RESUMEN
Natural killer (NK) cells hold promise for cancer therapy. NK cytotoxicity can be enhanced by expression of chimeric antigen receptors that re-direct specificity toward target cells by engaging cell surface molecules expressed on target cells. We developed a regulatory-compliant, scalable non-viral approach to engineer NK cells to be target-specific based on transfection of mRNA encoding chimeric receptors. Transfection of eGFP mRNA into ex vivo expanded NK cells (N=5) or purified unstimulated NK cells from peripheral blood (N=4) resulted in good cell viability with eGFP expression in 85+/-6% and 86+/-4%, 24 h after transfection, respectively. An mRNA encoding a receptor directed against CD19 (anti-CD19-BB-z) was also transfected into NK cells efficiently. Ex vivo expanded and purified unstimulated NK cells expressing anti-CD19-BB-z exhibited enhanced cytotoxicity against CD19(+) target cells resulting in > or =80% lysis of acute lymphoblastic leukemia and B-lineage chronic lymphocytic leukemia cells at effector target ratios lower than 10:1. The target-specific cytotoxicity for anti-CD19-BB-z mRNA-transfected NK cells was observed as early as 3 h after transfection and persisted for up to 3 days. The method described here should facilitate the clinical development of NK-based antigen-targeted immunotherapy for cancer.
Asunto(s)
Antígenos CD19/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de Antígenos/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Electroporación , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Receptores de Antígenos/genética , TransfecciónRESUMEN
Gene delivery can be accomplished using non-viral systems, and these have received increased attention These include ex vivo transfection of cells using an electric field to induce transient cell-membrane permeability (electroporation). This approach has the distinct advantage of not requiring the inclusion of a secondary agent (e.g. a lipid, viral package or carrier protein) any of which can be immunogenic or toxic. Available electroporation systems utilize a low volume (<1 mL) processing chamber and are open systems. The MaxCyte system employs a continuous flow design and can very rapidly process volumes ranging from 0.02 mL to >1 L. Transgenes for markers (eGFP) and functional proteins (e.g., cytokines, angiogenic factors) have been loaded in plasmids up to 14 kB in size. With appropriate application of pre- and post-processing cell manipulations, very satisfactory loading efficiencies and cell viability have been obtained. Cells can be processed with multiple plasmids, resulting in expression of the corresponding number of gene products. This capability has been considered for therapeutic and bioprocessing applications. The MaxCyte system was designed specifically for ex vivo clinical applications. The electrodes are manufactured of special materials and under precise conditions, in order to eliminate potential risks from electrolytic effects. The processing chamber and associated containers can be provided as disposable, sterile, closed (or functionally closed) systems-quite similar to the disposable harnesses used with cell separators. This system is thus suitable for integration into a current good manufacturing practice environment.
Asunto(s)
Electroporación/métodos , Plásmidos/genética , Transfección/métodos , Ensayos Clínicos como Asunto , Electroporación/instrumentación , Terapia Genética , Vectores Genéticos , HumanosRESUMEN
The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.
Asunto(s)
Cloroquina/farmacología , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Vacuolas/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Sistema Digestivo/metabolismo , Resistencia a Medicamentos , Exones , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plasmodium falciparum/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Transfección , Verapamilo/farmacologíaRESUMEN
We present the first single cell-level analysis of digestive vacuolar pH for representative chloroquine resistant (strain Dd2) versus sensitive (strain HB3) malarial parasites. Human red blood cells harboring intact intraerythrocytic parasites were attached to glass substrate, continuously perfused with appropriate buffer, and pH was analyzed via single cell imaging and photometry techniques. We find that digestive vacuolar pH (pH(vac)) is near 5.6 for HB3 parasites. Surprisingly, we also find that pH(vac) of Dd2 is more acidic relative to HB3. Notably, in vitro pH titration of hematin confirms a very steep transition between soluble heme (capable of binding chloroquine) and insoluble heme (not capable of binding chloroquine, but still capable of polymerization to hemozoin) with a distinct midpoint at pH 5.6. We suggest the similarity between the hematin pH titration midpoint and the measured value of HB3 pH(vac) is not coincidental, and that decreased pH(vac) for Dd2 titrates limited initial drug target (i.e. soluble heme) to lower concentration. That is, changes in pH(vac) for drug resistant Dd2 relative to drug sensitive HB3 are consistent with lowering drug target levels, but not directly lowering vacuolar concentrations of drug via the predictions of weak base partitioning theory. Regardless, lowering either would of course decrease the efficiency of drug/target interaction and hence the net cellular accumulation of drug over time, as is typically observed for resistant parasites. These observations contrast sharply with the common expectation that decreased chloroquine accumulation in drug resistant malarial parasites is likely linked to elevated pH(vac,) but nonetheless illustrate important differences in vacuolar ion transport for drug resistant malarial parasites. In the accompanying paper (Ursos, L. et al., following paper this issue) we describe how pH(vac) is affected by exposure to chloroquine and verapamil for HB3 versus Dd2.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Eritrocitos/parasitología , Plasmodium falciparum/efectos de los fármacos , Vacuolas/metabolismo , Naranja de Acridina/metabolismo , Animales , Antimaláricos/metabolismo , Cloroquina/metabolismo , Resistencia a Medicamentos , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Microscopía Confocal , Pruebas de Sensibilidad Parasitaria , Fotometría/instrumentación , Fotometría/métodos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/fisiologíaRESUMEN
In the preceding paper, we present a novel method for measuring the digestive vacuolar pH (pH(vac)) of the malarial parasite Plasmodium falciparum, and show that, surprisingly, pH(vac) is lower for chloroquine resistant (CQR) Dd2 parasites relative to chloroquine sensitive (CQS) HB3. These data may have important consequences for elucidating mechanisms of antimalarial drug resistance and for developing new antimalarial therapy. Additional issues central to a better understanding of antimalarial pharmacology and antimalarial drug resistance require detailed comparative data on the effects of key drugs and other compounds on parasite biophysical parameters such as pH(vac), measured under close-to-physiologic conditions. Since the methods we develop in the previous paper allow us to record fluorescence signals from spatially well-defined regions of the living parasite while they are under continuous perfusion, it is relatively straightforward for us to test how antimalarial drugs (e. g. chloroquine, CQ) and other compounds (e.g. the chemoreversal agent verapamil [VPL]) affect pH(vac). In this paper, we measure both short term (i.e. initial perfusion conditions) and longer-term effects of CQ and VPL for living, intraerythrocytic CQS (HB3) and CQR (Dd2) malarial parasites under constant perfusion with physiologically relevant buffers. We find that VPL normalizes pH(vac) for Dd2 to a value near that measured for HB3, but has no effect on pH(vac) for HB3. Longer term CQ exposure is found to alter pH(vac) for HB3 but not Dd2, and short-term exposure to the drug has no significant effect in either strain. The results may help resolve longstanding debate regarding the effects of CQ and VPL on parasite physiology, and further support our evolving hypothesis for the mechanism of CQ resistance.
Asunto(s)
Antimaláricos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cloroquina/farmacología , Plasmodium falciparum/efectos de los fármacos , Vacuolas/metabolismo , Verapamilo/farmacología , Naranja de Acridina/metabolismo , Animales , Humanos , Concentración de Iones de Hidrógeno , Microscopía Fluorescente , Pruebas de Sensibilidad Parasitaria , Fotometría/instrumentación , Fotometría/métodos , Plasmodium falciparum/fisiología , Vacuolas/efectos de los fármacosRESUMEN
In previous work (Weisburg, J. H., Curcio, M., Caron, P. C., Raghi, G., Mechetner, E. B., Roepe, P. D., and Scheinberg, D. A. (1996) J. Exp. Med. 183, 2699-2704), we showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV+) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pHi) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pHi by two independent methods (CO2 "conditioning" or isotonic Cl- substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pHi by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl-/HCO3- exchange upon Cl- substitution is blocked by treatment with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate. Continuous photometric monitoring of 2',7'-bis(carboxyethyl)-5, 6-carboxyfluorescein (BCECF), to assess changes in pHi or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pHi upon CO2 conditioning and Cl- substitution and release of BCECF upon formation of MAC pores. Antibody binding and internalization kinetics are similar in both the parental and resistant cell lines as measured by radioimmunoassay, but flow cytometric data showed that net complement deposition in the cell membrane is both delayed and reduced in magnitude in the MDR cells and in the cells with increased pHi. This interpretation is supported by comparison of BCECF release data for the different cells. Dual isotopic labeling of key complement components shows no significant change in molecular stoichiometry of the MACs formed at different pHi. The results are relevant to understanding clinical implications of MDR, the physiology of P-GP, and the biochemistry of the complement cascade and further suggest that the "drug pump" model of P-GP action cannot account for all of its effects.
Asunto(s)
Supervivencia Celular/fisiología , Proteínas del Sistema Complemento/fisiología , Resistencia a Múltiples Medicamentos , Concentración de Iones de Hidrógeno , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Dióxido de Carbono/química , Fluoresceínas , Colorantes Fluorescentes , Células HL-60 , Humanos , Células K562 , Cinética , RatonesRESUMEN
The biochemical mechanism of chloroquine resistance in Plasmodium falciparum remains unknown. We postulated that chloroquine-resistant strains could alter ion fluxes that then indirectly control drug accumulation within the parasite by affecting pH and/or membrane potential ('altered partitioning mechanism'). Two principal intracellular pH-regulating systems in many cell types are the amiloride-sensitive Na+/H+ exchanger (NHE), and the sodium-independent, stilbene-sensitive Cl-/HCO3- antiporter (AE). We report that under physiological conditions (balanced CO2 and HCO3-) chloroquine uptake and susceptibility are not altered by amiloride analogues. We also do not detect a significant difference in NHE activity between chloroquine-sensitive and chloroquine-resistant strains via single cell photometry methods. AE activity is dependent on the intracellular and extracellular concentrations of Cl- and HCO3- ions. Chloroquine-resistant strains differentially respond to experimental modifications in chloride-dependent homeostasis, including growth, cytoplasmic pH and pH regulation. Chloroquine susceptibility is altered by stilbene DIDS only on chloroquine-resistant strains. Our results suggest that a Cl(-)-dependent system (perhaps AE) has a significant effect on the uptake of chloroquine by the infected erythrocyte, and that alterations of this biophysical parameter may be part of the mechanism of chloroquine resistance in P. falciparum.
Asunto(s)
Antimaláricos/farmacocinética , Cloruros/metabolismo , Cloroquina/farmacocinética , Plasmodium falciparum/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Amilorida/farmacología , Animales , Antimaláricos/uso terapéutico , Diuréticos/farmacología , Resistencia a Medicamentos , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
The lateral intercellular spaces (LIS) of MDCK cell epithelia grown on permeable supports are about 0.4 pH units acidic to the bathing solutions, presumably because of buffering by the fixed negative charges on the surface of the lateral cell membranes. To test the hypothesis that fixed buffers are responsible for the acidity, a theoretical and experimental approach was developed for the determination of the concentration and pK of the fixed buffer constituted by the glycocalyx. The pH of the solution in the LIS was measured by ratiometric fluorescence microscopy while the buffer concentration or composition of the bathing solutions was altered. In addition, the divalent cation Sr2+ was added to the perfusion solutions to displace protons from the fixed buffer sites for the determination of the fixed buffer properties. We conclude that the LIS contain 3.7 mm of pK 6.2 fixed buffer and that this buffer is responsible for the acidic microenvironment in the LIS.
Asunto(s)
Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Espacio Extracelular/fisiología , Animales , Tampones (Química) , Línea Celular , Perros , Concentración de Iones de Hidrógeno , Microscopía FluorescenteRESUMEN
Application of an AC voltage to a model bilayer lipid membrane in the presence of electrogenic ionophores (valinomycin, protonophores CCCP and TTFB) was shown to change the steady-state current across the membrane induced by a DC voltage. The change in the DC current was proportional to the magnitude of the DC voltage and to the amplitude of the AC voltage and depended on both the AC frequency and the ionophore used. Application of this new experimental approach to the estimation of kinetic constants at individual stages of ionophore functioning is discussed.