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Biomedical subjects

B M Conti-Fine

Publications and source records attributed to B M Conti-Fine.

At least 55 records · Page 3Linked to original sources
PubMed1997

TCR V beta usage by acetylcholine receptor-specific CD4+ T cells in myasthenia gravis.

In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.

Amino Acid Sequence↗
PubMed1997

TCR vbeta usage of TSH receptor-specific CD4+ T cells in Graves' disease patients and healthy humans.

Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.

Adult↗
PubMed1997

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments.

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.

Adult↗
PubMed1997

Th1 epitope repertoire on the alpha subunit of human muscle acetylcholine receptor in myasthenia gravis.

In myasthenia gravis (MG), CD4+ T helper cells recognize the muscle acetylcholine receptor (AChR) alpha subunit. We investigated the epitope repertoire of anti-AChR blood CD4+ Th1 cells from 13 myasthenic patients and three healthy controls, using overlapping synthetic peptides screening the alpha subunit sequence and an enzyme-linked immunospot (ELISPOT) assay that detects antigen-induced interferon-gamma secretion of individual Th1 cells. All patients recognized a pool of the alpha subunit peptides. All but one patient recognized numerous peptides. Each patient had an individual pattern of peptide recognition, but most or all patients recognized four sequences (residues 48-67, 101-137, 304-322, and 403-437) that stimulated relatively large numbers of Th1 cells. They include previously identified "immunodominant" sequences recognized by AChR-specific CD4+ T cell lines from myasthenic patients. Peptide 1-14 was also recognized frequently. The controls recognized, with a low precursor frequency, the peptide pool and a few peptides that frequently included the immunodominant sequences described above. The present results demonstrate that Th1 cells are involved in the anti-AChR response in MG and that their epitope repertoire is very complex. This indicates that when MG is clinically evident, the AChR itself is the sensitizing antigen and the target of the autoimmune Th1 cells, although it does not exclude that molecular mimicry between one AChR epitope and a microbial structure may have triggered this autoimmune response. Although the complexity of the Th1 repertoire suggests that development of specific immunosuppressive treatments targeted on epitopes recognized by autoimmune T cells will be difficult, the existence of immunodominant T epitope sequences might facilitate that task.

Adolescent↗
PubMed1997

Expression of the alpha 7 subunit of the nicotinic acetylcholine receptor in normal and myasthenic human thymuses.

The nicotinic acetylcholine receptor (AChR) is a transmembrane glycoprotein composed of five homologous subunits. Different isoforms of the AChR alpha subunit exist (alpha 1 to alpha 9). Of them, alpha 1 is expressed in muscle, alpha 2 to alpha 9 in neuronal cells. Muscle AChR is the target autoantigen in the autoimmune disease myasthenia gravis (MG). The thymus is implicated in MG pathogenesis, and the anti-AChR autoimmune response may start in this tissue, that expresses the muscle-type alpha 1 subunit as well as other muscle AChR subunits. The thymus also expresses the "neuronal" alpha 3 and alpha 5 subunits. By using polymerase chain reaction and other molecular techniques, we demonstrate here expression of the AChR alpha 7 subunit transcript in thymuses from both myasthenic patients and normal subjects. The alpha 7 subunit can form homo-oligomeric functional AChR complexes that, like muscle AChR, bind alpha-bungarotoxin. The demonstration of expression of the alpha 7 subunit in the thymus suggests that alpha-bungarotoxin binding, functional AChRs of the neuronal type are normally present in the thymus.

Adolescent↗
PubMed1996

MHC class II presentation of human acetylcholine receptor in Myasthenia gravis: binding of synthetic gamma subunit sequences to DR molecules.

Embryonic muscle acetylcholine receptor (AChR) is composed of four subunits-alpha, beta, gamma, and delta. Upon innervation, the gamma subunit is substituted by a homologous epsilon subunit. Embryonic muscle AChR might be the original autoantigen in Myasthenia gravis (MG) because it is expressed in the thymus, where the anti-AChR response might start, as well as in adult extrinsic ocular muscles, which are a preferential target in MG. MG patients have antibodies specific for embryonic AChR and CD4+ T cells, which recognize epitopes on the gamma subunit, in association with DR molecules. In the present study, we investigated the binding to several purified DR molecules (DRB1*0101, DRB1*0201, DRB1*0401 and DRB1*0701) of overlapping synthetic peptides, screening the human gamma subunit sequence. Binding of the peptides to the DR molecules were determined from their ability to compete with radiolabelled peptide probes for binding to purified DR molecules. All peptides which were recognized by CD4+ cells bound to the relevant DR molecule. On the other hand, some AChR peptides not recognized by CD4+ cells of MG patients bound well to one or more DR molecules. In terms of relative ability to bind to the DR molecules tested, the gamma subunit behaved like an exogenous antigen: some AChR peptide sequences uniquely bound one DR molecule, others bound several DR alleles, while others did not bind any of the DR molecules tested.

Acetylcholine↗
PubMed1996

Epitope repertoire of human CD4+ lines propagated with tetanus toxoid or with synthetic tetanus toxin sequences.

The use of synthetic antigen sequences allows propagation in vitro of T cell lines and clones specific for rare antigens, or for individual epitopes. In the present study we investigated the extent of similarity of the epitope repertoire of CD4+ T cell line specific for the antigen tetanus toxin (TTX), propagated with the complete molecule of tetanus toxoid (TTD), and with the synthetic TTX peptides. We propagated from two healthy subjects CD4+ T cell lines specific for TTD, by cycles of stimulation in vitro with TTD or with pools of overlapping synthetic peptides, 20 residues long and overlapping by five residues, corresponding to all or part of the tetanus toxin (TTX) sequence. One pool corresponded to the complete TTX sequence (peptide pool). Two other pools corresponded to residues 1-305 of the TTX light chain and 476-780 of the TTX heavy chain (peptide minipools). The peptide pool-propagated lines recognized TTD vigorously, at levels comparable with those of the TTD-propagated lines. They recognized several peptides, most of which were also recognized by the TTD-propagated line from the same subject. They also recognized to a low extent a few peptides not recognized by the corresponding TTD-propagated line, which might contain cryptic epitopes. The TTD-propagated lines recognized also several peptides that did not elicit a detectable response by the lines propagated with the complete peptide pool. The peptide minipool propagated lines recognized most of the peptides recognized by the TTD-propagated lines. They also recognized several peptides that did not elicit a measurable response of the TTD-propagated line from the same subject, which might contain cryptic epitopes. Very few peptides recognized by the TTD-propagated line did not evoke a response from the peptide minipool propagated lines. To verify that the response to the TTD molecule of the lines propagated with the peptide pools reflected the response of clones recognizing different epitopes produced upon in vitro processing of the TTD molecule, we propagated from each of the two subjects CD4+ T cell lines by stimulation with individual peptide recognized by the TTD-specific lines of that subject (13 peptide-specific lines from subject #1, and 15 from subject #2). All lines responded to the presence of TTD to an extent comparable to the response induced by the same concentration of the relevant peptide, demonstrating that propagation by synthetic epitope sequences allows expansion of T cell clones specific for epitopes which result from processing of the complete TTD molecule. Therefore, whereas the use of very large pools of synthetic antigen peptides for propagation of antigen specific human CD4+ cell lines might lead to loss of clones recognizing less immunogenic sequence regions, peptide pools comprising a relatively limited number of synthetic sequences allow propagation of the majority of the antigen specific T cell clones. The use of peptide pools, and especially of limited peptide pools, results in propagation of polyclonal T cell lines having a more diverse repertoire than the lines propagated by stimulation with the complete antigen molecule. The T clones propagated by the use of the short peptide sequences, which are not expanded when the complete antigen molecule is used, may recognize poorly processed, cryptic epitopes. This approach may be adopted to propagate and detect minor clonal populations, recognizing less immunogenic parts of the antigen.

Adult↗
PubMed1996

Antibodies as tools to study the structure of membrane proteins: the case of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is the prototype of the ionotropic receptor superfamily of proteins, which includes the closely related gamma- aminobutyric acid type A and glycine receptors, and more distantly related serotonin type-3 and glutamate receptors. Several models of the transmembrane topology of the nicotinic acetylcholine receptor subunits were originally proposed based on hydropathy analysis of their deduced amino acid sequences. Antibodies specific to different epitopes of the nicotinic acetylcholine receptor have proven to be valuable probes for examining the validity of those models. Despite important caveats, a viable model for the transmembrane structure and functional topology of the nicotinic acetylcholine receptor subunits has been obtained from the antibody mapping studies. This model, and the associated methodological shortcomings and obstacles that were overcome in the process of its formulation, can legitimately be extended to other members of the ionotropic receptor superfamily and to other membrane proteins as well.

Animals↗
PubMed1995

Binding of monoclonal antibodies against the carboxyl terminal segment of the nicotinic receptor delta subunit suggests an unusual transmembrane disposition of this sequence region.

Monoclonal antibodies (mAbs) specific for the carboxyl terminal region of the delta subunit of Torpedo nicotinic acetylcholine receptor (AChR), derived from mice immunized with AChR or a synthetic carboxyl terminal sequence of the delta subunit (C delta-mAbs), were used to determine the transmembrane disposition of their epitope(s) by immunoelectron microscopy, using AChR-rich postsynaptic membrane fragments from Torpedo electroplax. Some C delta-mAbs recognized only the cytoplasmic side of the membranes, some both sides to a similar extent, and others bound mostly, but not exclusively, to the cytoplasmic side. Binding of C delta-mAbs to the membranes was specifically blocked by synthetic peptides containing the carboxyl terminal region of the delta subunit. Control anti-AChR mAbs specific for the alpha or the delta subunits, whose epitopes have known transmembrane topology, uniquely recognized the expected side of the postsynaptic membrane. Residues involved in C delta-mAb binding were identified using single residue substituted peptide analogues of the sequence delta 481-501. All C delta-mAbs recognized epitopes within the same sequence segment, delta 485-493, at the carboxyl terminal of the AChR delta subunit. These results suggest that the delta subunit of the AChR might have alternative conformations, leading to exposure of the same sequence region on the extracellular or the cytoplasmic surface. Several Pro residues are present in this region. The alternative cis or trans conformation of one or more of them might result in different folding patterns of the carboxyl terminal sequence of the delta subunit, as described for a viral protein [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L., & Harrison, S. C. (1991) Nature 354, 278-284.

Amino Acid Sequence↗
PubMed1995

Immunoregulatory CD8+ cells recognize antigen-activated CD4+ cells in myasthenia gravis patients and in healthy controls.

CD8+ cells inhibiting the response of CD4+ cells exist in rodents, recognizing epitopes unique to a CD4+ clone (Ids) or expressed by all activated CD4+ cell (ergotypes). Stimulation of CD8+ cells recognizing ergotypes shared by all Ag-activated CD4+ cells would be useful for treatment of diseases involving undesirable CD4+ responses to ill defined Ags, such as many autoimmune diseases and allergies. As a first step toward demonstrating the existence of anti-ergotype CD8+ immunoregulatory cells in humans, we investigated here whether CD8+ cells recognizing Ag-activated CD4+ cells exist in autoimmune and healthy humans. CD4+ cells specific for human muscle acetylcholine receptor, tetanus, or diphtheria toxoids were propagated from patients with myasthenia gravis patients and healthy controls. Ag-activated CD4+ cells were irradiated and used as Ag to test the response of CD(4+)-depleted CD(8+)-enriched PBMC (CD8+ PBMC) from myasthenic patients and controls and to propagate short-term CD8+ cell lines from CD8+ PBMC. In both patients and controls CD8+ PBMC and CD8+ lines responded vigorously to autologous Ag-activated CD4+ cells. The CD8+ lines responded equally well to the Ag-activated CD4+ cells of different Ag specificity, suggesting that they recognized CD4+ ergotypes. They did not seem to respond to CD4+ cells activated by PHA. The CD8+ cells recognized class I-restricted epitopes, as their response to activated CD4+ cells was suppressed by anti-class I Ab. CD8+ cells recognizing Ag-activated CD4+ were present cells in the controls for 5 to 12 wk after immunization. In myasthenic patients, CD8+ cells recognizing activated anti-acetylcholine receptor CD4+ cells seemed to be always present.

Adult↗
PubMed1995

Epitopes for human CD4+ cells on diphtheria toxin: structural features of sequence segments forming epitopes recognized by most subjects.

The sequence regions of diphtheria toxin (DTX) recognized by CD4+ T cells of seven healthy humans of different major histocompatibility complex haplotypes were identified. Overlapping synthetic peptides, screening the DTX sequence, were used to test in proliferation assays unselected blood CD4+ cells, or DTX-specific CD4+ lines propagated by stimulation with DTX of blood mononuclear cells. Blood CD4+ cells and DTX-specific CD4+ lines gave consistent results. Although each subject had an individual pattern of peptide recognition, six peptide sequences (residues 271-290, 321-340, 331-350, 351-370, 411-430 and 431-450) were recognized by all subjects. In the native DTX molecule, these sequence regions are flanked by sequence loops exposed on the DTX surface. They overlap uncharged segments of the DTX sequence. These structural properties may be general requirements for immunodominance in CD4+ cell sensitization in humans.

Adult↗
PubMed1995

TSH receptor sequences recognized by CD4+ T cells in Graves' disease patients and healthy controls.

Twenty-nine overlapping synthetic peptides, twenty residues long, representing the entire extracellular sequence of the human thyroid stimulating hormone receptor (hTSHr), were used to test the epitope repertoire of CD4+ T lymphocytes from patients with Graves' disease and from healthy subjects. The peptides were used to propagate and test short term CD4+ T cell lines specific for hTSHr epitopes, and to directly test CD8+ depleted, CD4+ enriched peripheral blood lymphocytes. Analysis of the response of short-term CD4+ T cells lines and CD8+ depleted peripheral blood lymphocytes to the individual peptides revealed that 14 of the 15 patients and nine of the ten controls responded to at least one hTSHr peptide. There was no common response pattern, nor any region of the hTSHr sequence that was predominantly recognized. Several peptides were recognized by both patients and controls. These results support the notion that immunological tolerance to hTSHr is due to peripheral tolerance of potentially autoreactive CD4+ T cells, not their clonal deletion. The presence of self-reactive, hTSHr-specific CD4+ T cells in healthy individuals implies that these cells are not permanently anergized, since they can be activated in vitro.

Adult↗
PubMed1995

A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.

Acetylcholine is synthesized and released by human epidermal keratinocytes and modulates the adhesion and motility of these cells. To understand the molecular basis of the effects of acetylcholine on keratinocytes, we investigated the presence, pharmacology, structure, and function of nicotinic acetylcholine receptors in human epidermal keratinocytes. Patch-clamp studies indicated that keratinocytes express acetylcholine receptors with ion gating and pharmacologic properties similar to those observed so far only in neurons, and containing the alpha 3 subunit. Specific binding of the receptor-specific ligand 125I-kappa-bungarotoxin revealed approximately 5500 binding sites per cell on undifferentiated keratinocytes in cell cultures and approximately 35,400 binding sites per cell on mature keratinocytes freshly isolated from human neonatal foreskins. Antibody binding and polymerase chain reaction experiments demonstrated the presence of alpha 3, beta 2, and beta 4 nicotinic receptor subunits. Binding of subunit-specific antibodies indicated that nicotinic receptors were associated with the suprabasal keratinocytes in epidermis and localized to the cell membranes of differentiated keratinocytes in cell cultures. Acetylcholine and the nicotinic agonist nicotine increased cell-substrate and cell-cell adherence of cultured keratinocytes and stimulated their lateral migration. The specific antagonists kappa-bungarotoxin and mecamylamine caused cell detachment and abolished migration. Thus, a nicotinic receptor expressed in keratinocytes may mediate acetylcholine control of keratinocyte adhesion and motility.

Acetylcholine↗
PubMed1995

Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor.

The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment.

Amino Acid Sequence↗
PubMed1995

Mechanisms by which the I-ABM12 mutation influences susceptibility to experimental myasthenia gravis: a study in homozygous and heterozygous mice.

The I-Abm12 mutation in C57B1/6 (B6) mice yields the B6.C-H-2bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4+ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR alpha subunit which dominate the CD4+ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12 x B6)F1 mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4+ cells recognized the same TAChR alpha subunit peptide sequences (T alpha 150-169, T alpha 181-200 and T alpha 360-378). CD4+ cells from TAChR-sensitized F1 mice were challenged with TAChR and alpha subunit epitope peptides, using F1, B6 or bm12 APC. B6 and F1 APC presented all these Ag efficiently, while bm12 APC presented TAChR and peptide T alpha 150-169 poorly and erratically. Anti-TAChR and anti-alpha subunit epitope CD4+ lines propagated from F1 and B6 mice had similar TcR V beta usage. All lines but those specific for the sequence T alpha 150-169 had unrestricted V beta usage. Anti-T alpha 150-169 lines from both B6 and F1 mice had a strong preferential usage of V beta 6. Anti-T alpha 150-169 lines from F1 mice had also a slightly higher V beta 14 usage. B6, bm12 and F1 mice developed similar anti-TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti-AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4+ cells sensitized to epitopes within the sequence T alpha 150-160, consequent to the inability of the I-Abm12 molecule to present this sequence.

Animals↗