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2_test

{"project":"2_test","denotations":[{"id":"19436703-15860753-86274485","span":{"begin":246,"end":248},"obj":"15860753"},{"id":"19436703-17277157-86274486","span":{"begin":1810,"end":1812},"obj":"17277157"},{"id":"19436703-15860753-86274487","span":{"begin":1964,"end":1966},"obj":"15860753"},{"id":"19436703-17277157-86274488","span":{"begin":2233,"end":2235},"obj":"17277157"},{"id":"19436703-11395507-86274489","span":{"begin":2940,"end":2942},"obj":"11395507"},{"id":"19436703-17277157-86274490","span":{"begin":3831,"end":3833},"obj":"17277157"},{"id":"19436703-10958685-86274491","span":{"begin":4595,"end":4597},"obj":"10958685"},{"id":"19436703-15860753-86274492","span":{"begin":6282,"end":6284},"obj":"15860753"},{"id":"T86367","span":{"begin":246,"end":248},"obj":"15860753"},{"id":"T38919","span":{"begin":1810,"end":1812},"obj":"17277157"},{"id":"T24071","span":{"begin":1964,"end":1966},"obj":"15860753"},{"id":"T69573","span":{"begin":2233,"end":2235},"obj":"17277157"},{"id":"T8362","span":{"begin":2940,"end":2942},"obj":"11395507"},{"id":"T57036","span":{"begin":3831,"end":3833},"obj":"17277157"},{"id":"T21764","span":{"begin":4595,"end":4597},"obj":"10958685"},{"id":"T87031","span":{"begin":6282,"end":6284},"obj":"15860753"}],"text":"Materials and Methods\n\nParticipants and Study Design\nWe studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.\n\nAntibodies and Reagents\nThe monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).\n\nCell Culture and PBMC Isolation\nA human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].\n\nReverse Transcription PCR\nTotal RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.\n\nCell Fractionation, Immunoprecipitation, and Western Blot Analysis\nNuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).\n\nChromatin Immunoprecipitation\nChromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C. Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse: 5′-TCATGGCTCTGAAACGTTCTG-3′. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.\n\nGR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)\nImmunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).\n\nNF-κB Activation\nNF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.\n\nImmunofluorescence Staining\nImmunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.\n\nGATA-3-GFP Construct\nThe GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C. Klenow was inactivated by incubation for 10 min at 75°C. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.\n\nTransfection\nHuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.\n\nTime-Lapse Microscopy\nHuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.\n\nDensitometric Analysis\nDensitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.\n\nStatistical Analysis\nData from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p\u003c0.05.\n"}

pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T6271","span":{"begin":7237,"end":7242},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T7168","span":{"begin":8588,"end":8593},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"}],"text":"Materials and Methods\n\nParticipants and Study Design\nWe studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.\n\nAntibodies and Reagents\nThe monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).\n\nCell Culture and PBMC Isolation\nA human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].\n\nReverse Transcription PCR\nTotal RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.\n\nCell Fractionation, Immunoprecipitation, and Western Blot Analysis\nNuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).\n\nChromatin Immunoprecipitation\nChromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C. Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse: 5′-TCATGGCTCTGAAACGTTCTG-3′. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.\n\nGR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)\nImmunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).\n\nNF-κB Activation\nNF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.\n\nImmunofluorescence Staining\nImmunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.\n\nGATA-3-GFP Construct\nThe GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C. Klenow was inactivated by incubation for 10 min at 75°C. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.\n\nTransfection\nHuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.\n\nTime-Lapse Microscopy\nHuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.\n\nDensitometric Analysis\nDensitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.\n\nStatistical Analysis\nData from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p\u003c0.05.\n"}

GO-BP

{"project":"GO-BP","denotations":[{"id":"T4298","span":{"begin":1169,"end":1172},"obj":"http://purl.obolibrary.org/obo/GO_0004707"},{"id":"T4299","span":{"begin":1499,"end":1511},"obj":"http://purl.obolibrary.org/obo/GO_0009293"},{"id":"T4599","span":{"begin":2161,"end":2173},"obj":"http://purl.obolibrary.org/obo/GO_0051179"},{"id":"T4829","span":{"begin":2239,"end":2260},"obj":"http://purl.obolibrary.org/obo/GO_0001171"},{"id":"T4830","span":{"begin":2247,"end":2260},"obj":"http://purl.obolibrary.org/obo/GO_0006351"},{"id":"T4831","span":{"begin":2295,"end":2300},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T4832","span":{"begin":2527,"end":2529},"obj":"http://purl.obolibrary.org/obo/GO_0003964"},{"id":"T4833","span":{"begin":2587,"end":2589},"obj":"http://purl.obolibrary.org/obo/GO_0003964"},{"id":"T5179","span":{"begin":2987,"end":2992},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T5180","span":{"begin":3214,"end":3222},"obj":"http://purl.obolibrary.org/obo/GO_0007349"},{"id":"T6713","span":{"begin":6997,"end":7003},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T7113","span":{"begin":7665,"end":7674},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T7114","span":{"begin":8314,"end":8323},"obj":"http://purl.obolibrary.org/obo/GO_0007586"},{"id":"T7596","span":{"begin":8930,"end":8936},"obj":"http://purl.obolibrary.org/obo/GO_0040007"}],"text":"Materials and Methods\n\nParticipants and Study Design\nWe studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.\n\nAntibodies and Reagents\nThe monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).\n\nCell Culture and PBMC Isolation\nA human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].\n\nReverse Transcription PCR\nTotal RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.\n\nCell Fractionation, Immunoprecipitation, and Western Blot Analysis\nNuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).\n\nChromatin Immunoprecipitation\nChromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C. Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse: 5′-TCATGGCTCTGAAACGTTCTG-3′. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.\n\nGR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)\nImmunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).\n\nNF-κB Activation\nNF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.\n\nImmunofluorescence Staining\nImmunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.\n\nGATA-3-GFP Construct\nThe GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C. Klenow was inactivated by incubation for 10 min at 75°C. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.\n\nTransfection\nHuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.\n\nTime-Lapse Microscopy\nHuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.\n\nDensitometric Analysis\nDensitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.\n\nStatistical Analysis\nData from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p\u003c0.05.\n"}

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GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T3954","span":{"begin":83,"end":89},"obj":"http://purl.bioontology.org/ontology/ICD10/J45.9"},{"id":"T3955","span":{"begin":276,"end":282},"obj":"http://purl.bioontology.org/ontology/ICD10/J45.9"},{"id":"T3956","span":{"begin":83,"end":89},"obj":"http://purl.bioontology.org/ontology/ICD10/J45"},{"id":"T3957","span":{"begin":276,"end":282},"obj":"http://purl.bioontology.org/ontology/ICD10/J45"}],"text":"Materials and Methods\n\nParticipants and Study Design\nWe studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 µg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.\n\nAntibodies and Reagents\nThe monoclonal antibodies against human CD3, CD28, GR, and importin-α were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).\n\nCell Culture and PBMC Isolation\nA human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 µg/ml each) for 1 h at 37°C to stimulate Th2 cytokine release in the presence or absence of FP (10−12 to 10−8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].\n\nReverse Transcription PCR\nTotal RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.\n\nCell Fractionation, Immunoprecipitation, and Western Blot Analysis\nNuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4°C for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 µl of antibody against GATA-3 or importin-α using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-α, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).\n\nChromatin Immunoprecipitation\nChromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 µg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4°C. Promoter sequences were detected with PCR primers for the IL-5 promoter (−445 to +4): forward 5′-TTAATCTAGCCACAGTCATAG-3′ and reverse: 5′-TCATGGCTCTGAAACGTTCTG-3′. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72°C for 10 min, 35 cycles at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s.\n\nGR-GATA-3 In Vitro Competition Assay for Importin-α (Far-Western ELISA)\nImmunoprecipitated importin-α (anti-importin-α, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10−8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 µl of importin-α solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-α antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-α-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).\n\nNF-κB Activation\nNF-κB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 µg of nuclear extracts were incubated with a plate coated with an NF-κB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.\n\nImmunofluorescence Staining\nImmunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1∶50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate–conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50∶50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.\n\nGATA-3-GFP Construct\nThe GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5′- EcoRI/3′-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37°C. Klenow was inactivated by incubation for 10 min at 75°C. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5′-EcoRI/3′–blunt end GATA-3 fragment was inserted into the 5′-EcoRI/3′–blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.\n\nTransfection\nHuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3×106 cells/4 µg DNA for 7–8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.\n\nTime-Lapse Microscopy\nHuT-78 cells expressing GATA-3-GFP were maintained at 37°C in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40×1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.\n\nDensitometric Analysis\nDensitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.\n\nStatistical Analysis\nData from three or more independent experiments are presented as the mean±standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 µg, and 500 µg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p\u003c0.05.\n"}

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