Human alpha-fetoprotein-rich fraction inhibits galactocerebroside antibody-mediated lysis of oligodendrocytes in vitro.

Polyclonal rabbit antiserum to galactocerebroside (anti-GalC) produces titer-dependent lysis of cultured Percoll-isolated bovine and rat oligodendrocytes. In this study anti-GalC produced complement-dependent lysis of 76% of the bovine cells and 65% of the rat cells maintained for 3 to 6 days in vitro. With the concomitant addition of human umbilical cord serum fractions containing fetal alpha-fetoprotein (AFP), lysis was decreased to 31% and 39%, respectively.

Metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocytes: studies with gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry was used to study the metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocyte monolayers. A steady-state level of 15NH3 was present by 1 min in both systems. Steady-state labeling in L-[amide-15N] glutamine, L-[15N]alanine, L-[15N]glutamate, and L-[15N]aspartate was attained by 1 min after 15NH3 addition in the organotypic cerebellar explants and by approximately 5 min in the cultured astrocytes.

Long-term cultures of human adult Schwann cells isolated from autopsy materials.

Enriched populations of adult human Schwann cells were obtained from trigeminal ganglia and roots of autopsy material. The cells, isolated by enzymatic procedure, were seeded on rat tail collagen-coated coverslips. Subcultures were established several weeks later, and secondary cells were grown on polylysine-coated coverslips and maintained in vitro for as long as 5 months.

Cultured human and rat oligodendrocytes and rat Schwann cells do not have immune response gene associated antigen (Ia) on their surface.

In order to determine if oligodendrocytes or Schwann cells had surface immune response gene associated antigen (Ia), we studied the binding of: (a) mouse monoclonal antibodies to rat Ia, to cultures of rat oligodendrocytes and Schwann cells; and, (b) mouse monoclonal antibodies to human Ia, to cultures of human oligodendrocytes employing radioimmunoassay and indirect immunofluorescence. Cells were identified using phenotypic markers; rabbit anti-galactocerebroside (GalC) for oligodendrocytes; rabbit anti-GalC and rabbit anti-Schwann cell for Schwann cells; rabbit anti-glial fibrillary acidic protein for astrocytes; rabbit anti-fibronectin for fibro-blasts and leptomeningeal cells, and the capacity to ingest latex particles for macrophage-microglia. Ia could not be detected on the surface of oligodendrocytes, Schwann cells, astrocytes, fibroblasts, or leptomeningeal cells.

Specific and potent mitogenic effect of axolemmal fraction on Schwann cells from rat sciatic nerves in serum-containing and defined media.

Using cultures of Schwann cells from neonatal rat sciatic nerves, we examined the mitogenic activity of an axolemmal fraction from adult rat CNS. Axolemmal fraction proved a potent mitogen, stimulating [3H]thymidine incorporation into Schwann cell DNA 13.5-fold over control values when axolemmal fraction equivalent to 16 micrograms of protein per culture microwell or more was added.

Long-term culture of human oligodendrocytes. Isolation, growth and identification.

Oligodendrocytes were isolated from adult human brains obtained at autopsy. The cells were prepared by Percoll density gradient centrifugation, seeded on plastic coverslips and were cultured for a period up to 6 months. The oligodendrocytes in culture expressed cell-type specific markers, galactocerebroside and myelin basic protein and revealed the ultrastructure of mature oligodendrocytes.

P2 protein-induced experimental allergic neuritis. An ultrastructural study.

Experimental allergic neuritis (EAN) was induced in 2 groups of inbred Lewis rats by sensitization with P2 protein and peripheral nervous system (PNS) myelin, both purified from bovine intradural roots. Light- and electronmicroscopic study of P2-induced EAN revealed demyelinative lesions in spinal ganglia and root nerves and less frequently in peripheral nerves and root entry zones. Both small and large myelinated fibers were demyelinated, contradictory to the reported selective binding of anti-P2 antibodies to myelin of large fibers.

Peripheral nervous system myelin and Schwann cell glycoproteins: identification by lectin binding and partial purification of a peripheral nervous system myelin-specific 170,000 molecular weight glycoprotein.

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities.

Culture of purified rat astrocytes in serum-free medium supplemented with mitogen.

We have obtained a highly purified astrocyte population in cultures originating from neonatal (2-5 days) rat cerebrum by use of the selection process provided by a serum-free chemically defined medium (DM). The addition of a glial growth factor isolated from bovine pituitary glands to DM induced in these astrocyte cultures both a stimulation of astrocytic proliferation and a morphological transformation of the astrocytes from flat fibroblastic form to multipolar stellate form.

[15N] leucine as a source of [15N] glutamate in organotypic cerebellar explants.

Approximately 26.0% of the [15N] glutamate and [alpha 15N] glutamine formed in organotypic cerebellar explants was derived from [15N] leucine. Approximately 14.

Sensory neuropathy from pyridoxine abuse. A new megavitamin syndrome.

We describe seven adults who had ataxia and severe sensory-nervous-system dysfunction after daily high-level pyridoxine (vitamin B6) consumption. Four were severely disabled; all improved after withdrawal. Weakness was not a feature of this condition, and the central nervous system was clinically spared.

CSF antibodies to myelin basic protein and oligodendrocytes in multiple sclerosis and other neurological diseases.

Cerebrospinal fluid (CSF) from 18 multiple sclerosis (MS) patients, 13 subacute sclerosing panencephalitis (SSPE) patients, 22 other neurological disease (OND) patients, and 7 neurotic patients as controls were tested in an 125I-labeled anti-human F(ab')2 binding assay for the presence of antibodies to normal human brain cells from tissue culture, human fibroblasts, plasma membranes of MS and normal human brain, myelin basic protein (MBP) and bovine oligodendrocytes. Antibodies to MBP and to oligodendrocytes were found in the CSF of MS, SSPE and OND patients. Absorption of CSF with bovine CNS myelin significantly diminished binding activity to oligodendrocytes.

Immune reactions to P2 protein in human inflammatory demyelinative neuropathies.

The significance of immune reactions against peripheral nervous system antigens in the human inflammatory polyneuropathies is still uncertain. Using a very sensitive assay, we found greatly increased levels of anti-P2 antibodies in sera of animals with experimental allergic neuritis (EAN) but no increases in humans with acute Guillain-Barré syndrome (GBS), chronic relapsing polyneuritis (CRIP), axonal neuropathy, or normals. P2 protein and CNS basic protein did not induce any increased proliferation in lymphocytes of GBS or CRIP patients.

Absence of expression of OKT8 antigen on cultured human, calf and rat oligodendrocytes.

Monoclonal antibodies which recognize human suppressor T cells (OKT8) have been reported by Oger and co-workers to bind to cultured sheep oligodendrocytes. These authors speculated that an immune response directed at determinants shared by suppressor lymphocytes and oligodendrocytes could explain the decrease in both circulating blood suppressor T cells and oligodendrocytes in patients with multiple sclerosis. In view of the vital issue of potential cross-reactivity between oligodendrocytes and lymphocytes, we studied the binding of viable cultured calf, rat and human oligodendrocytes using monoclonal antibodies to human T cells and monocytes.

Long-term culture of oligodendrocytes isolated from rat corpus callosum by Percoll density gradient. Lysis by polyclonal antigalactocerebroside serum.

Oligodendrocytes isolated from the corpus callosum of four-week-old rats by trypsimization and Percoll density gradient centrifugation were cultured on poly-1-lysine coated coverslips. Some cells extended short processes within 24 hours (h), and at that time up to 95% of the cells showed surface binding of rabbit antiserum to galactocerebroside (anti-GalC) as demonstrated by indirect immunofluorescence. Oligodendrocytes survived up to two months in culture, extending processes with membranous elaborations.

Antibodies to P2 and P1 myelin antigens in experimental allergic neuritis and allergic encephalomyelitis.

We confirmed earlier observations that experimental allergic neuritis (EAN) in Lewis rats induced by injection of bovine peripheral nerve myelin in complete Freund's adjuvant is not accompanied by development of experimental allergic encephalomyelitis. However, sera of these animals contained elevated titers of antibodies against central nervous system myelin basic protein (BP), likely induced by peripheral nerve P1 protein. Anti-BP antibodies were not seen in sera of rats with EAN induced by peripheral nerve P2 protein.

Oligoclonal IgG in the cerebrospinal fluid of guinea pigs with acute experimental allergic encephalomyelitis.

The cerebrospinal fluid (CSF) of guniea pigs with experimental allergic encephalomyelitis was examined for the presence of oligoclonal IgG using polyacrylamide gel electrophoresis. Oligoclonal IgG (greater than or equal to 2 bands) was seen in the CSF obtained from 3/4 animals with experimental allergic encephalomyelitis induced by myelin basic protein and 2/3 with spinal cord-induced disease. It was not seen in CSF of 3 non-sensitized, 4 adjuvant-sensitized and 7 liver-sensitized guinea pigs.

Long term culture of bovine oligodendroglia isolated with a Percoll gradient.

Oligodendroglia were isolated from calf central nervous system (CNS) white matter by trypsinization in phosphate buffered saline and separation by centrifugation through Percoll. Using antisera to phenotypic markers and double labelling experiments we were able to identify essentially all cells in the cultures. The cells obtained were: (1) viable; (2) had intact plasma membranes and well preserved organelles, ribosomes and mitochondria; and (3) were greater than or equal to 95% oligodendroglia 16-20 h after isolation as determined by ability to bind antigalactocerebroside antibodies (anti-GalC).

Oligodendroglial glycerophospholipid synthesis: incorporation of radioactive precursors into ethanolamine glycerophospholipids by calf oligodendroglia prepared by a Percoll procedure and maintained in suspension culture.

Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyrolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-3H]glycerol and either [1,2-14C]ethanolamine or L-[U-14C]serine.

Radioimmunoassay for detection of anti-oligodendrocyte antibodies.

A solid phase radioimmunoassay (RIA) for detection and quantitation of rabbit anti-oligodendrocyte antibody has been developed using bovine oligodendroglia preparation. The assay is simple, rapid, reproducible and economical. It is approximately 150 x as sensitive as immunofluorescence.

Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion.

A novel method of Schwann cell purification from neonatal rat sciatic nerve has been developed using differential adhesion. After enzymatic and mechanical dissociation, the cell digest is allowed to settle on polylysine-coated glass coverslips for 30 min with intermittent shaking. After an 18-h incubation, bipolar cells comprise greater than 95% of the non-adherent population.