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C C Bernard

Publications and source records attributed to C C Bernard.

At least 55 records · Page 3Linked to original sources
PubMed1994

Purification of histamine receptor proteins from detergent-solubilized human peripheral blood mononuclear cells.

Histamine is released from mast cells and basophils by either immunological or nonimmunological mechanisms. Histamine, which is the most potent short acting mediator released from these cells, exerts its diverse biological actions by binding to cell surface histamine receptors. We report the affinity purification of histamine receptor proteins from Triton X-100 solubilized peripheral human blood mononuclear cells which include lymphocytes and monocytes. Three different designs of histamine affinity columns were constructed; all three resulted in the same material being eluted. This consisted of bands which on SDS-PAGE after boiling and reduction had the following molecular weights: 193K, 84K, 58K, 48K, 37K, and 16K. The most abundant bands were of molecular weights 193K, 48K, and 16K, and these were disulfide bonded together to form a high molecular weight complex. (The 58K band was present in lower amounts than the others, and in only a few fractions. It had the same molecular weight as the dimeric form of histamine methyltransferase which is present in small amounts in mononuclear cells and may therefore have copurified.) The histamine binding proteins described in this report were purified by conventional affinity chromatography, rather than by an expression cloning approach which obviates the use of any protein chemistry. Consequently, we had the advantage of being able to verify the histamine binding specificity of our purified proteins directly and with several independent assays as follows. The histamine binding specificity of all three columns was established by specific elution with histamine, by preabsorption of crude cell extract with excess free histamine prior to column application, and by comparison with control columns. Independent determination of the binding specificity, using a radioreceptor dot blot assay, of the eluate containing only the 193K, 48K, and 16K disulfide-linked subunits confirmed that the purified material bound specifically to [3H]histamine and that a 300-500-fold degree of purification from tissue extract had been obtained. Following cell surface radioreceptor cross-linking of radiolabeled histamine to intact mononuclear cells, the 16K band was detected, indicating it to be the ligand-binding subunit for histamine. These same three proteins were purified from T lymphocyte and monocytoid cell lines, indicating that both lymphocyte and monocyte subsets of mononuclear cells express these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Proteins↗
PubMed1993

Identification of two human brain aryl sulfotransferase cDNAs.

A 1,179 bp and a 1,424 bp full-length aryl sulfotransferase cDNAs were isolated from a human brain cDNA library. Their coding domains are 93% identical, each encoding a cytosolic protein of 295 amino acids. Their deduced amino acid sequences of these cDNAs are also 93% identical. The 1179 bp brain cDNA has an identical coding domain to a previously reported human liver aryl sulfotransferase cDNA but it has a different 5' noncoding sequence. Northern blot analysis using a probe specific for the 1,424 bp cDNA identified a 1500 bp band in mRNA of human liver, colon, kidney and lung. In a human hepatocellular carcinoma the same band plus an extra larger band was also recognised. An intron of the gene encoding the 1424 bp cDNA was also identified.

Amino Acid Sequence↗
PubMed1993

Selection for T-cell receptor V beta-D beta-J beta gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which a restricted cellular immune response has been observed. In order to establish whether such T cell responses are likely to be antigen-specific particularly with regard to myelin basic protein (MBP), we analysed T-cell receptor (TCR) gene rearrangements directly from MS brain plaques, using the polymerase chain reaction on reverse transcribed messenger RNA, and compared these with TCR of previously described MBP-specific T cell clones from MS and the rat model experimental allergic encephalomyelitis. Rearranged V beta 5.2 genes were detected in the brains of all patients who were HLA DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401. The V beta 5.2-D beta-J beta sequences in these MS brain plaques revealed five motifs. One of the common motifs was identical to that described for the VDJ region of a V beta 5.2 T-cell clone. This clone was from an MS patient who was HLA DRB1*1501, DQB1*0602, DPB1*0401, and it was cytotoxic towards targets containing the MBP peptide 89-106 (ref. 1). The deduced amino-acid sequence of this VDJ rearrangement, Leu-Arg-Gly, has also been described in rat T cells cloned from experimental allergic encephalomyelitis lesions, which are specific for MBP peptide 87-99 (ref. 2). VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TCR V beta 5.2 N(D)N rearrangements in MS lesions. The capacity of rat T cells with these VDJ sequences to cause experimental allergic encephalomyelitis and the prevalence of such sequences in demyelinated human lesions indicate that T cells with this rearranged TCR may be critical in MS.

Amino Acid Sequence↗
PubMed1993

Purified antichick Thy-1 IgG abolishes intermediate and long-term memory.

One- to 2-day-old chicks administered purified antichick Thy-1 IgG showed substantial amnesia for a single-trial passive discriminated avoidance task. Amnesia was clearly evident at 30 min after learning, during the intermediate stage of a three-stage model of memory processing, and persisted for at least 3 h. A similar effect was also observed with Fab and F(ab')2 fragments. Fc fragments, the non-IgG fraction of ascites, and saline yielded normal retention levels at all times. These results contrast with our earlier reports that both polyclonal and monoclonal antibodies to chick Thy-1 inhibited long-term memory formation only. The present findings are interpreted as possibly representing a dual effect of the purified IgG.

Animals↗
PubMed1993

Gamma delta T cell receptor repertoire in brain lesions of patients with multiple sclerosis.

The identification of activated T cells in the brains of patients with multiple sclerosis (MS) suggests that these cells are critical in the pathogenesis of this disease. Recently we have used the PCR method to analyse rearrangements of V alpha and V beta genes of the T cell receptor (TCR) in samples of MS and control brains. The results of these studies showed that TCR V gene usage in MS brains may be restricted and in particular that V beta genes may be preferentially rearranged in certain HLA haplotypes associated with susceptibility to MS. In view of the recent evidence that T lymphocytes bearing the gamma delta chains may have autoreactive potential, we have assessed whether or not such TCR-bearing lymphocytes were also present in chronic MS lesions. TCR V gamma and V delta were analysed by the PCR method using a panel of V gamma and V delta primers paired with C gamma or C delta primers in 12 MS brains, as well as in brain samples of ten normal post-mortem cases and three neurological controls. TCR V gamma-C gamma and V delta-C delta rearrangements were confirmed using Southern blotting and hybridisation of the PCR products with specific C gamma and C delta probes. Only one to four rearranged TCR V gamma and V delta transcripts were detected in each of the 23 brain samples obtained from 12 MS patients, with the majority of gamma delta T cells expressing the V gamma 2 and V delta 2 chains. In marked contrast, V gamma and V delta transcripts could only be found in one of the ten non-neurological control brains analysed. To assess the clonality of V gamma 2 and V delta 2 T cell receptor chains in the brain samples of MS patients, we have sequenced the junctional regions of the TCR V gamma-N-J gamma-C gamma and V delta-N-D delta-N-J delta-C delta segments amplified from brain tissues, CSF and spleens of two MS patients and from the spleen of two control subjects. The sequence analysis obtained so far shows no compelling evidence of an MS specific expansion of one or more clones expressing particular types of gamma delta T cell receptors. In contrast, a clonal expansion of a different population of TCR gamma delta-bearing T cells was found in the spleen of both an MS patient and one of the control individuals.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular detection of interferon-alpha expression in multiple sclerosis brain.

The demonstration of intermittent interferonaemia in patients with multiple sclerosis prompted a molecular analysis of brain tissue for expression of interferon-alpha genes. A sensitive method was developed based on the polymerase chain reaction. Primer sets were used that could amplify all interferons-alpha or two particular subtypes, interferon-alpha 2 and interferon-alpha 4. The procedure was successful in detecting expression of interferons-alpha in brain and non-brain tissues in most patients with multiple sclerosis. However, expression was demonstrable also in a similar proportion of patients with other neural diseases, and patients with other illnesses. The data indicate that there can be constitutive expression of interferons-alpha in brain tissue, but the possibility that this becomes amplified in multiple sclerosis was not revealed by this study.

Adolescent↗
PubMed1993

Human myelin/oligodendrocyte glycoprotein: a new member of the L2/HNK-1 family.

Myelin/oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of CNS myelin. In this study, human MOG was found to express the L2/HNK-1 epitope on N-linked oligosaccharide structures. This carbohydrate epitope has been found previously in three other characterized human myelin glycoproteins: the myelin-associated glycoprotein, P0, and the oligodendrocyte-myelin glycoprotein. It seems, therefore, that the L2/HNK-1 epitope is expressed frequently in human myelin glycoproteins. Serial lectin affinity chromatography of 14C-glycopeptides indicated that MOG N-oligosaccharide structures are mainly of the complex type, accounting for 77.8% of total radioactivity. In contrast with myelin-associated glycoprotein and P0, which express the L2/HNK-1 epitope on fucosylated structures, in MOG the epitope was detected on all glycopeptide fractions obtained by serial lectin affinity chromatography, although a preferential expression of the L2/HNK-1 epitope was observed on fucosylated structures. Finally, the data indicated that, as for other human myelin glycoproteins, only a subpopulation of MOG molecules expresses the L2/HNK-1 epitope.

Animals↗
PubMed1993

Reactivity to myelin antigens in multiple sclerosis. Peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein.

Although T cell responses to the quantitatively major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), are likely to be of importance in the course of multiple sclerosis (MS), cell-mediated autoimmune responses to other myelin antigens, in particular quantitatively minor myelin antigens, such as myelin-associated glycoprotein (MAG) and the central nervous system-specific myelin oligodendrocyte glycoprotein (MOG), could also play a prevalent role in disease initiation or progression. Highly purified myelin antigens were used in this study to assess cell-mediated immune response to MOG in MS patients, in the context of the reactivity to other myelin antigens, MBP, PLP, and MAG. The greatest incidence of proliferative response by MS peripheral blood lymphocytes was to MOG, as 12 of 24 patients tested reacted and, of these, 8 reacted to MOG exclusively. In contrast, only 1 control individual of 16 tested reacted positively to MOG. The incidence of responses to MBP, PLP, and MAG did not differ greatly between MS patients and control individuals. A predominant T cell reactivity to MOG in MS suggests an important role for cell-mediated immune response to this antigen in the pathogenesis of MS.

Adolescent↗
PubMed1993

Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies.

Myelin oligodendrocyte glycoprotein, (MOG), a quantitatively minor central nervous system (CNS) myelin component, is a candidate target antigen for autoimmune-mediated demyelination. It is a highly hydrophobic protein present in very small amounts in CNS tissue and thereby difficult to purify. Our aim was to devise a purification procedure that would yield sufficient quantities of highly purified MOG to subsequently test its potential encephalitogenic activity, as well as investigate the humoral and cell-mediated responses to this antigen in naturally occurring and experimentally induced autoimmune demyelinating diseases. MOG was purified from human CNS white matter using immunoaffinity chromatography, a procedure that gave a final yield of MOG corresponding to 0.02% total white matter protein. The final product, which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as shown also by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for myelin basic protein, proteolipid protein and myelin-associated glycoprotein. Partial amino acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto PVDF. The sequence of the first 17 N-terminal amino acids had approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences obtained showed also very high homology. Our purified MOG preparations have been used to investigate T cell response to MOG by peripheral blood lymphocytes of multiple sclerosis patients and to induce a relapsing remitting demyelinating disease in Lewis rats.

Amino Acid Sequence↗
PubMed1992

Multiple sclerosis: an autoimmune disease of multifactorial etiology.

The etiology of multiple sclerosis is linked to a variety of genetic and environmental factors. Both cell-mediated and humoral immune responses, triggered by extraneous or autoantigens, are likely to contribute to the pathogenesis of this disease. A greater insight into the fundamental cause of multiple sclerosis has been provided by the recognition that certain immune response genes are associated with an increased susceptibility to the disease. Such knowledge should provide new opportunities for selective therapeutic interventions.

Animals↗
PubMed1992

Differing epitope selection of experimentally-induced and natural antibodies to a disease-specific autoantigen, the E2 subunit of pyruvate dehydrogenase complex (PDC-E2).

Naturally-occurring autoantibodies to a family of mitochondrial enzymes, the 2-oxoacid dehydrogenase complexes (2-OADC), characterize the human liver disease primary biliary cirrhosis. The immunodominant epitope for these autoantibodies is associated with the lipoyl-binding domain of the E2 subunit of the enzymes. The reactivity of these disease-associated autoantibodies was compared with that of antibodies raised in rats and rabbits, by immunization with various preparations derived from the 2-OADC enzymes, using immunization protocols that have successfully induced various organ-specific autoimmune diseases in animals. The immunogens included the intact pyruvate dehydrogenase complex (PDC) from bovine heart, human recombinant PDC-E2, and short synthetic peptides representing the immunodominant lipoic acid binding sequences of the 2-OADC enzymes. The techniques for antibody analysis included immunofluorescence, immunoblotting on mitochondrial extracts, ELISAs using entire PDC, PDC-E2, or synthetic peptides, epitope mapping by peptide scanning on overlapping octameric peptides representing the human PDC-E2 sequence, affinity purification on PDC-E2, and inhibition in vitro by sera of the catalytic function of PDC. Experimental immunization did not elicit any evidence of autoimmune disease. Moreover, the experimentally-induced antibodies in striking contrast to the natural autoantibodies showed preferential reactivity with PDC-E2 rather than with intact PDC, failed to inhibit in vitro the catalytic function of PDC, and, on peptide scanning, reacted with discrete epitopes, but at sites other than the lipoyl-binding region of PDC-E2. Our data indicate that 'multisystem' autoimmune diseases including primary biliary cirrhosis may not be elicitable experimentally because a critical disease-relevant autoepitope is not engaged by the immune system.

Amino Acid Sequence↗
PubMed1992

Influence of HLA-DR2, HLA-DPw4, and T cell receptor alpha chain genes on the susceptibility to multiple sclerosis.

In view of our recent report that T cell receptor (TCR) alpha chain restriction fragment length polymorphism (RFLP) is associated with multiple sclerosis (MS), we have assessed the possibility that HLA-DR2, HLA-DPw4, and TCR alpha chain RFLPs may interact to increase the risk of developing MS. Detection of TCR alpha chain polymorphisms, and HLA-DR and -DP typing were carried out by RFLP analysis on MS patients and healthy controls from the Melbourne metropolitan area. Interaction effects among these loci in producing susceptibility to MS were assessed by hierarchial log-linear analysis. Although HLA-DR2 was significantly associated with MS (chi 2 = 9.30, P = 0.002), no interactive effect between MS and HLA-DPw4 was observed. Significant interactions were observed with MS and both C alpha and V alpha, with the strongest effect seen with C alpha (chi 2 = 21.30, P less than 0.001). The combination of DR2/C alpha imparted a relative risk of 47. However, when the data were analysed for four and three way interactions, no significant effects were seen with MS, DR2, DPw4, V alpha, and C alpha, implying that the combined presence of these polymorphic markers is not essential for increasing susceptibility to MS.

Biomarkers↗
PubMed1991

Comparative study of myelin basic protein isoforms in developing vertebrate central nervous system: absence of 21.5- and 20.2-kilodalton myelin basic proteins in chicken may point to their importance in mammalian myelinogenesis.

Developmental appearance and accumulation pattern of myelin basic protein (MBP) isoforms were analyzed by quantitative immunoblotting in central nervous system of three mammalian (guinea pig, rabbit and bovine) and one avian (chicken) species. In these four species, myelination onset occurred in the spinal cord well before birth. In addition to the 18.5-kD MBP observed in all species studied, a 21.5- and a 20.2-kD MBPs were detected in the three mammalian species but not in chicken. In calf and chicken, a 17.3-kD MBP was also observed. The 18.5-kD and 17.3-kD MBPs were the major MBP isoforms of chicken central nervous system where a faint MBP-related 14.5-kD protein could also be seen. The major difference between mammalian and avian MBP profiles was indeed the presence only in mammals of the 21.5-kD and the 20.2-kD MBPs. The development patterns of these two isoforms as well as their rate of accumulation as compared to the 18.5-kD MBP suggest that their role in mammalian myelination may be of greater importance at early rather than late stages of this process. Differences in quantity as well as type of MBP isoforms present may indicate that in diverging animal species, the process of myelination may follow a different pathway and possibly involve different MBP isoforms at different stages.

Aging↗
PubMed1991

Immunopathological recognition of autoantigens in multiple sclerosis.

Although the aetiology of multiple sclerosis (MS) has not been established, circumstantial evidence points to the involvement of both cell-mediated and humoral immune responses in the formation of demyelinating lesions. In view of the current controversy regarding the reactivity of T lymphocytes from MS patients to myelin basic protein (MBP), we reassessed T cell reactivity in MS using a sensitive and specific indicator of cell-mediated immune response. No significant difference in the reactivity to MBP was observed between MS patients and healthy subjects. Interestingly, significant reactivity to MBP was detected in the control group comprising patients with other diseases. Nevertheless, our demonstration that polymorphism in T cell receptor (TcR) alpha chain are related to MS and that, in demyelinating lesions, TcR usage is limited, suggests that T cells may recognise particular epitopes of a critical antigen involved in this disease. The search for a specific MS antigen recognised by the intrathecally synthesized immunoglobulins typical of MS has so far been unsuccessful. However, recent work, which has focused more particularly onto myelin components with externally located epitopes accessible to the immune response, appears to be more promising. One such antigen, myelin-oligodendrocyte glycoprotein (MOG), is clearly a target for immune attack. Indeed, highly specific antibodies to MOG have been shown to cause demyelination not only in vivo but also in vitro, as demonstrated by our study of the demyelinating effects of a monoclonal anti-MOG antibody on aggregating fetal rat brain cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗