Donald H. Silberberg

GALACTOCEREBROSIDE IS A SPECIFIC CELL-SURFACE ANTIGENIC MARKER FOR OLIGODENDROCYTES IN CULTURE

A TEMPERATURE-SENSITIVE (ts) mutant of the highly oncogenic group A (ref. 1) human adenovirus type 12 (H12), H12ts401 (ref. 2) is unable to establish stable transformation of cells in restrictive conditions3. Cells transformed by ts401 at the permissive temperature and shifted to nonpermissive temperatures show a reversion to a normal phenotype; wild-type H12-transformed cells, in contrast, exhibit a transformed phenotype when grown in either restrictive or permissive conditions4. The temperature sensitivity of the transformed phenotype of the ts401 mutant-transformed cells suggests that the continued expression of gene 401 is required for maintenance of the transformed cell phenotype. This study was initiated to detect the H12 transformation-specific protein(s) in H12-transformed cell lines. We identify here a 60,000 molecular weight transformation-specific antigen in H12-transformed rat 3Y1 cells5,6 by immunoprecipitation of 35S-methionine labelled polypeptides with serum from H12 tumour-bearing hamsters. Furthermore, the expression of this antigen is temperature dependent in 3Y1 cells transformed by ts401. Further characterisation of the 60,000 MW antigen may lead to an understanding of the molecular mechanism of adenovirus cell transformation.

MHC antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of Ia antigen expression by γ-interferon

Abstract Macrophage-microglia were isolated from primary mixed brain cell cultures of normal newborn mice. They were successfully maintained in vitro for at least 8 weeks. Purity of the cultures was 97–100%, as determined by endocytosis of latex beads, non-specific staining through Fc receptors, EA and EAC rosette formation. These cells were non-specific esterase-positive, but peroxidase-negative. Electron-microscope observations revealed morphological similarities to mature macrophages. Isolated macrophage-microglia seldom incorporated [3H]thymidine in vitro. By means of 51Cr release assay, using monoclonal antibodies against mouse major histocompatibility complex (MHC) antigens and complement, we detected class I MHC (H-2) antigen on unstimulated macrophage-microglia, and both class I and class II (Ia) antigens on γ-interferon-treated cells. These observations suggest possible immunoregulatory functions of macrophage-microglia in the central nervous system, as is characteristics of other cells of monocyte lineage.

National Institutes of Health State-of-the-Science Conference statement: preventing alzheimer disease and cognitive decline.

The National Institute on Aging and the Office of Medical Applications of Research of the National Institutes of Health convened a State-of-the-Science Conference on 26-28 April 2010 to assess the available scientific evidence on prevention of cognitive decline and Alzheimer disease. This article provides the panels assessment of the available evidence.

Fibroblast growth factor is a mitogen for oligodendrocytes in vitro.

The effect of fibroblast growth factor (FGF) on oligodendrocyte development has been studied using dissociated mixed brain cells, cultured in a previously described serum-free medium. A greater number of galactocerebroside-positive oligodendrocytes could be demonstrated after 7 days in the presence of FGF than in control values. Using combined immunofluorescence and autoradiography an increased [3H]thymidine incorporation by galactocerebroside-positive oligodendrocytes was demonstrated after various times of exposure to FGF.

Antibodies to oligodendroglia in patients with multiple sclerosis

We demonstrated antibodies to isolated oligodendrocytes and to oligodendroglia in brain sections by indirect immunofluorescence technic in serums of 19 of 21 patients with multiple sclerosis. We also found such antibodies in three of five patients with subacute sclerosing panencephalitis and one of four patients with acute disseminated encephalomyelitis, but not in patients with other neurologic diseases or normal persons. The antibodies were absorbed by preincubation of serum with isolated oligodendrocytes or whole white matter, but not with purified myelin or liver tissue. Immunofluorescent staining was blocked by either rabbit anti-oligodendrocyte serum or non-fluoresceinated goat anti-human immunoglobulin. These findings suggest that antibodies to oligodendroglia are distinct from antibodies to myelin and that demyelination in multiple sclerosis could be a consequence of an immunopathologic reaction directed against oligodendroglial cells.

The differentiation of oligodendrocytes in a serum-free hormone-supplemented medium

Primary mixed cultures of trypsin-dissociated fetal and newborn rat brain and spinal cord have been grown in a serum-free medium. This medium, containing insulin, selenium, transferrin and triiodothyronine, was optimized for oligodendrocyte survival by determining the number of cells which expressed surface galactocerebroside. Comparison of cultures in serum-containing and serum-free media revealed that galactocerebroside positive (GalC+) oligodendrocytes could be detected earlier in the absence of serum. This early differentiation occurred in the absence of the added hormones and nutrients, whose main function appeared to be to prolong survival of the cells. The effect of serum on the differentiation of oligodendrocytes was studied by comparing the expression of surface GalC in media containing 2.5% or 10% fetal calf serum. At a given time a much greater number of GalC+ oligodendrocytes could be detected at the lower serum concentration. However, when cultures were transferred from 10% serum to serum-free medium (or 1% serum) large numbers of GalC+ oligodendrocytes subsequently appeared, showing that precursors were present in the high-serum medium, but that they were unable to differentiate. Possible explanations of the effect of serum on oligodendrocyte differentiation are discussed.

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.

The isolation and long-term culture of oligodendrocytes from newborn mouse brain.

Using differential adhesion we successfully isolated relatively pure populations of mouse oligodendrocytes which can be maintained in vitro for more than two months. The highest percentage of galactocerebroside (GalC)-positive oligodendrocytes was 95% at 3 days after isolation. Thereafter, proliferation of astrocytes and fibroblasts occurred more quickly than did oligodendrocyte precursor division. GalC-positive oligodendrocytes rarely incorporate [3H]thymidine so that the use of a mitotic inhibitor (5 X 10(-6)M AraC) reduced the number of non-oligodendrocytes so as to maintain the purity of oligodendrocytes at more than 75% for 14 days in culture. This system will be of use for immunological and virological studies which require viable cultured mouse oligodendrocytes.

The design of clinical studies to assess therapeutic efficacy in multiple sclerosis

Poorly designed trials of therapy for multiple sclerosis (MS) can waste time and money, and may lead either to false hopes or to the overlooking of a potentially effective treatment. A well-designed trial may well develop useful scientific information even if the putative therapy fails to show any therapeutic effect. The diagnosis, clinical course, and definitions of the stages of MS are discussed as they relate to trials of therapy. The goals of such trials include favorable modification of an exacerbation, favorable modification or prevention of future exacerbations, effective treatment of the progressive stage, and improvement of function in the stable-deficit stage. There should be an orderly progression from a small preliminary trial to a modest pilot trial and, when indicated, a full trial. All types of trials require careful organization and management, appropriate selection of patients, and properly planned and recorded observations. The treatment contrast-how the new treatment will be evaluated-provides the essential structure of the trial. The hypothesis being examined, the treatment contrast, and the observations being made in the designed clinical trial will govern the form of the analysis and the nature of the interpretations. Each goal requires that specific strategies and design considerations be applied to preliminary, pilot, and full trials.

The expression of MHC antigens on oligodendrocytes: Induction of polymorphic H-2 expression by lymphokines

Neither class I nor class II major histocompatibility complex (MHC) antigen has been demonstrated in native oligodendrocytes, the possible target of viral or immune damage in multiple sclerosis (MS). In this report, we show that H-2, but not Ia, antigen expression is induced on isolated mouse oligodendrocytes in vitro by crude supernatant from lectin-activated spleen cells, lectin-free interleukin 2, and cloned gamma-interferon. This induction of H-2 expression is not accompanied by proliferation of oligodendrocytes, whereas MHC induction in spleen cells is highly related to their proliferation, or blastoid transformation. Oligodendrocytes as well as other brain cells are probably isolated from these lymphokines by the blood-brain barrier (BBB). However, it is possible that oligodendrocytes express MHC class I antigen as a consequence of impairment of the BBB, or in the presence of activated T-cells which have been demonstrated in active MS lesions. This activation then renders oligodendrocytes possible target cells for MHC-restricted cytotoxic T-cells.