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Biomedical subjects

A C Roche

Publications and source records attributed to A C Roche.

At least 73 records · Page 4Linked to original sources
PubMed1986

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules.

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.

Animals↗
PubMed1985

Membrane lectins on human monocytes. Maturation-dependent modulation of 6-phosphomannose and mannose receptors.

Freshly isolated human monocytes, which do not contain cell-surface mannose-specific receptors, bind mannose 6-phosphate and actively endocytose mannose 6-phosphate-bearing neoglycoproteins (6-P-Man-F-BSA). Three days after isolation, human monocytes endocytose very actively 6-P-Man-F-BSA as well as Man-F-BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar-substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6-phosphate receptors are already present on freshly isolated human monocytes.

Carrier Proteins↗
PubMed1985

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice.

In vivo localization of a mouse monoclonal antibody (F2-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab')2 fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radio-localization studies were performed by using 125I- or 131I-labeled IgM monoclonal antibody or its F(ab')2 fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab')2 fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of 125I-radiolabeled F(ab')2 fragments and 125I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab')2 fragments was lower than with IgM, and so gamma-camera imaging was workable with F(ab')2 fragments without background subtraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab')2 fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab')2 fragments make them hardly suitable as carriers of toxic drugs.

Animals↗
PubMed1985

Macrophage activation by MDP bound to neoglycoproteins: metastasis eradication in mice.

Neoglycoproteins, which bind to membrane lectins of macrophages and are selectively endocytosed, were substituted with N-acetyl-muramyldipeptide (MDP). Such MDP-conjugates are shown to be able to induce macrophage tumoricity both in vitro and in vivo in a way much more efficient than free MDP does. These MDP-neoglycoprotein conjugates are shown to protect mice against metastatic growth.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Tumoricidal activation of murine alveolar macrophages by muramyldipeptide substituted mannosylated serum albumin.

Rat and mouse alveolar macrophages have almost no spontaneous tumoricidal activity and are only slightly activated by muramyldipeptide (MDP). When MDP was carried by serum albumin, the activation was higher than with free MDP but only at high concentration. When MDP was bound to a neoglycoprotein (mannosylated serum albumin) - which binds to the sugar binding receptor at the macrophage cell surface and is actively endocytosed - the activation of rat or mouse alveolar macrophages is dramatically enhanced even at very low concentration of neoglycoprotein -bound MDP. Furthermore, neoglycoprotein -bound MDP injected i.v. or i.p. was found to be able to activate alveolar macrophages, the activity of which was maximal after 48 hours in mice and 72 hours in rats. Such conjugates have so potential values as new immunostimulant agents in cancer and parasite therapy.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies.

Mouse 6D6 IgG2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of 125I- or 131I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG2) did not localize in the tumor. The amount of 6D6 IgG2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days. Imaging experiments confirmed the ability of 6D6 IgG2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4-6 h; after 48 h the background was quite low and the 6D6 IgG2a appeared to be specifically localized in the tumor.

Animals↗
PubMed1984

Selective macrophage activation by muramyldipeptide bound to monoclonal antibodies specific for mouse tumor cells.

IgM monoclonal antibodies directed against tumor cells which do not mediate antibody-dependent macrophage cytotoxicity (ADMC) even when they are cytotoxic in the presence of complement, have been shown to render macrophages tumoricidal when they carry an immunomodulating agent, i.e., muramyldipeptide (MDP). This statement is based on experiments using two IgM monoclonal antibodies selected for their ability to bind L1210 leukemia cells (F2-10-23-IgM) and 3LL Lewis lung carcinoma cells (6B6-IgM) specifically, as shown by flow cytofluorometry analysis. The MDP-IgM conjugates, containing 45 MDP molecules per IgM molecule, were prepared by allowing MDP-hydroxy-succinimide ester to react with IgM monoclonal antibodies. The MDP-IgM conjugates are shown to bind to relevant tumor cells and to induce the activation of thioglycolate-elicited peritoneal mouse macrophages leading to 80% growth inhibition of target cells at optimum concentrations of bound MDP. These concentrations of bound MDP were 10 times lower than the concentration of free MDP, giving a maximum activation that is limited to 20% growth inhibition. No macrophage activation was evidenced when tumor cells were incubated in the presence of irrelevant MDP-IgM conjugates and macrophages or when macrophages were preincubated in the presence of MDP-IgM conjugates and then incubated in the presence of relevant or irrelevant tumor cells but in the absence of the MDP-IgM conjugates. The reported results are discussed with reference to the mechanism of activation of macrophage by muramyldipeptide and to the usefulness of such MDP-IgM conjugates as potential antitumor agents in cancer therapy.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1984

Monoclonal antibodies (IgM) against a murine carcinoma, Lewis lung carcinoma (3LL): production, purification and in vitro selectivity.

C57BL/6 mice were immunized against a syngeneic murine carcinoma, Lewis lung carcinoma (3LL). Monoclonal antibodies were prepared by fusing spleen cells of immunized mice with SP2-O-Ag mouse myeloma cells. Hybridoma clones producing anti-3LL antibodies were selected on the basis of a solid-phase radioimmunoassay. Two clones were selected and subcloned to give the 5B5 and the 6B6 hybrid lines which were found to secrete IgM monoclonal antibodies. Large quantities of monoclonal antibodies were purified from ascitic fluids by gel filtration chromatography. Specificities of these antibodies were tested on 3LL tumor cells, 3LL metastases, lymphoid cells and leukemic L1210 cells. The 5B5 IgM monoclonal antibody was found to be selectively directed against primary tumor cells and metastatic cells.

Animals↗
PubMed1984

Uptake of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytofluorometry and drug targeting.

The presence and the sugar specificity of membrane lectins on the cell surface of mouse L1210 leukemia cells were demonstrated by using various neoglycoproteins (glycosylated serum albumin) substituted with fluorescein or methotrexate. Neoglycoproteins were prepared by reaction of glycosidophenylisothiocyanates with bovine serum albumin. The binding of neoglycoproteins to L1210 cells depends on the nature of the carried sugar and on the number of bound sugar residues per neoglycoprotein molecule. The best results were obtained with fucosylated serum albumin containing 25 +/- 5 residues of fucose. The amount of cell-associated fluorescein-labeled neoglycoproteins was several fold higher at 37 degrees C than at 4 degrees C suggesting a specific endocytotic process. The membrane lectin-mediated endocytosis was further demonstrated by showing that the cell-associated fluorescence upon cell incubation in the presence of fluorescein-labeled neoglycoproteins at 37 degrees C increased several fold after addition of monensin, a proton/sodium ionophore known to raise the pH of endosomes and lysosomes. The analysis of fluorescein-labeled neoglycoproteins association to the L1210 cells were achieved by quantitative flow cytofluorometry after standardization with calibrated polystyrene sulfonate beads carrying various amounts of 1-(fluoresceinylthioureido)-4,8-diazalicosane. In addition, the cytotoxicity of neoglycoprotein-bound methotrexate was shown to be related to the presence and to the nature of the carried sugar: fucosylated serum albumin was shown to be the most efficient neoglycoprotein carrier, and to have a cytotoxicity close to that of anti L1210 cell IgM monoclonal antibody carrying methotrexate.

Animals↗
PubMed1983

Sugar-specific endocytosis of glycoproteins by Lewis lung carcinoma cells.

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing alpha-D-glucose residues: This binding is competitively inhibited by neoglycoproteins containing alpha-D-glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell-associated neoglycoprotein was higher at 37 degrees C than at 4 degrees C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing alpha-D-glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar-specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate-binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and neoglycoprotein containing alpha-D-glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.

Animals↗
PubMed1981

Cell surface receptors for wheat germ agglutinin and limulin in baby hamster kidney cells and ricin resistant variants.

The cell surface glycoconjugates of Baby Hamster Kidney cells and of four ricin resistant variants were investigated by the use of 125I-substituted ricin (Ricinus communis toxin) which binds galactose residues, and by the use of fluorescein labelled lectins which bind N-acetylneuraminic acid and/or N-acetylglucosamine: Limulin (Limulus polyphemus agglutinin), wheat germ agglutinin (Triticum vulgare agglutinin) and succinylated wheat germ agglutinin. Striking differences in the number of lectin and/or ricin receptors were found between the cell surface of wild type cells and that of ricin resistant variants. The results are discussed on the basis of the main glycopeptide structure, and of the specificity of the sugar binding proteins used. The ricin resistance of variant cells is concomitant to modifications of the concentration of certain glycoconjugate structures which are accessible to the sugar binding proteins. Depending on the variants, N-asparaginyl glycopeptide types and/or O-glycosidic glycopeptide types are affected.

Animals↗
PubMed1981

Specificities of limulin and wheat-germ agglutinin towards some derivatives of GM3 gangliosides.

Lipid vesicles containing derivatives of GM3 ganglioside (II3-N-acetylneuraminosyllactosyl ceramide) were used to study the specificities of two lectins (limulin and wheat germ agglutinin) towards N-acetyl neuraminic acid and N-glycoloylneuraminic acid and some of their natural and chemically modified derivatives. The extent of the lectin binding to the gangliosides was related to the aggregation process of the lipid vesicles which was monitored as an absorbance increase. Limulin binds specifically to lipid vesicles containing N-glycoloyl derivatives of GM3. The hydroxyl group at C-4 and the carboxyl group of neuraminic acid have to be free for the binding to limulin. The side chain of neuraminic acid is not involved in the binding site of limulin. Wheat germ agglutinin binds to GM3 ganglioside only when the hydrophilic tail of the neuraminic group is cut off (C7 analogues). The acetamido group but not the carboxyl group is involved in the binding to wheat germ agglutinin. The wheat-germ-agglutinin-induced aggregation of vesicles containing derivatives of GM3-ganglioside is dependent on the pH, on the ionic strength and on the presence of Ca2+ ions. The dependence on ionic strength and Ca2+ is a consequence of the electrostatic repulsion of the vesicles. The wheat-germ-agglutinin-induced aggregation process of vesicles containing any suitable GM3-ganglioside derivative was reversed by the addition of N-acetylglucosamine showing that the N-acetylneuraminic acid derivatives bind to the N-acetylglucosamine binding site.

Calcium↗
PubMed1980

Sugar-lectin interactions: how does wheat-germ agglutinin bind sialoglycoconjugates?

The specific binding of N-acetylneuraminic acid to wheat-germ agglutinin is based on configuration similarities between N-acetylneuraminic acid and N-acetylglucosamine. The N-acetamido group and an adjacent hydroxyl group, both in an equatorial position are shown to be the main determinants. The N-acetylneuraminic acid--wheat-germ agglutinin interaction is increased by the removal of the last two carbons C8 and C9. The interaction between wheat-germ agglutinin and glycoconjugates containing N-acetylneuraminic acid is shown to be dependent on a charge effect and on an avidity effect. Succinylated wheat-germ agglutinin which is negatively charged at physiological pH, in contrast with wheat-germ agglutinin which is positively charged, does not bind cell surface glycoconjugates containing N-acetylneuraminic acid but does bind cell surface glycoconjugates containing N-acetylglucosamine. The use of wheat-germ agglutinin and of succinylated wheat-germ agglutinin leads to the determination of the number of cell surface receptors containing N-acetylneuraminic acid.

Acetylglucosamine↗
PubMed1979

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.

Fluoresceins↗