Robert P. Lisak

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.

Glatiramer acetate in primary progressive multiple sclerosis: Results of a multinational, multicenter, double-blind, placebo-controlled trial

To determine whether glatiramer acetate (GA) slows accumulation of disability in primary progressive multiple sclerosis.

Isolation of myelin basic protein-reactive T-cell lines from normal human blood

T-Cell lines which responded by proliferation to the autoantigen, myelin basic protein (MBP), were isolated from the blood of six of nine normal humans. These T-cell lines could be maintained in in vitro culture for up to 2 months through the use of Interleukin 2 and repeated MBP stimulation. Optimal antigen-induced proliferation required both antigen and antigen-presenting cells found in the adherent cell population of autologous peripheral blood mononuclear cells (PBM). The T-cell lines were predominantly of the helper phenotype (OKT3+, OKT4+, OKT8-) and responded to both human and guinea pig myelin basic protein.

Continuous long-term immunomodulatory therapy in relapsing multiple sclerosis : results from the 15-year analysis of the US prospective open-label study of glatiramer acetate

The ongoing US Glatiramer Acetate (GA) Trial is the longest evaluation of continuous immunomodulatory therapy in relapsing—remitting multiple sclerosis (RRMS). The objective of this study was to evaluate up to 15 years of GA as a sole disease-modifying therapy. Two hundred and thirty-two patients received at least one GA dose since study initiation in 1991 (mITT cohort), and 100 (43%, Ongoing cohort) continued as of February 2008. Patients were evaluated every 6 months using the Expanded Disability Status Scale (EDSS). Mean GA exposures were 8.6 ± 5.2, 4.81 ± 3.69, and 13.6 ± 1.3 years and mean disease durations were 17, 13, and 22 years for mITT, Withdrawn and Ongoing cohorts, respectively. For Ongoing patients, annual relapse rates (ARRs) maintained a decline from 1.12 ± 0.82 at baseline to 0.25 ± 0.34 per year; 57% had stable/improved EDSS scores (change ≤ 0.5 points); 65% had not transitioned to secondary progressive multiple sclerosis (SPMS); 38%, 18%, and 3% reached EDSS 4, 6, and 8. For all patients on GA therapy (the mITT cohort), ARRs declined from 1.18 ± 0.82 to 0.43 ± 0.58 per year; 54% had stable/improved EDSS scores; 75% had not transitioned to SPMS; 39%, 23%, and 5% reached EDSS 4, 6, and 8. In conclusion, multiple sclerosis patients with mean disease duration of 22 years administering GA for up to 15 years had reduced relapse rates, and decreased disability progression and transition to SPMS. There were no long-term safety issues.

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.

In Vitro Cell-Mediated Immunity of Cerebrospinal-Fluid Lymphocytes to Myelin Basic Protein in Primary Demyelinating Diseases

In an attempt to characterize the immunologic reactivity of cerebrospinal-fluid lymphocytes in demyelinating diseases, we compared the myelin-basic-protein-induced in vitro responses of these cells to peripheral blood lymphocytes from the same subjects with a variety of neurologic diseases. Peripheral blood lymphocytes from patients with acute disseminated encephalomyelitis and progressive multiple sclerosis had increased reactivity as compared to those of normal volunteers (P less than 0.01 and P less than 0.05, respectively). Cerebrospinal-fluid lymphocytes from patients with acute disseminated encephalomyelitis and acute and progressive (but not stable) multiple sclerosis were more reactive than cells from subjects with other neurologic diseases (P less than 0.005, P less than 0.02 and P less than 0.05, respectively). Cerebrospinal-fluid lymphocytes manifested a greater reactivity than peripheral blood lymphocytes in acute and progressive multiple sclerosis but not in acute disseminated encephalomyelitis. These findings demonstrate that lymphocytic cells reactive to myelin basic protein are present in the spinal fluid during active demyelinating disease; and that these cells may be more reactive than peripheral blood lymphocytes.

The thymus in myasthenia gravis. Evidence for altered cell populations

Abstract Comparisons of several types of immunologic reactivity were made in thymic cells from six patients with myasthenia gravis and thymic hyperplasia, four patients with myasthenia gravis and thymoma and six age-matched control cardiac-surgery patients. In mixed leukocyte reactions, thymic cells from the subjects with hyperplasia were capable of stimulating autologous peripheral blood lymphocytes. Such reactivity was not seen in thymocyte-blood lymphocyte mixtures from the other two groups. There was an increased number of B cells in the thymic-cell populations from the myasthenic patients as compared to that in the control group, carrying predominantly IgM receptors. The thymic cells from myasthenic patients also responded more vigorously to pokeweed mitogen. These findings suggest altered populations of lymphoid cells in the thymus of myasthenic patients that react with autologous lymphocytes from other cell compartments. The pathogenic implications of these findings remain to be determined. (N Engl...

Chronic Cerebrospinal Venous Insufficiency and Multiple Sclerosis

A chronic state of impaired venous drainage from the central nervous system, termed chronic cerebrospinal venous insufficiency (CCSVI), is claimed to be a pathologic phenomenon exclusively seen in multiple sclerosis (MS). This has invigorated the causal debate of MS and generated immense interest in the patient and scientific communities. A potential shift in the treatment paradigm of MS involving endovascular balloon angioplasty or venous stent placement has been proposed as well as conducted in small patient series. In some cases, it may have resulted in serious injury. In this Point of View, we discuss the recent investigations that led to the description of CCSVI as well as the conceptual and technical shortcomings that challenge the potential relationship of this phenomenon to MS. The need for conducting carefully designed and rigorously controlled studies to investigate CCVSI has been recognized by the scientific bodies engaged in MS research. Several scientific endeavors examining the presence of CCSVI in MS are being undertaken. At present, invasive and potentially dangerous endovascular procedures as therapy for patients with MS should be discouraged until such studies have been completed, analyzed, and debated in the scientific arena. ANN NEUROL 2010;67:286–290

Mitogens for glial cells: a comparison of the response of cultured astrocytes, oligodendrocytes and schwann cells

We have identified two growth factors for cultured rat astrocytes: fibroblast growth factor, a peptide derived from either whole bovine brain, or pituitaries, and a growth factor in extracts of bovine pituitary which was previously identified as a Schwann cell mitogen. Oligodendrocytes in primary cultures derived from neonatal rat central nervous system divide only rarely if at all. These growth factors did not stimulate primary oligodendrocytes to divide. Occasionally cells found in suspension in long-term cultures of the central nervous system were enriched for cells which were identified as oligodendrocytes by the presence of galactocerebroside on their surface and myelin basic protein in their cytoplasm. When provided with a monolayer of irradiated 3T3 cells, these oligodendrocytes were able to spread out and extend elaborate branched processes typical of oligodendrocytes in the primary cultures. Unlike their counterparts in the primary cultures, these suspension-derived oligodendrocytes are capable of cell division as demonstrated by the uptake of [3H]thymidine and autoradiography.

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. Indirect immunofluorescence with the cell-specific markers rabbit anti-galactocerebroside and rabbit anti-bovine-P-2 basic protein antiserum confirmed light microscopic identification of these bipolar cells as Schwann cells. Rabbit anti-human fibronectin specifically labeled fibroblasts which comprised less than 5% of the cell population, but did not bind to Schwann cells. Schwann cells isolated by differential adhesion were injected into a rabbit. When absorbed with cultured rat skin fibroblasts, serum from this rabbit specifically surface labeled greater than 99% of the bipolar and round cells after 18 h and 5 days in vitro and also labeled Schwann cells in fetal rat dorsal root ganglia cultures, but not fibroblasts or neurons.