blood or an acellular plasma substitute was achieved in an isolated rabbit lung model by hilum clamping. After reperfusion, we studied the isolated effects of vasoconstriction and inflammation on contralateral lung injury by using papaverine or hydrocortisone as vasodilator and anti-inflammatory, respectively. We observed that IR of one lung induces edema of the contralateral lung. Absence of leukocytes and platelets in the perfusate or use of hydrocortisone completely inhibits IR injury. Moreover, papaverine suppresses edema of the contralateral, but not that of the reperfused lung. We concluded that IR of one lung produces edema in the contralateral lung that requires vasoconstriction of the latter.


Subject(s)
Animals , Rabbits , Pulmonary Edema/pathology , Ischemia/complications , Reperfusion Injury , Vasoconstriction , Animals, Laboratory , Lung Diseases
17.
Cancer Lett ; 292(1): 133-9, 2010 Jun 01.
Article in English | MEDLINE | ID: mdl-20042273

ABSTRACT

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21(Cip1), initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21(Cip1) expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21(Cip1) in the response to imatinib in CML.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Drug Resistance, Neoplasm , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/antagonists & inhibitors , Piperazines/therapeutic use , Pyrimidines/antagonists & inhibitors , Pyrimidines/therapeutic use , Apoptosis/drug effects , Benzamides , Cell Line, Tumor , G2 Phase/drug effects , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
18.
Invest Clin ; 49(2): 181-93, 2008 Jun.
Article in Spanish | MEDLINE | ID: mdl-18717265

ABSTRACT

Hypocapnia/alkalosis is a consequence of several lung and metabolic pathologies. The aim of this study was to determine whether the increase of fluid filtration rate (FFR) that occurs during Hypocapnia/alkalosis circumstances is determined by hypocapnia, alkalosis or both. 7 groups were formed (N=36) using isolated rabbit lungs. Group 1: Control (PCO2 6%, pH: 7.35-7.45); Group 2 (n=6): Hypocapnia/Alkalosis (CO2 1%, pH: 7.9); Group 3 (n=6): Hypocapnia/Normo-pH (CO2 1% pH 7.35-7.45), Group 4 (n=6) Normocapnia/Alcalosis (CO2 6%, pH: 7.9). Fenoterol, papaverine and hydrocortisone were added to Groups 5, 6 and 7 (n=4) respectively, all under Normocapnia/Alkalosis. FFR and Pulmonary Arterial Pressure (Pap) were considerably higher in group 2 than in control (FFR: 1.92g/min +/- 0.6 vs 0.0 g/min +/- 0.006). A strong influence exerted by pH was observed when Group 3 and group 4 were compared (FFR: 0.02 g/min +/- 0.009 vs 2.3 g/min +/- 0.9) and (Pap: 13.5 cmH2O +/- 1.4 vs 90 cmH2O +/- 15). A reduced effect was observed in groups 5 and 6 (papaverine and hydrocorisone) and a totally abolished effect was observed in group 7 (fenoterol) (FFR: 0.001 +/- 0.0003 mL/min and Pap: 14 +/- 0.8 cmH2O). Pulmonary edema induced by Hypocapnia/alkalosis is a consequence of alkalosis and not of hypocapnia. This effect could be due to inflammatory damage in the lung parenchyma and alkalosis-mediated vasoconstriction.


Subject(s)
Alkalosis/physiopathology , Fluid Shifts/physiology , Hypocapnia/physiopathology , Lung/physiopathology , Pulmonary Edema/physiopathology , Adrenergic beta-Agonists/pharmacology , Alkalosis/complications , Animals , Anti-Inflammatory Agents/pharmacology , Blood Pressure/drug effects , Fenoterol/pharmacology , Fluid Shifts/drug effects , Hydrocortisone/pharmacology , Hydrogen-Ion Concentration , Hypocapnia/complications , Lung/blood supply , Lung/drug effects , Papaverine/pharmacology , Perfusion , Pulmonary Artery , Pulmonary Edema/etiology , Rabbits , Vasoconstriction/drug effects , Vasoconstriction/physiology , Vasodilator Agents/pharmacology
19.
Invest. clín ; 49(2): 181-193, jun. 2008. ilus, tab, graf
Article in Spanish | LILACS | ID: lil-518687

ABSTRACT

La hipocapnia/alcalosis es una situación que se presenta como consecuencia de diversas patologías pulmonares o metabólicas. El objetivo de este estudio fue determinar si el aumento de la tasa de filtración de liquido (TFL) que ocurre bajo estas circunstancias, está determinado por la hipocapnia, la alcalosis o la suma de ambas. Se realizaron 7 grupos (n=36), utilizando pulmones aislados de conejos. Grupo 1: Control (PCO2 6 por ciento, pH: 7,35-7,45); Grupo 2 (n=6): Hipocapnia/Alcalosis (CO2 1 por ciento, pH: 7,9); Grupo 3 (n=6): Hipocapnia/Normo-pH (CO2 1 por ciento pH 7,35-7,45), Grupo 4 (n=6) Normocapnia/Alcalosis (CO2 6 por ciento, pH: 7,9). En los grupos 5, 6 y 7 (n=4), todos bajo condición de Normocapnia/Alcalosis se añadió fenoterol, papaverina, e hidrocortisona respectivamente. La TFL y la presión de arteria pulmonar (Pap) fueron considerablemente mayores en el grupo 2 que en el control (TFL:1,92g/min ± 0,6 vs 0,0g/min ± 0,006), observándose una marcada influencia del pH, al comparar el grupo 3 y el grupo 4 (TFL: 0,02g/min ± 0,009 vs 2,3g/min ± 0,9) y (Pap: 13,5 cmH2O ± 1,4 vs 90 cmH2O ± 15). Se observó una disminución del efecto en los grupos 5 y 6 (papaverina e hidrocortisona) y su abolición total con fenoterol (grupo 7) (TFL: 0,001 ± 0,0003 g/min y Pap: 14 ± 0,8 cmH2O). El edema pulmonar inducido por Hipocapnia/Alcalosis es consecuencia principalmente de la alcalosis y no de la hipocapnia. Dicho efecto podría ser debido a un daño inflamatorio a nivel del parénquima y a la vasoconstricción causada por la alcalosis.


Subject(s)
Animals , Rabbits , Alkalosis , Pulmonary Edema/pathology , Fenoterol , Hydrocortisone , Hypocapnia , Papaverine
20.
Respirology ; 12(6): 799-806, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17986106

ABSTRACT

BACKGROUND AND OBJECTIVE: Mechanical obstruction has been considered the prime determinant of haemodynamic changes after pulmonary embolism (PE); however, the function of vasoconstrictive and inflammatory mediators in the physiopathology of this disease is unclear. The aim of this investigation was to study the effect of an anti-inflammatory and a vasodilator in a setting of induced PE. METHODS: A prospective, laboratory study was undertaken using 30 New Zealand white rabbits. A model of isolated and perfused rabbit lungs was used; PE was induced using autologous blood clots. Six study groups were established (each n = 5): PE without any drug (PG); PE + papaverine (PpG); PE + hydrocortisone (HG); PE in West's Zone III (ZIIIG); PE using acellular perfusate (AG) and PE using acellular perfusate + papaverine (APpG). The pulmonary artery pressure (PAP) and fluid filtration rate (FFR) were continuously measured during the experiments. RESULTS: Increases in PAP and oedema formation were observed in the PG after embolization. The PpG and the APpG showed neither oedema nor significant PAP increases. The HG group developed less oedema and less increase in PAP compared with the PG. The ZIIIG developed oedema the fastest. The AG developed less oedema and increases in PAP compared with the PG. CONCLUSION: These findings suggest that vasoconstriction and inflammatory mediators play an important role in the physiopathology of PE, as neither PAP increases nor oedema were observed in the PpG and a reduction of oedema and PAP was seen in the HG group. The decrease in oedema and PAP in the acellular group strongly suggests a key role of circulating blood cells.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Hydrocortisone/pharmacology , Papaverine/pharmacology , Pulmonary Embolism/physiopathology , Vasoconstriction/physiology , Vasodilator Agents/pharmacology , Animals , Disease Models, Animal , Hemodynamics , In Vitro Techniques , Prospective Studies , Pulmonary Edema/physiopathology , Rabbits , Respiratory Mechanics
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