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The glycolipids of Ascaris suum represent either neutral, zwitterionic or acidic structures. The acidic fraction comprises a sulphatide and an unusual phosphoinositolglycosphingolipid (Lochnit et al. 1998b). The sulphatide was previously localized to the hypodermis, contractile zone of somatic muscle cells and the external musculature of the female uterus, whereas the presence of the phosphoinositolglycosphingolipid species was restricted to the intestine. The neutral and zwitterionic components belong to the arthro-carbohydrate series, which are substituted in their zwitterionic structures by phosphocholine (PC) and in one glycolipid by an additional phosphoethanolamine residue. In previous immunohistochemical localization studies, however, the chemical nature of the PC-substituted biomolecules has not been investigated in detail. Here, we report on the immunohistochemical localization and differentiation of phosphocholine-containing structures into lipid- and protein-bound species in adult A. suum. The patterns of immunostaining, obtained with a PC-specific monoclonal antibody and anti-zwitterionic glycolipid hyperimmune serum in the female worm, indicated a parallel organ distribution for glycolipid- and protein-bound PC-epitopes. Immunoreactivity was localized to specific tissues of the body wall, intestine and reproductive tract. This is the first report of surface-located PC-epitopes for ascarids. The patterns of immunolabelling obtained with antibodies directed against the unsubstituted arthro-carbohydrate series backbone suggested that the glycolipid-bound epitope was restricted to the hypodermis, whilst the protein-bound antigenic determinant resembled that for PC.

The oligosaccharide structures of glycolipids from cercariae of the human blood fluke, Schistosoma mansoni, were analyzed in the form of their corresponding, pyridylaminated oligosaccharides by methylation analysis, partial hydrolysis, exoglycosidase treatment, on-target exoglyco­sidase cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The six, dominant chemical structures present have been determined as: GalNAc(β1-4)Glc1-ceramide; GlcNAc(β1-3)Gal­NAc(β1-4)Glc1-ceramide; Gal(β1-4)GlcNAc(β1-3)Gal­NAc(β1-4)Glc1-ceramide; Gal(β1-4)[Fuc(α1-3)]Glc­NAc(β1-3)Gal­NAc(β1-4)Glc1-ceramide (Lewis X pentasaccharide structure); Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Glc­NAc(β1-3)GalNAc(β1-4)Glc1-ceramide (Lewis X hexa­saccharide structure); and, Fuc(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)GalNAc(β1-4)Glc1-ceramide (pseudo-Lewis Y hexasaccharide structure). These structures belong to the characterized schisto-series of protostomial glycosphingolipids. The Lewis X and pseudo-Lewis Y glyco­lipids are stage-specifically expressed by the cercarial life-cycle stage, and not by the adult or egg.

The oligosaccharide structures of glycolipids from cercariae of the human blood fluke, Schistosoma mansoni, were analyzed in the form of their corresponding, pyridylaminated oligosaccharides by methylation analysis, partial hydrolysis, exoglycosidase treatment, on-target exoglyco-sidase cleavage and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The six, dominant chemical structures present have been determined as: GalNAc(beta1-4)Glc1-ceramide; GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)GlcNAc(beta1-3)Gal-NAc(beta1-4)Glc1-ceramide; Gal(beta1-4)[Fuc(alpha1-3)]Glc-NAc(beta1-3)Gal-NAc(beta1-4)Glc1-++ +ceram ide (Lewis X pentasaccharide structure); Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Glc-NAc(beta1-3)GalNAc(beta 1-4)Glc1-ceramide (Lewis X hexa-saccharide structure); and, Fuc(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4 )Glc1-ceramide (pseudo-Lewis Y hexasaccharide structure). These structures belong to the characterized schisto-series of protostomial glycosphingolipids. The Lewis X and pseudo-Lewis Y glyco-lipids are stage-specifically expressed by the cercarial life-cycle stage, and not by the adult or egg.

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.

The isolated neutral glycosphingolipid fraction from the pig parasitic nematode, Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor alpha, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below), [structure: see text] The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis(P. gingivalis), a keystone pathogen for the development of periodontitis.