Region Of Myelin Basic Protein Research Articles

Docking and molecular dynamics simulations of the Fyn-SH3 domain with free and phospholipid bilayer-associated 18.5-kDa myelin basic protein (MBP)-Insights into a noncanonical and fuzzy interaction.

The molecular details of the association between the human Fyn-SH3 domain, and the fragment of 18.5-kDa myelin basic protein (MBP) spanning residues S38-S107 (denoted as xα2-peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50-ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline-rich region (P93-P98) in both aqueous and membrane environments. In aqueous conditions, the xα2-peptide/Fyn-SH3 complex adopts a "sandwich""-like structure. In the membrane context, the xα2-peptide interacts with the Fyn-SH3 domain via the proline-rich region and the β-sheets of Fyn-SH3, with the latter wrapping around the proline-rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3-ligand. This study thus provides a more-detailed glimpse into the context-dependent interaction dynamics and importance of the β-sheets in Fyn-SH3 and proline-rich region of MBP. Proteins 2017; 85:1336-1350. © 2017 Wiley Periodicals, Inc.

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

Forced Abstinence from Cocaine Self-Administration is Associated with DNA Methylation Changes in Myelin Genes in the Corpus Callosum: a Preliminary Study

Background: Human cocaine abuse is associated with alterations in white matter integrity revealed upon brain imaging, an observation that is recapitulated in an animal model of continuous cocaine exposure. The mechanism through which cocaine may affect white matter is unknown and the present study tested the hypothesis that cocaine self-administration results in changes in DNA methylation that could result in altered expression of several myelin genes that could contribute to the effects of cocaine on white matter integrity. Methods: In the present study, we examined the impact of forced abstinence from cocaine self-administration on chromatin associated changes in white matter. To this end, rats were trained to self-administer cocaine (0.75 mg/kg/0.1 mL infusion) for 14 days followed by forced abstinence for 1 day (n = 6) or 30 days (n = 6) before sacrifice. Drug-free, sham surgery controls (n = 7) were paired with the experimental groups. Global DNA methylation and DNA methylation at specific CpG sites in the promoter regions ofmyelin basic protein (Mbp), proteolipid protein-1 (Plp1), and SRY-related HMG-box-10 (Sox10) genes were analyzed in DNA extracted from corpus callosum. Results: Significant differences in the overall methylation patterns of the Sox10 promoter region were observed in the corpus callosum of rats at 30 days of forced abstinence from cocaine self-administration relative to sham controls; the −189, −142, −93, and −62 CpG sites were significantly hypomethylated point-wise at this time point. After correction for multiple comparisons, no differences in global methylation or the methylation patterns of Mbp or Plp1 were found. Conclusion: Forced abstinence from cocaine self-administration was associated with differences in DNA methylation at specific CpG sites in the promoter region of the Sox10 gene in corpus callosum. These changes may be related to reductions in normal age related changes in DNA methylation and could be a factor in white matter alterations seen after withdrawal from repeated cocaine self-administration. Further research is warranted examining the effects of cocaine on DNA methylation in white matter.

Molecular dynamics investigation of myelin basic protein stability on lipid membranes

Simulation of proteins and membranes composed of synthetic lipids on computer clusters provides molecular information that complements experimental data. This paper describes molecular dynamics (MD) approaches to study the properties of biological membranes and proteins using the freely available GROMACS package on the C-terminal α-helical peptide of myelin basic protein (MBP). We simulated a mixed membrane – consisting of 2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC/DMPE), and a pure DMPC membrane, composed of 188 and 248 lipids, respectively – for 200 ns at 309K. The DMPC membrane was approximately three times more fluid compared to the DMPC/DMPE system, with the diffusion coefficients (D) being 0.0207x10-5 cm2/s and 0.0068x10-5 cm2/s, respectively. In addition, we simulated the 14-residue peptide representing the C-terminal α-helical region of murine MBP, with sequence NH2-A141YDAQGTLSKIFKL154-COOH, in both membrane systems for 200 ns. The negatively-charged N-terminal end of the peptide penetrated further into the DMPC bilayer than into the mixed DMPC/DMPE bilayer. Reduced peptide accessibility to a formal positive charge of the DMPC amine ‘N’ atom surrounded by methyl and methylene groups may be the cause [1]. The peptide lost its α-helical structure in DMPC/DMPE but not in the DMPC bilayer. These findings show that membrane composition affects MBP’s interaction with it, a phenomenon that provides insights into myelin structure – and that may eventually be relevant to understanding the pathogenesis of multiple sclerosis (MS).

SDSL-EPR Study of a C-terminal Segment of Myelin Basic Protein in a Myelin Mimetic Environment

Myelin basic protein (MBP) is a peripheral membrane protein and a major component of human central nervous system myelin. It is a multifunctional, highly positively charged, intrinsically disordered protein that undergoes extensive post-translational modifications. Its multifunctionality includes among others, binding of cytoskeletal proteins to a membrane surface and polymerization and bundling of cytoskeletal proteins. These interactions are modulated by interaction of MBP with Ca2+-calmodulin. We report here the use of site-directed spin labeling and continuous wave power saturation electron paramagnetic resonance spectroscopy (SDSL-EPR) to examine the conformation of segment A141-L154 of a recombinant 18.5 kDa murine isoform of MBP in a reconstituted membrane environment. This segment overlaps the primary calmodulin binding region (T147-D158) of MBP and is predicted, on the basis of helical wheel constructs, to form an amphipathic alpha helix. Solution NMR investigations also showed this region to exist as a transient alpha helix in 30% TFE. Our measurements using SDSL-EPR reveal that this region forms an extended helix with a period of ∼3.8 residues per turn and with a tilt angle of ∼4.3 degrees with respect to the plane of the lipid bilayer. The N-terminal end of the helix appears to be buried more deeply in the membrane bilayer than the C-terminal end. The C-terminal region of the segment bears two Lys residues whose side chains interact with the lipid head groups causing this end to be more exposed. The accessibility of the C-terminal region of A141-L154, which forms a part of the primary calmodulin binding segment, could facilitate interaction of this region with cytosolic proteins viz., calmodulin and modulate cytoskeletal binding to the oligodendrocyte plasma membrane.(Supported by CIHR, NSERC and MS Society of Canada)

Structural Insight into the Function of Myelin Basic Protein as a Ligand for Integrin αMβ2

Multiple sclerosis (MS) is an inflammatory disease where phagocytic cells infiltrate the nerve tissue and act as terminal agents in destruction of the myelin sheath. However, the mechanism that triggers the ability of these cells to recognize myelin remains obscure. We show that myelin basic protein (MBP), a major autoantigen in MS, is a potent and specific ligand for the integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) expressed mainly on phagocytic cells. MBP undergoes a dramatic conformational change when liberated from the lipid-rich environment of the myelin sheath. The MS drug glatiramer acetate mimics the conformationally labile regions of MBP, interacts in the unfolded state strongly with alpha(M)beta(2), and inhibits the MBP binding to alpha(M)beta(2). Our study reveals a link between MBP, glatiramer acetate, and the alpha(M)beta(2) integrin, and suggests a new model for MS pathogenesis based on the recognition of unfolded MBP by the alpha(M)beta(2) integrin.

Cytokine–neuroantigen fusion proteins: New tools for modulation of myelin basic protein (MBP)-specific T cell responses in experimental autoimmune encephalomyelitis

Fusion proteins incorporating anti-inflammatory cytokines and immunodominant self antigen as separate domains of a single protein may hold promise for development of antigen-specific tolerogenic vaccines. Proteins incorporating rat sequences of IL-1RA, IL-2, IL-4, IL-10, or IL-13 were expressed as fusion proteins containing the major encephalitogenic region of myelin basic protein (MBP). These fusion proteins were expressed via baculovirus (bv) expression systems and were shown to have cytokine-dependent and antigen-specific biological activity. In the case of the IL-2 and IL-4 fusion proteins, covalent linkage of the cytokine and neuroantigen domains resulted in synergistic antigen presentation. These data indicate that the cytokine domain may be able to modulate APC activity and simultaneously target the covalently tethered NAg for enhanced presentation by certain APC subsets. Cytokine/antigen fusion proteins may represent a novel tool for antigen-specific immune modulation in autoimmune disease.

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

Radiation hybrid mapping of genes and newly identified microsatellites in candidate regions for bovine arthrogryposis–palatoschisis and progressive ataxia based on comparative data from man, mouse and rat

A comparative pathology and mapping strategy was used to initiate a study on two bovine genetic diseases: arthrogryposis-palatoschisis and progressive ataxia, which affect mainly Charolais cattle. Bibliographic studies provided information on the pathology of these diseases, which helped to define similar diseases in other species. Animals affected by bovine arthrogryposis-palatoschisis display similar symptoms to those of muscular dysgenesis, mouse mutants and animals with progressive ataxia to those of Long Evans Shaker rat mutants. Candidate regions are respectively human chromosome 1q32 (BTA16) containing the gene CACNA1S and human chromosome 18q23 (BTA24) containing the gene myelin basic protein (MBP). Primer pairs were designed for 15 loci around each candidate gene, in a region of about 20 megabases and were used to screen a bovine Bacterial Artificial Chromosome (BAC) library. Eighteen microsatellites were found in the identified BAC clones, 11 on BTA24 and seven on BTA16. The genes and microsatellites were mapped by radiation hybrid (RH) analysis and a RH map was obtained for each region with 18 new localizations on BTA16 and 23 on BTA24. Comparative human-bovine analysis of the MBP region shows a good conservation of gene order while that of the CACNA1S region shows several breakpoints.

Ex vivo detection of myelin basic protein‐reactive T cells in multiple sclerosis and controls using specific TCR oligonucleotide probes

T cell reactivity to candidate myelin autoantigens, such as myelin basic protein (MBP), may play an important role in the pathogenesis of multiple sclerosis (MS). Although MBP-reactive T cells have been found to undergo in vivo activation in patients with MS, their true precursor frequency in MS is unknown as current frequency analysis is commonly based on the T cell functional responses to MBP. In this study, we developed a TCR sequence-based ex vivo detection system using colony hybridization with oligonucleotide probes specific for CDR3 of selected T cell clones for the analysis of true T cell precursor frequency in PBMC. The results revealed that the precursor frequency of five independent T cell clones recognizing the immunodominant MBP(83-99) region was found to be in the range of 1.6 x 10(-4) in total T cells in three HLA-DR2 patients with MS compared to that of 0.25 x 10(-4) in HLA-DR2 healthy individuals. The observed frequency of MBP(83-99)-reactive T cells in MS patients was considerably higher than those measured in parallel by cell culture-based analysis (2.3 x 10(-6)) or by enzyme-linked immunospot assay (3.9 x 10(-5)) in the same peripheral blood mononuclear cell specimens. Furthermore, the study showed that MBP(83-99)-reactive T cells detected ex vivo belonged to CD45RA+, CD25+ and CD95- T cell subsets as evidenced by preferential expression of specific TCR transcripts in these cell fractions.

Repertoire dynamics of autoreactive T cells in multiple sclerosis patients and healthy subjects: epitope spreading versus clonal persistence.

Autoantigen-specific T-lymphocytes are present in patients with autoimmune disease and in normal subjects. Little is currently known about the temporal variation (dynamics) of the immune repertoire of these autoreactive T cells. We analysed the long-term variation of the immune repertoire of T cells specific for myelin basic protein (MBP) in five untreated patients with multiple sclerosis and four normal control subjects over a mean observation period of 6 years. MBP-specific CD4(+) T-cell lines were selected with purified human MBP, and their epitope specificity was mapped with overlapping synthetic peptides. Three distinct patterns of repertoire development were observed. (i) Two patients and three control subjects maintained a broad epitope response with fluctuations over time. (ii) Two patients initially showed a focused response that broadened over the course of 6 years; this finding could be described as intramolecular epitope spreading. (iii) In one patient and one control subject, a strikingly focused response, which was directed to a cluster of nested epitopes in the MBP region 83-102, persisted over time. T-cell receptor Vbeta sequence analysis allowed us to trace individual clones of MBP-specific T cells for up to 7 years in the peripheral circulation in four of the five patients and three of the four controls, suggesting that the long-term persistence of MBP-specific T-cell clones is a common feature of the T-cell repertoire not unique to multiple sclerosis. The persisting MBP-specific T-cell clones were not detectable in the blood of one of the patients by complementarity-determining region (CDR)-3 spectratyping, indicating that their frequency does not exceed 1 in 5000 T cells. The temporal characteristics of the MBP-specific T-cell repertoire described here are relevant to therapeutic strategies targeting autoantigen-specific T cells in multiple sclerosis and other autoimmune diseases.

Myelin basic protein (MBP) and MBP peptides are mitogens for cultured astrocytes

After CNS demyelination, astrogliosis interferes with axonal regeneration and remyelination. We now provide evidence that myelin basic protein (MBP) can contribute to this observed astrocyte proliferation. We found that astrocytes grown in either serum-containing or serum-free medium proliferate in response to MBP. The mitogenic regions of MBP in both media were MBP(1-44), MBP(88-151) and MBP(152-167). The mitogenic effect of these individual peptides was potentiated by simultaneous treatment with microglia conditioned media (CM). MBP-induced proliferation was inhibited by suramin at concentrations known to block the fibroblast growth factor receptor (FGFR), whereas neither MBP(1-44), MBP(88-151) nor MBP(152-167) were affected. Cholera toxin B, that binds to ganglioside GM(1), inhibited the mitogenicity of MBP(1-44) and had no significant effect on the mitogenicity of MBP, MBP(88-151) or MBP(152-167). Treatment of astrocytes with MBP and MBP(152-167) caused a modest and transitory elevation of intracellular calcium, whereas treatment with MBP(1-44) resulted in a substantial and sustained increase in intracellular calcium. These results suggest that for cultured astrocytes 1) FGFR and extracellular calcium play a major role in MBP mitogenicity; 2) MBP(1-44), MBP(88-151) and MBP(152-167) are the mitogenic regions of MBP; 3) MBP(1-44) and MBP(152-167) interact with ganglioside GM(1) and FGFR, respectively; 4) Component(s) present in microglial CM potentiate the mitogenicity of MBP(1-44), MBP(88-151) and MBP(152-167). These data support the hypothesis that MBP related peptides in conjunction with microglial secreted factors may stimulate astrogliosis after demyelination in vivo.

Usage of vβ3.3 T‐cell receptor by myelin basic protein‐specific encephalitogenic T‐cell lines in the Lewis rat

T-cell receptor (TCR) β-chain usage in experimental autoimmune encephalomyelitis (EAE) seems to be much more heterogeneous than previously assumed even for a single autoantigen in an inbred animal strain. Owing to the lack of suitable antibodies, this has been demonstrated only at the RNA level so far. To characterize further the TCR elements used in the Lewis rat for the recognition of the main encephalitogenic region of myelin basic protein (MBP), BALB/c mice were immunized with T-cell hybridomas expressing non-Vβ8.2 TCR specific for guinea pig MBP peptide aa 68–88. Two B-cell hybridomas (clones C-A11 and F-D6) producing TCR Vβ3.3-specific monoclonal antibodies were selected. Specificity was demonstrated by RT-PCR and cloning of PCR products obtained from sorted T-cell lines and naive T cells. MBP-specific Vβ3.3 T cells used the L-S motif in the VDJ region, were associated with Vα2 or Vα8 chains, and specifically recognized MBP peptide aa 68–88. Vβ3.3 TCR-positive T cells were detected in all of a panel of six MBP-specific T-cell lines, although to a lesser degree than Vβ8.2 TCR-positive T cells. After intravenous injection of sorted Vβ3.3 T cells, animals developed EAE, and Vβ3.3-positive cells were found by immunocytochemical analyses in the spinal cord. Furthermore, treatment of EAE induced by immunization with MBP was more effective when a combination of anti-Vβ3.3 and anti-Vβ8.2 mAbs was used. These results confirm the functional role of TCR Vβ3.3 and thus underscore the heterogeneity of TCR usage in MBP-associated autoimmunity. J. Neurosci. Res. 58:214–225, 1999. © 1999 Wiley-Liss, Inc.

Usage of v?3.3 T-cell receptor by myelin basic protein-specific encephalitogenic T-cell lines in the Lewis rat

T-cell receptor (TCR) beta-chain usage in experimental autoimmune encephalomyelitis (EAE) seems to be much more heterogeneous than previously assumed even for a single autoantigen in an inbred animal strain. Owing to the lack of suitable antibodies, this has been demonstrated only at the RNA level so far. To characterize further the TCR elements used in the Lewis rat for the recognition of the main encephalitogenic region of myelin basic protein (MBP), BALB/c mice were immunized with T-cell hybridomas expressing non-Vbeta8.2 TCR specific for guinea pig MBP peptide aa 68-88. Two B-cell hybridomas (clones C-A11 and F-D6) producing TCR Vbeta3.3-specific monoclonal antibodies were selected. Specificity was demonstrated by RT-PCR and cloning of PCR products obtained from sorted T-cell lines and naive T cells. MBP-specific Vbeta3.3 T cells used the L-S motif in the VDJ region, were associated with Valpha2 or Valpha8 chains, and specifically recognized MBP peptide aa 68-88. Vbeta3.3 TCR-positive T cells were detected in all of a panel of six MBP-specific T-cell lines, although to a lesser degree than Vbeta8.2 TCR-positive T cells. After intravenous injection of sorted Vbeta3.3 T cells, animals developed EAE, and Vbeta3.3-positive cells were found by immunocytochemical analyses in the spinal cord. Furthermore, treatment of EAE induced by immunization with MBP was more effective when a combination of anti-Vbeta3.3 and anti-Vbeta8.2 mAbs was used. These results confirm the functional role of TCR Vbeta3.3 and thus underscore the heterogeneity of TCR usage in MBP-associated autoimmunity.

T cell responses to myelin basic protein in patients with spinal cord injury and multiple sclerosis

Autoimmune inflammation secondary to myelin destruction may play an inhibitory role in restoration of nerve functions in spinal cord injury (SCI). In this study, we demonstrated that T cells recognizing myelin basic protein (MBP) occurred at a high precursor frequency in patients with SCI, which was compatible to that in patients with multiple sclerosis (MS), a disease of presumed autoimmune pathology. The findings suggest of hyperactivity of MBP-reactive T cells in patients with SCI. MBP-reactive T cell lines derived from patients with SCI exhibited a preferential recognition pattern toward the 81–99 and the 151–169 regions of MBP. There were functional differences in the epitope recognition and cytokine profile between two panels of MBP-reactive T cell lines derived from patients with SCI and patients with MS. The study provides new evidence important for further investigation of the role of the inflammatory component in SCI.

Impaired apoptotic deletion of myelin basic protein-reactive T cells in patients with multiple sclerosis

T cell responses to myelin basic protein (MBP) may play an important role in the pathogenesis of multiple sclerosis (MS). If MBP-reactive T cells are involved in the disease processes and undergo clonal activation and expansion, their precursor frequency would be increased in patients with MS. The frequency of MBP-reactive T cells is also influenced by regulatory mechanisms in vivo, including apoptotic deletion. In this study, we examined changes in the frequency of MBP-reactive T cells in patients with MS as a function of the apoptotic deletional mechanism in vivo, using a cell culture-based assay. A significantly increased frequency of MBP-reactive T cells was found in patients with MS relative to healthy individuals only when Fas-ligand antibody was used to block apoptosis. This result indicates that a significant proportion of MBP-reactive T cells are sensitive to apoptosis and are not deleted in vivo in patients with MS, as opposed to healthy individuals, thus suggesting a functional deficit in apoptotic deletional mechanism. Surviving Fas-sensitive MBP-reactive T cell lines represent distinct subpopulations preferentially recognizing the 111–139 region of MBP and exhibiting a Th2 cytokine profile. The findings are relevant to our understanding of regulation of MBP-reactive T cells in vivo in MS.

Impaired apoptotic deletion of myelin basic protein-reactive T cells in patients with multiple sclerosis.

T cell responses to myelin basic protein (MBP) may play an important role in the pathogenesis of multiple sclerosis (MS). If MBP-reactive T cells are involved in the disease processes and undergo clonal activation and expansion, their precursor frequency would be increased in patients with MS. The frequency of MBP-reactive T cells is also influenced by regulatory mechanisms in vivo, including apoptotic deletion. In this study, we examined changes in the frequency of MBP-reactive T cells in patients with MS as a function of the apoptotic deletional mechanism in vivo, using a cell culture-based assay. A significantly increased frequency of MBP-reactive T cells was found in patients with MS relative to healthy individuals only when Fas-ligand antibody was used to block apoptosis. This result indicates that a significant proportion of MBP-reactive T cells are sensitive to apoptosis and are not deleted in vivo in patients with MS, as opposed to healthy individuals, thus suggesting a functional deficit in apoptotic deletional mechanism. Surviving Fas-sensitive MBP-reactive T cell lines represent distinct subpopulations preferentially recognizing the 111-139 region of MBP and exhibiting a Th2 cytokine profile. The findings are relevant to our understanding of regulation of MBP-reactive T cells in vivo in MS.

Myelin basic protein and myelin basic protein peptides induce the proliferation of Schwann cells via ganglioside GM1 and the FGF receptor.

Myelin basic protein (MBP) and two peptides derived from MBP (MBP(1-44) and MBP(152-167)) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of 125I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of 125I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP(1-44)) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP(1-44) and MBP(152-167) associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.

Sequence Determinants of Modified Cobra Venom Neurotoxin Which Induce Immune Resistance to Experimental Allergic Encephalomyelitis: Molecular Mechans for Immunologic Action

A nontoxic, iodoacetamide-modified cobratoxin derivative (CAM-NTX) induced resistance to experimental allergic encephalomyelitis (EAE) in guinea pigs. Resistance was retained after trypsin digestion and shown to reside in N-terminal and central peptides of CAM-NTX. A similarly modified protein cardiotoxin (CAM-CTX), representative of proteins homologous with cobratoxin, was not immunosuppressive. Depressed clinical symptoms in EAE-resistant animals correlated with reduced lymphocytic infiltration of the brain. Antibody to myelin basic protein (MBP) was reduced in immunosuppressed animals. The immunoinhibitory determinants in CAM-NTX may mimic immune response suppressor proteins (SIRS-α7) and the EAE-resistance region of MBP.

Restricted TCR Valpha gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis.

T cell responses to myelin basic protein (MBP) are thought to play an important role in the pathogenesis of multiple sclerosis (MS). The response to the 83-99 region of MBP represents a dominant response to MBP in patients with MS and is associated with HLA-DR2 that is linked with susceptibility to MS. Although T cell clones reactive to various regions of MBP have been found to exhibit heterogeneous TCR Vbeta gene usage in patients with MS, it is unclear whether T cell clones uniformly recognizing the 83-99 peptide of MBP in the context of the same DR molecule would have restricted TCR V gene rearrangements and recognition motifs. In this study, a panel of DR2- or DR4-restricted T cell clones specific for the MBP83-99 peptide were derived from 11 patients with MS and examined for TCR V gene usage by PCR and the recognition motifs using analog peptides. Our study revealed that despite a few T cell clone pairs having similar recognition motifs and shared sequence homology in the CDR3, the overall recognition motifs of MBP83-99-specific T cells were considerably diverse. Interestingly, the DR2-restricted T cell clones displayed a biased V gene usage for Valpha3 and Valpha8, while Vbeta gene rearrangements were highly heterogeneous. This study provided experimental evidence suggesting a limited heterogeneity in TCR Valpha gene rearrangements of MBP-reactive T cells in DR2 patients with MS.