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Biomedical subjects

C C Bernard

Publications and source records attributed to C C Bernard.

At least 73 records · Page 4Linked to original sources
PubMed1990

Limited heterogeneity of rearranged T-cell receptor V alpha transcripts in brains of multiple sclerosis patients.

The identification of activated T cells in the brain of individuals with multiple sclerosis (MS) indicates that these cells are critical in the pathogenesis of this disease. In an attempt to elucidate the nature of the lymphocytic infiltration, we used the polymerase chain reaction to amplify T-cell antigen receptor (TCR) V alpha sequences from transcripts derived from MS brain lesions. In each of three MS brains, only two to four rearranged TCR V alpha transcripts were detected. No V alpha transcripts could be found in control brains. Sequence analysis of transcripts encoded by the V alpha 12.1 region showed rearrangements to a limited number of J alpha region segments. These results imply that TCR V alpha gene expression in MS brain lesions is restricted.

Base Sequence↗
PubMed1990

HTLV-I sequences are not detected in peripheral blood genomic DNA or in brain cDNA of multiple sclerosis patients.

Human T-cell lymphotropic virus (HTLV-I) was recently reported to be etiologically associated with multiple sclerosis (MS). Genomic DNA from peripheral blood lymphocytes and brain plaques of patients with MS was analyzed for the presence of sequences homologous to the HTLV-I pol gene using the polymerase chain reaction and dot blot techniques. Comparison of DNA amplification patterns between patients with MS, and with control subjects who have other autoimmune conditions, with those in healthy control subjects and with an HTLV-I-infected cell line indicates that HTLV-I pol sequence is not present in the peripheral blood of patients with MS, and that the virus is not active in MS brain plaques.

Autoimmune Diseases↗
PubMed1990

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.

Ammonia↗
PubMed1990

Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine.

In order to detect histamine receptors on the surface of human peripheral blood mononuclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X-100 solubilized mononuclear cells, indicating it to be the ligand-binding subunit for the histamine receptor on these cells. For 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.

Autoradiography↗
PubMed1990

Cell-mediated immune response to copolymer I in multiple sclerosis measured by the macrophage procoagulant activity assay.

The macrophage/monocyte procoagulant activity (MPCA) assay, a sensitive and specific in vitro test for cell-mediated immunity, has been used to ascertain the reactivity of MS peripheral blood mononuclear cells (PBM) to copolymer I (Copl), a synthetic peptide analogue of myelin basic protein (MBP) currently being tested as a possible therapeutic agent in multiple sclerosis (MS). Because the suppressive effect of Copl is believed to lie in its possible cross-reactivity with MBP, the reactivity of PBM of MS patients to MBP was also tested. MS patients either at the stable phase of the relapsing/remitting form or with chronic progressive disease were investigated and compared with patients with other diseases and with healthy subjects. The reactivity to Copl was significantly increased in patients with chronic progressive disease but not in stable MS patients or in control subjects. No difference in reactivity to MBP between MS patients and healthy subjects was found regardless of disease status. However, in the control group comprising patients with other diseases, MBP reactivity was significantly elevated. In chronic progressive MS patients, a relationship was found between the response to Copl and that to MBP, supporting the possibility of an immunological cross-reactivity between these two antigens. There was no significant difference in reactivity to the non-specific stimulant, lipopolysaccharide, between the MS and control groups.

Adult↗
PubMed1989

Epidermal growth factor inhibits the expression of myelin basic protein in oligodendrocytes.

The major function of the oligodendrocyte is to myelinate axons in the central nervous system (CNS). Two of the components of myelin, galactocerebroside (galc) and myelin basic protein (MBP), have been used as markers of oligodendrocyte maturation in the developing CNS, and it has been found that galc+ cells arise initially, which then mature into MBP+ oligodendrocytes several days later. We have been interested in the control of expression of MBP and have followed its appearance in cultures of brain cells isolated from 4 day-old mice. In low serum (0.5% foetal bovine serum), approximately 330 MBP+ cells arise per 2 x 10(5) brain cells after 3 days incubation. We have examined the ability of several growth factors to influence the expression of MBP in these cultures, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and the fibroblast growth factors (acidic and basic FGF). EGF was found to suppress strongly the developmental expression of MBP in these cultures, but the suppression was reversible, since the number of MBP+ cells approached control numbers 3 days after removal of EGF from the cultures. It was also found that MBP could be down-regulated in mature MBP+ oligodendrocytes. The action of EGF in these cultures could be mimicked by transforming growth factor-alpha (TGF alpha). The effects of EGF appear to be associated primarily with MBP production in oligodendrocytes since expression of galc is unaffected by EGF.

Animals↗
PubMed1989

T-cell receptor V alpha and C alpha alleles associated with multiple and myasthenia gravis.

Polymorphic markers in genes encoding that alpha chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR alpha cDNA probe. These PCR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V alpha and C alpha markers were identified between patients and healthy individuals.

Alleles↗
PubMed1989

Developmental study of myelin basic protein variants in various regions of pig nervous system.

A developmental study of myelin basic protein (MBP) variants in eight regions of pig nervous system (NS) was performed using a quantitative electroimmunoblotting procedure. Four major MBP forms with apparent molecular weights of 21.5K, 20.2K, 18.5K, and 17.3K were identified in both the CNS and the PNS and were detected as early as 22 days before birth. Quantification of the most abundant forms, the 21.5K and 18.5K MBPs, revealed characteristic profiles of accumulation of these two variants in different regions of the NS. The ratio of 21.5K:18.5K MBP varied with developmental time as well as with the various NS regions, peaking 20 days postnatally. The 17.3K MBP was observed from embryonic stages to adulthood, as were the 21.5K and 18.5K forms. In contrast, the 20.2K variant appeared most abundant from 10 days before to 22 days after birth and thereafter decreased in intensity so as to be no longer detectable in the brain of a 5-year-old pig. A similar pattern was also observed with an anti-MBP-reacting protein with an apparent molecular weight of 23K. Taken together, these results suggest that in the pig NS, the expression of MBP variants may be regulated both regionally and developmentally.

Aging↗
PubMed1989

Multiple sclerosis brain immunoglobulins stimulate myelin basic protein degradation in human myelin: a new cause of demyelination.

Membrane-bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium-activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium-binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane-bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.

Brain↗
PubMed1988

Electroimmunoblotting of small peptides separated on urea-dodecyl sulphate (SUDS) gels. Application to myelin basic protein.

A method for the electroimmunoblotting and immunodetection of peptides of less than 50 amino acid residues is described. Excellent resolution of a mixture of myelin basic protein (MBP) peptides was achieved by electrophoresis in a polyacrylamide stacking, urea-dodecyl sulphate minislab gel. Following electrophoresis, the peptides were transferred to various matrices and probed with monoclonal and polyclonal antibodies. Variables such as transfer time, membrane type, fixation and the amount of peptide loaded on the gel have been optimized as a consequence native and synthetic peptides can now be visualized in gels and immunodetected on immobilizing matrices. This procedure is particularly suited to the analysis and identification of small MBP fragments arising in various neuropathological conditions as well as for the rapid characterization of antigenic determinants recognized by monoclonal and polyclonal anti-MBP antibodies.

Animals↗
PubMed1988

A nondenaturing vertical isoelectric focusing polyacrylamide slab gel system suitable for silver staining and electrophoretic blotting.

We have devised a nondenaturing vertical isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) system which is amenable to silver staining and electroblotting. Apart from being accessible, inexpensive, and simple to use, this new methodology overcomes problems inherent in current IEF methods, for example, pH gradient drift, nonuniform cooling, restricted sample volume, and inability to perform electroblotting. Two photopolymerization gel formulas were derived: a 5% acrylamide formula using bisacrylamide (Bis) as the crosslinker and a 6% acrylamide formula using diallyltartdiamide (DATD) as the crosslinker. The 5% acrylamide Bis gel gave excellent resolution and separation of proteins whereas the 6% acrylamide DATD gel expanded slightly during silver staining, resulting in mild band distortions. At least 80 ng of protein per band could be detected by the silver staining protocol devised. Both the DATD and the Bis gels were suitable for electroblot transfer. Parameters to ensure the optimum conditions for reproducible, high resolution vertical IEF-PAGE are described. IEF-PAGE silver staining and electroblotting procedures and silver staining of the nitrocellulose electroblot procedures are also described. The advantages of this methodology over previously published methods are discussed.

Animals↗
PubMed1988

Electroimmunoblotting of myelin basic protein peptides: a novel approach to the rapid characterisation of antigenic specificities of monoclonal and polyclonal anti-MBP antibodies.

A rapid and sensitive method for the identification of antigenic determinants recognised by monoclonal and polyclonal antibodies directed against myelin basic protein (MBP) is described. By electroimmunoblotting a series of overlapping peptides covering the entire MBP molecule with monoclonal anti-MBP antibodies, the binding pattern of immunoreactive peptides can be rapidly determined and the reactive antigenic determinant identified. This procedure, which can be performed with both native and synthetic peptides, can also with appropriate modification, be applied to the analysis of naturally occurring or experimentally induced polyclonal anti-MBP autoantibodies.

Antibodies↗
PubMed1987

Rapid characterization of protein epitopes recognized by monoclonal antibodies using direct probing on thin-layer and paper chromatograms.

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM paper.

Animals↗
PubMed1987

Correlation of the onset of experimental autoimmune encephalomyelitis-like clinical signs with oedema of the spinal cord in tunicamycin-poisoned rats.

The neurological signs induced by injection of tunicamycin are, in young adult rats, virtually identical to those typical of acute experimental autoimmune encephalomyelitis (EAE). Vasogenic exudation, of which the occurrence in the spinal cord of EAE rats has been shown to coincide with the onset of clinical signs, was investigated by quantitative electroimmunoblotting of central nervous system (CNS) tissue at various times following tunicamycin injection of young adult rats. Highly elevated levels of extravasated plasma proteins were observed in the spinal cord from 48 h after injection and, as in EAE rats, these increases coincided with the onset of neurological impairment. At 72 h post-injection, significant increases were also found in the brain of affected animals, albeit at much reduced levels. This is in contrast to previously reported findings in nursling rats where oedema was shown to be predominantly located in the brain. Quantitative electroimmunoblotting for myelin basic protein (MBP) in the CNS of tunicamycin-treated young adult rats indicated that, as in acute EAE, no extensive demyelination had occurred. These data provided further evidence that in both neurological diseases, vasogenic oedema of the spinal cord may be causally related to the appearance of neurological signs and suggested that its differential localization in the CNS may lead to differential neurological impairment.

Animals↗
PubMed1987

Solubilization and characterization of moderate and high affinity histamine binding sites on human blood mononuclear cells.

The Triton X-100 solubilized extract of human peripheral blood mononuclear cells, in direct binding studies with 10(-9)-10(-6) M [3H]histamine contained both high and moderate affinity sites whose dissociation constants (Kd 4.4 X 10(-9) and 6.7 X 10(-7) M) were commensurate with basal plasma histamine levels and plasma levels obtained following physiological or mild pathological stimuli, respectively. Binding was enhanced by mM concns of calcium cations and by the protease inhibitor Pepstatin A. It was inhibited by bacitracin, agents interfering with thiol groups, Triton X-100 concns greater than 0.2% and EDTA. Binding was optimal between the pH range of 7.0 and 8.5 and was enriched for in a plasma membrane preparation. Thus the histamine binding sites identified maintained their specific ligand binding properties after solubilization from the cell surface and displayed properties fulfilling the criteria for receptors.

Binding, Competitive↗
PubMed1987

Use of protein-bearing nitrocellulose as immunogen for in vitro production of monoclonal antibodies: application to myelin basic protein electrophoretically separated from a complex brain protein mixture.

A procedure for producing monoclonal antibody to myelin basic protein (MBP) using in vitro immunization with MBP transferred to nitrocellulose is described. Following the separation of brain proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer of the electrophoretogram onto nitrocellulose, the MBP band located by immunodetection was excised from the nitrocellulose, ground, and used as immunogen for in vitro stimulation of unprimed mouse spleen cells. While in vitro immunization with soluble MBP was able to generate many hybrids, all the wells in the fusions carried out with the immobilized MBP contained hybrids, 33 to 42% of which were positive to MBP. Among these, six were further characterized; all were IgM and all bound to epitopes common to the 18.5K and 21.5K MBP forms of several species. In view of its simplicity, this technique should have a wide application for the rapid production of monoclonal antibodies to selected proteins or their fragments present in small quantity or difficult to purify on a large scale.

Animals↗
PubMed1987

Inhibition of memory in the chick using a monoclonal anti-Thy-1 antibody.

One-day-old chicks trained on a single trial passive avoidance task were administered a monoclonal anti-chick Thy-1 antibody, either intracranially or subcutaneously, at various times before and after learning and retention tested at various times post-learning. This procedure resulted in profound amnesia when anti-Thy-1 antibody was administered immediately before learning (5 min) in the case of the subcutaneous injections or 5 min before until 5 min after the learning process with intracranial injections. Antibody administered at other times, either before or after learning had little or no effect on retention. Retention levels were normal until 50 min post-learning, then declined sharply and remained at control levels for the duration of the test period. Chicks injected with anti-chick cerebellum or anti-rat Thy-1 antibodies showed no evidence of amnesia for the concentration of the antibodies used.

Animals↗