Dose-Dependent ATP Depletion and Cancer Cell Death following Calcium Electroporation, Relative Effect of Calcium Concentration and Electric Field Strength

Background: Electroporation, a method for increasing the permeability of membranes to ions and small molecules, is used in the clinic with chemotherapeutic drugs for cancer treatment (electrochemotherapy). Electroporation with calcium causes ATP (adenosine triphosphate) depletion and cancer cell death and could be a novel cancer treatment. This study aims at understanding the relationship between applied electric field, calcium concentration, ATP depletion and efficacy. Methods: In three human cell lines — H69 (small-cell lung cancer), SW780 (bladder cancer), and U937 (leukaemia), viability was determined after treatment with 1, 3, or 5 mM calcium and eight 99 μs pulses with 0.8, 1.0, 1.2, 1.4 or 1.6 kV/cm. Fitting analysis was applied to quantify the cell-killing efficacy in presence of calcium. Post-treatment intracellular ATP was measured in H69 and SW780 cells. Post-treatment intracellular ATP was observed with fluorescence confocal microscopy of quinacrine-labelled U937 cells. Results: Both H69 and SW780 cells showed dose-dependent (calcium concentration and electric field) decrease in intracellular ATP (

ESOPE-Equivalent Pulsing Protocols for Calcium Electroporation: An In Vitro Optimization Study on 2 Cancer Cell Models

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs combined with electroporation represents the new frontier for electrochemotherapy applications; calcium electroporation has been recently proposed as a simple tool for anticancer therapy. In vitro investigations facilitate the optimization of electrical parameters and drugs for in vivo and clinical testing. In this optimization study, new pulsing protocols have been tested by increasing the pulse number and reducing the electric field with respect to the standard. European Standard Operating Procedure on Electrochemotherapy-equivalent protocols have been identified in HL-60 and A431 cancer cell models, and a higher sensitivity in terms of electroporation yield has been recorded in HL-60 cells. Moreover, cell killing efficacy of European Standard Operating Procedure on Electrochemotherapy-equivalent protocols has been demonstrated in the presence of increasing calcium concentrations on both cell lines. Equivalent European Standard Operating Procedure on Electrochemotherapy protocols can be used to optimize the therapeutic effects in the clinic, where different regions of the same cancer tissue, with different electrical properties, might result in a differential electroporation yield of the standard protocol over the same tissue, or, eventually, in an override of the operational limits of the instrument. Moreover, using calcium can help overcome the drawbacks of standard drugs (side effects, high costs, difficult handling, preparation, and storage procedures). These results support the possibility of new treatment options in both standard electrochemotherapy and calcium electroporation, with clear advantages in the clinic

Pattern of DAP12 Expression in Leukocytes from Both Healthy and Systemic Lupus Erythematosus Patients

DAP12 is an ITAM-bearing transmembrane adaptor originally identified on the surface of Natural Killer cells. A broad expression among other immune cells was later found in myeloid and lymphoid cells. However, data on DAP12 expression pattern rely only on immunoblot and microarray analysis. Here, we describe the generation and the characterization of an anti-DAP12 monoclonal antibody. Using this novel reagent, we show that DAP12 expression is restricted to innate immune cells in basal condition. Since a decreased expression of DAP12 has been suggested in NK cells of systemic lupus erythematosus patients, we have further investigated the NK cell receptor repertoire and leukocyte expression of DAP12 in these patients and no major changes were detectable when compared to controls

Les indicateurs de risque de transfert de produits phytosanitaires et leur validation : exemple de l'indicateur I-Phy

National audienceThe implementation of solutions to the problem of non-point source pollution due to pesticides requires the development of assessment tools of the risk associated to the application of those products. If pesticides risks indicators are currently recognised for the environmental assessment of farmers'practices, numerous studies during the last decade did not enable to conclude about their validity. The article aims at presenting results of an experimental validation of the surface water component of the I-Phy indicator from measurements of pesticide concentration in water. After a brief review of different types of indicators, we present the surface water component of the I-Phy indicator which was developed at INRA Colmar in the frame of the INDIGO® method. The construction is based on decision rules using fuzzy logic. In the surface water component, variable describing fields conditions, sensitivity to runoff and drift play a major role. In a second part, we present and discuss results of experimental validation using dataset of concentration measurement of active ingredient from three locations (Rouffach, Le Ruiné, Baslieux-sous-Châtillon). They yield a medium quality of prediction which can be partially explained by the upscaling of the indicator to watershed level.La mise en place de solutions au problème de pollution diffuse par les produits phytosanitaires nécessite le développement d'outils d'évaluation des risques liés à l'application de ces substances. Si les indicateurs phytosanitaires sont aujourd'hui des outils reconnus pour l'évaluation environnementale des pratiques agricoles, les nombreuses recherches menées dans la dernière décennie ne permettent pas encore de conclure sur leur validation complète. Le présent article a pour objectif de présenter les résultats d'un travail de validation expérimentale du module eau de surface de l'indicateur I-Phy, à partir de mesures de concentration dans les eaux. Après avoir brièvement revu différents groupes d'indicateurs, le module eau de surface de l'indicateur I-Phy développé à l'INRA de Colmar dans le cadre de la méthode INDIGO® est présenté en détail dans une première partie. Son mode de construction repose sur des règles de décision associant la logique floue. Les variables du milieu, la sensibilité au ruissellement et la dérive jouent un rôle prépondérant dans le module eau de surface. Dans une seconde partie, les résultats de validation expérimentale, utilisant notamment les jeux de mesures de concentration sur trois sites (Rouffach, Le Ruiné, Baslieux-sous-Châtillon), sont présentés et discutés. Ils montrent une performance moyenne de l'indicateur en termes de qualité prédictive qui peut s'expliquer en partie par la non-prise en compte des mécanismes de transfert au niveau d'un bassin versant

Les indicateurs de risque de transfert de produits phytosanitaires et leur validation : exemple de l'indicateur I-Phy

National audienceThe implementation of solutions to the problem of non-point source pollution due to pesticides requires the development of assessment tools of the risk associated to the application of those products. If pesticides risks indicators are currently recognised for the environmental assessment of farmers'practices, numerous studies during the last decade did not enable to conclude about their validity. The article aims at presenting results of an experimental validation of the surface water component of the I-Phy indicator from measurements of pesticide concentration in water. After a brief review of different types of indicators, we present the surface water component of the I-Phy indicator which was developed at INRA Colmar in the frame of the INDIGO® method. The construction is based on decision rules using fuzzy logic. In the surface water component, variable describing fields conditions, sensitivity to runoff and drift play a major role. In a second part, we present and discuss results of experimental validation using dataset of concentration measurement of active ingredient from three locations (Rouffach, Le Ruiné, Baslieux-sous-Châtillon). They yield a medium quality of prediction which can be partially explained by the upscaling of the indicator to watershed level.La mise en place de solutions au problème de pollution diffuse par les produits phytosanitaires nécessite le développement d'outils d'évaluation des risques liés à l'application de ces substances. Si les indicateurs phytosanitaires sont aujourd'hui des outils reconnus pour l'évaluation environnementale des pratiques agricoles, les nombreuses recherches menées dans la dernière décennie ne permettent pas encore de conclure sur leur validation complète. Le présent article a pour objectif de présenter les résultats d'un travail de validation expérimentale du module eau de surface de l'indicateur I-Phy, à partir de mesures de concentration dans les eaux. Après avoir brièvement revu différents groupes d'indicateurs, le module eau de surface de l'indicateur I-Phy développé à l'INRA de Colmar dans le cadre de la méthode INDIGO® est présenté en détail dans une première partie. Son mode de construction repose sur des règles de décision associant la logique floue. Les variables du milieu, la sensibilité au ruissellement et la dérive jouent un rôle prépondérant dans le module eau de surface. Dans une seconde partie, les résultats de validation expérimentale, utilisant notamment les jeux de mesures de concentration sur trois sites (Rouffach, Le Ruiné, Baslieux-sous-Châtillon), sont présentés et discutés. Ils montrent une performance moyenne de l'indicateur en termes de qualité prédictive qui peut s'expliquer en partie par la non-prise en compte des mécanismes de transfert au niveau d'un bassin versant

Supplemental Material, Fig_S1 - ESOPE-Equivalent Pulsing Protocols for Calcium Electroporation: An <i>In Vitro</i> Optimization Study on 2 Cancer Cell Models

<p>Supplemental Material, Fig_S1 for ESOPE-Equivalent Pulsing Protocols for Calcium Electroporation: An <i>In Vitro</i> Optimization Study on 2 Cancer Cell Models by Stefania Romeo, Anna Sannino, Maria Rosaria Scarfì, P. Thomas Vernier, Ruggero Cadossi, Julie Gehl, and Olga Zeni in Technology in Cancer Research & Treatment</p

Viability of H69 and SW780 cells after calcium electroporation.

<p>Viability, assessed using MTS assay, of H69, a human small cell lung cancer cell line (<b>A</b>); and SW780, a human bladder cancer cell line (<b>B</b>) 24 hours after treatment with calcium electroporation. Final extracellular calcium concentrations of 0 mM, 1 mM, 3 mM, or 5 mM and applied electric field of either 0.8 kV/cm, 1.0 kV/cm, or 1.2 kV/cm. Cells thrice thawed and frozen, then sonicated were used as control (dead cells). Results are illustrated as percentage of control (no electroporation, no added calcium), electroporation (EP), mean + S.D., n = 6.</p