Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.

Construction of bacteriophage expressing mouse monoclonal Fab fragments directed against the human MN glycophorin blood group antigens.

BACKGROUND: The MN human blood group antigens are complex glycopeptide antigens at the amino terminus of glycophorin A. Many different mouse monoclonal antibodies to these antigens have been produced and characterized. The construction of combinatorial immunoglobulin libraries displaying antibody Fab fragments on the surface of bacteriophage (Fab-phage) represents a novel approach for developing monoclonal reagents, for exploring the diversity of the immune response to specific antigens, and for understanding the molecular basis of the interaction of an antibody with its antigen. However, it is necessary to determine whether Fab fragments displayed on bacteriophage surfaces retain immunologic characteristics similar to the intact antibodies. STUDY DESIGN AND METHODS: Fab-phage were constructed from three anti-N (AH7, N61, and N92) and two anti-M (425/2B and M2A1) murine hybridomas. The Fab-phage and parental hybridomas were compared by enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. RESULTS: In each case, the Fab-phage and its parental hybridoma antibody had similar immunologic characteristics. In particular, their dependence on the pH of the buffer and on sialylation of the target antigen was similar. CONCLUSION: These results suggest that Fab-phage may provide novel reagents with applications in immunohematology and may be useful in the study of the immune response to human blood group antigens.

Biochemical characterization of the feline AB blood group system.

The biochemical nature of the feline AB blood group system was characterized by analysing red blood cells from homozygous (genotype A/A) and heterozygous (A/B) type A, type B (B/B), and type AB cats. High performance thin layer chromatography (HPTLC) of red cell glycolipids revealed that specific neuraminic acids (NA) on gangliosides, containing ceramide dihexoside (CDH) as a backbone, correlated with the feline AB blood group antigens. Although disialogangliosides predominated, mono- and trisialogangliosides were also isolated. B cats expressed solely N-acetyl-NA (NeuNAc) on these gangliosides. In addition to expressing N-glycolyl-NA (NeuNGc) containing gangliosides, A red cells have gangliosides with only NeuNAc or mixtures of both NA. HPTLC profiles of disialogangliosides from homozygous and heterozygous A cats differed slightly in the quantity of disialogangliosides. Equal amounts of NeuNAc and NeuNGc containing disialogangliosides, as well as two intermediary forms, were recovered from AB erythrocytes. Analysing disialogangliosides from red cells belonging to 17 genetically related cats, we consistently obtained the expected disialoganglioside profile, based on blood typing and pedigree information. SDS-PAGE of red cell membrane proteins and blotting with Triticum vulgaris, a lectin recognizing NeuNAc, revealed glycoproteins of approximately 51, 53, and 80 kD in B and AB cats but only a faint band of approximately 53 kD in A cats. By haemagglutination, Triticum vulgaris could also distinguish different blood types by specifically binding to B and AB cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The glycosphingolipid composition of the human hepatoma cell line,Hep-G2.

The origin of plasma glycosphingolipids in normal individuals and the mechanisms by which tumor-associated glycosphingolipid antigens enter the plasma in patients with cancer are largely unknown. The Hep-G2 human hepatoma cell line retains many of the characteristics of differentiated hepatocytes including the ability to synthesize and secrete lipoproteins. Preliminary results indicated that newly synthesized Hep-G2 cell glycosphingolipids are coupled to the secreted lipoproteins. This suggests that this cell line may offer an interesting model for studying glycosphingolipid secretion, transfer, and shedding. We now report on the chemical and immunological characterization of Hep-G2 cell glycosphingolipids. Five major glycosphingolipids were purified and biochemically characterized: glycosylceramide, lactosyl ceramide, ceramide trihexoside, ganglioside GM3, and lactosyl sulfatide. Four additional minor components (3-fucosyl-lactosamine containing glycolipids, asialo GM2, galactosylgloboside, and ganglioside GM1) were identified using a combination of exoglycosidase digestion and immunostaining of thin-layer chromatography plates with specific carbohydrate binding proteins. This demonstrates that although this cell line synthesizes a limited number of major glycosphingolipids, it retains the ability to produce at least small amounts of structures in the lactoneo, globo, and ganglio series of glycosphingolipids. These studies show that it will be possible to investigate the mechanisms of secretion by Hep-G2 cells of different classes of these molecules such as neutral glycosphingolipids, gangliosides, and sulfatides.

Carbohydrate-specific monoclonal antibodies bind to human granulocytes and stimulate antibody-dependent cellular cytotoxicity.

Mouse monoclonal antibodies which specifically recognize human granulocytes are used to study the classification, differentiation, and function of these cells. Mouse monoclonal antibody WEM-G1 specifically binds to human neutrophils and eosinophils. It also affects granulocyte function by stimulating granulocyte-mediated antibody-dependent cellular cytotoxicity. Biochemical studies presented here show that WEM-G1 recognizes the sugar sequence 3-fucosyllactosamine, Gal beta 1-4[Fuc alpha 1-3]GlcNAc. This sequence is present in granulocyte glycolipids and in glycoproteins of average approximate Mr 165,000 and 105,000. WEM-G1 is thus similar to other monoclonal antibodies that recognize this sequence on granulocytes and various other cells. Some of these 3-fucosyllactosamine-specific antibodies affect several other granulocyte functions. Knowledge of the biochemical structure of the WEM-G1 antigen suggested testing granulocyte function with other monoclonal antibodies of similar specificity. Antibodies recognizing both the identical oligosaccharide structure and a related sequence, Gal beta 1-4GlcNAc-R, were also found to stimulate granulocyte-mediated antibody-dependent cellular cytotoxicity.

Glycolipid antigen expression in human lung cancer.

Several mouse monoclonal antibodies which recognize carbohydrate sequences distinguish between different types of human lung cancer immunohistologically. These antibodies bind to glycolipid antigens produced by the cancer cells. When these glycolipids are separated by thin-layer chromatography, immunostaining of the chromatograms yields complex patterns of antigen-positive bands. To determine whether glycolipid patterns are useful in the classification of lung cancer, 16 human lung cancer cell lines comprising the major histological types of primary lung cancer were studied. Neutral glycolipids and gangliosides were isolated and separated by thin-layer chromatography. Six anti-carbohydrate antibodies which recognize structurally related antigens were used for immunostaining. Neuraminidase treatment of the chromatograms was used to detect "cryptic" sialylated antigens. All the cell lines were unique with regard to the type, amount, and chromatography pattern of the glycolipid antigens produced. Small cell lung cancer cell lines synthesized the greatest variety of antigens, whereas cell lines with large cell cytology synthesized the least. Interestingly, there was an inverse relationship between expression of some glycolipid antigens and DNA amplification of the c-myc oncogene. This suggests that enhanced c-myc expression may influence the types of glycolipids expressed at the surface of lung tumor cells.

Sialylated glycolipid antigens on human leukemic cell lines.

Many granulocyte-specific mouse monoclonal antibodies recognize the carbohydrate sequence 3-fucosyllactosamine, Ga 1 beta 1-4[Fuc alpha 1-3]GlcNAc, which occurs in cell-surface glycolipids and glycoproteins. In general, these antibodies bind to blast cells from most patients with acute myeloblastic leukemia, but not to those with acute lymphocytic leukemia. Neuraminidase treatment, however, increases exposure of this antigen on both myeloid and lymphoid cells. In the present study, the glycolipids from 13 lymphoid and nonlymphoid human cell lines were examined for the presence of unsialylated and sialylated 3-fucosyllactosamine sequences using a thin-layer chromatography immunostaining method. Nine of the cell lines were also tested by indirect immunofluorescence both before and after neuraminidase treatment. None of the six B-cell and T-cell lines had detectable neutral or sialylated glycolipid antigen. In contrast, six out of seven and five out of seven nonlymphoid cell lines had neutral and sialylated glycolipid antigens, respectively. These results agreed, in general, with those found by indirect immunofluorescence. They also represent the first direct demonstration of these sialylated glycolipids on human leukemic cells. Thus, in some cases increased antibody binding to neuraminidase-treated cells can be explained by the presence of sialylated glycolipid antigen.

Anti-My-28, an antigranulocyte mouse monoclonal antibody, binds to a sugar sequence in lacto-N-neotetraose.

Anti-My-28 is an IgM kappa monoclonal antibody produced by a hybridoma prepared from spleen cells of a mouse immunized with normal human granulocytes. By immunofluorescence it binds to human granulocytes but not to monocytes and lymphocytes. However, after treating cells with neuraminidase, the antibody also binds to lymphocytes and monocytes and to many leukemic cell lines and patient leukemic blast cells. Anti-My-28 binds to several neutral glycolipids and desialylated gangliosides of leukocytes and erythrocytes as detected by radioimmunoassay and immunostaining of thin-layer chromatograms. It recognizes a sugar sequence in lacto-N-neotetraose, Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc. This tetrasaccharide occurs in the glycolipids paragloboside and sialosylparagloboside, and its distal trisaccharide sequence is found in higher glycolipids and in glycoproteins.

Patterns of human thyroid parenchymal reaction following low-dose childhood irradiation.

Microscopic changes in the thyroids of 68 patients who had received low-dose childhood irradiation to the head and neck and who presented with palpable thyroid abnormalities culminating in surgery are compared to 34 control thyroids obtained from age- and sex-matched autopsy cases. Eighty-eight percent of irradiated thyroids showed moderate to severe focal hyperplasia, 51% contained single or multiple adenomas or adenomatous hyperplastic nodules, 68% exhibited chronic lymphocytic thyroiditis, 51% revealed colloid nodules, 42% presented with oxyphile change, 25% had mild fibrosis and 59% contained well-differentiated papillary, follicular or mixed thyroid carcinoma averaging 1.6 cm in diameter. Three small carcinomas were of the sclerosing type. The non irradiated thyroids showed 32% colloid nodule formation, 17% focal hyperplasia, 6% adenomatous hyperplasia and no identifiable carcinomas. Several nonspecific histologic abnormalities are now recognized as following low-dose radiation to the thyroid, the most important being focal hyperplasia, which may represent a pre-malignant change in thyroid parenchyma.