56 results on '"Pine PS"'
Search Results
2. Inhibition of HIV Infection of H9 Cells by Chlorpromazine Derivatives
- Author
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Pine Ps, Foldeak S, Weaver Jl, Lee S, Aszalos A, Hewlett I, and Molnar J
- Subjects
CD4-Positive T-Lymphocytes ,Anti-HIV Agents ,Chlorpromazine ,T-Lymphocytes ,Immunology ,HIV Envelope Protein gp120 ,Biology ,Polymerase Chain Reaction ,Virus ,Cell Line ,Zidovudine ,Virology ,medicine ,Humans ,Immunology and Allergy ,Infectivity ,Syncytium ,HIV ,virus diseases ,Biological activity ,In vitro ,Mechanism of action ,medicine.symptom ,medicine.drug - Abstract
The binding between the HIV surface protein, gp120, and the CD4 coreceptor is known to be initiated by electrostatic interactions. Because of the ability of chlorpromazine to interact with proteins by charge transfer, we tested several derivatives for their ability to block binding of HIV to CD4+ cells. We have shown that 7,8-dioxo-chlorpromazine blocks binding of fluorescein isothiocyanate-labeled anti-Leu3a and rgp120 to peripheral human blood T4 cells and blocks syncytia formation between gp120- and CD4-expressing cells. We also found that 7,8-dioxo-chlorpromazine blocks HIV infectivity of H9 cells and acts synergistically with zidovudine.
- Published
- 1997
3. Comparison of theIn Vitroand Biophysical Effects of Cyclosporine a, FK-506, and Mycophenolic Acid on Human Peripheral Blood Lymphocytes
- Author
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Pine Ps, Adorjan Aszalos, and James L. Weaver
- Subjects
Antigens, Differentiation, T-Lymphocyte ,CD3 Complex ,Immunology ,Cell ,Receptors, Antigen, T-Cell ,Stimulation ,Pharmacology ,Biology ,Lymphocyte Activation ,Toxicology ,Tacrolimus ,Mycophenolic acid ,Membrane Potentials ,medicine ,Humans ,Immunology and Allergy ,Lymphocytes ,Receptor ,Receptors, Interleukin-2 ,General Medicine ,Mycophenolic Acid ,Hyperpolarization (biology) ,In vitro ,medicine.anatomical_structure ,Mechanism of action ,Cyclosporine ,Verapamil ,Calcium ,medicine.symptom ,medicine.drug - Abstract
The immunosuppressive drugs FK-506 and mycophenolic acid (MPA) have recently been described, but their mode(s) of action are not well understood. We have compared them to cyclosporine A (CsA) in several assays. We have shown that CsA (1 microgram/ml), MPA (0.1 microgram/ml), and FK-506 (0.5 microgram/ml) all induce a state of unresponsiveness to anti-CD3 stimulation as measured by [3H]-thymidine uptake. This suggests that the target of these drugs may be present only after mitogenic stimulation. These drugs also cause a hyperpolarization of the plasma membrane of lymphocytes. This effect is blocked by quinine or verapamil. All three immunosuppressors only slightly modulate the increase in intracellular Ca++ caused by Con-A or by anti-CD3 stimulation but do not affect Ca++ levels alone. They also decrease expression of IL-2 receptors on alpha CD3-stimulated lymphocytes. Similarities in their modes of action, as measured by these biophysical and cell biological tests, indicate the possibility that these three drugs will show similarities in their clinical performance.
- Published
- 1991
4. Polyionic Compounds Selectively Alter Availability of CD4 Receptors for HIV Coat Protein rgp120
- Author
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James L. Weaver, Patzer E, Adorjan Aszalos, Gergely P, and Pine Ps
- Subjects
CD4-Positive T-Lymphocytes ,medicine.drug_class ,Immunology ,Receptors, Antigen, T-Cell ,HIV Envelope Protein gp120 ,Biology ,Monoclonal antibody ,Binding, Competitive ,Membrane Potentials ,law.invention ,Cell membrane ,chemistry.chemical_compound ,Cell surface receptor ,law ,Virology ,Aurintricarboxylic acid ,medicine ,Humans ,Receptor ,Cells, Cultured ,Evans Blue ,Aurintricarboxylic Acid ,Cell Membrane ,Dextran Sulfate ,Antibodies, Monoclonal ,Thiobarbiturates ,Recombinant Proteins ,In vitro ,Infectious Diseases ,medicine.anatomical_structure ,chemistry ,Biochemistry ,CD4 Antigens ,Recombinant DNA ,Calcium - Abstract
We studied the ability of several polyionic compounds, previously shown to have activity in vitro against human immunodeficiency virus (anti-HIV) to block binding of anti-CD4 and recombinant HIV gp120 to the CD4 receptor on human lymphocytes. We found that Evans blue and aurin tricarboxylic acid could completely inhibit binding of anti-CD4 (Leu3a) and rgp120 and have selectivity for the CD4 receptor. A number of other compounds, including dextran sulfate and heparin had no effect on binding of rgp120 and were shown to be nonspecific for inhibition of binding of monoclonal antibodies to different T-cell receptors. Studies using a number of membrane-active drugs showed that changes in membrane potential or ion fluxes were not involved in the inhibition of binding of rgp120 by Evans blue or aurin tricarboxylic acid.
- Published
- 1990
5. A semiautomated fluorescence-based cell-to-cell fusion assay for gp120-gp41 and CD4 expressing cells
- Author
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Oravecz T, Adorjan Aszalos, Ussery M, Pine Ps, James L. Weaver, and Pall M
- Subjects
CD4-Positive T-Lymphocytes ,Cytochalasin D ,Time Factors ,medicine.drug_class ,Cell ,Biology ,HIV Envelope Protein gp120 ,Microfilament ,Gp41 ,Monoclonal antibody ,Virus Replication ,Giant Cells ,Cell Line ,Cell Fusion ,chemistry.chemical_compound ,medicine ,Image Processing, Computer-Assisted ,Humans ,Fluorescent Dyes ,Nucleic Acid Synthesis Inhibitors ,chemistry.chemical_classification ,Cell fusion ,Antibodies, Monoclonal ,Cell Biology ,Fluoresceins ,Fluorescence ,Molecular biology ,HIV Envelope Protein gp41 ,medicine.anatomical_structure ,chemistry ,Biophysics ,HIV-1 ,Glycoprotein - Abstract
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably expressenv,the gp120–gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 andenvinitiated cell-to-cell fusion.
- Published
- 1998
6. Cytochalasin D modulates CD4 crosslinking sensitive mitogenic signal in T lymphocytes
- Author
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Rao Pe, Pine Ps, Weaver Jl, and A. Aszalos
- Subjects
CD4-Positive T-Lymphocytes ,Cytochalasin D ,Photochemistry ,CD3 ,Immunology ,Biology ,Microfilament ,Lymphocyte Activation ,Fluorescence ,Membrane Potentials ,chemistry.chemical_compound ,Humans ,Cytochalasin ,Receptor ,Membrane potential ,T-cell receptor ,Antibodies, Monoclonal ,Receptors, Interleukin-2 ,Flow Cytometry ,Molecular biology ,chemistry ,Receptor-CD3 Complex, Antigen, T-Cell ,CD4 Antigens ,Biophysics ,biology.protein ,Cyclosporine ,Calcium ,Intracellular ,Signal Transduction - Abstract
It has previously been shown that crosslinking of the CD4 molecule, either with anti-Leu3a mAb or with gp120 (the HIV coat protein) plus anti-gp120 mAb, suppresses activation induced by wt31, a TcR/CD3-specific mAb. This suppression was associated with hindrance of the necessary association of the p56lck kinase bearing CD4 molecule with the TcR/CD3 complex. In this paper we demonstrate that this crosslinking-induced suppression can be bypassed by perturbing the microfilament system of CD4+ cells by pretreatment with 1 μM cytochalasin D. Using the fluorescence resonance energy transfer method, we have shown that the cytochalasin D-affected increase of mitogenesis is not due to changes in the TcR/CD3 to CD4 distance. Likewise, other membrane biophysical parameters, membrane potential and lateral diffusion of surface receptors, cannot be associated with these cytochalasin D-affected mitogenic changes. Cytochalasin D treatment elevates intracellular Ca2+ levels induced by wt31 mAb plus crosslinking and generates a TcR/CD3-dependent signal which is cyclosporin sensitive.
- Published
- 1994
7. Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples
- Author
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Pine, PS, primary, Boedigheimer, M, additional, Rosenzweig, BA, additional, Turpaz, Y, additional, He, YD, additional, Delenstarr, G, additional, Ganter, B, additional, Jarnagin, K, additional, Jones, WD, additional, Reid, LH, additional, and Thompson, KL, additional
- Published
- 2008
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8. Aurin tricarboxylic acid, the anti-AIDS compound, prevents the binding of interferon-alpha to its receptor
- Author
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Yuxiang Gan, Pine Ps, K. C. Zoon, James L. Weaver, and Adorjan Aszalos
- Subjects
Biophysics ,Receptors, Antigen, T-Cell ,Alpha interferon ,Biology ,HIV Envelope Protein gp120 ,Biochemistry ,Antiviral Agents ,Membrane Potentials ,Antigen ,Interferon ,medicine ,Tumor Cells, Cultured ,Humans ,Receptors, Immunologic ,Receptor ,Molecular Biology ,Receptors, Interferon ,Membrane potential ,chemistry.chemical_classification ,Cell growth ,Aurintricarboxylic Acid ,HIV ,Cell Biology ,Tricarboxylic acid ,chemistry ,Cell culture ,Interferon Type I ,medicine.drug - Abstract
Binding of HIV to its receptor, the CD4 molecule of lymphocytes, can be prevented by chemical agents. These agents could be considered as potential anti-AIDS drugs. We have shown that aurin tricarboxylic acid (ATA, 3 microM) specifically blocks the binding of gp120, the HIV coat protein, to the CD4 molecule. We have also found that ATA prevents the binding of interferon-alpha to its receptor in a dose-dependent manner (12-50 microM range). Membrane potential shift, associated with binding of interferon-alpha to its receptor, was also blocked by ATA in a dose-dependent fashion. Our results indicate that potential anti-AIDS drugs should be screened for such undesired side effects.
- Published
- 1990
9. Cell-based reference samples designed with specific differences in microRNA biomarkers.
- Author
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Pine PS, Lund SP, Stass SA, Kukuruga D, Jiang F, Sorbara L, Srivastava S, and Salit M
- Subjects
- Analysis of Variance, Cell Line, Tumor, Gene Expression, Humans, Reference Standards, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction methods, Reverse Transcriptase Polymerase Chain Reaction standards, Biomarkers, Tumor genetics, Clinical Laboratory Techniques standards, MicroRNAs genetics, RNA, Small Untranslated genetics
- Abstract
Background: We demonstrate the feasibility of creating a pair of reference samples to be used as surrogates for clinical samples measured in either a research or clinical laboratory setting. The reference sample paradigm presented and evaluated here is designed to assess the capability of a measurement process to detect true differences between two biological samples. Cell-based reference samples can be created with a biomarker signature pattern designed in silico. Clinical laboratories working in regulated applications are required to participate in proficiency testing programs; research laboratories doing discovery typically do not. These reference samples can be used in proficiency tests or as process controls that allow a laboratory to evaluate and optimize its measurement systems, monitor performance over time (process drift), assess changes in protocols, reagents, and/or personnel, maintain standard operating procedures, and most importantly, provide evidence for quality results., Results: The biomarkers of interest in this study are microRNAs (miRNAs), small non-coding RNAs involved in the regulation of gene expression. Multiple lung cancer associated cell lines were determined by reverse transcription (RT)-PCR to have sufficiently different miRNA profiles to serve as components in mixture designs as reference samples. In silico models based on the component profiles were used to predict miRNA abundance ratios between two different cell line mixtures, providing target values for profiles obtained from in vitro mixtures. Two reference sample types were tested: total RNA mixed after extraction from cell lines, and intact cells mixed prior to RNA extraction. MicroRNA profiling of a pair of samples composed of extracted RNA derived from these cell lines successfully replicated the target values. Mixtures of intact cells from these lines also approximated the target values, demonstrating potential utility as mimics for clinical specimens. Both designs demonstrated their utility as reference samples for inter- or intra-laboratory testing., Conclusions: Cell-based reference samples can be created for performance assessment of a measurement process from biomolecule extraction through quantitation. Although this study focused on miRNA profiling with RT-PCR using cell lines associated with lung cancer, the paradigm demonstrated here should be extendable to genome-scale platforms and other biomolecular endpoints.
- Published
- 2018
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10. Summarizing performance for genome scale measurement of miRNA: reference samples and metrics.
- Author
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Pine PS, Lund SP, Parsons JR, Vang LK, Mahabal AA, Cinquini L, Kelly SC, Kincaid H, Crichton DJ, Spira A, Liu G, Gower AC, Pass HI, Goparaju C, Dubinett SM, Krysan K, Stass SA, Kukuruga D, Van Keuren-Jensen K, Courtright-Lim A, Thompson KL, Rosenzweig BA, Sorbara L, Srivastava S, and Salit ML
- Subjects
- Female, Gene Expression Profiling methods, High-Throughput Nucleotide Sequencing methods, Humans, Pregnancy, Reference Standards, Brain metabolism, Gene Expression Profiling standards, Genome, Human, Liver metabolism, MicroRNAs genetics, Placenta metabolism
- Abstract
Background: The potential utility of microRNA as biomarkers for early detection of cancer and other diseases is being investigated with genome-scale profiling of differentially expressed microRNA. Processes for measurement assurance are critical components of genome-scale measurements. Here, we evaluated the utility of a set of total RNA samples, designed with between-sample differences in the relative abundance of miRNAs, as process controls., Results: Three pure total human RNA samples (brain, liver, and placenta) and two different mixtures of these components were evaluated as measurement assurance control samples on multiple measurement systems at multiple sites and over multiple rounds. In silico modeling of mixtures provided benchmark values for comparison with physical mixtures. Biomarker development laboratories using next-generation sequencing (NGS) or genome-scale hybridization assays participated in the study and returned data from the samples using their routine workflows. Multiplexed and single assay reverse-transcription PCR (RT-PCR) was used to confirm in silico predicted sample differences. Data visualizations and summary metrics for genome-scale miRNA profiling assessment were developed using this dataset, and a range of performance was observed. These metrics have been incorporated into an online data analysis pipeline and provide a convenient dashboard view of results from experiments following the described design. The website also serves as a repository for the accumulation of performance values providing new participants in the project an opportunity to learn what may be achievable with similar measurement processes., Conclusions: The set of reference samples used in this study provides benchmark values suitable for assessing genome-scale miRNA profiling processes. Incorporation of these metrics into an online resource allows laboratories to periodically evaluate their performance and assess any changes introduced into their measurement process.
- Published
- 2018
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11. Two approaches for estimating the lower limit of quantitation (LLOQ) of microRNA levels assayed as exploratory biomarkers by RT-qPCR.
- Author
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Wolfinger RD, Beedanagari S, Boitier E, Chen T, Couttet P, Ellinger-Ziegelbauer H, Guillemain G, Mariet C, Mouritzen P, O'Lone R, Pine PS, Sharapova T, Yan J, Yuen PS, and Thompson KL
- Subjects
- Animals, Calibration, Genetic Markers, Rats, Reverse Transcriptase Polymerase Chain Reaction statistics & numerical data, Workflow, Limit of Detection, MicroRNAs genetics, Reverse Transcriptase Polymerase Chain Reaction methods
- Abstract
Background: Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation., Results: In this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates., Conclusions: Two workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.
- Published
- 2018
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12. A Bioinformatics 3D Cellular Morphotyping Strategy for Assessing Biomaterial Scaffold Niches.
- Author
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Florczyk SJ, Simon M, Juba D, Pine PS, Sarkar S, Chen D, Baker PJ, Bodhak S, Cardone A, Brady MC, Bajcsy P, and Simon CG Jr
- Abstract
Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L
1 -depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure-function relationships.- Published
- 2017
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13. Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.
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Thompson KL, Boitier E, Chen T, Couttet P, Ellinger-Ziegelbauer H, Goetschy M, Guillemain G, Kanki M, Kelsall J, Mariet C, de La Moureyre-Spire C, Mouritzen P, Nassirpour R, O'Lone R, Pine PS, Rosenzweig BA, Sharapova T, Smith A, Uchiyama H, Yan J, Yuen PS, and Wolfinger R
- Subjects
- Animals, Biomarkers blood, Biomarkers urine, Heart Injuries chemically induced, Isoproterenol toxicity, Male, Plasma chemistry, Rats, Rats, Wistar, Real-Time Polymerase Chain Reaction, Serum chemistry, Heart Injuries metabolism, MicroRNAs blood, MicroRNAs urine
- Abstract
Extracellular microRNAs (miRNAs) represent a promising new source of toxicity biomarkers that are sensitive indicators of site of tissue injury. In order to establish reliable approaches for use in biomarker validation studies, the HESI technical committee on genomics initiated a multi-site study to assess sources of variance associated with quantitating levels of cardiac injury induced miRNAs in biofluids using RT-qPCR. Samples were generated at a central site using a model of acute cardiac injury induced in male Wistar rats by 0.5 mg/kg isoproterenol. Biofluid samples were sent to 11 sites for measurement of 3 cardiac enriched miRNAs (miR-1-3p, miR-208a-3p, and miR-499-5p) and 1 miRNA abundant in blood (miR-16-5p) or urine (miR-192-5p) by absolute quantification using calibration curves of synthetic miRNAs. The samples included serum and plasma prepared from blood collected at 4 h, urine collected from 6 to 24 h, and plasma prepared from blood collected at 24 h post subcutaneous injection. A 3 parameter logistic model was utilized to fit the calibration curve data and estimate levels of miRNAs in biofluid samples by inverse prediction. Most sites observed increased circulating levels of miR-1-3p and miR-208a-3p at 4 and 24 h after isoproterenol treatment, with no difference seen between serum and plasma. The biological differences in miRNA levels and sample type dominated as sources of variance, along with outlying performance by a few sites. The standard protocol established in this study was successfully implemented across multiple sites and provides a benchmark method for further improvements in quantitative assays for circulating miRNAs., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)
- Published
- 2016
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14. External RNA Controls Consortium Beta Version Update.
- Author
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Lee H, Pine PS, McDaniel J, Salit M, and Oliver B
- Abstract
Spike-in RNAs are valuable controls for a variety of gene expression measurements. The External RNA Controls Consortium developed test sets that were used in a number of published reports. Here we provide an authoritative table that summarizes, updates, and corrects errors in the test version that ultimately resulted in the certified Standard Reference Material 2374. We have noted existence of anti-sense RNA controls in the material, corrected sub-pool memberships, and commented on control RNAs that displayed inconsistent behavior., Competing Interests: The authors declare no competing interests.
- Published
- 2016
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15. Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design.
- Author
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Pine PS, Munro SA, Parsons JR, McDaniel J, Lucas AB, Lozach J, Myers TG, Su Q, Jacobs-Helber SM, and Salit M
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- Algorithms, Gene Expression Profiling methods, High-Throughput Nucleotide Sequencing methods, Humans, Reference Values, Reproducibility of Results, Sensitivity and Specificity, Gene Expression Profiling standards, High-Throughput Nucleotide Sequencing standards, RNA genetics, RNA standards, Sequence Analysis, RNA standards
- Abstract
Background: Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background., Results: ERCC pools were analyzed on four different microarray platforms: Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms: the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate., Conclusions: The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics: linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies.
- Published
- 2016
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16. An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.
- Author
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Devonshire AS, Sanders R, Whale AS, Nixon GJ, Cowen S, Ellison SL, Parkes H, Pine PS, Salit M, McDaniel J, Munro S, Lund S, Matsukura S, Sekiguchi Y, Kawaharasaki M, Granjeiro JM, Falagan-Lotsch P, Saraiva AM, Couto P, Yang I, Kwon H, Park SR, Demšar T, Žel J, Blejec A, Milavec M, Dong L, Zhang L, Sui Z, Wang J, Viroonudomphol D, Prawettongsopon C, Partis L, Baoutina A, Emslie K, Takatsu A, Akyurek S, Akgoz M, Vonsky M, Konopelko LA, Cundapi EM, Urquiza MP, Huggett JF, and Foy CA
- Abstract
Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
- Published
- 2016
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17. Using mixtures of biological samples as process controls for RNA-sequencing experiments.
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Parsons J, Munro S, Pine PS, McDaniel J, Mehaffey M, and Salit M
- Subjects
- Gene Expression Profiling, RNA chemistry, RNA genetics, Sequence Analysis, RNA methods
- Abstract
Background: Genome-scale "-omics" measurements are challenging to benchmark due to the enormous variety of unique biological molecules involved. Mixtures of previously-characterized samples can be used to benchmark repeatability and reproducibility using component proportions as truth for the measurement. We describe and evaluate experiments characterizing the performance of RNA-sequencing (RNA-Seq) measurements, and discuss cases where mixtures can serve as effective process controls., Results: We apply a linear model to total RNA mixture samples in RNA-seq experiments. This model provides a context for performance benchmarking. The parameters of the model fit to experimental results can be evaluated to assess bias and variability of the measurement of a mixture. A linear model describes the behavior of mixture expression measures and provides a context for performance benchmarking. Residuals from fitting the model to experimental data can be used as a metric for evaluating the effect that an individual step in an experimental process has on the linear response function and precision of the underlying measurement while identifying signals affected by interference from other sources. Effective benchmarking requires well-defined mixtures, which for RNA-Seq requires knowledge of the post-enrichment 'target RNA' content of the individual total RNA components. We demonstrate and evaluate an experimental method suitable for use in genome-scale process control and lay out a method utilizing spike-in controls to determine enriched RNA content of total RNA in samples., Conclusions: Genome-scale process controls can be derived from mixtures. These controls relate prior knowledge of individual components to a complex mixture, allowing assessment of measurement performance. The target RNA fraction accounts for differential selection of RNA out of variable total RNA samples. Spike-in controls can be utilized to measure this relationship between target RNA content and input total RNA. Our mixture analysis method also enables estimation of the proportions of an unknown mixture, even when component-specific markers are not previously known, whenever pure components are measured alongside the mixture.
- Published
- 2015
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18. Assessing technical performance in differential gene expression experiments with external spike-in RNA control ratio mixtures.
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Munro SA, Lund SP, Pine PS, Binder H, Clevert DA, Conesa A, Dopazo J, Fasold M, Hochreiter S, Hong H, Jafari N, Kreil DP, Łabaj PP, Li S, Liao Y, Lin SM, Meehan J, Mason CE, Santoyo-Lopez J, Setterquist RA, Shi L, Shi W, Smyth GK, Stralis-Pavese N, Su Z, Tong W, Wang C, Wang J, Xu J, Ye Z, Yang Y, Yu Y, and Salit M
- Subjects
- Gene Expression Profiling standards, Humans, Reference Standards, Reproducibility of Results, Gene Expression Profiling methods, RNA, Messenger genetics
- Abstract
There is a critical need for standard approaches to assess, report and compare the technical performance of genome-scale differential gene expression experiments. Here we assess technical performance with a proposed standard 'dashboard' of metrics derived from analysis of external spike-in RNA control ratio mixtures. These control ratio mixtures with defined abundance ratios enable assessment of diagnostic performance of differentially expressed transcript lists, limit of detection of ratio (LODR) estimates and expression ratio variability and measurement bias. The performance metrics suite is applicable to analysis of a typical experiment, and here we also apply these metrics to evaluate technical performance among laboratories. An interlaboratory study using identical samples shared among 12 laboratories with three different measurement processes demonstrates generally consistent diagnostic power across 11 laboratories. Ratio measurement variability and bias are also comparable among laboratories for the same measurement process. We observe different biases for measurement processes using different mRNA-enrichment protocols.
- Published
- 2014
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19. Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films.
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Baker BA, Pine PS, Chatterjee K, Kumar G, Lin NJ, McDaniel JH, Salit ML, and Simon CG Jr
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- Biocompatible Materials chemistry, Cell Adhesion physiology, Cell Differentiation physiology, Cells, Cultured, Humans, Microarray Analysis, Nanofibers chemistry, Osteogenesis physiology, Polymers chemistry, RNA, Messenger genetics, RNA, Messenger metabolism, Signal Transduction, Tissue Engineering methods, Transcriptome, Transforming Growth Factor beta genetics, Transforming Growth Factor beta metabolism, Gene Expression, Mesenchymal Stem Cells metabolism, Tissue Scaffolds chemistry
- Abstract
Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-β and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation., (Published by Elsevier Ltd.)
- Published
- 2014
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20. The determination of stem cell fate by 3D scaffold structures through the control of cell shape.
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Kumar G, Tison CK, Chatterjee K, Pine PS, McDaniel JH, Salit ML, Young MF, and Simon CG Jr
- Subjects
- Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Bone Marrow Cells ultrastructure, Cell Count, Cells, Cultured, DNA metabolism, Gene Expression Profiling, Humans, Stem Cells metabolism, Stem Cells ultrastructure, Stromal Cells cytology, Stromal Cells metabolism, Stromal Cells ultrastructure, Cell Lineage, Cell Shape, Stem Cells cytology, Tissue Scaffolds chemistry
- Abstract
Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage., (Published by Elsevier Ltd.)
- Published
- 2011
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21. An adaptable method using human mixed tissue ratiometric controls for benchmarking performance on gene expression microarrays in clinical laboratories.
- Author
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Pine PS, Rosenzweig BA, and Thompson KL
- Subjects
- Area Under Curve, Benchmarking, Clinical Laboratory Techniques, Gene Expression Regulation, Humans, Liver metabolism, Muscle, Skeletal metabolism, Organ Specificity, ROC Curve, Gene Expression Profiling methods, Oligonucleotide Array Sequence Analysis methods, RNA metabolism
- Abstract
Background: Molecular biomarkers that are based on mRNA transcripts are being developed for the diagnosis and treatment of a number of diseases. DNA microarrays are one of the primary technologies being used to develop classifiers from gene expression data for clinically relevant outcomes. Microarray assays are highly multiplexed measures of comparative gene expression but have a limited dynamic range of measurement and show compression in fold change detection. To increase the clinical utility of microarrays, assay controls are needed that benchmark performance using metrics that are relevant to the analysis of genomic data generated with biological samples., Results: Ratiometric controls were prepared from commercial sources of high quality RNA from human tissues with distinctly different expression profiles and mixed in defined ratios. The samples were processed using six different target labeling protocols and replicate datasets were generated on high density gene expression microarrays. The area under the curve from receiver operating characteristic plots was calculated to measure diagnostic performance. The reliable region of the dynamic range was derived from log(2) ratio deviation plots made for each dataset. Small but statistically significant differences in diagnostic performance were observed between standardized assays available from the array manufacturer and alternative methods for target generation. Assay performance using the reliable range of comparative measurement as a metric was improved by adjusting sample hybridization conditions for one commercial kit., Conclusions: Process improvement in microarray assay performance was demonstrated using samples prepared from commercially available materials and two metrics - diagnostic performance and the reliable range of measurement. These methods have advantages over approaches that use a limited set of external controls or correlations to reference sets, because they provide benchmark values that can be used by clinical laboratories to help optimize protocol conditions and laboratory proficiency with microarray assays.
- Published
- 2011
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22. Overview of laser microbeam applications as related to antibody targeting.
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Pine PS
- Subjects
- Ablation Techniques instrumentation, Ablation Techniques methods, Epitopes analysis, Fluorescence Recovery After Photobleaching instrumentation, Fluorescence Recovery After Photobleaching methods, Fluorescence Resonance Energy Transfer instrumentation, Fluorescence Resonance Energy Transfer methods, Membrane Proteins analysis, Micromanipulation instrumentation, Micromanipulation methods, Microscopy instrumentation, Microscopy methods, Antibodies analysis, Lasers
- Abstract
This chapter reviews several techniques which combine the use of laser microbeams with antibodies to study molecular and cellular biology. An overview of the basic properties of lasers and their integration with microscopes and computers is provided. Biophysical applications, such as fluorescence recovery after photobleaching to measure molecular mobility and fluorescence resonance energy transfer to measure molecular distances, as well as ablative applications for the selective inactivation of proteins or the selective killing of cells are described. Other techniques, such as optical trapping, that do not rely on the interaction of the laser with the targeting antibody, are also discussed.
- Published
- 2010
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23. Comparison of the diagnostic performance of human whole genome microarrays using mixed-tissue RNA reference samples.
- Author
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Thompson KL and Pine PS
- Subjects
- Animals, Biomarkers, Gene Expression Profiling, Humans, Predictive Value of Tests, Rats, Reference Standards, Reproducibility of Results, Clinical Medicine methods, Diagnostic Techniques and Procedures, Genome, Human, Genomics, Oligonucleotide Array Sequence Analysis, RNA, Messenger analysis
- Abstract
Universal approaches for assessing the diagnostic performance of microarray assays are essential for the application of microarray technology to clinical and regulatory settings. Reference systems for diagnostic assays in laboratory medicine typically involve the utilization of reference samples, metrics, and reference datasets to ensure that measurements are comparable and true. For microarray performance evaluation and process improvement, reference samples can be composed of mixes of different tissue or cell line RNAs that contain tissue-selective analytes at defined target ratios. The diagnostic accuracy of detected changes in expression, measured as the area under the curve from receiver-operating characteristic plots, can provide a single commutable value for comparing assay specificity and sensitivity. Examples of applying this method for assessing overall performance are provided using public datasets generated on five commercial human whole genome microarray platforms for the MicroArray Quality Control project, a community-wide effort to address issues surrounding microarray data reliability.
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- 2009
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24. Assessment of repeated microarray experiments using mixed tissue RNA reference samples.
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Mongan MA, Higgins M, Pine PS, Afshari C, and Hamadeh H
- Subjects
- Animals, Area Under Curve, Benchmarking methods, Brain Chemistry, Kidney chemistry, Liver chemistry, Male, Organ Specificity, RNA isolation & purification, ROC Curve, Rats, Rats, Sprague-Dawley, Reference Standards, Sensitivity and Specificity, Testis chemistry, Evaluation Studies as Topic, Gene Expression Profiling methods, Oligonucleotide Array Sequence Analysis methods, RNA chemistry
- Abstract
Genome-scale gene expression technologies are increasingly being applied for biological research as a whole and toxicological screening in particular. In order to monitor data quality and process drift, we adopted the use of two rat-tissue mixtures (brain, liver, kidney, and testis) previously introduced as RNA reference samples. These samples were processed every time a microarray experiment was hybridized, thereby verifying the comparability of the resulting expression data for cross-study comparison. This study presents the analysis of 21 technical replicates of these two mixed-tissue samples using Affymetrix RAE230_2 GeneChip over a period of 12 months. The results show that detection sensitivity, measured by the number of present and absent sequences, is robust, and data correlation, indicated by scatter plots, varies little over time. Receiver operating characteristic (ROC) curves show the sensitivity and specificity of the current measurements are consistent with arrays previously classified as well performing. Overall, this paper shows that the inclusion of standard samples during microarray labeling and hybridization experiments is useful to benchmark the performance of microarray experiments over time and allows discovery of any process drift that, if it occurs, may confound the comparison of these datasets.
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- 2008
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25. Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.
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Thompson KL, Pine PS, Rosenzweig BA, Turpaz Y, and Retief J
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- Animals, Gene Expression Profiling, Male, RNA chemistry, RNA metabolism, RNA Stability, Rats, Rats, Sprague-Dawley, Reproducibility of Results, Specimen Handling methods, Liver metabolism, Oligonucleotide Array Sequence Analysis methods, RNA genetics
- Abstract
Background: The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course., Results: Incubation of tissue at 37 degrees C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip(R) arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold., Conclusion: Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.
- Published
- 2007
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26. Improvement in the reproducibility and accuracy of DNA microarray quantification by optimizing hybridization conditions.
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Han T, Melvin CD, Shi L, Branham WS, Moland CL, Pine PS, Thompson KL, and Fuscoe JC
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- Animals, Computational Biology, Mice, Organ Specificity, Rats, Reproducibility of Results, Nucleic Acid Hybridization methods, Oligonucleotide Array Sequence Analysis methods
- Abstract
Background: DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data., Results: Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements., Conclusion: The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.
- Published
- 2006
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27. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.
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Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC, Collins PJ, de Longueville F, Kawasaki ES, Lee KY, Luo Y, Sun YA, Willey JC, Setterquist RA, Fischer GM, Tong W, Dragan YP, Dix DJ, Frueh FW, Goodsaid FM, Herman D, Jensen RV, Johnson CD, Lobenhofer EK, Puri RK, Schrf U, Thierry-Mieg J, Wang C, Wilson M, Wolber PK, Zhang L, Amur S, Bao W, Barbacioru CC, Lucas AB, Bertholet V, Boysen C, Bromley B, Brown D, Brunner A, Canales R, Cao XM, Cebula TA, Chen JJ, Cheng J, Chu TM, Chudin E, Corson J, Corton JC, Croner LJ, Davies C, Davison TS, Delenstarr G, Deng X, Dorris D, Eklund AC, Fan XH, Fang H, Fulmer-Smentek S, Fuscoe JC, Gallagher K, Ge W, Guo L, Guo X, Hager J, Haje PK, Han J, Han T, Harbottle HC, Harris SC, Hatchwell E, Hauser CA, Hester S, Hong H, Hurban P, Jackson SA, Ji H, Knight CR, Kuo WP, LeClerc JE, Levy S, Li QZ, Liu C, Liu Y, Lombardi MJ, Ma Y, Magnuson SR, Maqsodi B, McDaniel T, Mei N, Myklebost O, Ning B, Novoradovskaya N, Orr MS, Osborn TW, Papallo A, Patterson TA, Perkins RG, Peters EH, Peterson R, Philips KL, Pine PS, Pusztai L, Qian F, Ren H, Rosen M, Rosenzweig BA, Samaha RR, Schena M, Schroth GP, Shchegrova S, Smith DD, Staedtler F, Su Z, Sun H, Szallasi Z, Tezak Z, Thierry-Mieg D, Thompson KL, Tikhonova I, Turpaz Y, Vallanat B, Van C, Walker SJ, Wang SJ, Wang Y, Wolfinger R, Wong A, Wu J, Xiao C, Xie Q, Xu J, Yang W, Zhang L, Zhong S, Zong Y, and Slikker W Jr
- Subjects
- Equipment Design, Equipment Failure Analysis, Gene Expression Profiling methods, Quality Control, Reproducibility of Results, Sensitivity and Specificity, United States, Gene Expression Profiling instrumentation, Oligonucleotide Array Sequence Analysis instrumentation, Quality Assurance, Health Care methods
- Abstract
Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.
- Published
- 2006
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28. Using RNA sample titrations to assess microarray platform performance and normalization techniques.
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Shippy R, Fulmer-Smentek S, Jensen RV, Jones WD, Wolber PK, Johnson CD, Pine PS, Boysen C, Guo X, Chudin E, Sun YA, Willey JC, Thierry-Mieg J, Thierry-Mieg D, Setterquist RA, Wilson M, Lucas AB, Novoradovskaya N, Papallo A, Turpaz Y, Baker SC, Warrington JA, Shi L, and Herman D
- Subjects
- Algorithms, Reference Values, Reproducibility of Results, Sensitivity and Specificity, United States, Equipment Failure Analysis methods, Gene Expression Profiling instrumentation, Gene Expression Profiling standards, Oligonucleotide Array Sequence Analysis instrumentation, Oligonucleotide Array Sequence Analysis standards, RNA analysis, RNA genetics
- Abstract
We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.
- Published
- 2006
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29. Workgroup report: Review of genomics data based on experience with mock submissions--view of the CDER Pharmacology Toxicology Nonclinical Pharmacogenomics Subcommittee.
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Leighton JK, Brown P, Ellis A, Harlow P, Harrouk W, Pine PS, Robison T, Rosario L, and Thompson K
- Subjects
- Animals, Database Management Systems, Education, Oligonucleotide Array Sequence Analysis, Genomics, Pharmacogenetics, Toxicology
- Abstract
Over the past few years, both the U.S. Food and Drug Administration (FDA) and the pharmaceutical industry have recognized the potential importance of pharmacogenomics and toxicogenomics to drug development. To resolve the uncertainties surrounding the use of microarray technology and the presentation of genomics data for regulatory purposes, several pharmaceutical companies and genomics technology providers have provided the FDA with reports of genomics studies that included supporting toxicology data (e.g., serum chemistry, histopathology). These studies were not associated with any active drug application and were exploratory or hypothesis generating in nature. For training purposes, these reports were reviewed by the Nonclinical Pharmacogenomics Subcommittee consisting of the Center for Drug Evaluation and Research pharmacology and toxicology researchers and reviewers. In this article, we describe some of these submissions and report on our assessment of data content, format, and quality control metrics that were useful for evaluating these nonclinical genomics submissions, specifically in relation to the proposed MIAME/MINTox (minimum information about a microarray experiment/minimum information needed for a toxicology experiment) recommendations. These genomics submissions allowed both researchers and regulators to gain experience in the process of reviewing and analyzing toxicogenomics data. The experience will allow development of recommendations for the submission and review of these data as the state of the science evolves.
- Published
- 2006
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30. Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.
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Thompson KL, Rosenzweig BA, Pine PS, Retief J, Turpaz Y, Afshari CA, Hamadeh HK, Damore MA, Boedigheimer M, Blomme E, Ciurlionis R, Waring JF, Fuscoe JC, Paules R, Tucker CJ, Fare T, Coffey EM, He Y, Collins PJ, Jarnagin K, Fujimoto S, Ganter B, Kiser G, Kaysser-Kranich T, Sina J, and Sistare FD
- Subjects
- Animals, Gene Expression Profiling methods, Male, Oligonucleotide Array Sequence Analysis methods, Oligonucleotide Probes, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Reference Standards, Reproducibility of Results, Tissue Distribution, Gene Expression Profiling standards, Oligonucleotide Array Sequence Analysis standards, RNA, Messenger standards
- Abstract
The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.
- Published
- 2005
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31. Determination of plasma and brain levels of isotretinoin in mice following single oral dose by high-performance liquid chromatography.
- Author
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Yang Y, Faustino PJ, Pine PS, Davis H, Grunberg N, Phillips J, Lyon RC, Yu LX, Ciavarella AB, Del Grosso AV, and Hanig JP
- Subjects
- Administration, Oral, Animals, Brain drug effects, Brain metabolism, Chromatography, High Pressure Liquid methods, Isotretinoin chemistry, Male, Mice, Brain Chemistry drug effects, Brain Chemistry physiology, Isotretinoin administration & dosage, Isotretinoin blood
- Abstract
An isocratic reversed-phase high-performance liquid chromatographic method was established and validated according to FDA's Guidance for Industry, "Bioanalytical Method Validation", for the determination of isotretinoin in plasma and brain tissue from mice following single and multiple oral doses of Accutane. Plasma sample preparation included deproteination with acetonitrile-perchloric acid followed by centrifugation. Brain tissue was homogenized and extracted with acetonitrile-perchloric acid followed by centrifugation. The supernatants were analyzed by high-performance liquid chromatography (HPLC). Benz[alpha]anthrancene-7,12-dione was used as the internal standard. Chromatographic separation was achieved on a C18 column using an acetonitrile-aqueous 0.5% acetic acid (85:15, v/v) elution. The average extraction efficiency was >95% for plasma and >82% for brain. The lower limit of quantification was 30 ng/mL for plasma and was 30 ng/0.1g for brain tissue, respectively. The linear range for plasma was 30-600 ng/mL, and 15-300 ng/0.1g for brain. Maximum concentrations of isotretinoin in both plasma and brain were observed at 1h after single oral dosing (25 mg/kg). The maximum concentrations in plasma and brain were 2.36 microg/mL and 0.34 microg/g, respectively. The mean area under curve (AUC) in plasma was 6.13 microg h/mL. The mean eliminate half-life in plasma was estimated as 46 min.
- Published
- 2005
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32. Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.
- Author
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Scallet AC, Schmued LC, Slikker W Jr, Grunberg N, Faustino PJ, Davis H, Lester D, Pine PS, Sistare F, and Hanig JP
- Subjects
- Animals, Animals, Newborn, Brain pathology, Brain Chemistry drug effects, Dose-Response Relationship, Drug, Excitatory Amino Acid Antagonists blood, Female, Fluoresceins, Fluorescent Dyes, Ketamine blood, Male, Nervous System drug effects, Neurons drug effects, Organic Chemicals, Rats, Rats, Sprague-Dawley, Silver Staining, Thalamus pathology, Apoptosis drug effects, Excitatory Amino Acid Antagonists toxicity, Ketamine toxicity, Nervous System growth & development, Nervous System pathology, Neurons pathology, Neurotoxicity Syndromes pathology
- Abstract
Ketamine is a widely used pediatric anesthetic recently reported (C. Ikonomidou et al., 1999, Science 283, 70-74) to enhance neuronal death in neonatal rats. To confirm and extend these results, we treated four groups of PND 7 rats with seven sc doses, one every 90 min, of either saline, 10 mg/kg ketamine, 20 mg/kg ketamine, or a single dose of 20 mg/kg ketamine. The repeated doses of 20 mg/kg ketamine increased the number of silver-positive (degenerating) neurons in the dorsolateral thalamus to a degree comparable to previous results (Ikonomidou et al., 1999, Science 283, 70-74), i.e., 28-fold vs. 31-fold respectively. However, blood levels of ketamine immediately after the repeated 20 mg/kg doses were about 14 micrograms/ml, about seven-fold greater than anesthetic blood levels in humans (J. M. Malinovsky et al., 1996, Br. J. Anaesth. 77, 203-207; R. A. Mueller and R. Hunt, 1998, Pharmacol. Biochem. Behav. 60, 15-22). Levels of ketamine in blood following exposure to the multiple 10 mg/kg doses of ketamine or to a single 20 mg/kg dose ranged around 2-5 micrograms/ml; although these blood levels are close to an anesthetic level in humans, they failed to produce neurodegeneration. To investigate the mode of ketamine-induced neuronal death, coronal sections were stained with both Fluoro-Jade B (a green fluorescent stain selective for neurodegeneration) and DAPI (a blue DNA stain), as well as for caspase-3 (using an antisera labeled red with rhodamine). These histochemical results confirmed the developmental neurotoxicity of ketamine, demonstrated that Fluoro-Jade B (FJ-B), like silver methods, successfully stained degenerating neurons in neonatal rats, and indicated that ketamine acts by increasing the rate of neuronal apoptosis.
- Published
- 2004
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33. Dye bias correction in dual-labeled cDNA microarray gene expression measurements.
- Author
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Rosenzweig BA, Pine PS, Domon OE, Morris SM, Chen JJ, and Sistare FD
- Subjects
- Cell Culture Techniques, Control Groups, Gene Expression Profiling statistics & numerical data, Humans, Reproducibility of Results, Thymidine Kinase, Transcription, Genetic, Coloring Agents, Gene Expression Profiling standards, Oligonucleotide Array Sequence Analysis
- Abstract
A significant limitation to the analytical accuracy and precision of dual-labeled spotted cDNA microarrays is the signal error due to dye bias. Transcript-dependent dye bias may be due to gene-specific differences of incorporation of two distinctly different chemical dyes and the resultant differential hybridization efficiencies of these two chemically different targets for the same probe. Several approaches were used to assess and minimize the effects of dye bias on fluorescent hybridization signals and maximize the experimental design efficiency of a cell culture experiment. Dye bias was measured at the individual transcript level within each batch of simultaneously processed arrays by replicate dual-labeled split-control sample hybridizations and accounted for a significant component of fluorescent signal differences. This transcript-dependent dye bias alone could introduce unacceptably high numbers of both false-positive and false-negative signals. We found that within a given set of concurrently processed hybridizations, the bias is remarkably consistent and therefore measurable and correctable. The additional microarrays and reagents required for paired technical replicate dye-swap corrections commonly performed to control for dye bias could be costly to end users. Incorporating split-control microarrays within a set of concurrently processed hybridizations to specifically measure dye bias can eliminate the need for technical dye swap replicates and reduce microarray and reagent costs while maintaining experimental accuracy and technical precision. These data support a practical and more efficient experimental design to measure and mathematically correct for dye bias.
- Published
- 2004
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34. Identification of platform-independent gene expression markers of cisplatin nephrotoxicity.
- Author
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Thompson KL, Afshari CA, Amin RP, Bertram TA, Car B, Cunningham M, Kind C, Kramer JA, Lawton M, Mirsky M, Naciff JM, Oreffo V, Pine PS, and Sistare FD
- Subjects
- Animals, Rats, Rats, Sprague-Dawley, Reproducibility of Results, Antineoplastic Agents toxicity, Cisplatin toxicity, Gene Expression Profiling methods, Kidney drug effects, Kidney pathology, Oligonucleotide Array Sequence Analysis methods
- Abstract
Within the International Life Sciences Institute Committee on Genomics, a working group was formed to focus on the application of microarray technology to preclinical assessments of drug-induced nephrotoxicity. As part of this effort, Sprague-Dawley rats were treated with the nephrotoxicant cisplatin at doses of 0.3-5 mg/kg over a 4- to 144-hr time course. RNA prepared from these animals was run on a variety of microarray formats at multiple sites. A set of 93 differentially expressed genes associated with cisplatin-induced renal injury was identified on the National Institute of Environmental Health Sciences (NIEHS) custom cDNA microarray platform using quadruplicate measurements of pooled animal RNA. The reproducibility of this profile of statistically significant gene changes on other platforms, in pooled and individual animal replicate samples, and in an independent study was investigated. A good correlation in response between platforms was found among the 48 genes in the NIEHS data set that could be matched to probes on the Affymetrix RGU34A array by UniGene identifier or sequence alignment. Similar results were obtained with genes that could be linked between the NIEHS and Incyte or PHASE-1 arrays. The degree of renal damage induced by cisplatin in individual animals was commensurate with the number of differentially expressed genes in this data set. These results suggest that gene profiles linked to specific types of tissue injury or mechanisms of toxicity and identified in well-performed replicated microarray experiments may be extrapolatable across platform technologies, laboratories, and in-life studies.
- Published
- 2004
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35. Virtual neuropathology: three-dimensional visualization of lesions due to toxic insult.
- Author
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Lester DS, Pine PS, Delnomdedieu M, Johannessen JN, and Johnson GA
- Subjects
- Animals, Brain pathology, Kainic Acid analogs & derivatives, Kainic Acid toxicity, Magnetic Resonance Imaging, Microscopy, Neurotoxins toxicity, Oxidopamine toxicity, Parkinson Disease, Secondary chemically induced, Parkinson Disease, Secondary pathology, Rats, Sympatholytics toxicity, Image Processing, Computer-Assisted methods, Nervous System Diseases pathology, Neurotoxicity Syndromes pathology
- Abstract
A first-pass approach incorporating high-field magnetic resonance imaging (MRI) was used for rapid detection of neuropathologic lesions in fixed rat brains. This inherently 3-dimensional and nondestructive technique provides high-resolution, high-contrast images of fixed neuronal tissue in the absence of sectioning or staining. This technique, magnetic resonance microscopy (MRM), was used to identify diverse lesions in 2 well-established rat neurotoxicity models. The intrinsic contrast in the images delineated lesions that were identified using a battery of histologic stains, some of which would not be used in routine screening. Furthermore, the MRM images provided the locations of lesions, which were verified upon subsequent sectioning and staining of the same samples. The inherent contrast generated by water properties is exploited in MRM by choosing suitable pulse sequences, or proton stains. This approach provides the potential for a comprehensive initial MRM screen for neurotoxicity in preclinical models with the capability for extrapolation to clinical analyses using classical MRI.
- Published
- 2000
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36. Overview of laser microbeam applications as related to antibody targeting.
- Author
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Pine PS
- Subjects
- Animals, Antibodies immunology, Lasers, Microscopy, Fluorescence methods
- Published
- 1999
- Full Text
- View/download PDF
37. A semiautomated fluorescence-based cell-to-cell fusion assay for gp120-gp41 and CD4 expressing cells.
- Author
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Pine PS, Weaver JL, Oravecz T, Pall M, Ussery M, and Aszalos A
- Subjects
- Antibodies, Monoclonal, CD4-Positive T-Lymphocytes immunology, Cell Fusion physiology, Cell Line, Cytochalasin D pharmacology, Fluoresceins pharmacokinetics, Fluorescent Dyes pharmacokinetics, Giant Cells physiology, HIV-1 growth & development, Humans, Image Processing, Computer-Assisted, Nucleic Acid Synthesis Inhibitors pharmacology, Time Factors, Virus Replication drug effects, CD4-Positive T-Lymphocytes cytology, CD4-Positive T-Lymphocytes virology, HIV Envelope Protein gp120 analysis, HIV Envelope Protein gp41 analysis, HIV-1 isolation & purification
- Abstract
A novel fluorescence-based method was developed to measure HIV envelope glycoprotein (env)-CD4-mediated cell fusion. This method measures the spread of a fluorescent dye as the cytosolic compartments of adjacent cells become contiguous upon cell-to-cell fusion. Calcein-labeled CD4+ Sup-T1 cells were seeded onto a monolayer of unlabeled TF228.1.16 cells, which stably express env, the gp120-gp41 complex. Changes in the following parameters were measured using a stage-scanning laser microscope: total fluorescent area, average fluorescent area, and average shape factor. Anti-CD4 monoclonal antibodies, anti-Leu3a, and OKT4E were shown to block fusion in a dose-dependent manner, while OKT4 had no effect. Aurin tricarboxylic acid, a compound that interferes with the binding of anti-Leu3a mAb and gp120 to CD4+ human peripheral blood lymphocytes, T20, a peptide that interferes with gp41, and cytochalasin D, a microfilament disrupter, all blocked fusion in a dose-dependent manner. This semiautomated assay can be used to quickly assess the effectiveness of compounds acting at different sites to block CD4 and env initiated cell-to-cell fusion.
- Published
- 1998
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38. Inhibition of HIV infection of H9 cells by chlorpromazine derivatives.
- Author
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Hewlett I, Lee S, Molnar J, Foldeak S, Pine PS, Weaver JL, and Aszalos A
- Subjects
- CD4-Positive T-Lymphocytes metabolism, Cell Line, Chlorpromazine analogs & derivatives, HIV Envelope Protein gp120 metabolism, Humans, Polymerase Chain Reaction, T-Lymphocytes virology, Anti-HIV Agents pharmacology, Chlorpromazine pharmacology, HIV drug effects
- Abstract
The binding between the HIV surface protein, gp120, and the CD4 coreceptor is known to be initiated by electrostatic interactions. Because of the ability of chlorpromazine to interact with proteins by charge transfer, we tested several derivatives for their ability to block binding of HIV to CD4+ cells. We have shown that 7,8-dioxo-chlorpromazine blocks binding of fluorescein isothiocyanate-labeled anti-Leu3a and rgp120 to peripheral human blood T4 cells and blocks syncytia formation between gp120- and CD4-expressing cells. We also found that 7,8-dioxo-chlorpromazine blocks HIV infectivity of H9 cells and acts synergistically with zidovudine.
- Published
- 1997
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39. Effect of combination of suboptimal concentrations of P-glycoprotein blockers on the proliferation of MDR1 gene expressing cells.
- Author
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Hwang M, Ahn CH, Pine PS, Yin JJ, Hrycyna CA, Licht T, and Aszalos A
- Subjects
- Cell Division drug effects, Cell Membrane drug effects, Glycerol pharmacology, Humans, Leukemia, Transfection, Tumor Cells, Cultured, ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors, ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Calcium Channel Blockers pharmacology, Cell Membrane metabolism, Cyclosporins pharmacology, Glycerol analogs & derivatives, Verapamil pharmacology
- Abstract
Pharmacologically active in vivo doses of P-glycoprotein (Pgp) blockers, specifically verapamil, Cremophor EL and PSC833 cause toxicity in addition to that from the concomitantly used cancer chemotherapeutic drugs. It was shown before that these blockers cause different types of toxicities in vivo. We found that these 3 chemically distinct Pgp blockers exert different biophysical effects on the membranes of L1210 MDR cells. They also affect the general metabolism of these cells differently, but all block affinity labeling of Pgp. We could also show that the combination of suboptimal doses of these blockers can restore the uptake of the Pgp substrate rhodamine 123 into L1210MDR, 3T3MDR and KB-VI cells and can reduce the survival rate of these cells when treated in combination with daunorubicin. Our results suggest that the combination of suboptimal doses of these Pgp blockers may be advantageous in clinical practice.
- Published
- 1996
- Full Text
- View/download PDF
40. Behavior of N-acylated daunorubicins in MDR1 gene transfected and parental cells.
- Author
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Aszalos A, Pine PS, Pandey R, and Gottesman MM
- Subjects
- 3T3 Cells, ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors, ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Animals, Cell Division drug effects, Cyclosporine pharmacology, Daunorubicin metabolism, Dose-Response Relationship, Drug, Drug Interactions, Humans, Mice, Transfection, Tumor Cells, Cultured, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Daunorubicin analogs & derivatives, Drug Resistance, Multiple genetics
- Abstract
The substrate specificity of the P-glycoprotein (P-170), a multidrug transporter, was studied using N-acylated daunorubicin derivatives and four MDR1 cDNA transfected cell lines. Results showed that N-acetyl-daunorubicin is a substrate, but the longer fatty acid derivatives, N-octanoyl and N-dodecanoyl daunorubicins, are not. This conclusion was reached by flow cytometric drug uptake assay, cell proliferation assays, and confocal microscopy. It was concluded that the longer fatty acid derivatives interact with plasma membranes in a way that affected P-glycoprotein function.
- Published
- 1995
- Full Text
- View/download PDF
41. Cross-linking of CD4 in a TCR/CD3-juxtaposed inhibitory state: a pFRET study.
- Author
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Szabó G Jr, Weaver JL, Pine PS, Rao PE, and Aszalos A
- Subjects
- Biophysical Phenomena, Biophysics, CD4-Positive T-Lymphocytes chemistry, CD4-Positive T-Lymphocytes immunology, Cross-Linking Reagents, Energy Transfer, Epitopes, Humans, In Vitro Techniques, Photochemistry, Spectrometry, Fluorescence, CD3 Complex chemistry, CD4 Antigens chemistry
- Abstract
Instances when T cell activation via the T cell receptor/CD3 complex is suppressed by anti-CD4 Abs are generally attributed either to the topological separation of CD4-p56lck from CD3, or their improper apposition. Photobleaching fluorescence resonance energy transfer measurements permitted direct analysis of these alternatives on human peripheral blood lymphocytes. Distinction between changes of relative antigen densities or positioning was made possible by simultaneously recording donor and acceptor fluorescence in the energy transfer experiment performed on homogeneous populations of flow-sorted cells. We show here that CD4 stays in the molecular vicinity of CD3, while anti-CD3 stimulation is suppressed by anti-CD4 or cross-linked HIV gp120. Our data suggest that cross-linking of CD4 through particular epitopes is capable of inhibiting activation driven by Abs binding to specific sites on CD3 without major topological sequestration of the Ags, in such a way that additional positive signals will also be affected. Thus, these and other related cases of negative signaling via CD4 may be interpreted in terms of functional uncoupling rather than a wide physical separation of CD4 from the T cell receptor-CD3 complex.
- Published
- 1995
- Full Text
- View/download PDF
42. The L-selectin (Leu8) molecule is associated with the TcR/CD3 receptor; fluorescence energy transfer measurements on live cells.
- Author
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Szabo G Jr, Pine PS, Weaver JL, Rao PE, and Aszalos A
- Subjects
- CD3 Complex immunology, Calcium metabolism, Cell Adhesion Molecules immunology, Cell Division, DNA Replication, Energy Transfer, Epitopes immunology, Humans, L-Selectin, Lasers, Lymphocyte Activation, Muromonab-CD3 immunology, Photochemistry, Protein Binding, Receptors, Fc metabolism, Signal Transduction, Spectrometry, Fluorescence, T-Lymphocytes immunology, Cell Adhesion Molecules analysis, Receptor-CD3 Complex, Antigen, T-Cell analysis, T-Lymphocytes ultrastructure
- Abstract
Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-Leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecules.
- Published
- 1994
- Full Text
- View/download PDF
43. Cytochalasin D modulates CD4 crosslinking sensitive mitogenic signal in T lymphocytes.
- Author
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Aszalos A, Pine PS, Weaver JL, and Rao PE
- Subjects
- Antibodies, Monoclonal, CD4-Positive T-Lymphocytes drug effects, Cyclosporine pharmacology, Flow Cytometry, Fluorescence, Humans, Lymphocyte Activation drug effects, Membrane Potentials drug effects, Membrane Potentials physiology, Photochemistry, Receptor-CD3 Complex, Antigen, T-Cell drug effects, Receptor-CD3 Complex, Antigen, T-Cell physiology, Receptors, Interleukin-2 immunology, Signal Transduction drug effects, CD4 Antigens physiology, CD4-Positive T-Lymphocytes physiology, Calcium physiology, Cytochalasin D pharmacology, Signal Transduction immunology
- Abstract
It has previously been shown that crosslinking of the CD4 molecule, either with anti-Leu3a mAb or with gp120 (the HIV coat protein) plus anti-gp120 mAb, suppresses activation induced by wt31, a TcR/CD3-specific mAb. This suppression was associated with hindrance of the necessary association of the p56lck kinase bearing CD4 molecule with the TcR/CD3 complex. In this paper we demonstrate that this crosslinking-induced suppression can be bypassed by perturbing the microfilament system of CD4+ cells by pretreatment with 1 microM cytochalasin D. Using the fluorescence resonance energy transfer method, we have shown that the cytochalasin D-affected increase of mitogenesis is not due to changes in the TcR/CD3 to CD4 distance. Likewise, other membrane biophysical parameters, membrane potential and lateral diffusion of surface receptors, cannot be associated with these cytochalasin D-affected mitogenic changes. Cytochalasin D treatment elevates intracellular Ca2+ levels induced by wt31 mAb plus crosslinking and generates a TcR/CD3-dependent signal which is cyclosporin sensitive.
- Published
- 1994
- Full Text
- View/download PDF
44. The interaction of immunosuppressive compounds in tandem stimulated peripheral human lymphocytes.
- Author
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Weaver JL, Pine PS, and Aszalos A
- Subjects
- Antibodies, Monoclonal immunology, Benzoquinones, CD28 Antigens immunology, Cells, Cultured, Cyclosporine pharmacology, Drug Interactions, Humans, Immunosuppressive Agents administration & dosage, Interleukin-2 immunology, Lactams, Macrocyclic, Mycophenolic Acid pharmacology, Polyenes pharmacology, Quinones pharmacology, Receptors, Antigen, T-Cell immunology, Rifabutin analogs & derivatives, Sirolimus, Suramin pharmacology, Tacrolimus pharmacology, Immunosuppressive Agents pharmacology, Lymphocytes drug effects
- Abstract
We have developed an in vitro system to model the interactions of drugs used to treat transplant rejection. This system consists of stimulation of human lymphocytes with a primary mitogen (anti-T-cell receptor complex antibodies (OKT3 or wt31)) and treatment with a primary immunosuppressive drug (ISD) (Cyclosporine A (CsA) or FK-506)). This is later followed by stimulation with a secondary mitogen (Interleukin-2 or anti-CD28), and treatment with a second ISD. This system allows a variety of concentrations and compounds to be rapidly tested. We have used this system to study the effect of various compounds when used as either primary or secondary ISDs. Our results show that when CsA is used as the primary ISD, further proliferation can be inhibited by rapamycin, mycophenolic acid, or suramin. When FK-506 is the primary ISD, inhibition of proliferation by rapamycin is variable depending on the primary and secondary mitogens. If rapamycin is the primary ISD, both CsA and FK-506 show antagonistic interactions. These results suggest that the order in which combinations of ISDs are administered in transplantation may have significant effects on the clinical outcome.
- Published
- 1994
- Full Text
- View/download PDF
45. Overview of laser microbeam applications as related to antibody targeting.
- Author
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Pine PS
- Subjects
- Animals, Biophysical Phenomena, Biophysics, Cell Death, Cell Membrane metabolism, Cell Separation methods, Energy Transfer, Flow Cytometry methods, Fluorescent Dyes, Humans, Immunohistochemistry instrumentation, Membrane Proteins metabolism, Microscopy, Fluorescence instrumentation, Optics and Photonics instrumentation, Antibodies, Immunohistochemistry methods, Lasers, Microscopy, Fluorescence methods
- Published
- 1994
- Full Text
- View/download PDF
46. Specific disengagement of cell-bound anti-LAM-1 (anti-L-selectin) antibodies by aurintricarboxylic acid.
- Author
-
Szabó G Jr, Weaver JL, Pine PS, and Aszalos A
- Subjects
- Binding Sites, Antibody, Binding, Competitive, Flow Cytometry, Humans, L-Selectin, Antigen-Antibody Reactions drug effects, Aurintricarboxylic Acid pharmacology, Cell Adhesion Molecules immunology, Lymphocytes drug effects
- Abstract
Brief treatment of human peripheral blood lymphocytes with the potential anti-HIV compound aurintricarboxylic acid (ATA) prompts the selective release of already bound L-selectin-specific anti-Leu8 and anti-LAM1-1 antibodies from the cells. Two other anti-LAM1 antibodies, anti-LAM1-3 and anti-LAM1-5 stay antigen-bound at the same time. Interestingly, the ATA-sensitive anti-Leu8 strongly competes with the ATA-resistant anti-LAM1-3 for binding. Photobleaching fluorescence resonance energy transfer (pFRET) measurements on flow-sorted cells suggests that these two antibodies compete for the same epitope, while anti-LAM1-5-FITC and anti-Leu8-PE bind to distinct sites, although they also compete for binding. Combining the data on competition, pFRET and ATA effect, we suggest that the ATA sensitive anti-Leu8 and resistant anti-LAM1-3 bind to overlapping but non-identical epitopes. This remarkably specific effect may be exploited for designing anti-inflammatory drugs that modulate leukocyte adhesion.
- Published
- 1993
- Full Text
- View/download PDF
47. The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter.
- Author
-
Weaver JL, Szabo G Jr, Pine PS, Gottesman MM, Goldenberg S, and Aszalos A
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1, Amino Acid Sequence, Animals, Benzofurans pharmacology, Biological Transport, Active drug effects, Carrier Proteins metabolism, Cell Membrane drug effects, Cell Membrane metabolism, Cell Membrane physiology, Daunorubicin metabolism, Daunorubicin pharmacokinetics, Drug Resistance, Ethers, Cyclic pharmacology, Flow Cytometry, Fluorescent Dyes, Hydrogen-Ion Concentration, Intracellular Fluid chemistry, Leukemia L5178 drug therapy, Leukemia L5178 metabolism, Lymphoma, T-Cell drug therapy, Lymphoma, T-Cell metabolism, Membrane Glycoproteins metabolism, Membrane Potentials drug effects, Membrane Potentials physiology, Mice, Molecular Sequence Data, Potassium, Potassium Channels drug effects, Potassium Channels metabolism, Rhodamine 123, Rhodamines metabolism, Rhodamines pharmacokinetics, Tumor Cells, Cultured, Carrier Proteins drug effects, Carrier Proteins physiology, Immunosuppressive Agents pharmacology, Ion Channels drug effects, Ion Channels metabolism, Membrane Glycoproteins drug effects, Membrane Glycoproteins physiology
- Abstract
The MDRI protein is an energy-dependent transport protein responsible for the multi-drug resistance seen in many tumors. A variety of drugs have been shown to inhibit the function of this pump, including compounds known to block various ion channels. The mouse lymphoma cell line L5178Y has been transduced with the human mdrI gene. Using this cell line, we have tested a number of compounds to determine whether there is a correlation between the ability to block a specific type of ion channel, or shift membrane potential, and the ability to act as an MDR-reversing agent using the fluorescent substrates Rhodamine 123 and daunorubicin as test compounds. Our results show no apparent correlation between the ability to block a specific ion channel and reversal of MDR transport ability. We have found active MDR inhibitors in compounds that affect K+, Na+, Ca++, H+, but not Cl- channels. Our data suggest that Cl- channel activity may be distinct from MDR activity. Several immunosuppressive compounds and analogs were also tested and found to be active reversing agents. Measurements suggest a significant difference in resting membrane potential between the L5178YvMDR line and the L5178Y parental cell line used in these experiments. No correlation was found between the ability of drugs to alter membrane potential and to inhibit MDR transport activity. Our results suggest that MDR transport function may be independent of the physiological movement of ions and show that a wide variety of compounds can inhibit MDR transport.
- Published
- 1993
- Full Text
- View/download PDF
48. CD4 changes conformation upon ligand binding.
- Author
-
Szabò G Jr, Pine PS, Weaver JL, Rao PE, and Aszalos A
- Subjects
- Antibodies, Monoclonal immunology, CD4 Antigens metabolism, Humans, In Vitro Techniques, Ligands, Membrane Fluidity, Microscopy, Fluorescence, Protein Conformation, Antigen-Antibody Reactions drug effects, Aurintricarboxylic Acid pharmacology, CD4 Antigens chemistry
- Abstract
Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with > or = 1 micrograms/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its Ag.
- Published
- 1992
49. Prevention of binding of rgp120 by anti-HIV active tannins.
- Author
-
Weaver JL, Pine PS, Dutschman G, Cheng YC, Lee KH, and Aszalos A
- Subjects
- Drug Design, Humans, Lymphocytes drug effects, Lymphocytes immunology, Protein Binding drug effects, Recombinant Proteins metabolism, Antiviral Agents pharmacology, CD4 Antigens metabolism, HIV Envelope Protein gp120 metabolism, Tannins pharmacology
- Abstract
Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.
- Published
- 1992
- Full Text
- View/download PDF
50. Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system.
- Author
-
Szabà G Jr, Pine PS, Weaver JL, Kasari M, and Aszalos A
- Subjects
- Antibodies, Monoclonal, Cell Line, Fluorescein-5-isothiocyanate, Humans, Immunoglobulin Fab Fragments, Lasers, Leukemia, T-Cell, Microscopy, Electron, Scanning methods, Microscopy, Fluorescence methods, Receptors, Antigen, T-Cell immunology, Spectrometry, Fluorescence, Epitopes analysis, Lymphocytes immunology, Receptors, Antigen, T-Cell analysis
- Abstract
The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.
- Published
- 1992
- Full Text
- View/download PDF
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