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Biomedical subjects

H Wekerle

Publications and source records attributed to H Wekerle.

At least 127 records · Page 7Linked to original sources
PubMed1991

Acetylcholine receptor-specific T-lymphocyte clones in the normal human immune repertoire: target epitopes, HLA restriction, and membrane phenotypes.

Potentially autoimmune T-lymphocyte lines specific for the nicotinic acetylcholine receptor of the neuromuscular junction have been isolated previously from patients with myasthenia gravis. We report on the isolation and expansion of T cells specific for the acetylcholine receptor of Torpedo californica or for a recombinant mammalian acetylcholine receptor alpha chain peptide (X4), from the peripheral blood of 11 healthy donors. Two major T-cell epitopes, located between amino acid positions 44-104 and 141-172, were identified using a panel of overlapping mammalian alpha chain fusion proteins. Most T lines recognized the acetylcholine receptor epitopes in the molecular context of HLA-DR molecules. Unexpectedly, all the T. californica acetylcholine receptor-specific T lines obtained from one DR4 (DRw53), DQw3 donor and two DR4, w8 (DRw53), DQw3 donors were restricted by DRw53 product(s). Using DR gene-transfected L cells as antigen presenters, in 4 lines, a close relationship between the recognized epitope and the restricting DR element was revealed. The membrane phenotype of the T. californica acetylcholine receptor-and X4-specific T lines was predominantly CD4+CD8-, with some CD4+CD8+ components. It did not significantly differ from that of control, tuberculin purified protein derivate-specific T lines raised from the same donors. These findings are in harmony with previous ones demonstrating the presence of potentially autoimmune T-lymphocyte clones within normal immune repertoires.

Antigens, CD↗
PubMed1991

Neurological diseases.

Recent advances have improved our understanding of the T-cell recognition process in neuroimmunological autoimmune disease as well as the involvement of major histocompatibility complex molecules and cell adhesion molecules in the autoimmune attack.

Autoimmune Diseases↗
PubMed1991

Monoclonal antibodies against myelin proteolipid protein: identification and characterization of two major determinants.

This report describes the preparation and characterization of a panel of monoclonal antibodies (mAbs) against the myelin proteolipid protein (PLP). A Lewis rat was immunized with bovine proteolipid apoprotein and 27 mAbs were selected based on their reactivity against bovine PLP on enzyme-linked immunosorbent assays. Eleven mAbs recognized the PLP carboxyl-terminal sequence when tested against a panel of synthetic peptides in a solid-phase assay. A carboxyl-terminal pentapeptide (residues 272-276) was sufficient for antibody binding and the terminal phenylalanine residue was found particularly important. Deletion, modification, or replacement of this residue markedly reduced or obliterated antigen-antibody interaction. Nine mAbs reacted with a second antigenic determinant, residues 209-217, but these could be identified only by competitive immunoassays. This peptide was a more effective inhibitor than the longer peptides 202-217 and 205-221, suggesting that flanking residues may interfere with peptide-antibody interaction. Seven antibodies did not react with any of the synthetic peptides tested and their determinants remain unidentified. Immunoblot analysis showed that the mAbs reacted with both the PLP and the DM-20 isoforms. Twenty-three of the mAbs were of the immunoglobulin G2a or b isotype; the remaining antibodies were immunoglobulin M and all of these were specific for residues 209-217. Cultured murine oligodendrocytes were stained by most of the mAbs tested, but the most intense reactivity was observed with the carboxyl-terminus-specific mAbs. The immunocytochemical analyses demonstrate that the mAbs react with the native PLP in situ and show their potential usefulness for studies of the cell biology of myelin and oligodendrocytes.

Amino Acid Sequence↗
PubMed1991

Interaction of T lymphocytes with cerebral endothelial cells in vitro.

As a prerequisite of inflammatory lesion formation in (auto-)immune disease of the central nervous system, lymphocytes have to interact with brain endothelia. In recent years much progress has been made towards a better understanding of mechanisms and factors involved in organ specific homing of lymphocytes. Many lines of evidence indicate that T lymphocytes recognizing antigens which are exclusively beyond the blood-brain barrier cross this barrier only when they are in an activated state, irrespective of their antigen specificity. Antigen presentation by blood-brain barrier endothelia, however, may play a role in later stages of florid inflammation.

Animals↗
PubMed1991

Myasthenia gravis: measurement of anti-AChR autoantibodies using cell line TE671.

Anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) can be quantitated using AChR extracted from the human rhabdomyosarcoma cell line TE671 (AChRTE671) as a practical alternative to AChR from human amputated limbs (AChRAMP). We compared the two antigen preparations using serum samples from different clinical groups of MG patients (n = 112) and various controls (n = 189). With two exceptions, both tests were positive or negative in the same patients. However, in the generalized MG group, the TE671 assay yielded significantly lower titers than the AChRAMP assay.

Adolescent↗
PubMed1991

Immunopathogenesis of multiple sclerosis.

The immunopathogenesis of MS is discussed in the light of data recently obtained in Experimental Autoimmune Encephalomyelitis (EAE), a myelin specific experimental autoimmune disease reflecting the first, inflammatory phase of MS plaque generation. EAE is caused by CD4+ T cells specific for defined myelin protein, like MBP and PLP. In rats and mice there is a marked dominance of the autoantigenic peptide epitopes on the one hand, and the T cell receptor V regions on the other. During T cell mediated EAE, clonotypically counterregulatory CD8+ T cells are induced, which specifically neutralize the encephalitogenic T clones.

Animals↗
PubMed1991

The thymus in myasthenia gravis. Changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat.

In human myasthenia gravis (MG) formation of autoantibodies against acetylcholine receptor (AChR) is commonly associated with thymic changes termed lymphofollicular hyperplasia (LFH). To learn whether the thymic lesions of human MG are primary changes in the autoimmune pathogenesis, or rather secondary events caused by peripheral autoimmunization, the authors compared the pathologic changes of MG thymuses with the thymuses of Lewis rats with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced either actively by immunization with AChR, or transferred passively with monoclonal antibodies (mAb) binding to AChR. The clinical diagnosis of EAMG was confirmed by electromyography. Germinal centers, which are typical for human MG thymuses, were not detectable in the thymus of EAMG rats. Scattered B cells were seen as normal components of the thymic medulla. In EAMG their number was not augmented, nor were they accumulated focally. The perivascular spaces (PVS) were not distended and the amount of reticulin was not increased. Thymic myoid cells were identified in EAMG as well as in control thymuses; their cellular microenvironment was inconspicuous. Both in normal and in EAMG thymuses, a subpopulation of myoid cells expressed the main immunogenic region of the AChR. Heavily affected rats showed a severe cortical involution, but no specific changes of the medulla. The fact that none of the thymic lesions characteristic for human MG was found in EAMG is compatible with the concept that the thymic changes in MG are primary events in the autoimmune pathogenesis of this disease.

Animals↗
PubMed1990

Rapid method based on reversed-phase high-performance liquid chromatography for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides.

Reversed-phase high-performance liquid chromatography was applied to isolate myelin basic protein from human brain, followed by separation of proteolytic peptides thereof on the same chromatographic system. Brain tissue was delipidated under conditions that keep copurifying proteases inactive. The crude brain protein fraction was applied directly to a C4 column. The homogeneous protein obtained in this way was digested with thrombin and endoproteinase Lys-C in order to produce short defined myelin basic protein peptides. The purified peptides were used to determine the antigen fine specificity of myelin basic protein recognizing T lymphocyte lines isolated from multiple sclerosis patients.

Brain Chemistry↗
PubMed1990

Establishment of a permanent rat brain-derived glial cell line as a source of purified oligodendrocyte-type 2 astrocyte lineage cell populations.

A permanent glial cell line (L3) has been established from mixed glial cultures obtained from neonatal rat forebrain by repetitive passaging and selection of the process-bearing cells growing on top of a flat cell monolayer. Continuous propagation of the process-bearing cells was supported by the flat cells, of presumed astroglial origin, which were present in negligible amounts following each passage but then grew and formed a basal, feeder layer. Throughout a culture period of over 2 years, the L3 cells have maintained a stable morphological and antigenic phenotype. In serum-containing culture medium, most of the process-bearing cells expressed at the same time features of immature oligodendrocytes (O4 positivity) and of astrocytes [glial fibrillary acidic protein (GFAP) positivity]. A smaller proportion of them was labeled by the monoclonal antibody LB1. LB1+ or O4+ cells were rarely GFAP-, and GFAP+ cells were rarely LB1- or O4-. GalC+ oligodendrocytes were seen only occasionally, but the proportion of these cells increased up to 30% upon culturing in chemically defined medium containing 0.5% fetal calf serum. The L3 process-bearing cells accumulated the neurotransmitter gamma-aminobutyric acid (GABA), expressed the proteoglycan chondroitin sulfate, and responded to the mitogenic action of platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). All these properties are characteristic of cells belonging to the O-2A (oligodendrocyte-type 2 astrocyte) cell lineage. The L3 flat cells were largely negative for the glial markers tested, but resembled type 1 astrocytes in their ability to support the growth of O-2A lineage cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes of multiple-sclerosis patients and healthy donors.

A panel of 20 human myelin basic protein (hMBP)-specific T-lymphocyte lines was generated from the peripheral blood of eight multiple sclerosis (MS) patients and two healthy donors, most of them expressing the HLA-DR2 haplotype, which is associated with an increased susceptibility to MS. Using HLA-DR gene-transfected mouse L-cell lines as antigen-presenting cells, we established that of the 20 hMBP-specific T-lymphocyte lines, 7 were restricted by the DR2a gene products of the DR2Dw2 haplotype. Four T-cell lines recognized hMBP in the context of the DR2b products of the DR2Dw2 haplotype. DR2b-restricted T-cell responses were demonstrable only in T-cell lines derived from MS patients. The hMBP epitopes presented by the DR2a heterodimer were mapped to peptides covering amino acid residues 1-44, 76-91, 131-145, or 139-153 and to a region spanning the thrombin-cleaved bond at Arg130-Ala131. DR2b-restricted T-cell lines recognized epitopes within amino acids 80-99 and 148-162. Peptide 139-153 was also presented in the context of HLA-DR1 molecules. Our data show that (i) in MS patients both the DR2a and DR2b products of the DR2Dw2 haplotype function as restriction elements for the myelin autoantigen hMBP, (ii) the DR2a molecule presents at least five different epitopes to hMBP-specific T lymphocytes, and (iii) anti-hMBP T-cell lines derived from individual donors can differ in their antigen fine specificity as well as in their HLA restriction.

Adult↗
PubMed1990

Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.

Animals↗
PubMed1990

Myelin basic protein-specific T lymphocyte lines from MS patients and healthy individuals.

We derived a total of 146 T lymphocyte lines specific for human myelin basic protein (MBP) from the peripheral blood of 20 MS patients and from a control group of 12 healthy donors, and determined the reactivities of T cell lines by [3H]thymidine incorporation on exposure to MBP and MBP peptides 1-44, 45-89, and 90-170. We defined HLA restriction of the T lines by using monoclonal antibodies against monomorphic determinants on human HLA-DR, HLA-DQ, and HLA-DP molecules. MBP-specific T cell lines could be isolated with a comparable efficiency from MS patients and healthy individuals. In both groups, MBP-specific T lymphocytes recognized at least 4 different epitopes in the MBP molecule, and specificities showed comparable patterns for different MBP peptides. MBP-specific T cell lines derived from MS patients and controls were restricted by DR products of the human major histocompatibility class II locus. Notable phenotypic differences of T cell lines existed between the 2 groups. Lines isolated from MS patients expressed predominantly the CD3+ CD4+ CD8- phenotype, while some control lines were composed of up to 87% CD3+CD4+CD8+ T lymphocytes. These findings illustrate the presence of MBP-specific T cells in MS patients and controls that are similarly sensitized to MBP and restricted by HLA-DR products.

Adult↗
PubMed1990

Schwann cells and myasthenia gravis. Preferential uptake of soluble and membrane-bound AChR by normal and immortalized Schwann cells, and immunogenic presentation to AChR-specific T line lymphocytes.

The normal neuromuscular synapse is formed by the intimate association of nerve endings, postsynaptic end-plate foldings in the muscle fiber, and nonmyelinating Schwann cells (SC) sealing the synaptic ramifications. Because SC have been recognized recently to have an immunogenic potential inducible to present protein autoantigens to autoimmune T lymphocytes, and considering their close proximity to the acetylcholine receptor (AChR)-bearing postsynaptic membranes, presentation of soluble and membrane vesicle-bound AChR to appropriate T cells was investigated. Short-term monolayer cultures of SC isolated from neonatal rat sciatic nerves, as well as cells of an immortalized SC line of similar origin, were fully able to present the relevant molecular epitopes to major histocompatibility complex (MHC) compatible AChR-specific T line lymphocytes immunogenically. Presentation of AChR was restricted by RT1.B (I-A) MHC class II products. Both types of cultured rat SC were inducible to expression of MHC class I and II products, and they were able to phagocytose AChR-enriched membrane vesicles preferentially. In contrast, phagocytosis of latex particles by SC was negligible. These data qualify perisynaptic SC as potential presenter cells of autoimmunogenic AChR in myasthenia gravis. Thus, SC may play a critical and as-yet unpredicted regulatory role in the cellular pathogenesis of myasthenia gravis.

Animals↗
PubMed1990

Experimental autoimmune encephalomyelitis in the maturing central nervous system. Transfer of myelin basic protein-specific T line lymphocytes to neonatal Lewis rats.

We analyzed the effects of myelin basic protein (MBP)-specific encephalitogenic T line cells in neonatal syngeneic recipients before and after formation of central nervous system myelin. Lewis rat pups (postnatal days 0, 1, 2, 4, 8) were injected intraperitoneally with MBP-specific T cell line in doses which cause clinical and histologic changes of experimental autoimmune encephalomyelitis (EAE) in adult rats. We correlated the susceptibility to transferred EAE with the developmental appearance of MBP as demonstrated by immunohistochemical staining. MBP was barely demonstrable until postnatal day 2 but became definitely apparent on postnatal day 4. All newborn rats that were injected on postnatal day 0, 1, 2 failed to develop any apparent clinical signs, but histologically some recipients displayed slight inflammatory cell infiltration of the meninges and subpial lesions limited to the lower spinal cord. In striking contrast, rats that were injected on postnatal day 4 or 8, developed clinical signs and, in particular, many of the older recipients became moribund. Histologically, these animals displayed marked inflammatory cell infiltrations and white matter destruction within the spinal cord. Clinical and histologic severity clearly increased with the recipient's age. Histologically, there were some differences between adult rat EAE and newborn rat EAE. In contrast to adult rat EAE which displayed grey and white matter involvement with marked perivascular mononuclear cell infiltrations, newborn rat EAE typically showed very severe changes with edema formation selectively in the white matter and the cellular infiltrates were dominated by polymorphonuclear cells and macrophages. Newborn T line-mediated EAE thus strikingly resembles hyperacute EAE induced in immunocompromised (irradiated) recipients.

Animals↗
PubMed1990

[Mercaptopurine and silylated mercaptopurine the treatment of experimental allergic encephalomyelitis].

The therapeutic effects of mercaptopurine (6-mercaptopurine, 6-MP) and a silylated derivative (6-trimethylsilylthio-9-trimethyl-silylpurine, S-MP) were compared on the course of T lymphocyte line mediated experimental allergic encephalomyelitis (t-EAE). This transferred EAE is a passively induced model disease in Lewis rats easy to elicit, reliably reproducible and characterized by a dose-dependent lethality. If given at different points in single injections the silicon derivative is shown to be more efficient than 6-MP (43/125 surviving animals compared to 26/129), severity of disease is attenuated and number of survivors increased. Silylation is able to improve blood-brain barrier and cellular permeability; S-MP suppresses intrathecal inflammatory cells more effectively than the original immunosuppressant.

Animals↗
PubMed1989

T cell receptor beta chain usage in myelin basic protein-specific rat T lymphocytes.

Myelin basic protein (MBP)-specific T cell lines and clones have been established from rats of the major histocompatibility complex (MHC)-compatible Lewis and BS strains. All lines and clones are MHC class II restricted and share the CD4+ phenotype. The cells proliferate specifically in response to either a peptide representing amino acids #68-88 of guinea pig MBP, to residues #47-67 or to an unidentified myelin antigen which is distinct from MBP. All lines and clones specific for MBP express the same T cell receptor (TcR) variable (V) beta chain element, which is homologous to the mouse V beta 8.2 gene segment. Three lines/clones with the same antigen fine specificity have identical V beta D beta J beta junctions on the protein level, a region which represents part of the potential antigen-binding portion of the TcR; two of the lines express members of the V alpha 2 family. These results suggest biased usage of TcR V beta elements in rat T cells specific for MBP. Our findings broaden the basis for a rational therapeutic strategy to specifically intervene in the rodent model system of experimental allergic encephalomyelitis.

Amino Acid Sequence↗