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Biomedical subjects

M P Rols

Publications and source records attributed to M P Rols.

At least 19 recordsLinked to original sources
PubMed2002

Electrochemotherapy of horses. A preliminary clinical report.

Sarcoids are skin spontaneous tumours detected in horses. It can be cured by chemotherapy by using cisplatin. A multisequence treatment must be performed. Problems are present due to the poor diffusion of the hydrophilic product in the tumours. Electropulsation is known to drastically enhance the effect of antitumoral drugs in vivo. Taking into account the very successful results of the group in Ljubljana (Slovenia), we started a research clinical program where electropulsation was applied after local cisplatin injection. The size of sarcoids is large (several centimeters). A specially designed set of wire contact electrodes was built. The distance between the electrodes was 0.9 cm and their length was 0.9 cm. The contact with the skin was obtained by a conductive paste. A PS15 Jouan Electropulsator was used to deliver eight pulses of 0.1 ms at a 1-Hz frequency with a 1.3-kV voltage. The animal was anesthesized. Intratumoral cisplatin injections were operated every 0.6 cm (0.2 ml at a 1-mg/ml concentration). Five minutes after the first drug injection, multiple electrotreatments were applied by moving the electrodes between the pulse applications. This allows the treatment of all the tumour surface. Several successive treatments were performed with a delay of 2 weeks between each. All lesions completely responded. The sarcoids disappear after only 2 or 3 electrochemotherapies. Objective responses were obtained in 100% of the treated lesions. All horses tolerated the treatment well. No adverse effect from the electric pulses was observed even in the case of a high number of pulses, or when several consecutive treatments were applied. No regrowth was observed in the 18 months follow-up period.

Animals↗
PubMed2001

Control by membrane order of voltage-induced permeabilization, loading and gene transfer in mammalian cells.

Cells can be transiently permeabilized by application of electric pulses. A direct consequence of this treatment is to create a new state in the membrane leading to DNA and protein transfers. A key step, in the interaction between macromolecules and the electropermeabilized membrane, is involved. We previously reported that membrane and DNA associated hydration and undulation forces appeared to be involved in this process by studying the effects of osmotic pressure. Effects of ethanol (EtOH) and L-alpha-lysophosphatidylcholine (lyso-PC), molecules known to affect membrane order and therefore undulation forces, were investigated on Chinese hamster ovary (CHO) cells. We used millisecond square wave pulses, conditions giving high efficiency for gene transfer. No effect was observed on cell permeabilization for small sized molecules. Only little change on electroloading of proteins such as R-phycoerythrin was obtained in presence of EtOH. But, a decrease (increase) in electrotransfection was observed for cells treated with EtOH (lyso-PC). Under our conditions, no additional effects of the chemical treatment were observed on cell viability and on membrane resealing. These results tentatively explained in terms of the effect of membrane order on membrane organization and interaction between molecules and membrane supports the existence of the plasmid-membrane interaction in the mechanism of electrically mediated gene transfer.

Animals↗
PubMed2001

Antitumor activity of 2',3'-dideoxycytidine nucleotide analog against tumors up-regulating DNA polymerase beta.

DNA polymerase beta (Pol beta), an error-prone DNA-synthesizing enzyme tightly down-regulated in healthy somatic cells, has been shown to be overexpressed in many human tumors. In this study, we show that treatment with the 2',3'-dideoxycytidine (ddC) nucleoside analog inhibited in vitro and in vivo the proliferation of Pol beta-transfected B16 melanoma cells, which up-regulate Pol beta compared with control isogenic cells. The administration of ddC also increased specifically the survival of mice bearing Pol beta-overexpressing B16 melanoma. When the phosphorylated form of ddC was electrotransfered into Pol beta-transfected melanoma, the cell growth inhibition was strengthened, strongly suggesting that the cytotoxic effect results from incorporation of the chain terminator into DNA. Using in vitro single- and double-stranded DNA synthesis assays, we demonstrated that excess Pol beta perturbs the replicative machinery, favors ddC-TP incorporation into DNA, and consequently promotes chain termination. Therefore, the use of chain terminator anticancer agents could be suitable for the treatment of tumors with a high level of Pol beta.

Animals↗
PubMed2000

Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells.

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.

Animals↗
PubMed2000

Electrochemotherapy of cutaneous metastases in malignant melanoma.

Electrochemotherapy is a new anticancer therapy in which transient permeabilization of cells by an electric field induces a significant increase in the bleomycin concentration and toxicity in tumour cells. We report a clinical study of electrochemotherapy in malignant melanoma. The main issues addressed were the effect of the size of the nodules, the optimization of the electrical parameters, and posttreatment clinical observations. Four patients were enrolled in the study. They received a 10 mg/m2 dose of bleomycin administered intravenously, followed by short, intense electric pulses applied directly to the skin at the tumour sites. Antitumour effects were obtained, especially in the smallest nodules. Objective responses were obtained in more than 90% of the 55 nodules treated, with a complete response rate of 9%. All patients tolerated the treatment well. No residual effects from the electric pulses were observed, even when a high number of pulses were required or when two consecutive treatments were applied. These results are encouraging and the study should be continued.

Adult↗
PubMed1998

Control by ATP and ADP of voltage-induced mammalian-cell-membrane permeabilization, gene transfer and resulting expression.

We have permeabilized and transfected mammalian cells by transient alteration of their native transmembrane electrical potential difference. Chinese hamster ovary cells were chosen as a model in order to study the electropermeabilization and electrotransfection processes. Propidium iodide was used to monitor permeabilization. A plasmid carrying the beta-galactosidase gene was used to follow direct gene transfer and expression by determining transient expression of the electrotransfered activity at the single-cell level. The effect of nucleotides on cell permeabilization and transfection was studied by altering the cytosolic ATP and ADP contents of cells either during the pulsation or during the period following it. Permeabilization and transfection are not regulated in the same way by the ATP and ADP levels. The permeabilization efficiency remains unaffected. Cell viability and the transfection yield are dramatically affected. While ADP is involved in the step of DNA transfer across the electropermeabilized plasma membrane, ATP controls other steps (cytoplasmic DNA migration towards the nucleus, expression). Our results prove, firstly, that membrane resealing is required but is not sufficient to preserve cell viability and, secondly, that transfection is a cell-mediated process and not only an electrophoretic step driven by the external field.

Adenosine Diphosphate↗
PubMed1998

Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.

Membrane electropermeabilization to small molecules depends on several physical parameters (pulse intensity, number, and duration). In agreement with a previous study quantifying this phenomenon in terms of flow (Rols and Teissié, Biophys. J. 58:1089-1098, 1990), we report here that electric field intensity is the deciding parameter inducing membrane permeabilization and controls the extent of the cell surface where the transfer can take place. An increase in the number of pulses enhances the rate of permeabilization. The pulse duration parameter is shown to be crucial for the penetration of macromolecules into Chinese hamster ovary cells under conditions where cell viability is preserved. Cumulative effects are observed when repeated pulses are applied. At a constant number of pulses/pulse duration product, transfer of molecules is strongly affected by the time between pulses. The resealing process appears to be first-order with a decay time linearly related to the pulse duration. Transfer of macromolecules to the cytoplasm can take place only if they are present during the pulse. No direct transfer is observed with a postpulse addition. The mechanism of transfer of macromolecules into cells by electric field treatment is much more complex than the simple diffusion of small molecules through the electropermeabilized plasma membrane.

Animals↗
PubMed1998

Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells.

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.

Animals↗
PubMed1998

Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy.

Electrochemotherapy (ECT) enhances the effectiveness of chemotherapeutic agents by administering the drug in combination with short intense electric pulses. ECT is effective because electric pulses permeabilize tumour cell membranes and allow non-permeant drugs, such as bleomycin, to enter the cells. The aim of this study was to demonstrate the anti-tumour effectiveness of ECT with bleomycin on cutaneous and subcutaneous tumours. This article summarizes results obtained in independent clinical trials performed by five cancer centres. A total of 291 cutaneous or subcutaneous tumours of basal cell carcinoma (32), malignant melanoma (142), adenocarcinoma (30) and head and neck squamous cell carcinoma (87) were treated in 50 patients. Short and intense electric pulses were applied to tumours percutaneously after intravenous or intratumour administration of bleomycin. The tumours were measured and the response to the treatment evaluated 30 days after the treatment. Objective responses were obtained in 233 (85.3%) of the 273 evaluable tumours that were treated with ECT. Clinical complete responses were achieved in 154 (56.4%) tumours, and partial responses were observed in 79 (28.9%) tumours. The application of electric pulses to the patients was safe and well tolerated. An instantaneous contraction of the underlying muscles was noticed. Minimal adverse side-effects were observed. ECT was shown to be an effective local treatment. ECT was effective regardless of the histological type of the tumour. Therefore, ECT offers an approach to the treatment of cutaneous and subcutaneous tumours in patients with minimal adverse side-effects and with a high response rate.

Adenocarcinoma↗
PubMed1998

In vivo electrically mediated protein and gene transfer in murine melanoma.

We show that efficient permeabilization of murine melanoma can be obtained in vivo by applying electric pulses. More than 80% of the cell population is affected as shown by the penetration of propidium iodide. A protein, beta-galactosidase, can be transferred and expressed into the cells by incorporating either the protein or a plasmid carrying the reporter gene with respective efficiencies of 20% and 4%. This is obtained by a direct injection of either the protein or the plasmid in the tumor, followed by the application of electric pulses with surface electrodes in contact with the skin. This approach is simple and safe to use, reproducible, and specific; moreover, it is potentially applicable to a wide variety of tissues, cell types, and animals.

Animals↗
PubMed1995

Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance.

The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long-term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

Adaptation, Physiological↗
PubMed1995

Long-lived macropinocytosis takes place in electropermeabilized mammalian cells.

Electropermeabilization is a technique which allows free access of molecules to cytosol. In the present study, we report on results dealing with the penetration of macromolecules. Under electric conditions that allow maintenance of cell viability at a high level, i.e., at low electric field intensity but long time duration (ms time range), all the pulsed cells become permeable to macromolecules. By loading beta-galactosidase the electro-transferred activity in the cells is maintained over 24 hours. Transfer mediated during the pulse occurs by free diffusion into the cytoplasm, while post pulse transfer takes place by a different pathway. When added a few minutes after application of the electric field, the enzyme enters the cell via a macropinocytosis-like process. This is a new long-term effect of the electric field pulse on the membrane.

Animals↗
PubMed1994

Control by pulse parameters of electric field-mediated gene transfer in mammalian cells.

Electric field-mediated gene transfer in mammalian cells (electrotransformation) depends on the pulsing conditions (field intensity, pulse duration, number of pulses). The effect of these parameters was systematically investigated using the transient expression of the chloramphenicol acetyltransferase and the beta-galactosidase activities in Chinese hamster ovary cells. Pulsing conditions inducing reversible permeabilization of the cell plasma membrane are not sufficient to induce gene transfer. The plasmid must be present during the electric pulse if it is to be transferred across the membrane into the cytoplasm. Only the localized part of the cell membrane brought to the permeabilized state by the external field is competent. Pulse duration plays a key role in the magnitude of the transfer. The field induces a complex reaction between the membrane and the plasmid that is accumulated at the cell interface by electrophoretic forces. This leads to an insertion of the plasmid, which can then cross the membrane.

Animals↗
PubMed1994

Two complementation groups account for most cases of inherited MHC class II deficiency.

MHC class II immuno-deficiency is a rare autosomal recessive disease due to a defect in transacting genes, which control the expression of the entire family of MHC alpha and beta class II genes. Previous analyses classified cells from eight MHC class II-deficient patients and four experimental mutant cell lines into four complementation groups, pointing to the existence of a large number of regulatory genes. We conducted fusion experiments with cell lines from two-thirds of all known patients and found that two complementation groups accounted for 20 of the 22 cases studied. These two complementation groups correspond closely to two ethnic groups: most patients of north African origin were classified into one group, while all patients originating from Spain were classified into a second main group. This suggests the existence of restricted number of ancestor mutations leading to this disease.

Africa, Northern↗