Jean-Bernard Le Pecq

A new fluorometric method for RNA and DNA determination

Abstract Previous studies have shown that a dye, ethidium bromide, markedly increases its fluorescence when it binds to double-stranded DNA or double-stranded regions of RNA. It is shown that, in the given conditions, a linear relationship between fluorescence and nucleic acid concentration is obtained. Thus, a new spectrofluorometric method for determining DNA and RNA using this principle is described. This method is specific for nucleic acids, can be used over a wide range of salt concentrations, and has a good sensitivity (0.01 μg/ml of DNA). No biological compounds able to interfere have been found. An adaptation of this method for determining both DNA and RNA in a mixture is also described.

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

BackgroundDC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome.Patients and methodsExosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression.ResultsThe GMP process allowed to harvest about 5 × 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood.ConclusionThe first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.

Production and characterization of clinical grade exosomes derived from dendritic cells

We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50-90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class I, MHC class II, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. Biol. Chem. 273 (1998) 20121; J. Cell. Sci. 113 (2000) 3365; J. Immunol. Methods 247 (2001) 163; J. Immunol. 166 (2001) 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4-6 h of a 2-3 l culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/deuterium oxide (D2O) (98%) cushion (density 1.210 g/cm3) reduced the volume and protein concentration approximately 200- and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5-25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class II) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11a].

Mechanisms of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity.

Genistein has been shown to inhibit specifically in vitro the epidermal growth factor (EGF)-receptor tyrosine protein kinase activity (Akiyama et al., J Biol Chem 262: 5592-5597, 1987). When the effects of genistein on NIH-3T3 cells were studied, a cytostatic effect was observed at low concentration (less than 40 microM) and a cytotoxic effect at higher concentration (greater than 40 microM). Genistein was able to block the mitogenic effect mediated by EGF on NIH-3T3 cells (IC50 = 12 microM) or by insulin (IC50 = 19 microM). Genistein was also able to block the mitogenic effect mediated by thrombin (IC50 = 20 microM) although the thrombin receptor does not involve a protein tyrosine kinase activity. Genistein at cytostatic concentration (less than 40 microM) did not prevent the induction of c-myc mRNA synthesis caused by the activation of EGF receptor by EGF. Therefore the cytostatic effect of genistein on NIH-3T3 cells did not appear to be mediated by EGF receptor tyrosine kinase inhibition. This hypothesis was also supported by the absence of effect of genistein on the EGF-stimulated phosphorylation of several proteins and particularly of a cytosolic 80 kD protein. The stimulation of S6 kinase activity of cells treated by EGF was prevented by genistein. The stimulation by EGF of in situ S6 phosphorylation was also prevented by genistein. In addition, S6 kinase extracted from cells treated by EGF was inhibited by genistein. These effects occur at similar doses and maximal inhibition of S6 kinase was obtained at about 15 microM.

Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids.

Abstract A dimeric derivative of ethidium is used for fluorimetric assay of nucleic acids. Due to the very high binding affinity of this derivative for DNA and RNA, a significant increase of sensitivity of the ethidium fluorimetric technique for nucleic acids determination is obtained. Using a commercially available instrument, DNA concentrations in the nanogram per milliliter range are determined. In addition, we have found that an acridine ethidium dimer can also be used for a sensitive fluorimetric assay of DNA concentration, while simultaneously providing a determination of the DNA base composition.

Corrections for instrumental errors in measurement of fluorescence and polarization of fluorescence

Abstract Calculations have been made to allow corrections for instrumental errors in the measurement of fluorescence and polarization coefficient. These errors are due to differences in transmittivities of the instruments for the horizontal and vertical components of the light. The relative error made in the quantum efficiency determination can be as large as 12%. When natural light is used for polarization measurement the relative error can be 15%. Special attention has been given to the case in which polarization measurements are used for measurements of binding of small molecules to macromolecules.

The change of the torsion of the DNA helix caused by intercalation: I — A discussion of the two different possibilities, winding or unwinding

Summary The four basic assumptions made by Lerman to establish his intercalation model are discussed. It is explained how according to this model intercalation can only lead to an unwinding of the DNA helix. On the other hand, it is shown that by changing one of the four basic assumptions made by Lerman , one can build an intercalation model leading possibly to a limited winding of the DNA helix and to the exclusion of one site per each intercalated site.

Binding of bifunctional ethidium intercalators to transfer RNA

Abstract Two bifunctional intercalating dimers, an ethidium homodimer and an acridine ethidium heterodimer, bind to yeast tRNA phe through two classes of sites, I and II (K I ≥ 10 9 M −1 , K II ∼ 10 6 M −1 ), as indicated by fluorescence titration, fluorescence lifetime, “contact” energy transfer and equilibrium dialysis measurements. Binding appears to involve mono-intercalation of the phenanthridinium moiety of these dimers and it is sensitive to, or possibly coupled with, conformational changes within the tRNA macromolecule. These observations raise the possibility that tRNA may represent a pharmacological target of the bifunctional intercalators.

Effect of membrane potential on the cellular uptake of 2-N-methyl-ellipticinium by L1210 cells

Some quaternary ammonium derivatives of ellipticine are active antitumor drugs on both experimental and human tumors. Because of their positive charge, the cellular uptake of these molecules is expected to be influenced by the electric membrane potential. Experimental variations of the potential were produced by changing the external potassium concentration and the potassium permeability by the addition of valinomycin. Using the fluorescent lipophilic cationic dye 3,3-dihexyloxacarbocyanine iodide, the L1210 cell membrane potential was estimated at -35 mV by flow cytometric analysis, and the same technique was then used to study the effects of the membrane potential variations on 2-N-methyl-ellipticinium (NME) cellular uptake. Our results show that indeed NME uptake depends on the cell membrane potential, which might then influence its pharmacological properties.

Determination of antibody-complement mediated cytotoxicity using ATP release induced by a monoclonal antibody against the Burkitt lymphoma associated globotriaosylceramide antigen

The kinetics of ATP release from Burkitt lymphoma target cells following complement activation by rat monoclonal IgM antibody have been used to develop a standard assay for antibody-complement mediated cytotoxicity. This assay permitted the study of the reactivity of the monoclonal IgM with Burkitt and non-Burkitt cell lines and to perform IgM-complement mediated cytotoxicity inhibition experiments with oligosaccharides. This led to a precise definition of the antigenic determinant recognized by the monoclonal IgM on globotriaosylceramide.