The glycosidic antigen recognised by a novel monoclonal antibody, 75.12, is developmentally regulated on mouse embryonal carcinoma cells

Monoclonal antibody 75.12 raised against the human ovarian teratocarcinoma cell line PA1 detects a 'Y' or iso-leb glycosidic structure. Using the 75.12 antibody we have established that the Y antigen is expressed on some but not all mouse embryonal carcinoma (EC) lines. The Y or 75.12 antigen-positive EC cell lines F9 and PCC4 cease to express the antigen after differentiation induced with retinoic acid and this decreased expression parallels the morphological differentiation of the EC cells. These results support not only the idea that carbohydrate structures present on embryonic cells undergo marked alteration during differentiation, but also that established mouse EC cells may differ in their differentiation states.     

An anti-carbohydrate monoclonal antibody inhibits cell-substratum adhesion of F9 embryonal carcinoma cells

A monoclonal rat IgM antibody (4C9) raised against F9 embryonal carcinoma cells reacted with fucosyl residues in poly-N-acetyllactosamine-type large carbohydrates of these cells (embryoglycan). The chemical properties and distribution of the antigen resembled those of SSEA-1. The monoclonal antibody was found to inhibit cell-substratum adhesion of F9 cells: in the presence of the antibody, cells grew as spherical cell aggregates on plastic dishes. When the antibody was added to the already spread cells, they displayed the initial sign of rounding up within 3 h; the rounding process was largely completed within 6 h. After removal of the antibody, cells resumed their normal morphology. The antibody could act in the presence of 2,4-dinitrophenol. In serum-free medium, F9 cells spread on plastic dishes coated with fibronectin or with laminin, and the process was also inhibited by the antibody. Immuno-electronmicroscopy revealed that 4C9 antigen was diffusely distributed over the cell surface of F9 cells. The distribution of the antigen was not altered generally after culturing with the antibody for 6 h. Another monoclonal rat IgM antibody, which did not react with embryoglycan and resembled anti-Forssman, did not inhibit cell-substratum adhesion of F9 cells, in spite of its reactivity to the cells. Thus, a glycoprotein with fucosyl (poly)-N-acetyllactosamine structure appears to be involved in cell-substratum adhesion of F9 cells.     

A novel tumor-associated, developmentally regulated glycolipid antigen defined by monoclonal antibody ACFH-18

A mouse IgM monoclonal antibody, ACFH-18, was established after immunization of mice with the human gastric cancer cell line MKN74. The antibody reacts strongly with gastrointestinal carcinoma and showed a clear dependence on the degree of differentiation of gastric cancer cells. The antibody defines a series of glycolipid species with extremely slow TLC mobility present in both acidic and neutral glycolipid fractions of the extract from gastrointestinal adenocarcinoma and the original MKN74 cells. Isolation and structural study of the active glycolipids present in the acidic and neutral fractions and comparison of the antibody reactivity with glycolipids having related structures revealed a novel specificity. The minimum requirement for the maximal reactivity of ACFH-18 was identified as VII3Fuc-nLc10 (Structure A below and Fig. 8 in text). Since the antibody did not react with III3Fuc-nLc6 (Structure B below), which shares the same terminal sequence as VII3Fuc-nLc10, and since it cross-reacted with VII3Fuc-nLc8 (Z1 glycolipid) and VII3Fuc,V3-Fuc,III3Fuc-nLc8 (Z3 glycolipid), antibody ACFH-18 is capable of recognizing a fucosyl residue plus an internal repeating N-acetyllactosamine proximal to ceramide, as indicated by lines in Fig. 8 (Structures A, B, and E-G). (Formula: see text).     

The epitope of mouse embryonic antigen(s) recognized by monoclonal antibody TEC-02 is a carbohydrate carried by high-molecular-weight glycoconjugates

The expression and properties of mouse embryonic antigens, recognized by monoclonal antibody TEC-02, were analyzed in teratocarcinoma-derived cell lines. TEC-2 antigens were found in the majority of the parietal endoderm cells PYS-2 and in a fraction of cultured embryonal carcinoma cells but not in other cell lines tested. During the course of retinoic acid-induced differentiation of embryonal carcinoma cells F9, the expression of TEC-2 was transiently increased. Immunolabeling of extracts from F9 and PYS-2 cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydisperse glycoconjugates of high molecular weight (mostly greater than 100,000). The TEC-2 epitope was shown to be carbohydrate which in F9 cells might be attached to the same carrier as another developmentally regulated carbohydrate epitope TEC-1. The TEC-2 antigens, isolated by indirect immunoprecipitation, were degraded by extensive pronase digestion or mild alkaline treatment to mostly large products. Immunostaining of glycolipid standards suggested that TEC-2 epitope involves the GalNAc beta 1----4Gal beta 1----4R sequence. Combined data indicate that TEC-2 is a new developmentally regulated carbohydrate epitope carried in embryonal carcinoma cells predominantly on glycoprotein-bound large carbohydrates.     

Inhibition of embryonic neural retina cell-substratum adhesion with a monoclonal antibody

The C1H3 monoclonal antibody recognizes two distinct developmentally regulated cell surface antigens, with molecular masses of 170,000 and 140,000 daltons, in embryonic chick neural retina (Cole, G. J., and Glaser, L. (1984) Proc. Natl. Acad. Sci. U. S. A., in press). In vitro, the 170,000-dalton polypeptide is released by retinal cells into the surrounding culture medium and is present in material sedimentable at 100,000 X g. This pelletable material contains particles designated as adherons (Schubert, D., LaCorbiere, M., Klier, F. G., and Birdwell, G. (1983) J. Cell Biol. 96, 990-998) which promote cell-substratum adhesion of chick neural retina cells. In the present study, evidence is provided that the C1H3 monoclonal antibody inhibits cell adhesion to adheron-coated dishes when bound either to cells or to the adherons. The failure of other monoclonal antibodies, that bind to retinal cells with equal abundance, to disrupt adhesion demonstrates that the effect is specific. These data suggest that the neural-specific 170,000-dalton C1H3 polypeptide is the neural cell-adhesion molecule which is responsible for the ability of adherons to bind to cells.     

Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody.

The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.     

Induction of murine 8-cell blastomere polarity by F9 embryonal carcinoma cells

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage of development. This polarity forms as a result of a specific cell-cell interaction that has been termed induction. The ability of embryonal carcinoma (EC) cells to induce 8-cell blastomere polarization has been investigated by aggregating nonpolar 8-cell blastomeres with various types of EC cells. F9, a nullipotent stem cell, induced polarization of a nonpolar 8-cell companion in 80% of the aggregates. Stimulation of differentiation of F9 cells with retinoic acid (RA), with or without dibutyryl cAMP, caused a reduction in the polarity-inducing ability of these cells. Other EC cells, PSA-1, NULLI-SCC1, 3TDM, C3HNE, and P10, all displayed less polarity-inducing activity than F9. In addition, it was observed that when any of these cell types assumed a more differentiated phenotype, either spontaneously or in response to specific stimuli, they displayed a decrease in their ability to induce 8-cell polarization. As a control, the inducing ability of cells from normal mouse tissues was examined. It was found that neither STO mouse fibroblasts nor primary cultures of mouse lymphocytes were able to induce significant polarization of 8-cell stage blastomeres. These data support the hypothesis that while undifferentiated stem cell populations retain the ability to induce 8-cell blastomere polarization, it is apparently lost upon cellular differentiation.     

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.     

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.     

Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

A series of human erythrocyte glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen SSEA-1

A series of glycolipid antigens reacting with the monoclonal antibody directed to the stage-specific embryonic antigen 1 was isolated and characterized from group O human erythrocyte membranes. A ceramide heptasaccharide (Structure 1), ceramide nonasaccharide (Structure 2), and ceramide decasaccharide (Structure 3) have been characterized (formula, see text) The main feature of this glycolipid series is its long core sugar chain with a nonbranched repeating N-acetyllactosamine (norpolylactosamine). This characteristic is in contrast to that of co-existing H-active glycolipid series in which the longer core structures are branched type repeating N-acetyllactosamine (isopolylactosamine). The reactivity of these glycolipids to monoclonal anti-stage-specific embryonic antigen 1 antibody varied proportionately to the length of their core sugar chains. A possible significance of these glycolipids as developmentally regulated antigens and as cancer-associated antigens was discussed.     

A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM-2

A mouse monoclonal antibody, VIM-2, specific for human blood cells of myelomonocytic lineage, was found to bind to a series of minor gangliosides isolated from the cells of patients with chronic myelogenous leukemia (Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W., and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36). TLC immunostaining studies with the VIM-2 antibody of gangliosides from normal human neutrophils, acute myeloid leukemia, and chronic myelogenous leukemia cells showed that the total amount and the ratio of the VIM-2 gangliosides varies among these different myeloid cells and appears to be related to the level of cellular differentiation. Purification of these gangliosides from chronic myelogenous leukemia cells was aided by a sensitive enzyme-linked immunosorbent assay procedure used in conjunction with high performance liquid chromatography. Structures for two of the immunoreactive gangliosides (a ceramide decasaccharide, VIII3NeuAcV3-Fuc-nLc8Cer and a ceramide dodecasaccharide X3-NeuAcVII3Fuc-nLc10Cer) are proposed from negative ion fast atom bombardment mass spectrometry of the native gangliosides, methylation analysis, and the combined use of glycosidase treatment and TLC immunostaining with carbohydrate sequence specific antibodies. The VIM-2 antigen was thus characterized as involving the sialofucooligosaccharide sequence.     

Wnt6 induces the specification and epithelialization of F9 embryonal carcinoma cells to primitive endoderm

Epithelial-to-mesenchymal transitions (EMTs) play key roles in the normal development of an organism as well as its demise following the metastasis of a malignant tumour. An EMT during early mouse development results in the differentiation of primitive endoderm into the parietal endoderm that forms part of the parietal yolk sac. In the embryo, primitive endoderm develops from cells in the inner cell mass, but the signals that instruct these cells to become specified and adopt an epithelial fate are poorly understood. The mouse F9 teratocarcinoma cell line, a model that can recapitulate the in vivo primitive to parietal endoderm EMT, has been used extensively to elucidate the signalling cascades involved in extraembryonic endoderm differentiation. Here, we identified Wnt6 as a gene up-regulated in F9 cells in response to RA and show that Wnt6 expressing cells or cells exposed to Wnt6 conditioned media form primitive endoderm. Wnt6 induction of primitive endoderm is accompanied by beta-catenin and Snail1 translocation to the nucleus and the appearance of cytokeratin intermediate filaments. Attenuating glycogen synthase kinase 3 activity using LiCl gave similar results, but the fact that cells de-differentiate when LiCl is removed reveals that other signalling pathways are required to maintain cells as primitive endoderm. Finally, Wnt6-induced primitive endodermal cells were tested to determine their competency to complete the EMT and differentiate into parietal endoderm. Towards that end, results show that up-regulating protein kinase A activity is sufficient to induce markers of parietal endoderm. Together, these findings indicate that undifferentiated F9 cells are responsive to canonical Wnt signalling, which negatively regulates glycogen synthase kinase 3 activity leading to the epithelialization and specification of primitive endoderm competent to receive additional signals required for EMT. Considering the ability of F9 cells to mimic an in vivo EMT, the identification of this Wnt6-beta-catenin-Snail signalling cascade has broad implications for understanding EMT mechanisms in embryogenesis and metastasis.