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P Machy

Publications and source records attributed to P Machy.

30 records · Page 2Linked to original sources
PubMed1987

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies.

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.

Animals↗
PubMed1986

Interferon sensitive and insensitive MHC variants of a murine thymoma differentially resistant to methotrexate-containing antibody-directed liposomes and immunotoxin.

We evaluated the ability of methotrexate-containing liposomes or a ricin alpha-chain immunotoxin, both associated with monoclonal antibodies specific for the major histocompatibility complex-encoded class I molecule H-2Kk, to kill cells of the murine k haplotype thymoma RDM4. Cells were incubated with liposomes or immunotoxin in the presence or absence of interferon-gamma, which is known to augment the expression of the target class I molecules. The great majority of cells were killed by either of these reagents. Two types of mutant cells were obtained: type 1 cells, selected by methotrexate-containing liposomes, failed to express sufficient target H-2k molecules to be killed by liposomes in the absence of interferon-gamma. In the presence of interferon-gamma, these cells increased expression of all H-2 class I molecules and could be killed by targeted liposomes. Type 2 cells were immunoselected from cloned type 1 cells by liposomes in the presence of interferon. These cells failed to respond to interferon with expression of the H-2Kk molecule, but continued to augment H-2Dk expression in response to interferon. A third variant (type 3) selected from the wild type population by an H-2Kk specific immunotoxin in the absence of interferon phenotypically resembled type 1 cells. Type 1 but not type 2 cells respond to interferon by augmented synthesis of H-2Kk specific mRNA. The results suggest that for interferon-sensitive cell surface molecules of tumor cells, use of interferon improves the efficacy of targeted chemotherapy, but does not prevent development of mutants lacking the target molecule.

Adjuvants, Immunologic↗
PubMed1984

Weak acid-induced release of liposome-encapsulated carboxyfluorescein.

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form: 9 X 10(-9) cm X s-1), acetic acid (greater than 1 X 10(-7) cm X s-1) and glycine (cation: 6 X 10(-9) cm X s-1). These results are consistent with low effective proton permeability of liposomes (less than 5 X 10(-12) cm X s-1) and with the permeability coefficient of HCl (3 X 10(-3) cm X s-1) reported by Nozaki and Tanford ( (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324-4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.

Acids↗
PubMed1984

Elimination or rescue of cells in culture by specifically targeted liposomes containing methotrexate or formyl-tetrahydrofolate.

Methotrexate (Mtx) inhibits cellular proliferation by blocking formyl tetrahydrofolate (THF) production. Mtx- or THF-containing liposomes were covalently coupled to Staphylococcus aureus protein A. These liposomes bound specifically in vitro to cells pre-incubated with relevant monoclonal antibodies. When liposomes contained Mtx, targeted cells died. When liposomes contained THF, targeted cells survived in the presence of quantities of Mtx in the medium sufficient to eliminate non-targeted cells. These negative or positive selection liposomes allow the recovery of either a small proportion of cells not bearing a given antigen, or a small proportion of cells bearing a given antigen, from heterogeneous cell populations.

Animals↗
PubMed1983

Endocytosis of liposomes bound to cell surface proteins measured by flow cytofluorometry.

A new technique for the quantification of cellular receptor-mediated endocytosis has been developed based on the analysis by flow cytometry of ligand-bearing liposomes containing the fluorochrome carboxyfluorescein. Carboxyfluorescein encapsulated at high concentrations in protein A-bearing liposomes is self-quenched. Binding and internalization of such liposomes by cells via antibodies directed towards membrane surface determinants results in the release of the liposome-encapsulated carboxyfluorescein into the cytoplasm causing an increase in cell-associated fluorescence. This increase can be quantified on a flow cytofluorometer.

Animals↗
PubMed1983

Small liposomes are better than large liposomes for specific drug delivery in vitro.

We have compared drug transfer into target cells in vitro from liposomes of different sizes. Liposomes of mean diameter 800 A, 2000 A or 4000 A, containing the folate analogue, methotrexate, and the fluorophore, carboxyfluorescein, were covalently coupled to Staphylococcus aureus protein A. Cells of the murine k haplotype were preincubated with an anti-H-2Kk monoclonal antibody. Excess antibody was removed and then cells were incubated with liposomes. The number of cell-bound liposomes was determined by fluorimetry. The drug effect was assayed by the methotrexate-mediated inhibition of radiolabeled deoxyuridine uptake. The drug effect was more important in the case of the 800 A vesicles than for the larger liposomes, despite the fact that the quantity of drug bound to cells was several-fold greater for large liposomes than for small ones. Since fusion is excluded by the non-proportionality of drug binding and drug effect, the predominant manner of liposome entry seems to be endocytosis. At least for these in vitro studies, the endocytosis by target cells of small liposomes seems to be more efficient than that of large liposomes.

Animals↗
PubMed1983

Endocytosis of HLA and H-2 molecules on transformed murine cells measured by fluorescence dequenching of liposome-encapsulated carboxyfluorescein.

We have studied internalization of cell surface proteins encoded by genes of the human major histocompatibility complex (HLA) transferred into murine L cells, in comparison with mouse (H-2) histocompatibility determinants. This internalization was measured by the use of monoclonal antibodies directed at these determinants, to which were bound methotrexate- and carboxyfluorescein-containing liposomes covalently coupled to protein A. In addition to the effect of methotrexate on the cells, the technique of fluorescence self-quenching release was used to measure the kinetics of internalization for single cells using a flow cytofluorometer. The native and foreign gene products were internalized in an apparently identical manner. These techniques can be applied to cells expressing genes with altered or deleted segments thus providing a basis for the analysis of the effect of sequence modifications on the internalization of the encoded molecule.

Animals↗
PubMed1982

Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate.

Antibody-bearing fluorescent liposomes containing methotrexate became bound to cells expressing determinants recognized by the antibody. The number of bound liposomes could be evaluated by fluorometry, and the internalization of liposomes was evaluated by the methotrexate-mediated inhibition of radio-labeled deoxyuridine incorporation. The effect of methotrexate transferred from the liposomes into the cells was a function not of the number of liposomes bound but of the nature of the cells and of the target molecules. Liposomes bearing antibodies with specificity for the H-2K or Mr 94,000 and 180,000 molecules were much more effective at drug delivery into T than B cells, even though these determinants were expressed by both cell types. B cells were more sensitive to the effect of methotrexate in anti-H-2 I-A and I-E liposomes than in anti-H-2K liposomes. Inhibition of the methotrexate effect by NH4Cl suggested that methotrexate entered the cell by endocytosis of the liposomes. The results are consistent with differential internalization of H-2K, I-A, I-E, and Mr 94,000 and 180,000 cell surface molecules by mitogen-stimulated T and B cells.

Ammonium Chloride↗
PubMed1982

Drug transfer into lymphoblasts mediated by liposomes bound to distinct sites on H-2 encoded I-A, I-E, and K molecules.

Protein A from S. aureus was covalently coupled to small sonicated liposomes containing methotrexate (MTX) and carboxyfluorescein (CF). These liposomes were incubated with mitogen-stimulated CBA T or B cells preincubated with one of a panel of murine IgG2 monoclonal antibodies directed at distinct sites on H-2Kk, I-Ak, or I-Ek molecules. Liposomes became bound to the cells as a consequence of the interaction of protein A on the liposome and antibody on the cell. The number of bound liposomes was measured by fluorescence, and the endocytosis of liposomes was evaluated by the MTX-mediated reduction in radiolabeled deoxyuridine incorporation. T cells were sensitive to MTX in liposomes bound to all anti-H-2Kk antibodies tested, but B cells were essentially insensitive, even though they expressed more H-2Kk, bound more liposomes directed at this molecule, and were equally sensitive to free MTX. By contrast, B cells were sensitive to liposome-encapsulated MTX when liposomes were bound to any antibody directed at I-Ak. The situation was more complex with respect to I-Ek. Antibodies directed at one part of the molecule were quite effective at drug delivery, while antibodies directed at other, spatially separate domains were much less effective, even though the number of liposomes bound to these various determinants was the same. The results suggest differential regulation of the internalization of H-2K by T and B cells, and are compatible with the existence of functionally distinct I-E molecules.

Animals↗
PubMed1981

Monoclonal antibody covalently coupled to liposomes: specific targeting to cells.

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar liposomes. Coupling of an IgG2 alpha monoclonal anti-beta 2-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter:580 A). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human beta 2-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigens specificity confirm that the technique can be generally applied.

Antibodies, Monoclonal↗
PubMedDate not supplied

Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes.

Endocytosis of the major histocompatibility complex (MHC)-encoded class I and class II molecules has been the subject of recent investigations. Class I molecules, which are key elements in T cell-mediated cytotoxicity, are differentially endocytosed by different cell types. Fibroblasts internalize their class I molecules via uncoated cell surface vesicles and tubular invaginations when these molecules are cross-linked with multivalent ligands. T lymphocytes internalize their class I molecules spontaneously, but B lymphocytes do not internalize them at all. Here we describe a morphological investigation of the mechanism by which class I molecules are endocytosed by T lymphocytes. We show that, unlike fibroblasts, T lymphocytes spontaneously internalize 20-40% of their class I molecules in a process involving coated pits and coated vesicles. Thus, the endocytic pathway of class I molecules in T lymphocytes is similar to those of other more classical cell-surface receptors involved in receptor-mediated endocytosis. In contrast, the same class I molecules remained on the cell surface in B lymphocytes. These data show that class I molecules are differentially regulated in T and B lymphocytes and fibroblasts.

Animals↗