Masanaga Yamawaki

Anti-GM1 antibody facilitates leakage in an in vitro blood–nerve barrier model

Article abstract Using an in vitro blood–nerve barrier (BNB) model, the authors tested the effect of various monoclonal antiganglioside antibodies on BNB function. Only anti-GM1 antibody significantly facilitated BNB leakage in a concentration-dependent, complement-independent manner. This study provided evidence that anti-GM1 antibody, frequently detected in sera from patients with inflammatory neuropathies, may participate BNB dysfunction and contribute to development of neuropathy.

Glycosphingolipid antibodies and blood-nerve barrier in autoimmune demyelinative neuropathy.

Objective: To evaluate morphologic changes in small endoneurial vessels in patients with autoimmune demyelinative neuropathy and antiglycosphyngolipid antibodies. Background: Although a humoral immune cause has been postulated for various demyelinating neuropathies, the mechanism by which large molecules like immunoglobulins can traverse the blood-nerve barrier (BNB) to enter the endoneurium is not understood. Methods: We examined histologic and ultrastructural changes in small endoneurial vessels in sural nerve biopsy specimens from autoimmune demyelinative neuropathy patients, with or without anti-glycosphingolipid (GSL) antibodies. Density of small endoneurial vessels, mean endothelial area, mean luminal area, and the percentage of endothelial cell junctions (that make up the BNB) that showed evidence of disruption were evaluated. Results: Only junctional disruption showed a significant difference: autoimmune demyelinative neuropathy patients with anti-GSL antibodies showed more BNB disruption than autoimmune demyelinative neuropathy patients without antibodies or control patients with nonautoimmune neuropathies. The most commonly observed endoneurial change in antibody-positive autoimmune demyelinative neuropathy patients was the finding of continuous, patent spaces lacking tight junctions between endothelial cells. Conclusions: Brain endothelial cells and endoneurial endothelial cells share various GSL antigens, including GM1 and GD1b gangliosides, with peripheral nerve tissues. Circulating anti-GSL antibodies could damage cell-to-cell attachments in endoneurial endothelium. This barrier disruption may permit the large molecules like immunoglobulins to enter the endoneurial space, contributing to development of autoimmune demyelinative neuropathy.

Sera from Guillain-Barré patients enhance leakage in blood–nerve barrier model

Background: In Guillain-Barré syndrome (GBS), the destruction or malfunction of blood–nerve barrier (BNB) has been considered to be the beginning of the disease process. It is unclear whether sera from patients with GBS can open the BNB, and which component of patient sera is most important in the dysregulation of the BNB. Methods: The authors evaluated the effect of sera from patients with GBS on permeability of an in vitro BNB model using bovine endoneurial microvascular endothelial cells (PnMEC) cultured on the luminal side of a collagen-coated culture insert (pore size: 0.4 μm). Results: PnMEC monolayers challenged by GBS sera showed significantly lower transendothelial electrical resistance and higher clearance of [carboxyl-14C]-inulin with or without complement. Sera with anti-GM1 antibody showed greater loosening of the barrier than others. This effect decreased significantly after incubation with pure GM1 antigen, suggesting the importance of anti-GM1 antibody in BNB dysregulation. Serial analyses of [carboxyl-14C]-inulin clearance in four patients disclosed a favorable effect of plasmapheresis in restoring BNB function in some cases. Conclusions: The authors found an unfavorable effect of sera from patients with GBS on BNB function, supporting involvement of humoral factors causing BNB derangement in the acute stage. Serial evaluation of permeability change using the authors’ in vitro system might be useful for the clinical assessment of BNB derangement in individual patients.

Generation and characterization of anti‐sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glucuronic acid‐containing glycolipid (SGGL) family found primarily in peripheral nerves. These glycolipids contain the HNK‐1 carbohydrate epitope and are recognized by monoclonal IgM from patients with chronic demyelinating neuropathy and paraproteinemia. Recent studies indicate that SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellular adhesion. To elucidate the biological function of these glycolipids, we produced a murine monoclonal antibody (mAb) and studied its antigenic specificity. Using an enzyme‐linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG2a subclass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG as the antigen. Thin‐layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), which is a structural analogue of the former, but not with other glycolipids. Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50. Western blot analysis showed crossreactivity with human myelin‐associated glycoprotein (MAG), but not with rat MAG or rat glycoprotein P0. Unlike anti‐HNK‐1 monoclonal antibody, however, NGR50 reacted only weakly with several proteins in the 20–30‐kD regions, including human P0, suggesting that mAb50 has a different fine specificity as an anti‐HNK‐1 antibody. Immunocytochemical study of rat sciatic nerve using mAb NGR50 revealed positive staining at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues. Faint staining was also visible at the axolemmal‐myelin interface; however, compact myelin was not stained.

Subcellular Distribution of Sulfated Glucuronyl Glycolipids in Human Peripheral Motor and Sensory Nerves.

Sulfated glucuronyl glycolipids (SGGL) have been implicated as important target antigens in patients with demyelinating polyneuropathy and IgM paraproteinemia. Sulfated glucuronyl paragloboside (SGPG), a major species of SGGL, was identified in the subcellular fractions of human peripheral motor and sensory nerves using a simple and quantitative method. SGPG was found to be concentrated in the myelin-enriched fractions of both motor and sensory nerves (1.3 +/- 0.3 and 1.5 +/- 0.4 &mgr;g/mg protein, respectively), whereas its concentration was 0.9 +/- 0.2 and 1.8 +/- 0.6 &mgr;g/mg protein in the axolemma-enriched fractions of motor and sensory nerves, respectively. Our finding that SGPG is more abundant in the human sensory nerve axolemma-enriched fraction may account for the clinical and pathological observations that the lesions are more heavily concentrated in the sensory nerve than in other parts of the nerve tissues in this disorder. Copyright 1994 S. Karger AG, Basel

Effect of Nerve Growth Factor and Forskolin on Glycosyltransferase Activities and Expression of a Globo‐Series Glycosphingolipid in PC12D Pheochromocytoma Cells

Abstract: The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK‐dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo‐series neutral GSL, Galα1‐3Galα1‐4Galβ1‐4Glcβ1‐1′Cer (GalGb3), and the activity of GalGb3‐synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)‐, GM3‐, GD1a‐, and GM2‐synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti‐GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3‐synthase activity may be closely related to the cellular differentiation process in this cell line.

Rapid Communication: GM3 Regulates Protein Kinase Systems in Cultured Brain Microvascular Endothelial Cells

Abstract: The barrier function of endothelial cells is known to be positively regulated by protein kinase A (PKA) and negatively regulated by protein kinase C (PKC). We found that exogenously administered GM3(NeuAc) promoted PKA activity in cultured brain microvascular endothelial cells (BMECs). Other glycolipids, including GM1, sulfoglucuronyl paragloboside, and GM3(NeuGc), did not have any effect on the PKA activity of BMECs. PC12 cells did not respond to exogenously applied GM3(NeuAc). GM3(NeuAc) also suppressed the PKC activity of BMECs. Thus, GM3(NeuAc) may function as a modulator of blood‐brain barrier function via the two different kinase systems.

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewisx (fucosylneolactonorpentaosyl ceramide; Lex), difucosylneolactonorhexaosyl ceramide (dimeric Lex), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Lex and the complex type of sialyl-Lex derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Lex glycolipids and sialyl-Lex were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Lex glycolipid was located on the tip of fine cellular processes. The unique localization of the Lex glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.

Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.

Glycosyltransferase Activities in Cultured Endothelial Cells of Bovine Brain Microvascular Origin

Bovine brain microvascular endothelial cells (BMECs) express GM3 (NeuAc) and GM3 (NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b as well as sialosylparagloboside and sialosyllactosaminylparagloboside as the minor species. To investigate the metabolic basis of this ganglioside pattern, the activities of eight glycosyltransferases (GM3-, GD1a-, GD3-, LM1-, GM2 (NeuAc)-, GM2 (NeuGc)-, LacCer-, and GM1-synthases) in cultured BMECs were studied. It was found that BMECs possessed high activities of GM3- and GD1a-synthases, and low activities of GM2-, GM1-, and GD3-synthases. Thus, the present study provides evidence that endothelial cells are capable of synthesizing gangliosides in situ and that the high content of GM3 in BMEC is closely associated with high activities of GM3-synthase and low activities of GM2-, GM1-, and GD3-synthases.