Characterization of a reverse gyrase from the extremely thermophilic hydrogen-oxidizing eubacterium Calderobacterium hydrogenophilum

Abstract Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae. The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae. Gangliosides capable of binding K99 fimbriated E. coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG. No binding was detected to NeuAc-GM3 and NeuGc-GM1.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

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. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.     

An anti-idiotype vaccine elicits a specific response to N-glycolyl sialic acid residues of glycoconjugates in melanoma patients

We generated the 1E10 gamma-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM(3) ganglioside.     

Analysis of anti-tumor antibodies in mice and rabbits induced by monoclonal anti-idiotope antibodies

Eight different mouse monoclonal anti-idiotope antibodies (mAb2) generated against a mouse monoclonal anti-human melanoma proteoglycan Ag (MPG) antibody (mAb1), MEM136, were tested for their ability to induce anti-MPG responses in mice and rabbits. All Ab2 were idiotypically cross-reactive and combining site-specific as demonstrated by competitive cross-inhibition studies and their ability to inhibit the binding of MEM136 to the melanoma cells, Colo38. However, only two Ab2, IM32 and IM06, were able to induce specific anti-TAA-specific (Ab1') responses in rabbits. When IM32 and IM06 were tested in allogeneic stains of mice for the induction of anti-MPG responses, only IM32 produced an Ab1' response. In mice, the Ab3 response induced by IM32 is idiotypically cross-reactive with its Ab1. Furthermore, the IM32-induced murine Ab3 and MEM136 recognized a similar MPG epitope on the melanoma cells because the Ab3 inhibited the binding of MEM136 to melanoma cells. The Ab3 induced by IM32 and IM06 in rabbits also recognized a similar epitope as the Ab1. In rabbits, the Ab3 response induced by IM32 and IM06 were idiotypically cross-reactive with each other. However, additional studies indicated that the majority of Ab3 induced by IM32 were IM32 Id-specific and lacked IM06 idiotopes. Further experimentation indicated that IM32-induced rabbit Ab3 were biologically active as demonstrated by the ability of the Ab3 to inhibit melanoma cell invasion in a Matrigel assay.     

Immunosensitization of melanoma tumor cells to non-MHC Fas-mediated killing by MART-1-specific CTL cultures

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.     

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.     

Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and antiangiogenic effect in a metastatic lung carcinoma

Anti-idiotype monoclonal antibody (mAb) 1E10 was generated by immunizing BALB/c mice with an Ab1 mAb which recognizes NeuGc-containing gangliosides, sulfatides and some tumor antigens. 1E10 mAb induces therapeutic effects in a primary breast carcinoma and a melanoma model. However, the tumor immunity mechanisms have not been elucidated. Here we show that aluminum hydroxide-precipitated 1E10 mAb immunization induced anti-metastatic effect in the 3LL-D122 Lewis Lung carcinoma, a poorly immunogenic and highly metastatic model in C57BL/6 mice. The therapeutic effect was associated to the increment of T cells infiltrating metastases, the reduction of new blood vessels formation and the increase of apoptotic tumor cells in lung nodules. Interestingly, active immunization does not induce measurable antibodies to the 1E10 mAb, the NeuGc-GM3 or tumor cells, which may suggest a different mechanism which has to be elucidated. These findings may support the relevance of this target for cancer biotherapy.     

Influence of gangliosides or LPS-like gangliosides on the tumoricidal activity of adherent leukocytes

We previously showed that highly metastatic clones derived from the poorly metastatic human melanoma cell line M4Be are very radiosensitive provided that they are deficient in complex gangliosides. Here, we report that the highly metastatic clone 4 appears more sensitive to activated adherent leukocytes than M4Be via a transmembrane TNF-alpha-dependent mechanism. Adherent leukocytes (AL) were freshly isolated from different blood donors and were activated with Esherichia coli lipopolysaccharide (LPS). These AL contain 80% (73-93%) monocytes, 15% (6-20%) B lymphocytes and 5% (1-8%) T lymphocytes. The tumour cell survival following contact with AL was estimated with a clonogenic assay where isolated tumour cells were plated for 14 days with AL. We show on the one hand that either exogenous bovine brain GM1 gangliosides or Campylobacter jejuni LPS with GM1-like structure (LPS-like GM1) significantly decrease the hypersensitivity of clone 4 to AL. On the other hand, the cleaving with neuraminidase of more than 50% of the sialic residues bound to endogenous gangliosides in resistant M4Be cells significantly increases their sensitivity to AL. Thus, our highly metastatic cells appear both very sensitive to activated AL when they are deficient in complex gangliosides and resistant to AL when they are transiently exposed to exogenous gangliosides or LPS-like gangliosides. These in vitro data may reflect the paradoxidal behaviour of highly metastatic cells in vivo which appear both very sensitive to physiological stresses and able to survive to form secondary tumours.     

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. Although C18 sphingosine was the only long chain base detected, ceramide fatty acid ranged from C16 to C24 carbon moieties. Based on gas-liquid chromatographic and antibody analyses, all major tetraosyl structure gangliosides were ganglio series types. Comprising 43 to 60% of thioglycollate-stimulated cells and 60 to 70% of Escherichia coli-activated cells, monosialosyl-gangliotetraosyl ceromides (Gm1 gangliosides) were the major monosialo species of which four were present: sialidase-resistant NeuGc-GM1a and NeuAc-GM1a and sialidase sensitive NeuGc-GM1b and NeuAc-GM1b. Analyses of thioglycollate-elicited murine peritoneal macrophage ganglioside patterns from four strains of mice, including the C3H/HeJ strain, indicated that, in the absence of any expression of a genetic defect, the pattern is conserved. However, when E. coli was used as the activating agent, the normal C3H/HeN macrophage contained little Gm1a with the sialidase-sensitive Gm1b predominant; the converse was true for the congenic endotoxin hyporesponsive C3H/HeJ strain. Therefore, C3H/HeJ mice are not defective in ganglioside metabolism per se but in the processing of an endotoxin stimulus such that one manifestation is an altered macrophage ganglioside pattern deficient in Gm1b.     

Isolation of the melanoma-associated antigen p23 using antibody phage display

The general responsiveness of human melanoma to immunotherapy has been well established, but active immunotherapy of melanoma has been hampered by insufficient information on the immunogenicity of melanoma-associated Ags in patients. In this study, we isolated a recombinant phage-Fab clone (A10-5) from a phage-Fab library derived from the B cells of a melanoma patient in remission after immunotherapy. Purified A10-5 Fab bound at high levels to cultured melanoma cell lines and to tissue sections of metastatic and vertical growth phase primary melanoma, but not to radial growth phase primary melanoma, nevi, or normal skin. A10-5 Fab bound to both the surface and the cytoplasm of cultured melanoma cells, but only to the cytoplasm of cultured fibroblasts. Western blot analysis revealed A10-5 Fab reactivity with a 33- and a 23-kDa glycoprotein under nonreducing conditions, and with a 23-kDa protein only under reducing conditions. A cDNA with an open reading frame predicted to encode a 23-kDa protein was cloned by screening a melanoma cell cDNA library with A10-5 Fab. This protein (p23) is the human homologue of the murine tumor transplantation Ag P198 that interacts with the cytoplasmic domain of ErbB-3 expressed by melanoma cells. Thus, the Ab phage display method has identified a novel, stage-specific melanoma-associated Ag that may have therapeutic and diagnostic value.     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.     

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.     

Effect of exogenous gangliosides on the lipid composition of chick neurons in culture

When exogenous gangliosides are added to the growth medium of neuronal cell cultures they are inserted into their plasma membranes and are afterwards metabolized in the cytoplasmic interior. The action of exogenous gangliosides brings important morphological and biochemical changes to neurons in culture. The present report shows that the treatment with exogenous gangliosides of a primary culture of chick neurons modified the distribution of fatty acids in phosphatidylinositol (PI), mainly that of arachidonic acid and the fatty acids of the (n - 3) series without affecting the other phospholipids. The composition of neutral lipids did not change but their content was increased up to 2-3-fold depending upon the concentration of gangliosides. The change of the growth medium from one containing fetal calf serum to a chemically defined one reduced dramatically the content of free fatty acids while the addition of gangliosides raised this content to normal levels. The increase in the amount of diacylglycerol (DG) confirmed the finding that gangliosides stimulate phosphoinositide degradation. Finally the fatty acid composition of DG suggests indirectly that this compound might be produced also by degradation of phosphatidylcholine and not only of PI.     

Effect of exogenous gangliosides on human neural cell division

Human neural cells in exponential growth phase were transferred to a serum-free medium and maintained for 72 hr without any detectable loss in viability. The two normal fetal cell lines (CH_I and CH_II) showed a serum-dependent cell proliferation, but the glioblastoma multiforme cells (12–18) were able to continue proliferating in this totally synthetic medium. The incorporation of [³H] thymidine into the acid-precipitable fraction of both normal and neoplastic human neural cells was assayed in the presence and the absence of exogenous gangliosides by a convenient new method. In serum-free medium, gangliosides (50 μM) inhibited the thymidine incorporation into the normal fetal cells within 24 hr and, in serum containing medium, reduced their proliferation within 48 hr. No such effects were detectable in the glioma cells. The inhibition of thymidine incorporation in the normal cells was reversible upon removal of the gangliosides. These results indicate a role of gangliosides in the postmitotic phase of normal human neural cells resulting in the regulation of cell proliferation.     

Structural and immunological characterization of O-acetylated GD2. Evidence that GD2 is an acceptor for ganglioside O-acetyltransferase in human melanoma cells.

We have shown previously that Golgi-enriched vesicles from the human melanoma cell line Melur can transfer [3H]acetate from [acetyl-3H]acetyl-CoA to endogenous GD3 to form [acetyl-3H]O-acetyl-GD3 (Manzi, A. E., Sjoberg, E. R., Diaz, S., and Varki, A. (1990) J. Biol. Chem. 265, 13091-13103). Applying the same approach in the human melanoma cell line M21, label was found in [acetyl-3H]O-acetyl-GD3 and also in a species co-migrating with unsubstituted GD3 on TLC. Both were sialidase-sensitive and alkali-labile, indicating incorporation as [3H]O-acetyl esters on sialic acids. Immunological reactivity, sialidase sensitivity, chromatographic behavior, and the known ganglioside pattern of M21 cells suggested that the slower migrating species might be [acetyl-3H]O-acetyl-GD2. Sialic acids released from this labeled molecule by sialidase showed esterification with [3H]acetate at both C7 and C9 hydroxyls. Lipid extracts from cells metabolically labeled with [3H]galactose showed a corresponding ganglioside, which upon alkali treatment yielded a species migrating with GD2. Analysis of purified ganglioside by high performance thin layer chromatography immuno-overlays, fast atom bombardment-mass spectrometry in positive and negative ion modes, periodate oxidation resistance, linkage analysis by permethylation and gas chromatography-mass spectrometry, and 500 MHz 1H NMR was consistent with the following structure: 9-O Ac-Neu5Ac alpha 2-8Neu5Ac alpha 2-3(GalNAc beta 1-4) Gal beta 1-4Gluc beta 1-1' ceramide Total gangliosides from M21 were analyzed by high performance thin layer chromatography immuno-overlay with monoclonal antibodies D1.1, JONES, 27A, and 8A2, all known to, or suspected of reacting with 9-O-acetylated gangliosides. The first three bound well to 9-O-acetyl-GD3 and a slower migrating 9-O-acetylated ganglioside, which was distinct from 9-O-acetyl-GD2. Antibody 8A2 reacted weakly with purified 9-O-acetyl-GD2 and strongly with two other 9-O-acetylated gangliosides migrating slower than 9-O-acetyl-GD2. Thus, the family of O-acetylated gangliosides in melanoma cells is much more complex than previously appreciated.     

Alteration of gangliosides in plasma and red cells of humans bearing melanoma tumors

In melanoma tumor-bearing humans, the levels of lipid-bound sialic acid were significantly elevated in both plasma and erythrocytes. Disialosyllactosylceramide in the plasma and sialosyllactosylceramide in red cells were the gangliosides mainly concerned by the increase, as compared to their concentration in the blood of healthy humans. In surgically treated patients, the plasma gangliosides remained higher than the controls, whereas a downward trend was noticeable in red cells. It is suggested that the occurrence of increased amounts of disialosyllactosylceramide in patients' plasma reflects the previously shown presence of this ganglioside as a major component in the sialic acid-containing glycolipid fraction of malignant melanocytes.     

Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.     

Glycolipids of the bovine pineal organ and retina

Neutral and acidic glycolipids from the bovine pineal organ and neutral glycolipids from the bovine retina were characterized. The chemical structures of the isolated glycolipids were determined by means of carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, long chain base analysis, mass spectrometry, NMR spectroscopy, and IR spectroscopy. GM3, GD3, and GT1 were the major bovine pineal organ gangliosides, GD3 accounting for 75% of the total gangliosides. Galactosylceramide, glucosylceramide, and lactosylceramide were found in both the bovine pineal organ and retina. Sulfatide was also present in both tissues. It had already been reported that the major bovine retina ganglioside was GD3 (Handa, S. & Burton, R.M. (1969) Lipids 4, 205-208). The glycolipid patterns of the two tissues were very similar to each other and quite different from those of other tissues.     

Characterization of major glycolipids in bovine erythrocyte membrane

Several neutral glycolipids and gangliosides were isolated from bovine erythrocyte stroma. Their structures were determined by partial acid hydrolysis, methylation analysis, periodate oxidation and CrO3 oxidation. Two major neutral glycolipids were characterized as lactosylceramide and galactosyl(alpha1--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Two major gangliosides were N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide and N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Minor glycolipids were glucosyl- and galactosylceramide, glucosamine-containing tri- and tetraglycosylceramide, glucosamine-containing disialosylhexaglycosylceramide, and gangliosides containing N-acetylneuraminic acid. The ceramide moiety of each glycolipid contained perdominantly d18:1 sphingosine, and normal fatty acids of C16:0, C22:0, C24:0, and C24:1.