RESUMEN
One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Vacunas contra el Cáncer/inmunología , Linfocitos T Citotóxicos/citología , Antígenos Virales/inmunología , Técnicas de Cultivo de Célula , Citomegalovirus/inmunología , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo/métodos , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Recuento de Linfocitos , Sensibilidad y Especificidad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: Celiac plexus neurolysis, although effective in relieving pain associated with upper abdominal malignancy, occasionally results in paraplegia. Diffusion of the neurolytic agent to arteries supplying the spinal cord has been postulated as a cause, and previous studies with isolated lumbar segmental arteries have demonstrated contraction in response to ethanol and phenol. The mechanism of this contractile effect is unknown, but a role for insular free calcium (Ca2+i) is suggested by the known involvement of Ca2+i in both smooth muscle vasoconstriction and toxic cell injury. The authors sought to determine whether nontoxic concentrations of ethanol cause a direct elevation of Ca2+i in arterial smooth muscle and endothelium. METHODS: Primary cultures of human aortic smooth muscle and endothelial cells were studied to determine the direct effect of ethanol independent of interactions with agonists or contractile proteins. Ca2+i levels were determined in single cells with digitized video fluorescence microscopy, using ratio imaging of the Ca2+i-sensitive fluorophore fura-2. RESULTS: In aortic smooth muscle cells, initial Ca2+i was 98 +/- 41 nM (n = 59 cells). Histamine (10 microM) as a positive control caused an increase in Ca2+i, as expected. Ethanol alone, at doses of 2-5% (v/v) also caused a sustained elevation in Ca2+i of physiologically significant magnitude. Ethanol at doses of 5% or lower did not cause any visibly apparent injury within 30 minutes. In contrast, 10% or higher ethanol doses quickly caused membrane blebbing, a sign of toxic injury, followed by cell death within 20 minutes. Aortic endothelial cells were more resistant to ethanol than smooth muscle cells, in terms of both Ca2+i elevation and cell death. CONCLUSIONS: Ethanol, even at nontoxic concentrations, has a direct effect on aortic smooth muscle Ca2+i, large enough to be associated with significant vasoconstriction. The findings suggest a possible role for pharmacologic agents that preserve Ca2+i homeostasis in protecting against neurolysis-induced paraplegia, although additional study is required before clinical application is appropriate.
Asunto(s)
Calcio/metabolismo , Plexo Celíaco/efectos de los fármacos , Endotelio Vascular/metabolismo , Etanol/efectos adversos , Músculo Liso Vascular/metabolismo , Bloqueo Nervioso/efectos adversos , Células Cultivadas , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
BACKGROUND: Oxidant damage to endothelial cells occurs during inflammation and reperfusion after ischemia, mediated in part by endogenously produced hydrogen peroxide (H2O2). Previous studies have established a role for increased cytosolic calcium in the mechanism of endothelial oxidant injury, and have suggested that volatile anesthetics may exacerbate oxidant injury in pulmonary endothelium. However, the effect of volatile anesthetics on oxidant injury to systemic arterial endothelial cells, and their effect on oxidant-related changes in cytosolic calcium homeostasis, have not been reported previously. METHODS: Primary cultures of human aortic and pulmonary arterial endothelial cells were studied. The rate of cell death after H2O2 exposure was determined in cell suspension by propidium iodide fluorimetry and lactate dehydrogenase release. The final extent of cell death 24 h after H2O2 exposure was determined in monolayer cultures by methyl thiazolyl tetrazolium reduction. Cytosolic calcium and cell death were determined in single cells using fura-2 and propidium iodide imaging with digitized, multiparameter, fluorescent video microscopy. RESULTS: In aortic endothelial cells, clinical concentrations of halothane (1.0%) and isoflurane (1.5%) decreased both the rate of cell death and the final extent of cell death after H2O2 exposure, with halothane being more protective. Supraclinical concentrations of halothane (2.7%) and isoflurane (4.0%) were less protective. In pulmonary arterial endothelial cells, halothane and isoflurane had essentially no effect on H2O2-mediated cell death. The protective effect of anesthetic in aortic endothelial cells was not due to an enhanced removal of H2O2 by endogenous enzymes. Hydrogen peroxide exposure caused a large increase in cytosolic calcium well before cell death, and this was moderated by anesthetic treatment. CONCLUSIONS: The effect of volatile anesthetics on oxidant injury to endothelial cells may differ between cells derived from systemic and pulmonary vascular beds. Halothane, and to a lesser extent, isoflurane, protects against oxidant injury in aortic endothelial cells. The mechanism of protection may involve modulation of the interaction of H2O2 with vital cellular constituents, and/or amelioration of the toxic increase in cytosolic calcium that follows such interaction.
Asunto(s)
Anestésicos por Inhalación/farmacología , Endotelio Vascular/efectos de los fármacos , Halotano/farmacología , Peróxido de Hidrógeno/toxicidad , Isoflurano/farmacología , Arteria Pulmonar/efectos de los fármacos , Aorta/efectos de los fármacos , Aorta/lesiones , Aorta/metabolismo , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Células Cultivadas , Citoplasma/metabolismo , Endotelio Vascular/lesiones , Endotelio Vascular/metabolismo , Humanos , Peróxido de Hidrógeno/farmacocinética , Hipoxia/metabolismo , Inactivación Metabólica , Microscopía Fluorescente , Arteria Pulmonar/lesiones , Arteria Pulmonar/metabolismo , Sensibilidad y EspecificidadRESUMEN
Elevated free cytosolic Ca2+ (Ca2+i) has been implicated as a mechanism of hypoxic neuronal death. The calcium hypothesis postulates that the basic metabolic response to hypoxic ATP depletion is a toxic increase in free cytosolic Ca2+i in all cell types. This inherent response then creates the environment in which subsequent derangements of Ca2+i may occur, for example, from glutamate excitotoxicity. Although the effect of glutamate on neuronal Ca2+i has been extensively studied, the basic neuronal response to hypoxia independent of glutamate receptor activation is not well defined. We therefore assayed both Ca2+i and plasma membrane integrity in fura-2-loaded, single SK-N-SH neuroblastoma cells, using digitized video microscopy and metabolic inhibition (2.5 mM NaCN, 10 mM 2-deoxyglucose) to model the ATP depletion of hypoxia. Median time to cell death was 90 min (n = 51 cells). Initial Ca2+i was 121 +/- 67 nM. Ca2+i increased by 50 nM after 5-10 min of metabolic inhibition. Blebbing of the cell membrane was evident within 30 min. Ca2+i did not appreciably increase further until the time of cell death, when the loss of plasma membrane integrity allowed unimpeded influx of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Neuronas/metabolismo , Muerte Celular , Cianuros/farmacología , Citoplasma/metabolismo , Desoxiglucosa/farmacología , Fura-2 , Humanos , Peróxido de Hidrógeno/farmacología , Neuronas/efectos de los fármacos , Receptores de Glutamato/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: In hypoxic rats, halothane causes hepatotoxicity at oxygen levels that would cause minimal hepatotoxicity in the absence of halothane. Using a model that excludes systemic and extrahepatic effects of halothane, the authors tested the hypothesis that halothane hepatotoxicity in the whole-rat model is caused by a direct hepatotoxic effect of halothane, which is mediated by halothane-derived free radicals. METHODS: Rat hepatocyte monolayer cultures were exposed to defined gas phases for 2 h. Three experimental variables were present or absent: hypoxia (1% O2), halothane (2%), and cytochrome P-450 induction (by phenobarbital). Two experimental outcomes were measured: aspartate aminotransferase release, a measure of cell death, and reduced glutathione, an endogenous free radical scavenger whose levels are decreased by physiologically significant free radical injury. RESULTS: As anticipated, hypoxia increased cell death. Cytochrome P-450 induction by itself increased cell death during hypoxia. However, halothane had no effect on cell death during hypoxia, with or without cytochrome P-450 induction. Halothane had no toxic effect, even when glutathione was depleted before the onset of hypoxia. Glutathione was decreased moderately by hypoxia alone. Neither halothane nor cytochrome P-450 induction had any effect on glutathione levels. CONCLUSIONS: Halothane was not toxic, and it did not generate a physiologically significant free radical insult during hypoxia in the isolated rat hepatocyte under the experimental conditions used in testing.
Asunto(s)
Hipoxia de la Célula/efectos de los fármacos , Glutatión/metabolismo , Halotano/farmacología , Animales , Muerte Celular , Células Cultivadas , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
In vitro drug sensitivity testing, both by optical colony counting and by a [3H]-TdR incorporation assay, was performed on human tumour cells proliferating in soft agar cultures. Cells from two different human tumour cell lines, 5 different human tumour xenografts, and 94 different primary human tumour specimens of various histologic types were studied. Regression analysis comparing the results of the colony counting assay and the [3H]-TdR assay revealed good to excellent correlations between the two assay endpoints for quantitating the effect of in vitro anticancer drug exposure for a large number of different agents. The presence of pre-existing tumour cell aggregates complicates the performance of the optical colony counting assay. The [3H]-TdR incorporation assay is more sensitive and reproducible than the colony counting assay when performed on samples containing a large number of initially seeded tumour cell aggregates.
Asunto(s)
Antineoplásicos/farmacología , Ensayo de Unidades Formadoras de Colonias , Timidina/metabolismo , Ensayo de Tumor de Célula Madre , Recuento de Células , Línea Celular , Femenino , Humanos , Trasplante de Neoplasias , Neoplasias/metabolismo , Sefarosa , Trasplante Heterólogo , TritioRESUMEN
The photosensitizing/cytotoxic effects of four porphyrin compounds derived from hematoporphyrin were compared using two human tumor cell lines with a soft-agar colony-forming assay. Reproducible dose- and time-dependent increases in reproductive cell death were observed for each porphyrin tested. Two fractions derived from hematoporphyrin derivative (HPD) were found to be better photosensitizers than HPD itself, indicating the potential value of this in vitro assay for detecting the most active component from a heterogeneous mixture.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Hematoporfirinas/farmacología , Luz , Fármacos Sensibilizantes a Radiaciones/farmacología , Agar , Línea Celular , Supervivencia Celular/efectos de los fármacos , Derivado de la Hematoporfirina , Humanos , Neoplasias Pulmonares/patología , Melanoma/patologíaRESUMEN
Use of a metabolizable tetrazolium salt was observed to facilitate assessments of tumor cell drug sensitivity in the soft-agar colony formation assay. Enzyme-mediated staining permits discrimination between viable and non-viable groups of cells so that drug-induced cytotoxicity is clearly identifiable by visual inspection as well as by computerized image analysis. The technique appears to be especially useful in the evaluation of primary tumor cell cultures which often contain substantial numbers of non-viable cellular aggregates.