Neuraminidase-sensitive Receptors Research Articles

Lectin reactivity of murine fibrosarcoma lines with a different metastatic potential

Lectins are suitable tools for investigating the glycoconjugate characteristics of metastatic cells. In the present study, we investigated whether there were differences between high metastatic T3 cells and a low metastatic isolate in their reactivities to several lectins specific for galactosyl and sialyl groups. Analysis of reactivity of the two cell lines to wheat germ agglutinin revealed a complex pattern. In fact, T3 cells had high-affinity, neuraminidase-resistant as well as low-affinity, neuraminidase-sensitive receptors. Instead, the low metastatic isolate showed only high-affinity receptors, both neuraminidase-resistant and neuraminidase-sensitive. The two cell lines reacted similarly to galactose-specific lectins. These findings indicate that sialyl groups, rather than galactosyl groups, affect the metastatic behavior of our cell system.

Mechanisms of neutralization of influenza virus in tracheal epithelial and BHK cells vary according to IgG concentration

The interaction of IgG-neutralized type A influenza virus with differentiated epithelial cells of mouse trachea, BHK cells, and chicken erythrocytes was studied using three mouse monoclonal antibodies (IgG2a) each directed against a different antigenic site on the hemagglutinin. At high HIU:HAU ratios virus was neutralized >99%, monodisperse, and attached to tracheal epithelial and BHK cells in normal amounts. The majority (70–80%) of neutralized virus failed to attach to erythrocytes. At low HIU:HAU ratios the virus was aggregated by each of the antibodies, and attachment to tracheal epithelial and BHK cells was inhibited by up to 75%. Combined aggregation and inhibition of attachment could theoretically account for up to 96% loss of infectivity but this corresponded with the observed degree of neutralization with only one of the antibodies. With increasing antibody:virus ratios, aggregation and inhibition of attachment contributed ever diminishingly to the observed neutralization and eventually not at all. Both neutralized and infectious virus attached to neuraminidase-sensitive receptors. After attachment neutralized virus became increasingly resistant to removal by neuraminidase suggesting that it had been internalized by the cell.

Mechanisms of Neutralization of Influenza Virus on Mouse Tracheal Epithelial Cells by Mouse Monoclonal Polymeric IgA and Polyclonal IgM Directed Against the Viral Haemagglutinin

The mechanism of neutralization of a type A influenza virus by polyclonal IgM was similar for both tracheal epithelial and BHK cells. Maximum neutralization was only 90% and most (70%) of the virus failed to attach to inoculated cells. The remainder attached to N-acetylneuraminic acid receptors but was not internalized. IgM aggregated virus, but only at an IgM: virus ratio below the level required for neutralization. Failure to detect any loss of infectivity associated with aggregation suggested that aggregates were unstable. Monoclonal polymeric IgA neutralized virus more efficiently on BHK cells (99.9%) than the equivalent amounts of IgM (90%). Otherwise the mechanisms of IgA and IgM neutralization were similar, except that IgA-induced aggregation was coincident with loss of infectivity and may thus have contributed to it. However, IgA-neutralized virus attached to tracheal epithelial cells more efficiently than infectious virus, initially using a neuraminidase-sensitive receptor, but then becoming neuraminidase-resistant. Whether the latter IgA-virus complexes were internalized or attached to a neuraminidase-resistant receptor is not known. This use of differentiated murine cells with murine IgA gave neutralization data that differed qualitatively from those obtained with the same antibody and undifferentiated hamster cells.

Elucidation of the receptors in macrophage and Plasmodium yoelii interactions.

Binding of hyperimmune serum opsonized merozoites of Plasmodium Yoelii nigerensis to trypsinized macrophages suggested it to be mediated by FcII receptor. Receptor blocking inhibition with monoclonal antibody 2.4G2 directed against Fc receptor for IgG1/IgG2b provided evidence that Fc delta 2b on macrophage played an important role in the merozoite-macrophage interactions. In addition, a neuraminidase sensitive receptor was noted to mediate the binding of P. yeelii merozoites in the absence of serum. Binding inhibition studies with two monosaccharides, D-mannose and alpha-methyl mannoside, indicated the role of Mannose/Fucose receptor on macrophage in this interaction.

Cytochemical and biochemical characterization of galactose-bearing membrane glycoprotiens in differentiating erthroid cells in rabbits

Lectins are suitable tools for investigating the glycoconjugate characteristics of metastatic cells. In the present study, we investigated whether there were differences between high metastatic T3 cells and a low metastatic isolate in their reactivities to several lectins specific for galactosyl and sialyl groups. Analysis of reactivity of the two cell lines to wheat germ agglutinin revealed a complex pattern. In fact, T3 cells had high-affinity, neuraminidase-resistant as well as low-affinity, neuraminidase-sensitive receptors. Instead, the low metastatic isolate showed only high-affinity receptors, both neuraminidase-resistant and neuraminidase-sensitive. The two cell lines reacted similarly to galactose-specific lectins. These findings indicate that sialyl groups, rather than galactosyl groups, affect the metastatic behavior of our cell system.

Adherence of Streptococcus sanguis to saliva-coated hydroxyapatite: evidence for two binding sites.

The characteristics of bacterial adherence to saliva-coated hydroxyapatite were examined for a salivary aggregating strain of Streptococcus sanguis, strain 12, and for its nonaggregating variant, strain 12na. Both strains were found to adhere in similar numbers to saliva-coated hydroxyapatite that had been preincubated at 4 degrees C overnight. Preincubation of saliva-coated hydroxyapatite overnight at 37 degrees C reduced subsequent adherence of S. sanguis 12 by approximately 10%, whereas adherence of S. sanguis 12na was reduced by over 80%. Preincubation at 37 degrees C in the presence of neuraminidase reduced adherence of S. sanguis 12 by over 90% and caused some additional reduction in adherence of S. sanguis 12na. The data were analyzed with Langmuir isotherms, Scatchard plots, and Hill plots. Some evidence of cooperativity was seen. A peak in the Scatchard plot for S. sanguis 12 binding to saliva-coated hydroxyapatite preincubated at 4 degrees C disappeared after preincubation at 37 degrees C, suggesting the loss of a salivary receptor. Many more organisms were found to bind when adherence was measured by assays counting the number of organisms remaining in suspension after the beads had settled. These weakly binding organisms, which were removed by washing, demonstrated adherence characteristics similar to those of the firmly bound organisms. Both strains were strongly hydrophobic. It is proposed that the binding of S. sanguis 12 and 12na involves two types of receptor on the salivary pellicle. One type of receptor is stable at 37 degrees C, but sensitive to neuraminidase; the second type is inactivated by prolonged incubation at 37 degrees C. S. sanguis 12 may bind to both types of receptor, whereas S. sanguis 12na binds only to the second type. The neuraminidase-sensitive receptor might be involved in saliva-mediated aggregation.

Association of neuraminidase-sensitive receptors and putative hydrophobic interactions with high-affinity binding sites for Streptococcus sanguis C5 in salivary pellicles.

The relationship of neuraminidase-sensitive receptors and putative hydrophobic interactions to high- and low-affinity binding sites in experimental salivary pellicles for Streptococcus sanguis C5 was investigated. NaSCN, an inhibitor of hydrophobic interactions, reduced the number of cells which adsorbed to pellicles to a greater extent than NaCl or KCl when both low and high streptococcal concentrations were used in assays. However, NaSCN was not more effective than NaCl or KCl in desorbing 3H-labeled salivary pellicle components from hydroxyapatite, and NaSCN pretreatment of either strain C5 cells or the salivary pellicles did not destroy or remove either the streptococcal adhesions or the pellicle receptors. Neuraminidase treatment of pellicles or the presence of sialic acid-containing gangliosides only inhibited S. sanguis adsorption when low streptococcal concentrations were used. At these concentrations, S. sanguis adsorbs primarily to high-affinity pellicle binding sites. Adsorption isotherms indicated that neuraminidase-sensitive interactions were mainly responsible for the high affinity of these binding sites, whereas putative hydrophobic interactions inhibitable by NaSCN were mainly associated with the numbers of binding sites available. Sugar inhibition studies suggested that the two classes of binding sites previously demonstrated in untreated salivary pellicles for S. sanguis C5 are not the result of a partial conversion of high-affinity sites to low-affinity sites due to removal of sialic acid residues.

Hemadsorption and virulence are separable properties of Mycoplasma pneumoniae.

A selective enrichment technique was used to isolate a hemadsorption-positive revertant of a hemadsorption-negative mutant strain of Mycoplasma pneumoniae. This hemadsorption-positive revertant was shown to have simultaneously regained both the ability to attach to neuraminidase-sensitive receptors on the tracheal ring respiratory epithelium in vitro and the ability to synthesize three virulent-strain-specific proteins which were not synthesized by the hemadsorption-negative mutant. Despite the persistence of the revertant in hamster lung tissue for 9 to 12 weeks postinfection, no cytopathology was observed. Intranasal inoculation of the revertant provided limited protection against a challenge dose of virulent M. pneumoniae.

Immunochemical studies of infectious mononucleosis—X. Characterization of a glycoprotein from horse erythrocytes which reacts with Paul-Bunnell antibody

A highly purified preparation of horse erythrocyte glycoprotein was prepared from an aqueous ethanolic extract of hemoglobin-free membranes. The subunit apparent mol. wt was 30,000. In aqueous solution the glycoprotein formed globular aggregates of 93 ± 16 Å diameter. The glycoprotein had a receptor for the Paul-Bunnell antibody of infectious mononucleosis which was associated with an O-glycosidically linked oligosaccharide and dependent on the presence of N-glycolylneuraminic acid. In addition the glycoprotein had a neuraminidase-sensitive receptor for human peripheral blood lymphocytes. Fifty per cent inhibition of the rosetting of sheep red cells by 4 × 10 5 lymphocytes was caused by 30 μg of glycoprotein.

Cellular immunity of mice to Leishmania donovani in vitro: lymphokine-mediated killing of intracellular parasites in macrophages.

Leishmania donovani, an intracellular protozoan, causes kala-azar by parasitizing the macrophages of its mammalian host. Outbred NCS and CD-1 mice develop immunity to this parasite. This immunity was demonstrable when supernatant fluids from cultured splenic lymphocytes were added to infected macrophages. Only the lymphokine preparations from infected mice showed significant leishmanicidal activity. Mice receiving multiple inocula were more potent producers of leishmanicidal lymphokines than were those receiving single inocula. The expression of leishmanicidal activity in our system required continuous presence of the lymphokine preparation and was independent of trypsin- or neuraminidase-sensitive receptors of the macrophages. Light and electron microscopy revealed that, in the presence of lymphokines, macrophages appeared to be "activated," and intracellular leishmanias developed specific subcellular lesions in the kinetoplast-mitochondria. A time-course study showed that cultivation of the lymphocytes for 1 1/2 days completed the release of their leishmanicidal lymphokines which were heat-labile molecules larger than 50,000 daltons.

Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae.

Previously isolated mutants of Mycoplasma pneumoniae incapable of hemadsorption were characterized with respect to specific protein content, tracheal ring attachment capability, and virulence for both in vitro and in vivo model systems. Two-dimensional gel electrophoresis revealed both quantitative and qualitative differences between the protein complements of two different mutant strains and that of the virulent parent strain. Studies of mycoplasma attachment to hamster tracheal rings in vitro demonstrated that only one of these mutant strains still possessed the ability to attach to the respiratory epithelium via neuraminidase-sensitive receptors. Measurement of [3H]orotic acid uptake in mycoplasma-infected tracheal rings indicated that infection with the hemadsorption-negative mutants resulted in only slight reductions of ribonucleic acid synthesis, similar to levels observed for tracheal rings infected with an avirulent strain of M. pneumoniae. The virulence potential of the two mutant strains was further investigated by utilizing the hamster model system. Both mutant strains were rapidly cleared from the lungs of infected animals and produced little or no microscopic pneumonia.

Interaction of Mycoplasma pneumoniae with human lung fibroblasts: characterization of the in vitro model.

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.

Adherence of erythrocytes to Mycoplasma pneumoniae.

The human pathogen Mycoplasma pneumoniae adheres to a variety of cells, including erythrocytes. A hemadsorption technique was developed to quantitate adherence by photometric measurement of lysates of erythrocytes that attached to sheets of M. pneumoniae grown in cups of Linbro plates. Attachment of sheep erythrocytes (SE) increased with higher ionic strength, was unaffected by minor pH variations (6 to 9), and was blocked by anti-M. pneumoniae antiserum, but was not inhibited by a variety of sugars, amino acids, and bovine serum albumin. The reaction was time and temperature dependent. The temperature curve showed peaks at 14 and 28 degrees C with untreated SE but only one peak at about 38 degrees C with glutaraldehyde-treated SE. The temperature dependence indicated involvement of either metabolic or membrane activities in the binding process. Trypsin treatment of the M. pneumoniae sheet abolished adherence of SE but was only partially effective with human erythrocytes and noneffective with rabbit erythrocytes. The binding capacity of the mycoplasma cells for SE was restored by incubation in growth medium for 3 to 4 h; this restoration was inhibited by 10 mug of chloramphenicol per ml. Neuraminidase treatment of SE removed their attachment capacity but had no effect on attachment of rabbit erythrocytes and only a slight effect on attachment of human erythrocytes. Pretreatment of M. pneumoniae with neuraminic acid partially blocked the adherence of SE, whereas rabbit erythrocyte attachment was not affected. Attached SE could be detached by trypsin, but not by neuraminidase. For human and rabbit erythrocytes, the results suggest binding mechanisms other than the interaction between neuraminidase-sensitive receptors and protein-containing binding sites shown for SE.