RESUMEN
Proteomics has the potential to identify pharmacodynamic (PD) biomarkers for similarity assessment of proposed biosimilars without relying on clinical efficacy end points. In this study, with 36 healthy participants randomized to therapeutic doses of interferon-beta 1a products (IFNß-1a) or pegylated-IFNß-1a (pegIFNß-1a) approved to treat multiple sclerosis or placebo, we evaluated the utility of a proteomic assay that profiles > 7,000 plasma proteins. IFNß-1a and pegIFNß-1a resulted in 248 and 528 differentially expressed protein analytes, respectively, between treatment and placebo groups over the time course. Thirty-one proteins were prioritized based on a maximal fold change ≥ 2 from baseline, baseline adjusted area under the effect curve (AUEC) and overlap between the 2 products. Of these, the majority had a significant AUEC compared with placebo in response to either product; 8 proteins showed > 4-fold maximal change from baseline. We identified previously reported candidates, beta-2microglobulin and interferon-induced GTP-binding protein (Mx1) with ~ 50% coefficient of variation (CV) for AUEC, and many new candidates (including I-TAC, C1QC, and IP-10) with CVs ranging from 26%-129%. Upstream regulator analysis of differentially expressed proteins predicted activation of IFNß1 signaling as well as other cytokine, enzyme, and transcription signaling networks by both products. Although independent replication is required to confirm present results, our study demonstrates the utility of proteomics for the identification of individual and composite candidate PD biomarkers that may be leveraged to support clinical pharmacology studies for biosimilar approvals, especially when biologics have complex mechanisms of action or do not have previously characterized PD biomarkers.
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Biosimilares Farmacéuticos , Esclerosis Múltiple , Humanos , Interferón beta/uso terapéutico , Biosimilares Farmacéuticos/uso terapéutico , Proteómica , Interferón beta-1a/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , BiomarcadoresRESUMEN
Unexpected mortality occurred in a group of 12 NOD.Cg-NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and 12 NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) immunodeficient mice. At 10 d after routine bone marrow-liver-thymus humanization surgery, 9 mice were found dead without observation of initiating clinical signs; 1 d later (day 11), 3 additional mice showed signs of morbidity, including severe hunching, lateral recumbency, slow movement, shallow respiration, and decreased response to external stimulus. All remaining mice rapidly decompensated and were found dead or were euthanized within 4 d after the first death. Histopathology revealed severe ascending pyelonephritis with numerous yeast. Cultures in some mice were positive for Enterococcus faecalis or Staphylococcus xylosus, 2 bacteria considered commensals in rodents. In addition, Candida albicans was cultured from some animals. Further investigation revealed that a restraining device used for tail vein injections was the likely fomite harboring Candida organisms. These findings indicate that ascending pyelonephritis, with Candida as the etiologic agent, can cause significant mortality in NSG and NRG immunodeficient mice.
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Candidiasis/veterinaria , Infecciones Oportunistas/veterinaria , Pielonefritis/veterinaria , Enfermedades de los Roedores/microbiología , Animales , Candidiasis/complicaciones , Candidiasis/epidemiología , Brotes de Enfermedades/veterinaria , Femenino , Humanos , Huésped Inmunocomprometido , Ratones Endogámicos NOD , Infecciones Oportunistas/complicaciones , Pielonefritis/complicaciones , Pielonefritis/epidemiología , Pielonefritis/microbiología , Enfermedades de los Roedores/epidemiología , Trasplante de TejidosRESUMEN
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.
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Encéfalo/metabolismo , Perfilación de la Expresión Génica/normas , Genoma Humano , Hígado/metabolismo , MicroARNs/genética , Placenta/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Embarazo , Estándares de ReferenciaRESUMEN
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.
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Lesiones Cardíacas/metabolismo , MicroARNs/sangre , MicroARNs/orina , Animales , Biomarcadores/sangre , Biomarcadores/orina , Lesiones Cardíacas/inducido químicamente , Isoproterenol/toxicidad , Masculino , Plasma/química , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Suero/químicaRESUMEN
Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection. Circulating proinflammatory cytokines and/or miRNAs that track with the onset and progression of lethal A/Puerto Rico/8/34 (PR8) influenza A virus infection and their response to oseltamivir treatment were investigated up to 10 days after infection. Changes in plasma cytokines (IL-1ß, IL-10, IL-12p70, IL-6, KC, TNF-α, and IFN-γ) and several candidate miRNAs were profiled. Among the cytokines analyzed, IL-6 and KC/GRO cytokines appeared to correlate with peak viral titer. Over the selected 48 miRNAs profiled, certain miRNAs were up- or downregulated in a manner that was dependent on the oseltamivir treatment and disease severity. Our findings suggest that IL-6 and KC/GRO cytokines can be a potential disease severity biomarker and/or marker for the progression/remission of infection. Further studies to explore other cytokines, miRNAs, and lung injury proteins in serum with different subtypes of influenza A viruses with varying disease severity may provide new insight into other unique biomarkers.
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Antivirales/administración & dosificación , Citocinas/sangre , MicroARNs/genética , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/administración & dosificación , Animales , Antivirales/farmacología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Virus de la Influenza A/efectos de los fármacos , Ratones , MicroARNs/sangre , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Oseltamivir/farmacología , Resultado del Tratamiento , Carga Viral/efectos de los fármacosRESUMEN
Phospholipidosis (PLD), an abnormal accumulation of phospholipids within tissues, is observed during the preclinical testing of many pharmaceutical drugs. Diagnosis of PLD is currently based on morphologic criteria. An understanding of the clinical incidence of PLD and its possible relationship to adverse drug reactions has been hampered by the absence of noninvasive biomarkers for PLD. The uncommon phospholipid di-docosahexaenoyl bis(monoacylglycerol) phosphate (di-22:6-BMP) has been proposed as a potential urinary biomarker for PLD, but data on the utility of serum di-22:6-BMP measurements in diagnosing PLD is more limited. In this report, we compared the performance of serum and urinary di-22:6-BMP as biomarkers for PLD in rats treated with the PLD-inducing drugs amiodarone and 4,4'-diethylaminoethoxyhexestrol dihydrochloride or the hepatotoxicant acetaminophen (APAP). Serum levels of di-22:6-BMP showed a higher correlation to a generalized PLD incidence score than did levels in urine, but were unexpectedly elevated in rats with marked levels of APAP-induced liver necrosis. When samples were filtered based on serum ALT or liver histopathology thresholds, the diagnostic accuracy of serum di-22:6-BMP for PLD improved to the high level observed for urinary di-22:6-BMP without sample exclusion. These data help define the potential context-of-use of serum di-22:6-BMP as a non-clinical biomarker of PLD.
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Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/orina , Lipidosis/sangre , Lipidosis/orina , Lisofosfolípidos/sangre , Lisofosfolípidos/orina , Acetaminofén/toxicidad , Amiodarona/toxicidad , Animales , Biomarcadores/sangre , Biomarcadores/orina , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Histocitoquímica , Masculino , Microscopía Electrónica de Transmisión , Fosfolípidos/sangre , Fosfolípidos/orina , Ratas , Ratas Endogámicas F344RESUMEN
Cationic amphiphilic drugs and aminoglycoside antibiotics can induce phospholipidosis (PLD), an abnormal accumulation of phospholipids in lysosome-derived vesicles, in preclinical studies. The incidence of PLD in patients and its clinical relevance are difficult to assess without noninvasive biomarkers. Di-docosahexaenoyl bis(monoacylglycerol)phosphate (di-22:6-BMP) is a phospholipid that is enriched in lysosomal membranes and a proposed urinary biomarker of drug-induced PLD. The specificity of di-22:6-BMP for PLD was compared to other phospholipid species that can increase in urine with nephrotoxicity. Using liquid chromatography coupled to mass spectrometry, 12 phospholipids were assayed in the urine of rats treated with drugs that induced PLD or caused renal or skeletal muscle injury. In receiver operating curve analyses, urinary di-22:6-BMP was a significantly better predictor of PLD and the least predictive of tissue injury of the phospholipids assayed. The data provide evidence supporting the use of di-22:6-BMP as a urinary biomarker of PLD in rats.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Enfermedades Renales/inducido químicamente , Lisofosfolípidos/orina , Fosfolípidos/orina , Animales , Biomarcadores/orina , Moléculas de Adhesión Celular/orina , Cisplatino/efectos adversos , Femenino , Gentamicinas/efectos adversos , Hexestrol/efectos adversos , Hexestrol/análogos & derivados , Enfermedades Renales/patología , Enfermedades Renales/orina , Lipocalina 2 , Lipocalinas/orina , Hígado/efectos de los fármacos , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Osteopontina/orina , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Simvastatina/efectos adversos , Bazo/efectos de los fármacos , Bazo/patología , Troponina I/sangreRESUMEN
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.
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Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/metabolismo , Área Bajo la Curva , Benchmarking , Técnicas de Laboratorio Clínico , Regulación de la Expresión Génica , Humanos , Hígado/metabolismo , Músculo Esquelético/metabolismo , Especificidad de Órganos , Curva ROCRESUMEN
We designed a study to provide reversibility and comparative injury data for several candidate urinary biomarkers of kidney injury in the United States Food and Drug Administration biomarker qualification process. The nephrotoxin gentamicin was given to rats once on each of three days and the animals were killed during dosing or over the following 42 days. Between days one and three, all biomarkers except albumin were elevated, peaked at day 7, and returned to control levels by day 10 (µ- and α-glutathione S-transferases, and renal papillary antigen-1) or day 15 (kidney injury molecule-1, lipocalin-2, osteopontin, and clusterin). All biomarkers performed better during injury than during recovery except osteopontin, which performed equally well in both time periods. During the evolution of injury, kidney injury molecule-1, renal papillary antigen-1, and clusterin best mirrored the histopathologic lesions. During injury resolution, kidney injury molecule-1, osteopontin, and blood urea nitrogen best reflected recovery. Based on histopathology, necrosis, or apoptosis scoring, kidney injury molecule-1 was the best biomarker of overall renal injury. Evaluation by regeneration score showed that renal papillary antigen-1 best reflected tubular and/or collecting duct regeneration, especially during recovery. Thus, these biomarkers performed with different effectiveness when evaluated by individual pathological processes such as necrosis, apoptosis, and regeneration.
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Lesión Renal Aguda/orina , Biomarcadores/orina , Túbulos Renales Proximales/metabolismo , Proteómica , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Apoptosis , Biomarcadores/sangre , Moléculas de Adhesión Celular/orina , Clusterina/orina , Gentamicinas , Glutatión Transferasa/orina , Inmunohistoquímica , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Lipocalinas/orina , Masculino , Necrosis , Osteopontina/orina , Valor Predictivo de las Pruebas , Proteómica/métodos , Curva ROC , Ratas , Ratas Sprague-Dawley , Juego de Reactivos para Diagnóstico , Regeneración , Factores de TiempoRESUMEN
PURPOSE: The antineoplastic anthracycline doxorubicin can induce a dose-dependent cardiomyopathy that limits the total cumulative dose prescribed to cancer patients. In both preclinical and clinical studies, pretreatment with dexrazoxane, an intracellular iron chelator, partially protects against anthracycline-induced cardiomyopathy. To identify potential additional cardioprotective treatment strategies, we investigated early doxorubicin-induced changes in cardiac gene expression. METHODS: Spontaneously hypertensive male rats (n = 47) received weekly intravenous injections of doxorubicin (3 mg/kg) or saline 30 min after pretreatment with dexrazoxane (50 mg/kg) or saline by intraperitoneal injection. Cardiac samples were analyzed 24 h after the first (n = 20), second (n = 13), or third (n = 14) intravenous injection on days 1, 8, or 15 of the study, respectively. RESULTS: Rats receiving three doses of doxorubicin had minimal myocardial alterations that were attenuated by dexrazoxane. Cardiac expression levels of genes associated with the Nrf2-mediated stress response were increased after a single dose of doxorubicin, but not affected by cardioprotectant pretreatment. In contrast, an early repressive effect of doxorubicin on transcript levels of genes associated with mitochondrial function was attenuated by dexrazoxane pretreatment. Dexrazoxane had little effect on gene expression by itself. CONCLUSIONS: Genomic analysis provided further evidence that mitochondria are the primary target of doxorubicin-induced oxidative damage that leads to cardiomyopathy and the primary site of cardioprotective action by dexrazoxane. Additional strategies that prevent the formation of oxygen radicals by doxorubicin in mitochondria may provide increased cardioprotection.
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Antibióticos Antineoplásicos , Doxorrubicina , Cardiopatías , Miocardio , Animales , Masculino , Ratas , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Doxorrubicina/toxicidad , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Cardiopatías/inducido químicamente , Cardiopatías/metabolismo , Cardiopatías/prevención & control , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas Endogámicas SHR , Razoxano/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Troponina T/sangre , Troponina T/metabolismo , Factor 2 Relacionado con NF-E2/biosíntesis , Factor 2 Relacionado con NF-E2/genéticaRESUMEN
Sensitive biomarkers are needed to detect kidney injury at the earliest stages. The objective of this study was to determine whether the appearance of kidney injury molecule-1 (Kim-1) protein ectodomain in urine and kidney injury molecule-1/hepatitis A viral cellular receptor-1 (Kim-1/Havcr1) gene expression in kidney tissue may be more predictive of renal injury after exposure to nephrotoxicants when compared to traditionally used biomarkers. Male Sprague-Dawley rats were injected with a range of doses of gentamicin, mercury (Hg; HgCl2), or chromium (Cr; K2Cr2O7). The results showed that increases in urinary Kim-1 and kidney Kim-1/Havcr1 gene expression paralleled the degree of severity of renal histopathology and were detected at lower doses of nephrotoxicants when compared to blood urea nitrogen (BUN), serum creatinine, and urinary N-acetyl-beta-D-glucosaminidase (NAG). In a time course study, urinary Kim-1 was elevated within 24 h after exposure to gentamicin (100 mg/kg), Hg (0.25 mg/kg), or Cr (5 mg/kg) and remained elevated through 72 h. NAG responses were nephrotoxicant dependent with elevations occurring early (gentamicin), late (Cr), or no change (Hg). At 72 h, after treatment with any of the three nephrotoxicants, there was increased Kim-1 immunoreactivity and necrosis involving approximately 50% of the proximal tubules; however, only urinary Kim-1 was significantly increased, while BUN, serum creatinine, and NAG were not different from controls. In rats treated with the hepatotoxicant galactosamine (1.1 mg/kg), serum alanine aminotransferase was increased, but no increase in urinary Kim-1 was observed. Urinary Kim-1 and kidney Kim-1/Havcr1 expression appear to be sensitive and tissue-specific biomarkers that will improve detection of early acute kidney injury following exposure to nephrotoxic chemicals and drugs.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Cromo/toxicidad , Gentamicinas/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/orina , Riñón/química , Proteínas de la Membrana/metabolismo , Mercurio/toxicidad , Inhibidores de la Síntesis de la Proteína/toxicidad , Acetilglucosamina/orina , Animales , Biomarcadores , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/orina , Enfermedad Hepática Inducida por Sustancias y Drogas/orina , Relación Dosis-Respuesta a Droga , Galactosamina/toxicidad , Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Riñón/patología , Enfermedades Renales/patología , Pruebas de Función Renal , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/orina , ARN/biosíntesis , ARN/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/genética , Animales , Perfilación de la Expresión Génica , Masculino , ARN/química , ARN/metabolismo , Estabilidad del ARN , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Manejo de Especímenes/métodosRESUMEN
A multi-age rat model was used to identify potential age-related differences in renal injury following exposure to gentamicin (GM). In this study, 10-, 25-, 40-, and 80-day-old Sprague-Dawley rats were dosed with GM at 0, 50, or 100 mg kg(-1) body weight per day (mkd) sc for 6 or 14 days. Urine samples were collected up to 72 h after initial dosing. The maximum tolerated dose was lower in 10-day-old rats than for other ages (none survived 11 days of treatment). Eighty-day-old rats given the highest dose showed a diminished rate of growth and an increase in serum creatinine, blood urea nitrogen (BUN), urinary kidney injury molecule-1 (Kim-1), and renal pathology. Ten- and 40-day-old rats given 100 mkd of GM for 6- or 14 days also had increased levels of serum BUN and Cr and renal pathology, whereas only mild renal alterations were found in 25-day-old rats. After 6 days of treatment with 100 mkd GM, significant increases in Havcr-1 (Kim-1) gene expression were detected only in 10- and 80-day-old rats. In urine samples, nuclear magnetic resonance and ultra performance liquid chromatography/mass spectrometry analysis detected changes related to GM efficacy (e.g., hippurate) and increases in metabolites related to antioxidant activity, which was greatest in the 80-day-old rats. The magnitude of the genomic, metabonomic, and serum chemistry changes appeared to correlate with the degree of nephropathy. These findings indicate that an experimental animal model that includes several developmental stages can detect age-related differences in drug-induced organ toxicities and may be a useful predictor of pediatric drug safety in preclinical studies.
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Riñón/efectos de los fármacos , Pediatría , Factores de Edad , Animales , Corazón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Espectrometría de Masas , Modelos Animales , Osteopontina/genética , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Necrosis Tumoral/genética , Bazo/efectos de los fármacos , Receptor de TWEAKRESUMEN
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.
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Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , ARN Mensajero/normas , Animales , Perfilación de la Expresión Génica/métodos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los Resultados , Distribución TisularRESUMEN
Identifying genes that are differentially expressed in response to DNA damage may help elucidate markers for genetic damage and provide insight into the cellular responses to specific genotoxic agents. We utilized cDNA microarrays to develop gene expression profiles for ionizing radiation-exposed human lymphoblastoid TK6 cells. In order to relate changes in the expression profiles to biological responses, the effects of ionizing radiation on cell viability, cloning efficiency, and micronucleus formation were measured. TK6 cells were exposed to 0.5, 1, 5, 10, and 20 Gy ionizing radiation and cultured for 4 or 24 hr. A significant (P < 0.0001) decrease in cloning efficiency was observed at all doses at 4 and 24 hr after exposure. Flow cytometry revealed significant decreases in cell viability at 24 hr in cells exposed to 5 (P < 0.001), 10 (P < 0.0001), and 20 Gy (P < 0.0001). An increase in micronucleus frequency occurred at both 4 and 24 hr at 0.5 and 1 Gy; however, insufficient binucleated cells were present for analysis at the higher doses. Gene expression profiles were developed from mRNA isolated from cells exposed to 5, 10, and 20 Gy using a 350 gene human cDNA array platform. Overall, more genes were differentially expressed at 24-hr than at the 4-hr time point. The genes upregulated (> 1.5-fold) or downregulated (< 0.67-fold) at 4 hr were those primarily involved in the cessation of the cell cycle, cellular detoxification pathways, DNA repair, and apoptosis. At 24 hr, glutathione-associated genes were induced in addition to genes involved in apoptosis. Genes involved in cell cycle progression and mitosis were downregulated at 24 hr. Real-time quantitative PCR was used to confirm the microarray results and to evaluate expression levels of selected genes at the low doses (0.5 and 1.0 Gy). The expression profiles reflect the cellular and molecular responses to ionizing radiation related to the recognition of DNA damage, a halt in progression through the cell cycle, activation of DNA-repair pathways, and the promotion of apoptosis.
Asunto(s)
Daño del ADN , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Timidina Quinasa/genética , Análisis de Varianza , Ciclo Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Cartilla de ADN , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Humanos , Pruebas de Micronúcleos , Análisis de Secuencia por Matrices de Oligonucleótidos , Radiación Ionizante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Colorantes , Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Célula , Grupos Control , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Reproducibilidad de los Resultados , Timidina Quinasa , Transcripción GenéticaRESUMEN
Understanding the strengths and limitations of alternative models, such as the Tg.AC assay, for evaluation of the potential carcinogenicity of pharmaceuticals requires assessment of assay specificity through studies that specifically target biologically active compounds that are known to not be carcinogens in rodents. To identify drugs that might provoke a false positive response in the Tg.AC assay, we screened pharmaceuticals for in vitro induction of the gadd153 promoter and the zeta-globin promoter. We have previously found a high correlation between induction of the gadd153 promoter in HepG2 cells and activity in the Tg.AC assay. The three drugs selected through screening 99 noncarcinogenic pharmaceuticals were amiloride, dipyridamole, and pyrimethamine. A 26-week skin paint study was conducted in hemizygous Tg.AC mice with the three drugs at two doses selected by a 4-week dose range finding study. Evidence of systemic toxicity was observed in animals dosed chronically with pyrimethamine or amiloride, but no skin papillomas were observed in mice treated with amiloride, dipyridamole, or pyrimethamine for 26 weeks. All male mice and 80% of female mice treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in acetone developed a maximal tumor burden. However, mice treated with TPA in a vehicle containing 2.4% DMSO had greatly reduced incidences of papillomas. In summary, the correct negative response was shown in the Tg.AC assay for three noncarcinogenic pharmaceuticals, which adds further favorable evidence of appropriate specificity of this model system. However, vehicle composition must be carefully selected because the outcome of this assay can be confounded by certain commonly used solvents.