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Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K.
Elajaili HB, Hernandez-Lagunas L, Ranguelova K, Dikalov S, Nozik-Grayck E
(2019) J Vis Exp :
MeSH Terms: Animals, Antimycin A, Bleomycin, Bronchoalveolar Lavage Fluid, Cattle, Cell Compartmentation, Electron Spin Resonance Spectroscopy, Lung, Mice, Mitochondria, Oxidation-Reduction, RAW 264.7 Cells, Superoxides, Temperature
Show Abstract · Added March 26, 2019
The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments. In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide). Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 °C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 °C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.
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14 MeSH Terms
Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates.
Bates JT, Pickens JA, Schuster JE, Johnson M, Tollefson SJ, Williams JV, Davis NL, Johnston RE, Schultz-Darken N, Slaughter JC, Smith-House F, Crowe JE
(2016) Vaccine 34: 950-6
MeSH Terms: Alphavirus, Animals, Antibodies, Neutralizing, Antibodies, Viral, Bronchoalveolar Lavage Fluid, Chlorocebus aethiops, Encephalitis Virus, Venezuelan Equine, Immunoglobulin G, Metapneumovirus, Neutralization Tests, Nose, Paramyxoviridae Infections, Replicon, Respiratory Syncytial Virus Infections, Respiratory Syncytial Virus, Human, Viral Fusion Proteins, Viral Vaccines
Show Abstract · Added January 26, 2016
Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.
Copyright © 2016 Elsevier Ltd. All rights reserved.
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17 MeSH Terms
Myeloid-related protein-8/14 facilitates bacterial growth during pneumococcal pneumonia.
Achouiti A, Vogl T, Endeman H, Mortensen BL, Laterre PF, Wittebole X, van Zoelen MA, Zhang Y, Hoogerwerf JJ, Florquin S, Schultz MJ, Grutters JC, Biesma DH, Roth J, Skaar EP, van 't Veer C, de Vos AF, van der Poll T
(2014) Thorax 69: 1034-42
MeSH Terms: Animals, Bronchoalveolar Lavage Fluid, Calgranulin B, Disease Models, Animal, Female, Humans, Lung, Male, Mice, Mice, Inbred C57BL, Pneumonia, Pneumococcal, Streptococcus pneumoniae
Show Abstract · Added January 24, 2015
BACKGROUND - Streptococcus pneumoniae is the most commonly identified pathogen in community-acquired pneumonia (CAP). Myeloid-related protein (MRP) 8/14 is a major component of neutrophils that is released upon infection or injury. MRP8/14 is essential for protective immunity during infection by a variety of micro-organisms through its capacity to chelate manganese and zinc. Here, we aimed to determine the role of MRP8/14 in pneumococcal pneumonia.
METHODS - MRP8/14 was determined in bronchoalveolar lavage fluid (BALF) and serum of CAP patients, in lung tissue of patients who had succumbed to pneumococcal pneumonia, and in BALF of healthy subjects challenged with lipoteichoic acid (a component of the gram-positive bacterial cell wall) via the airways. Pneumonia was induced in MRP14 deficient and normal wildtype mice. The effect of MRP8/14 on S. pneumoniae growth was studied in vitro.
RESULTS - CAP patients displayed high MRP8/14 levels in BALF, lung tissue and serum. Healthy subjects challenged with lipoteichoic acid demonstrated elevated MRP8/14 in BALF. Likewise, mice with pneumococcal pneumonia had high MRP8/14 levels in lungs and the circulation. MRP14 deficiency, however, was associated with reduced bacterial growth and lethality, in the absence of notable effects on the inflammatory response. High zinc levels strongly inhibited growth of S. pneumoniae in vitro, which was partially reversed by MRP8/14.
CONCLUSIONS - In sharp contrast to its previously reported host-protective role in several infections, the present results reveal that in a model of CAP, MRP8/14 is misused by S. pneumoniae, facilitating bacterial growth by attenuating zinc toxicity toward the pathogen.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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12 MeSH Terms
Glutathione S-transferase M1 modulates allergen-induced NF-κB activation in asthmatic airway epithelium.
Polosukhin VV, Polosukhin IV, Hoskins A, Han W, Abdolrasulnia R, Blackwell TS, Dworski R
(2014) Allergy 69: 1666-72
MeSH Terms: Adult, Allergens, Animals, Asthma, Bronchoalveolar Lavage Fluid, Cytokines, Enzyme Activation, Eosinophils, Female, Genotype, Glutathione Transferase, Humans, Male, Middle Aged, NF-kappa B, Neutrophils, Respiratory Mucosa, Young Adult
Show Abstract · Added February 19, 2015
BACKGROUND - Glutathione S-transferase M1 (GSTM1) is a phase II enzyme and regulator of inflammatory signaling in airway epithelial cells. We have found upregulation of neutrophilic airway inflammation in atopic asthmatics expressing GSTM1 gene (GSTM1+) compared to GSTM1null asthmatics. We hypothesized that GSTM1 modulates NF-κB activation in bronchial epithelium in atopic asthmatics. We determined regulation of allergen-induced NF-κB activation in bronchial epithelium by GSTM1 in human atopic asthmatics in vivo.
METHODS - Endobronchial biopsies and bronchoalveolar lavage fluid samples were collected from 13 GSTM1+ and 12 GSTM1null human atopic asthmatics at baseline and 24 h after segmental allergen challenge. A quantitative analysis of NF-κB activation in airway epithelium was accomplished using a polyclonal antibody against the phosphorylated p65 component of NF-κB. Elastase-positive neutrophils in the bronchial wall were quantified.
RESULTS - Postallergen neutrophilia in airway subepithelium and epithelial lining fluid was greater in GSTM1+ compared to GSTM1null asthmatics. Airway eosinophilia was similar in GSTM1+ and GSTM1null asthmatics. Allergen-provoked NF-κB induction in bronchial epithelium was significantly greater in GSTM1+ compared to GSTM1null asthmatics. Activation of NF-κB activation in airway epithelial cells correlated with interleukin-8 concentrations and absolute neutrophil numbers in bronchoalveolar lavage fluid in GSTM1+ but not GSTM1null asthmatics.
CONCLUSIONS - Allergen-induced neutrophilic airway inflammation in GSTM1+ asthmatics is associated with NF-κB activation in airway epithelial cells in vivo. These novel data provide a potential mechanism of the genomic link between GSTM1 polymorphism and airway neutrophilia in atopic asthma.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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18 MeSH Terms
Differential role for p120-catenin in regulation of TLR4 signaling in macrophages.
Yang Z, Sun D, Yan Z, Reynolds AB, Christman JW, Minshall RD, Malik AB, Zhang Y, Hu G
(2014) J Immunol 193: 1931-41
MeSH Terms: Acute Lung Injury, Adaptor Proteins, Vesicular Transport, Animals, Bronchoalveolar Lavage Fluid, Catenins, Cells, Cultured, Endocytosis, Interferon Regulatory Factor-3, Interferon-beta, Interleukin-6, Leukocyte Count, Lipopolysaccharides, Macrophages, Alveolar, Male, Mice, Mice, Inbred C57BL, Myeloid Differentiation Factor 88, NF-kappa B, Neutrophils, Protein Transport, RNA Interference, Signal Transduction, Toll-Like Receptor 2, Toll-Like Receptor 3, Toll-Like Receptor 4, Tumor Necrosis Factor-alpha, rhoA GTP-Binding Protein
Show Abstract · Added May 2, 2016
Activation of TLR signaling through recognition of pathogen-associated molecular patterns is essential for the innate immune response against bacterial and viral infections. We have shown that p120-catenin (p120) suppresses TLR4-mediated NF-кB signaling in LPS-challenged endothelial cells. In this article, we report that p120 differentially regulates LPS/TLR4 signaling in mouse bone marrow-derived macrophages. We observed that p120 inhibited MyD88-dependent NF-κB activation and release of TNF-α and IL-6, but enhanced TIR domain-containing adapter-inducing IFN-β-dependent IFN regulatory factor 3 activation and release of IFN-β upon LPS exposure. p120 silencing diminished LPS-induced TLR4 internalization, whereas genetic and pharmacological inhibition of RhoA GTPase rescued the decrease in endocytosis of TLR4 and TLR4-MyD88 signaling, and reversed the increase in TLR4-TIR domain-containing adapter-inducing IFN-β signaling induced by p120 depletion. Furthermore, we demonstrated that altered p120 expression in macrophages regulates the inflammatory phenotype of LPS-induced acute lung injury. These results indicate that p120 functions as a differential regulator of TLR4 signaling pathways by facilitating TLR4 endocytic trafficking in macrophages, and support a novel role for p120 in influencing the macrophages in the lung inflammatory response to endotoxin.
Copyright © 2014 by The American Association of Immunologists, Inc.
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27 MeSH Terms
Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples.
Bastarache JA, Koyama T, Wickersham NE, Ware LB
(2014) J Immunol Methods 408: 13-23
MeSH Terms: Animals, Biomarkers, Bronchoalveolar Lavage Fluid, Electrochemical Techniques, Enzyme-Linked Immunosorbent Assay, Humans, Inflammation Mediators, Interleukin-6, Interleukin-8, Interleukins, Limit of Detection, Luminescent Measurements, Mice, Mice, Inbred C57BL, Predictive Value of Tests, Reagent Kits, Diagnostic, Reproducibility of Results, Species Specificity
Show Abstract · Added May 19, 2014
BACKGROUND - Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance.
METHODS - In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD®) and compare this platform to validated, singleplex immunoassays (R&D Systems®) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BAL) and plasma) samples.
RESULTS - We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD were 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5 to 28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations.
CONCLUSIONS - In conclusion, we validated a multiplex electrochemiluminescent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISAs.
Published by Elsevier B.V.
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18 MeSH Terms
Utility of bronchoscopy in pulmonary Langerhans cell histiocytosis.
Baqir M, Vassallo R, Maldonado F, Yi ES, Ryu JH
(2013) J Bronchology Interv Pulmonol 20: 309-12
MeSH Terms: Adult, Aged, Antigens, CD1, Biopsy, Bronchoalveolar Lavage, Bronchoalveolar Lavage Fluid, Bronchoscopy, Female, Histiocytosis, Langerhans-Cell, Humans, Male, Middle Aged, Predictive Value of Tests, Retrospective Studies, Smoking, Staining and Labeling, Young Adult
Show Abstract · Added February 1, 2016
BACKGROUND - Pulmonary Langerhans cell histiocytosis (PLCH) is an uncommon form of interstitial lung disease and is usually smoking-related when seen in adults. There are relatively little data regarding the utility of bronchoscopic lung biopsy for this disorder.
METHODS - A computer-assisted search was carried out to identify patients with PLCH seen at Mayo Clinic Rochester, MN from 1997 to 2012 and who underwent bronchoscopy with lung biopsy. Approval was obtained from the Mayo Foundation Institutional Review Board before beginning the study. Medical records of these patients were reviewed to extract data with regard to demographic and clinical features, imaging studies, and biopsy results.
RESULTS - Thirty-eight patients with PLCH underwent diagnostic bronchoscopy with biopsies. Their median age was 39.5 years (range, 21 to 66 y) and included 24 women. Thirty-two patients (84%) were current smokers at the time of the diagnosis, 5 were ex-smokers (13%), and 1 was a never-smoker (3%). The diagnosis of PLCH required the presence of typical histopathologic features on surgical or bronchoscopic lung biopsy, >5% CD1a-positive cells in the bronchoalveolar lavage (BAL), and/or biopsy of an extrapulmonary site in the presence of clinical and chest computed tomographic findings compatible with the diagnosis. Bronchoscopic biopsy yielded diagnostic specimens that allowed the diagnosis of PLCH in 19 patients (50%). CD1a immunostaining of BAL cells had been performed in 8 patients and demonstrated ≥ 5% CD1a-positive BAL cells in 3 additional patients (8%).
CONCLUSIONS - We conclude that bronchoscopic lung biopsy is useful in the diagnosis of PLCH and should be the initial method of obtaining diagnostic specimens.
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17 MeSH Terms
Loss of mitochondrial protein Fus1 augments host resistance to Acinetobacter baumannii infection.
Hood MI, Uzhachenko R, Boyd K, Skaar EP, Ivanova AV
(2013) Infect Immun 81: 4461-9
MeSH Terms: Acinetobacter Infections, Acinetobacter baumannii, Animals, Bronchoalveolar Lavage Fluid, Interleukin-10, Interleukin-17, Lung, Macrophages, Male, Membrane Potential, Mitochondrial, Mice, Mice, Knockout, NF-kappa B, Neutrophils, Phosphatidylinositol 3-Kinase, Pneumonia, Bacterial, Proto-Oncogene Proteins c-akt, Reactive Oxygen Species, TOR Serine-Threonine Kinases, Tumor Suppressor Proteins
Show Abstract · Added March 7, 2014
Fus1 is a tumor suppressor protein with recently described immunoregulatory functions. Although its role in sterile inflammation is being elucidated, its role in regulating immune responses to infectious agents has not been examined. We used here a murine model of Acinetobacter baumannii pneumonia to identify the role of Fus1 in antibacterial host defenses. We found that the loss of Fus1 in mice results in significantly increased resistance to A. baumannii pneumonia. We observed earlier and more robust recruitment of neutrophils and macrophages to the lungs of infected Fus1(-/-) mice, with a concomitant increase in phagocytosis of invading bacteria and more rapid clearance. Such a prompt and enhanced immune response to bacterial infection in Fus1(-/-) mice stems from early activation of proinflammatory pathways (NF-κB and phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin [mTOR]), most likely due to significantly increased mitochondrial membrane potential and mitochondrial reactive oxygen species production. Significant early upregulation of interleukin-17 (IL-17) in Fus1(-/-) immune cells was also observed, together with significant downregulation of IL-10. Depletion of neutrophils eliminates the enhanced antibacterial defenses of the Fus1(-/-) mice, suggesting that ultimately it is the enhanced immune cell recruitment that mediates the increased resistance of Fus1(-/-) mice to A. baumannii pneumonia. Taken together, our data define the novel role for Fus1 in the immune response to A. baumannii pneumonia and highlight new avenues for immune modulating therapeutic targets for this treatment-resistant nosocomial pathogen.
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20 MeSH Terms
Investigation of complement activation product c4d as a diagnostic and prognostic biomarker for lung cancer.
Ajona D, Pajares MJ, Corrales L, Perez-Gracia JL, Agorreta J, Lozano MD, Torre W, Massion PP, de-Torres JP, Jantus-Lewintre E, Camps C, Zulueta JJ, Montuenga LM, Pio R
(2013) J Natl Cancer Inst 105: 1385-93
MeSH Terms: Adult, Aged, Biomarkers, Tumor, Bronchoalveolar Lavage Fluid, Complement Activation, Complement C4b, Complement Pathway, Classical, Early Detection of Cancer, Female, Humans, Lung Neoplasms, Male, Middle Aged, Neoplasm Staging, Peptide Fragments, Predictive Value of Tests, Prognosis
Show Abstract · Added March 7, 2014
BACKGROUND - There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments.
METHODS - We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test.
RESULTS - Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 ± 0.87 vs 0.16 ± 0.11 µg/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 ± 2.02 vs 1.86 ± 0.95 µg/mL, P < 0.001; 3.18 ± 3.20 vs 1.13 ± 0.69 µg/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93).
CONCLUSIONS - Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.
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17 MeSH Terms
Reversal of global CD4+ subset dysfunction is associated with spontaneous clinical resolution of pulmonary sarcoidosis.
Oswald-Richter KA, Richmond BW, Braun NA, Isom J, Abraham S, Taylor TR, Drake JM, Culver DA, Wilkes DS, Drake WP
(2013) J Immunol 190: 5446-53
MeSH Terms: Adult, Aged, Bronchoalveolar Lavage Fluid, CD4-Positive T-Lymphocytes, Clonal Anergy, Female, Humans, Interleukin-2, Male, Middle Aged, Receptors, Antigen, T-Cell, Remission, Spontaneous, Sarcoidosis, Pulmonary, Signal Transduction, T-Lymphocyte Subsets, T-Lymphocytes, Helper-Inducer, T-Lymphocytes, Regulatory, Young Adult
Show Abstract · Added May 27, 2014
Sarcoidosis pathogenesis is characterized by peripheral anergy and an exaggerated, pulmonary CD4(+) Th1 response. In this study, we demonstrate that CD4(+) anergic responses to polyclonal TCR stimulation are present peripherally and within the lungs of sarcoid patients. Consistent with prior observations, spontaneous release of IL-2 was noted in sarcoidosis bronchoalveolar lavage CD4(+) T cells. However, in contrast to spontaneous hyperactive responses reported previously, the cells displayed anergic responses to polyclonal TCR stimulation. The anergic responses correlated with diminished expression of the Src kinase Lck, protein kinase C-θ, and NF-κB, key mediators of IL-2 transcription. Although T regulatory (Treg) cells were increased in sarcoid patients, Treg depletion from the CD4(+) T cell population of sarcoidosis patients did not rescue IL-2 and IFN-γ production, whereas restoration of the IL-2 signaling cascade, via protein kinase C-θ overexpression, did. Furthermore, sarcoidosis Treg cells displayed poor suppressive capacity indicating that T cell dysfunction was a global CD4(+) manifestation. Analyses of patients with spontaneous clinical resolution revealed that restoration of CD4(+) Th1 and Treg cell function was associated with resolution. Conversely, disease progression exhibited decreased Th1 cytokine secretion and proliferative capacity, and reduced Lck expression. These findings implicate normalized CD4(+) T cell function as a potential therapeutic target for sarcoidosis resolution.
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18 MeSH Terms