Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I).

LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAcalpha2-3Galbeta1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope.

Reduced inflammatory potential of peritoneal macrophages recruited in mice pretreated with a glycolipid synthesis inhibitor.

Integral components of mammalian cell membranes, glycosphingolipids (GSL) reside in specialized plasma membrane microdomains critical for cell signaling. N-alkylated nojirimycins are compounds developed for GSL substrate deprivation therapy, blocking GSL synthesis by specifically inhibiting an essential enzyme, ceramide glucosyltransferase. Peritoneal macrophages recruited in mice pretreated with an inhibitory N-alkylnojirimycin displayed a reduced capacity to release either TNFalpha or interleukin-6 when re-exposed to whole killed E.

Nontypeable Haemophilus influenzae-binding gangliosides of human respiratory (HEp-2) cells have a requisite lacto/neolacto core structure.

Nontypeable Haemophilus influenzae (NTHI) are a major cause of human infections. We previously demonstrated high affinity and high specificity binding of NTHI to minor gangliosides of human respiratory (HEp-2) cells and macrophages, but not to brain gangliosides. We further identified the NTHI-binding ganglioside of human macrophages as alpha2,3-sialylosylparagloboside (IV3NeuAc-nLcOse4Cer, nLM1), which possesses a neolacto core structure that is absent in brain gangliosides.

The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures.

Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events. Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage. Macrophages were isolated from healthy human volunteers by two different methods.

Differences in splenic B-lymphocyte ganglioside expression and accessibility in normal and endotoxin-hyporesponsive mice.

Endotoxin-responsive (C3H/HeN) and -hyporesponsive (C3H/HeJ) murine B lymphocytes purified by adherence to anti-immunoglobulin ("antibody panning") possess identical gangliosides but different ganglioside surface accessibilities. We investigated the distribution and surface accessibility of gangliosides of B lymphocytes purified by adherence to plastic ("plastic panning") or by subtraction of non-B-lymphocyte components. As with antibody panning, there were no entirely new or absent gangliosides in plastic-panned or subtraction-purified B lymphocytes of each strain.

Expression of mouse sialic acid on gangliosides of a human glioma grown as a xenograft in SCID mice.

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides.

Tumor-infiltrating macrophages influence the glycosphingolipid composition of murine brain tumors.

A procedure was developed to analyze glycosphingolipids (GSLs) in tumor-infiltrating macrophages (TIMs). The procedure entailed dissociating tumors into single cell suspensions with a concurrent metabolic labeling of GSLs using [14C]galactose. TIMs were then separated from tumor cells and other host cells by magnetic activated cell sorting and CD11b (Mac-1) microbeads.

Ganglioside composition of a mouse brain tumor grown in the severe combined immunodeficiency (SCID) mouse.

The content and composition of gangliosides were examined in an experimental mouse brain tumor, EPEN, that was grown subcutaneously in the flank of the syngeneic C57BL/6J (B6) host and in the B6 severe combined immunodeficiency (SCID) host. SCID mice lack functional T- and B-lymphocytes, but have a normal complement of macrophages. The content and distribution of the brain tumor gangliosides were similar whether the tumor was grown in the immunocompetent B6 host or in the B6-SCID host.

Structural characterization of the disialogangliosides of murine peritoneal macrophages.

Sialoglycosphingolipids (gangliosides) have been increasingly implicated as regulators of membrane signaling events. Macrophage ganglioside patterns dramatically increase in complexity when murine peritoneal macrophages are stimulated in vivo with the appearance of the sialidase-sensitive monosialoganglioside GM1b (cisGM1) as a major component. Gangliosides from stimulated murine peritoneal macrophages were separated into monosialo and polysialo fractions and the polysialo fraction structurally characterized by enzymatic, chemical, and mass spectra methods.

Ganglioside biosynthetic gene expression in experimental mouse brain tumors.

The genes for cytidine monophospho-N-acetylneuraminic acid hydroxylase (NeuAc-H) and beta-1,4-N-acetylgalactosaminyl transferase (GalNAc-T) were examined using reverse transcription-PCR in two experimental mouse brain tumors, EPEN and CT-2A. NeuAc-H is required for the synthesis of gangliosides containing N-glycolylneuraminic acid, whereas GalNAc-T is required for the synthesis of ganglioside GM2. The genes were analyzed in solid tumors grown in vivo and in tumor cells grown in vitro.

Influence of host cell infiltration on the glycolipid content of mouse brain tumors.

Previous studies showed that levels of some glycosphingolipids (GSLs) expressed in solid brain tumors grown in vivo were reduced or undetectable in cultured cells prepared from the tumors. This phenomenon has been attributed either to suppressed glycolipid synthesis from unknown forces of the tissue culture environment or to the absence of host cells that normally infiltrate the solid tumors growing in vivo. To test further the host cell hypothesis, we examined host cell markers in two experimental mouse brain tumors, the ependymoblastoma and the CT-2A, that were grown as subcutaneous solid tumors in the flank of C57BL/6J (B6) mice or as cultured cells in vitro.

In situ accessibility of murine macrophage gangliosides.

Gangliosides are implicated in cell signal transduction. Prior to investigating this phenomenon in macrophages, the in situ accessibility of gangliosides to macromolecules was assessed for peritoneal macrophages isolated from normal C3H/HeN and endotoxin-hyporesponsive C3H/HeJ mice. C3H/HeJ resident and thioglycolate-elicited macrophage ganglioside patterns are the same as normal strains, and no strain differences in galactose oxidase accessibility for resident or thioglycolate-elicited macrophage gangliosides were found.

Removal of borate from tritiated gangliosides via the mannitoborate complex.

Purified gangliosides were radiolabeled by exposure to sodium boro [3H]hydride in the presence of palladium on barium sulfate. After centrifugation to remove the catalyst, borate was complexed with mannitol and the resulting mannitoborate complex and salts were removed by reversed phase chromatography. The labeled gangliosides were repurified by ion exchange and silica gel chromatographies.

GM1b and GM1b-GalNAc: major gangliosides of murine-derived macrophage-like WEHI-3 cells.

WEHI-3 cells, derived from a BALB/c mouse, are a myelomonocytic leukemic cell line with macrophage-like properties. We have isolated, purified and characterized the monosialogangliosides from WEHI-3 cells by 1D-HPTLC, 2D-HPTLC, enzymatic degradation, HPTLC-immunostaining, gas-liquid chromatography and fast atom bombardment-mass spectrometry (FAB-MS). Quantitative 2D-HPTLC shows two monosialogangliosides are the major components, constituting 77% of the total, with a third monosialoganglioside being 3%.

The presence of sialidase-sensitive sialosylgangliotetraosyl ceramide (GM1b) in stimulated murine macrophages. Deficiency of GM1b in Escherichia coli-activated macrophages from the C3H/HeJ mouse.

The stimulated murine macrophage was found to contain 11 major gangliosides of which 8 were determined to be monosialylated. The thin-layer chromatographic patterns were complicated by the presence of both sialic acid and ceramide fatty acid heterogeneity. N-glycolyl and N-acetylneuraminic acid-containing species were present for each ganglioside characterized.

Altered B-lymphocyte membrane architecture indicated by ganglioside accessibility in C3H/HeJ mice.

We have analyzed both the total ganglioside composition and the surface accessibility of C3H/HeN B lymphocytes and C3H/HeJ B lymphocytes. Seventeen individual resorcinol-positive moieties were visualized by two-dimensional thin-layer chromatography of the purified gangliosides from both strains. Complete homology between strains was seen in the patterns of total gangliosides purified from the endotoxin-responsive and -hyporesponsive strains, with only minor differences in the relative concentrations of four gangliosides.

Factors mediating lipopolysaccharide-induced ganglioside expression in murine peritoneal macrophages.

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.

Comparison of thymocyte and T lymphocyte gangliosides from C3H/HeN and C3H/HeJ mice.

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28-30 discrete gangliosides of which eight represented major gangliosides.

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice.

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice.

Further evidence for an intrinsic neuraminidase in CNS myelin.

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.

High-resolution proton NMR studies of gangliosides. III. Elucidation of the structure of ganglioside GM3 lactone.

Ganglioside GM3 lactone (1) was prepared in 95% yield from the parent ganglioside by incubation at 25 degrees C for 4 days in glacial acetic acid. Inspection of the 500 MHz proton NMR spectra of 1 and its precursor in dimethylsulfoxide-d6-deuterium oxide at 30 degrees C revealed a large deshielding (+1.42 ppm) of the H-2 resonance of the galactosyl residue.

Lack of specificity of brain gangliosides in the modulation of lymphocyte activation.

A potential role for glycolipid gangliosides to act as immunomodulating agents has been suggested. Most studies have employed brain gangliosides. We have systematically investigated highly purified murine brain gangliosides for their ability to modulate lymphocyte activation.

Ganglioside alterations in stimulated murine macrophages.

A two-dimensional thin-layer chromatographic technique has been used to separate and display gangliosides from murine peritoneal macrophages in different functional states. Resident macrophages have a relatively simple ganglioside pattern with about 15 resorcinol-positive spots. Gangliosides from resident cells contained mostly (90%) N-glycolylneuraminic acid.

Changes in membrane gangliosides: differentiation of human and murine monocytic cells.

Membrane ganglioside changes in murine peritoneal macrophages and the human promyelocytic leukemia cell line HL-60 have been assessed by two-dimensional thin-layer chromatography. C3H/HeJ mice respond to protein-containing endotoxin but are hyporesponsive to protein-free endotoxin preparations. Compared to unstimulated resident cells, protein-containing endotoxin produced an alteration in the C3H/HeJ macrophage ganglioside pattern whereas protein-free endotoxin did not.