Detection Of Myelin Basic Protein Research Articles

Role for TGF-beta1, FGF-2 and PDGF-AA in a myelination of CNS aggregate cultures enriched with macrophages.

The increase in myelin basic protein (MBP) synthesis observed in brain aggregate cultures supplemented with macrophages is reflected in elevated supernatant protein levels of the key promoters of oligodendrocyte proliferation, fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-AA (PDGF-AA), during the premyelinating phase. Although supernatant levels of transforming growth factor-beta1 (TGF-beta1), the most abundant growth factor produced at the transcriptional and translational levels by phagocytic macrophages, were reduced at this stage, it was the only growth factor for which mRNA expression was increased significantly in macrophage-enriched cultures. TGF-beta1, which supports oligodendrocyte differentiation, was increased in the supernatant of macrophage-enriched cultures only after the onset of myelinogenesis. Hence, standard cultures treated with TGF-beta1 during the premyelinating period reproduced effects of macrophage supplementation, inducing an increase in MBP synthesis and in PDGF-AA and FGF-2 bioavailability. A similar increase in MBP synthesis in PDGF-AA treated cultures emphasises its central role in oligodendrocyte progenitor proliferation. In contrast, FGF-2 blocked MBP synthesis in the cultures. In cultures treated with anti-TGF-beta1 antibody before or after the first detection of MBP, supernatant levels of TGF-beta1, FGF-2, and PDGF-AA were reduced with resultant inhibition of myelination. Paradoxically, supraphysiological TGF-beta1 treatment after the onset of myelination had the same effect on myelin accumulation. These results indicate an enabling and regulatory role for TGF-beta1 in oligodendrocyte development and, as a source of TGF-beta1, macrophages in the inflammatory multiple sclerosis lesion, may have the potential to promote remyelination by modulating the growth factor repertoire in demyelinating disease.

The Trophic Role of Oligodendrocytes in the Basal Forebrain

Traditionally, the primary function of oligodendrocytes (OLGs) in the CNS has been considered to be myelination. Here, we investigated whether OLGs may play a trophic role, particularly during development. Neurotrophin expression was assessed in postnatal day 7 basal forebrain (BF) OLGs, using in situ hybridization and detection of myelin basic protein. Nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs were revealed in OLGs in vivo and in culture. To determine whether OLGs support nearby neurons, we examined the influence of OLGs on BF cholinergic neurons. Neuronal function was enhanced by cocultured OLGs and OLG conditioned medium. Moreover, trophic effects of OLG conditioned medium were partially blocked by K252a, a trk tyrosine kinase inhibitor, and by neutralizing anti-BDNF or anti-NT-3 antisera, indicating that neurotrophins may mediate these effects, perhaps in concert with other signals. Our studies support a novel role for OLGs in providing local trophic support for neurons in the CNS.

Detection of myelin basic protein in cerebrospinal fluid and serum from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

BackgroundHuman T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) is a chronic inflammatory disease that primarily affects the spinal cord and may be a neurological syndrome that is clinically similar to multiple sclerosis (MS). Myelin basic protein (MBP) in the cerebrospinal fluid (CSF) of MS patients is generally measured by radio-immunoassay. We have recently established a sensitive enzyme-linked immunosorbent assay (ELISA) and measured the MBP concentrations in CSF and serum of HAM/TSP patients. MethodsA sensitive two-site ELISA capable of measuring MBP at a concentration as low as 30pg/mL in serum and CSF samples was used. ResultsSignificantly higher CSF MBP concentrations were detected in 61% of HAM/TSP patients than in patients with non-neurological diseases. Serum MBP concentrations were also higher in 9% of HAM/TSP patients compared with patients with non-neurological diseases or healthy controls. ConclusionsUsing our ELISA system, we detected MBP in CSF and serum not only in patients with central active demyelination as in MS, but also in patients with spinal cord demyelination as in HAM/TSP

Detection of myelin basic protein in cerebrospinal fluid and serum from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis.

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) is a chronic inflammatory disease that primarily affects the spinal cord and may be a neurological syndrome that is clinically similar to multiple sclerosis (MS). Myelin basic protein (MBP) in the cerebrospinal fluid (CSF) of MS patents is generally measured by radioimmunoassay. We have recently established a sensitive enzyme-linked immunosorbent assay (ELISA) and measured the MBP concentrations in CSF and serum of HAM/TSP patients. A sensitive two-site ELISA capable of measuring MBP at a concentration as low as 30 pg/mL in serum and CSF samples was used. Significantly higher CSF MBP concentrations were detected in 61% of HAM/TSP patients than in patients with non-neurological diseases. Serum MBP concentrations were also higher in 9% of HAM/TSP patients compared with patients with non-neurological diseases or healthy controls. Using our ELISA system, we detected MBP in CSF and serum not only in patients with central active demyelination as in MS, but also in patients with spinal cord demyelination as in HAM/TSP.

Detection of myelin basic protein in cerebrospinal fluid

Radioimmunoassay for myelin basic protein in cerebrospinal fluid is commonly used as a biochemical marker of demyelination in multiple sclerosis patients. A sensitive enzyme-linked immunosorbent assay for myelin basic protein has been recently developed, which can make a clinical evaluation of myelin basic protein in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. Most multiple sclerosis patients with acute exacerbation had markedly high myelin basic protein. Longitudinal studies of multiple sclerosis patients showed that myelin basic protein in CSF increases rapidly in agreement with acute relapse and then rapidly declines and disappears. Significantly higher cerebrospinal fluid myelin basic protein levels in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis patients were also detected. This enzyme-linked immunosorbent assay system can be used routinely to measure myelin basic protein in cerebrospinal fluid as a useful diagnostic indicator, not only for central active demyelination as in multiple sclerosis but, also for spinal cord demyelination as in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis.

Temporal analysis of growth factor mRNA expression in myelinating rat brain aggregate cultures: increments in CNTF, FGF-2, IGF-I, and PDGF-AA mRNA are induced by antibody-mediated demyelination.

Myelinogenesis in rat brain aggregate cultures is associated with a pattern of growth factor mRNA expression comparable to that of the developing brain. The rate of increase in platelet-derived growth factor-AA (PDGF-AA) expression was greatest just before the detection of myelin basic protein (MBP) mRNA in the cultures and remained high thereafter, consistent with in vivo observations. Levels of fibroblast growth factor-2 (FGF-2) and of ciliary neurotrophic factor (CNTF) mRNA increased continuously over the period of MBP accumulation. High rates of transforming growth factor beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and neurotrophin-3 (NT-3) expression at early time points during the culture gradually decreased over time, indicative of a key regulatory role during oligodendrocyte development. The addition of demyelinative anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibody resulted in a significant increase in MBP peptide fragments with a C-terminus at phenylalanine 89 indicating proteolytic breakdown of MBP after myelin phagocytosis. Immediately after antibody treatment the expression of CNTF mRNA was significantly increased, compared with controls, while that of FGF-2 and IGF-I, and of PDGF-AA peaked during the early and later stages of recovery respectively. Thus, specific growth factors combine to regulate myelination and remyelination in the aggregates; these data have implications for demyelinating disease in which protective growth factor secretion may be central to regeneration.

Immunohistochemical detection of myelin basic protein is a sensitive marker of myelination in second trimester human fetal spinal cord.

The Luxol fast blue (LFB) technique is widely used for the assessment of myelination. Lectin histochemistry using peanut agglutinin (PNA) has also been employed for this purpose. Recently, immunohistochemical methods using antibodies to several myelin-related proteins have been adopted to study myelination in humans. However, the relative sensitivities of these different methods for the detection of early myelination in the human fetal central nervous system have not been determined. Vibratome sections of cervical spinal cord from 15 human abortuses ranging in age from 15 to 24 gestational weeks (GW) were probed with immunohistochemical methods using antibodies to myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG). In addition, LFB and PNA histochemistry was employed. The degree of myelination observed in immunohistochemically stained sections was compared to that found in corresponding LFB- and PNA-stained paraffin-embedded tissues. The intensity of myelination was graded by two observers on a scale of 0 (none), +1 (mild), +2 (moderate), and +3 (marked). At all ages examined, the MBP immunohistochemical method revealed more myelin than LFB or MAG staining. CNPase could not be reliably detected until after 18 GW. Peanut agglutinin stained myelin, but subpial astrocytes and the intervening neuropil were also stained. These results suggest that MBP is a more sensitive marker for early human fetal myelination than CNPase, MAG, PNA or LFB.

The sequence of myelination in the brainstem of the rat monitored by myelin basic protein immunohistochemistry

The temporal order which is observed by nervous pathways in their myelination was investigated in the brainstem of neonatal to 16-day-old albino rats. The immunohistochemical detection of myelin basic protein in newly forming myelin sheaths provided a highly sensitive and specific criterion to judge the time of onset and the progress of myelination in its initial stages. Immunohistochemistry was performed on paraffin-embedded tissue according to the unlabeled peroxidase-antiperoxidase method. A small number of immunoreactive fibers was already present in neonate rats in the ventral funiculus of the cervical spinal cord and in the medial longitudinal fascicle of the medulla oblongata. By the 4th postnatal day myelination had commenced in many sensory tracts and in the trigeminal root, in the tectospinal tract, in the inferior cerebellar peduncle and in the roots of most cranial motor nerves. The auditory pathway up to the inferior colliculus, the gracile, spinocerebellar and rubrospinal tracts and the superior cerebellar peduncle had acquired some myelin on the 7th day. Phylogenetic late fiber systems also appeared to myelinate comparatively late: the transverse pontine fibers and the medial cerebellar peduncle started on the 10th day, and in the corticospinal tract a conspicuously synchronized myelination appeared on the 13th day. All pathways of the brainstem possessed myelinated fibers on the 16th day and some tracts had approached their relative maturity.

Three possible laboratory indexes of disease activity in multiple sclerosis

In a search for an objective measure of disease activity in MS, we studied three laboratory indexes in 15 patients over 12 months, relating them to the occurrence of relapse and the development of increased disability. Relapse was associated with detection of myelin basic protein (MBP) in the CSF (p less than 0.01), but not with decreased numbers of peripheral blood T lymphocytes. Persistently low T-cell numbers and more frequent detection of MBP in remission were associated with increased disability (p less than 0.01). There were no associations between other CSF abnormalities and either relapse or increased disability.

Solid phase radioimmunoassay for human myelin basic protein using a monoclonal antibody

A solid phase competitive assay for human myelin basic protein (MBP) has been developed using a monoclonal antibody to MBP. The assay has been applied to the detection of antigenic peptides derived from MBP, the measurement of anti-idiotypic antibody and the detection of MBP in human serum.

Comparison of a non-competitive two-site RIA and an inhibition RIA for the detection of myelin basic protein in cerebrospinal fluid

Two radioimmunoassay (RIA) produces were used to measure human myelin basic protein (HBP) in cerebrospinal fluid (CSF): (1) an inhibition RIA, with the use of TNP-conjugated anti-BP IgG, 125I-labelled HBP, and anti-TNP-coated polystyene beads, and (2) a non-competitive two-site RIA, with the use of Sepharose-coupled anti-BP antibodies and 125I-labeled anti-BP IgG.The two-site RIA detects less HBP in CSF than the inhibition RIA, partly due to the presence of HBP fragments in CSF that are detected by the inhibition asasy, but less by the two-site RIA. The correlation was improved when in the two-site RIA Sepharose-coupled anti-BP antibodies were changed. Because certain substrates (such as autoantibodies to HBP) may give false-positive results in the competitive RIA but not in the two-site RIA, we conclude that a combination of the (more sensitive) inhibition RIA with the (more specific) two-site assay provides a more reliable HBP assay than either assay alone.