GM2-GM3 gangliosides ratio is dependent on GRP94 through down-regulation of GM2-AP cofactor in brain metastasis cells.

GRP94 is an ATP-dependent chaperone able to regulate pro-oncogenic signaling pathways. Previous studies have shown a critical role of GRP94 in brain metastasis (BrM) pathogenesis and progression. In this work, an untargeted lipidomic analysis revealed that some lipid species were altered in GRP94-deficient cells, specially GM2 and GM3 gangliosides. The catalytic pathway of GM2 is affected by the low enzymatic activity of ß-Hexosaminidase (HexA), responsible for the hydrolysis of GM2 to GM3. Moreover, a deficiency of the GM2-activator protein (GM2-AP), the cofactor of HexA, is observed without alteration of gene expression, indicating a post-transcriptional alteration of GM2-AP in the GRP94-ablated cells. One plausible explanation of these observations is that GM2-AP is a client of GRP94, resulting in defective GM2 catabolic processing and lysosomal accumulation of GM2 in GRP94-ablated cells. Overall, given the role of gangliosides in cell surface dynamics and signaling, their imbalance might be linked to modifications of cell behaviour acquired in BrM progression. This work indicates that GM2-AP could be an important factor in ganglioside balance maintenance. These findings highlight the relevance of GM3 and GM2 gangliosides in BrM and reveal GM2-AP as a promising diagnosis and therapeutic target in BrM research.

Liquid chromatography/electrospray ionisation-tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma.

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted ß-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.

Gangliosides and ceramides change in a mouse model of blast induced traumatic brain injury.

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced "mild" traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5-5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology.

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Storage of gangliosides GM2 and fucosyl GM1 in the kidney of MCC strain of mastomys (Praomys coucha).

Previously, we histochemically examined the kidney of the MCC strain of mastomys (Praomys coucha) and found the storage of gangliosides. In the present studies, the lipid-bound sialic acid content of gangliosides in the MCC kidney was about 9- to 14-fold higher than that of the control (MWC strain). In the MCC kidney, sialic acids of male gangliosides were composed of N-acetylneuraminic acid at 91.5%; sialic acids of female gangliosides, however, were composed almost entirely of N-glycolylneuraminic acid. TLC of gangliosides showed that the MCC kidney contained four abundant gangliosides (two gangliosides each in males and females). These gangliosides isolated by HPLC were identified to be GM2(NeuAc) and fucosyl GM1(NeuAc) in the male MCC kidney and GM2(NeuGc) and fucosyl GM1(NeuGc) in the female MCC kidney by secondary ion mass spectrometry, TLC/immunostaining and TLC after enzyme treatments. Although the MCC kidney contained control levels of the activities of beta-N-acetylhexosaminidase, alpha-l-fucosidase, N-acetylgalactosaminyltransferase and fucosyltransferase, the activity of beta-galactosidase in the MCC kidney was increased to 400-500% of that in the MWC kidney. Therefore, we discussed the possibility that in the MCC kidney, GM2 was abundantly produced by the effect of increased beta-galactosidase activity.

Obesity causes a shift in metabolic flow of gangliosides in adipose tissues.

Obesity is associated with insulin resistance and a mild chronic inflammation in adipose tissues. Recent studies suggested that GM3 ganglioside mediates dysfunction in insulin signaling. However, it has not been determined the ganglioside profiling in adipose tissues of obese animals. Here, we for the first time examined semi-quantitative ganglioside profiles in the adipose tissues of high fat- and high sucrose-induced obese, diabetic C57BL/6J mice by TLC and HPLC/mass spectrometry. In control adipose tissues GM3 dominated with traces of GM1 and GD1a; obesity led to a dramatic increase in GM2, GM1, and GD1a with the GM3 content unchanged. Similar results were obtained in KK and KKAy mice. Adipocytes separated from stromal vascular cells including macrophages contained more of those gangliosides in KKAy mice than in KK mice. These results underscore those gangliosides in the pathophysiology of obesity-related diseases.

Interface of an array of five capillaries with an array of one-nanoliter wells for high-resolution electrophoretic analysis as an approach to high-throughput chemical cytometry.

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1-nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/m). In this example, AtT-20 cells were used that had been treated with TMR-G(M1). The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.98 cell/well. This system provides an approach to high-throughput chemical cytometry.

Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease.

The quality of tissue imaging by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) depends on the effectiveness of the matrix deposition, especially for lipids that may dissolve in the solvent used for the matrix application. This article describes the use of an oscillating capillary nebulizer (OCN) to spray small droplets of matrix aerosol onto the sample surface for improved matrix homogeneity, reduced crystal size, and controlled solvent effects. This system was then applied to the analysis of histological slices of brains from mice with homozygous disruption of the hexb gene (hexb-/-), a model of Tay-Sachs and Sandhoff disease, versus the functionally normal heterozygote (hexb+/-) by imaging MALDI-MS. This allowed profiling and localization of many different lipid species, and of particular interest, ganglioside GM2, asialo-GM2 (GA2), and sulfatides (ST). The presence of these compounds was confirmed by analysis of brain extracts using electrospray ionization in conjunction with tandem mass spectrometry (MS/MS). The major fatty acid of the ceramide backbone of both GM2 and GA2 was identified as stearic acid (18:0) versus nervonic acid (24:1) for ST by both tissue-imaging MS and ESI-MS/MS. GM2 and GA2 were highly elevated in hexb-/- and were both localized in the granular cell region of the cerebellum. ST, however, was localized mainly in myelinated fiber (white matter) region of the cerebellum as well as in the brain stem with a relatively uniform distribution and had similar relative signal intensity for both hexb+/- and hexb-/- brain. It was also observed that there were distinct localizations for numerous other lipid subclasses; hence, imaging MALDI-MS could be used for "lipidomic" studies. These results illustrate the usefulness of tissue-imaging MALDI-MS with matrix deposition by OCN for histologic comparison of lipids in tissues such as brains from this mouse model of Tay-Sachs and Sandhoff disease.

Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

Mammalian sialidase Neu4, ubiquitously expressed in human tissues, is located in the lysosomal and mitochondrial lumen and has broad substrate specificity against sialylated glycoconjugates. To investigate whether Neu4 is involved in ganglioside catabolism, we transfected beta-hexosaminidase-deficient neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid and demonstrated the correction of storage due to the clearance of accumulated GM2 ganglioside. To further clarify the biological role of Neu4, we have generated a stable loss-of-function phenotype in cultured HeLa cells and in mice with targeted disruption of the Neu4 gene. The silenced HeLa cells showed reduced activity against gangliosides and had large heterogeneous lysosomes containing lamellar structures. Neu4(-/-) mice were viable, fertile and lacked gross morphological abnormalities, but showed a marked vacuolization and lysosomal storage in lung and spleen cells. Lysosomal storage bodies were also present in cultured macrophages preloaded with gangliosides. Thin-layer chromatography showed increased relative level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in brain of Neu4(-/-) mice suggesting that Neu4 may be important for desialylation of brain gangliosides and consistent with the in situ hybridization data. Increased levels of cholesterol, ceramide and polyunsaturated fatty acids were also detected in the lungs and spleen of Neu4(-/-) mice by high-resolution NMR spectroscopy. Together, our data suggest that Neu4 is a functional component of the ganglioside-metabolizing system, contributing to the postnatal development of the brain and other vital organs.

Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 ganglioside antibodies.

BACKGROUND: GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 ganglioside activator protein associated with accumulation of GM2 ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. METHODS: We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. RESULTS: We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. CONCLUSIONS: This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.

Clinical and molecular analysis of GM2 gangliosidosis in two apparent littermate kittens of the Japanese domestic cat.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.

Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease.

BACKGROUND: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically. METHODS: Cultured fibroblasts from I-cell disease patients were double-stained with a monoclonal antibody to lysosome-associated membrane protein-1 (LAMP-1) and that to GM2 ganglioside, or a series of lectins that specifically bind to sugar moieties. RESULTS: The patients' cells were granularly stained with the antibody to GM2 ganglioside and the lectins including Maakia amurensis, Datura stramonium, and concanavalin A. Their localization was coincident with that of LAMP-1. CONCLUSIONS: GM2 ganglioside and various kinds of glycoconjugates having sialic acidalpha2-3galactose, galactosebeta1-4N-acetylglucosamine and mannose residues accumulate in enlarged lysosomes in I-cell disease fibroblasts.

Mutation of the GM2 activator protein in a feline model of GM2 gangliosidosis.

The G(M2) activator protein is required for successful degradation of G(M2) ganglioside by the A isozyme of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52). Deficiency of the G(M2) activator protein leads to a relentlessly progressive accumulation of G(M2) ganglioside in neuronal lysosomes and subsequent fatal deterioration of central nervous system function. G(M2) activator deficiency has been described in humans, dogs and mice. This manuscript reports the discovery and characterization of a feline model of G(M2) activator deficiency that exhibits many disease traits typical of the disorder in other species. Cats deficient in the G(M2) activator protein develop clinical signs at approximately 14 months of age, including motor incoordination and exaggerated startle response to sharp sounds. Affected cats exhibit central nervous system abnormalities such as swollen neurons, membranous cytoplasmic bodies, increased sialic acid content and elevated levels of G(M2) ganglioside. As is typical of G(M2) activator deficiency, hexosaminidase A activity in tissue homogenates appears normal when assayed with a commonly used synthetic substrate. When the G(M2) activator cDNA was sequenced from normal and affected cats, a deletion of 4 base pairs was identified as the causative mutation, resulting in alteration of 21 amino acids at the C terminus of the G(M2) activator protein.

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats.

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

Detection of serum anti-ganglioside antibodies by latex agglutination assay in Guillain-Barré syndrome: comparison with ELISA.

OBJECTIVE: Rapid detection of serum anti-ganglioside antibodies in Guillain-Barré syndrome (GBS) could facilitate early diagnosis and early initiation of treatment, which might shorten the term of illness and reduce sequelae. We examined serum anti-ganglioside antibodies in patients with GBS using the latex agglutination assay developed by Alaedini and Latov (J Immunoassay 21: 377-386, 2000) with some modifications. MATERIALS AND METHODS: We used 75 sera from GBS patients, which exhibited IgG anti-GM1, GD1b, or GQ1b, or IgM anti-GM2 antibodies on previous enzyme-linked immunosorbent assay (ELISA). Blue latex beads (2.5% solution of 0.3 microm) were coated with 1 mg/ml of GM1, GD1b, GQ1b or GM2. Aliquots (4 microl) of serum and the ganglioside-coated particles were mixed and rocked on a glass slide for 30 to 40 seconds. The reaction was observed under a microscope and compared with the antiganglioside antibody titers determined with ELISA. RESULTS: Agglutination was strong in sera of which the IgM or IgG titers of anti-GM1, GD1b, GQ1b or GM2 antibodies were found to be more than 1:6,400 on ELISA except for 2 samples, but weak or absent in sera with titers of 1:3,200. Agglutination was absent in sera of which the antibody titers were less than 1:3,200 on ELISA. CONCLUSION: We could rapidly detect serum IgM and IgG anti-GM1, GD1b, GQ1b and GM2 antibodies in patients with GBS by means of the latex agglutination assay when sera exhibited high titers of the respective antibodies on ELISA. The sensitivity of our agglutination assay was much lower than that of ELISA.

Correlation of gangliosides GM2 and GM3 with metastatic potential to lungs of mouse B16 melanoma.

Ten B16 mouse melanoma cell lines with increasing metastatic potential to lungs (B16LuF1 to B16LuF10) were generated by in-vitro & in-vivo selection technique starting with B16F1 melanoma cell line. The number of metastatic tumor nodules in lungs rose with increasing metastatic potential. Tumor cell gangliosides of B16LuF1 to B16LuF10 cell lines, analysed and compared with TLC, showed eight major ganglioside bands. Band1 to band6 corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively. Band7 and Band8 could not be identified. The concentration of total as well as individual ganglioside bands of B16LuF1 to B16LuF10 cells appeared to rise with increasing metastatic potential. Gangliosides from the plasma of these cell lines (B16LuF1 to B16LuF10) maintained in-vivo in C57BL/6 mice on TLC analysis gave eight major ganglioside bands, similar to those of cells. Plasma gangliosides appeared to rise with increasing metastatic potential. However, it was interesting to see that only band5 and band6 gangliosides in plasma increased almost linearly with increasing metastatic potential. The remaining six ganglioside bands in the plasma did not show such correlation. Band5 and Band6 gangliosides corresponded with standard gangliosides GM2 and GM3 respectively. Gangliosides of the spent culture media, secreted by these cell lines in-vitro in tissue culture also gave eight major ganglioside bands, similar to that of cells. Spent culture media gangliosides appeared to increase with increasing metastatic potential. However, concentration of only band5 and band6 gangliosides of spent culture media increased almost linearly with increasing metastatic potential, thus further confirming the role of band5(GM2) and Band6(GM3) gangliosides in regulating metastatic potential of B16-melanoma cells to lung.

Distribution of enzyme-bearing cells in GM2 gangliosidosis mice: regionally specific pattern of cellular infiltration following bone marrow transplantation.

Tissue distribution of beta-hexosaminidase was investigated using 5-bromo-4-chloro-3-indolyl N-acetyl beta-D-glucosaminide (X-Hex) as substrate in wild-type mice, four GM2 gangliosidosis model mice (Hexa-/-, Hexb-/-, Gm2a-/- and Hexa-/-Hexb-/-) and Hexb-/- mice that received bone marrow transplantation (BMT). In wild-type mice histochemical localization of beta-hexosaminidase was detected in the perikarya of the majority of neurons, small process-bearing microglial cells, perivascular macrophages, and macrophages in the choroid plexus and leptomeninges. X-Hex positivity was also noted in the renal tubular epithelium and macrophages in the liver and spleen. The staining pattern in the Gm2a-/- and Hexa-/- mice was generally similar to those of wild type, but in these mice, X-Hex stain was also noted in some storage neurons with swollen perikarya. No X-Hex-positive cells were detected in Hexb-/- or Hexa-/-Hexb-/- (DKO) mice. In Hexb-/- mice that received wild-type BMT (Hexb-/- +WBMT), many X-Hex-positive cells were detected in the spleen, and to a far lesser extent, in liver and kidney. In the CNS of these mice, X-Hex-positive cells were largely detected in the leptomeninges and choroid plexus. Some positive cells were also detected, mostly in the perivascular regions of the cerebrum, in particular in the regions of the posterior thalamus, brain stem and spinal cord. Some of X-Hex-positive cells were immunoreactive with Mac-1 and F4/80 antibodies and, thus, were cells of microglia/macrophage lineage. X-Hex-positive staining was not detected in neurons in these mice despite clinical improvement following BMT. This is the first time, as far as we know, that the regional distribution of the donor cells in the CNS has been investigated in a model of neuronal storage disease. Our study indicated that donor-derived cells of microglia/macrophage lineage infiltrated the CNS in a regionally specific manner following the BMT.

Apoptosis induction of human lung cancer cell line in multicellular heterospheroids with humanized antiganglioside GM2 monoclonal antibody.

The chimeric antiganglioside GM2 monoclonal antibody (MAb) KM966, which showed high effector functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), potently suppressed growth and metastases of GM2-positive human cancer cells inoculated into mice. To further improve the therapeutic efficacy of the anti-GM2 MAb in humans, we constructed a humanized anti-GM2 MAb, KM8969. The humanized KM8969 was more efficient in supporting ADCC against GM2-positive human cancer cell lines than the chimeric KM966, whereas complement-dependent cytotoxicity was slightly reduced in the humanized KM8969. In addition, the humanized KM8969 was shown to exert potent ADCC mediated by both lymphocytes and monocytes. To investigate the effect of the humanized KM8969 on the biological function of GM2 in the condition physiologically mimicking formation and growth of cancer masses, the heterospheroids composed of normal human dermal fibroblasts and GM2-positive human lung cancer cells were developed. Interestingly, the humanized KM8969 gave rise to growth inhibition of heterospheroids without dependence of the effector functions. Morphological and immunocytochemical analysis suggested that the inhibitory effect was due to the apoptosis of GM2-positive cancer cells in the heterospheroids. The result indicates that GM2 captured by the antibody on the cell surface loses its physiological function that plays a critical role in maintaining the three-dimensional growth of cancer cells in contact with its own cells or other type of cells in a microenvironment. The humanized KM8969, which can destroy the cancer cells via blocking functional GM2 on the cell surface as well as the effector functions, would have extraordinary potential in human cancer therapy.

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.

Anti-GM2 IgM antibodies: clinical correlates and reactivity with a human neuroblastoma cell line.

Anti-GM2 IgM antibodies have been reported in some patients with dysimmune neuropathy or lower motor neuron syndrome, in whom they were often associated with a concomitant reactivity with GM1. To investigate the possible clinical and pathogenetic relevance of these antibodies we measured serum anti-GM2 IgM titers by ELISA in 224 patients with different neuropathies and motor neuron disease and examined their binding to SK-N-SH neuroblastoma cells by indirect immunofluorescence (IIF). High titers of anti-GM2 IgM antibodies were found in eight patients with dysimmune neuropathies including two with multifocal motor neuropathy (MMN), two with purely motor demyelinating neuropathy without conduction block (MN) and four with Guillain-Barré syndrome (GBS). In two MMN patients reactivity with GM2 was associated with anti-GM1 reactivity and in one MN patient with anti-GM1, -GD1a and -GD1b reactivity. All but one patient had a concomitant reactivity with GalNAc-GD1a. Serum IgM from all positive patients intensely stained by IIF the surface of SK-N-SH neuroblastoma cells. This reactivity was blocked by serum pre-incubation with GM2, was not observed with sera from patients without anti-GM2 antibodies including those with high anti-GM1 or other anti-glycolipid antibodies, and correlated with the presence of GM2 in the SK-N-SH neuroblastoma cells. These findings indicate that anti-GM2 antibodies, though infrequent, are strictly associated with dysimmune neuropathies and suggest that SK-N-SH neuroblastoma cells can be a suitable in vitro model to study the functional and biological effects of these antibodies.