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B M Conti-Fine

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

At least 37 records · Page 2Linked to original sources
PubMed1999

CD4+ T cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects.

These studies have begun to clarify the complex cellular mechanisms involved in the immune response to factor VIII. Although vigorous sensitization of CD4+ cells occurs in healthy subjects, the absence of clinically significant levels of inhibitor antibodies is likely related to the prompt down-regulation of the immune response. It may also be possible that the specific epitope repertoire recognized by CD4+ cells plays a role in the outcome of the immune response to factor VIII. Further characterization and comparison of the CD4+ repertoire in healthy subjects with that of hemophilia patients with and without inhibitors will help clarify which mechanism explains the absence of productive inhibitor synthesis in certain individuals. Also, it might identify CD4+ epitopes recognized by T helper cells that are essential for inhibitor synthesis. Additional studies to further characterize the role of Th1 and Th2 cells in the immune response to factor VIII may also be needed for the design of novel therapeutic strategies aimed at preventing inhibitor development.

Antibodies↗
PubMed1998

CD4+ T cell repertoire on the epsilon subunit of muscle acetylcholine receptor in myasthenia gravis.

We have identified sequence regions of the human muscle acetylcholine receptor epsilon subunit recognized by CD4+ T cells from myasthenia gravis patients. We tested the proliferative response in vitro of blood CD4+ cells from 18 myasthenic patients and 5 controls, to individual overlapping synthetic peptides spanning the epsilon subunit sequence. All patients recognized a complex epitope repertoire. The peptides recognized by the CD4+ cells included sequence regions of the epsilon subunit that were diverged as compared to the homologous sequences of the other receptor subunits. Recognition of epitopes formed by sequence regions unique to the epsilon subunit suggests a direct role of this subunit in sensitizing the CD4+ cells. Several epsilon subunit peptides were recognized by many patients. Thus the epsilon subunit, like other acetylcholine receptor subunits, forms 'universal' CD4+ epitopes. The healthy subjects recognized some epsilon subunit peptides sporadically and at a low level.

Adult↗
PubMed1998

Amino acids within residues 181-200 of the nicotinic acetylcholine receptor alpha1 subunit involved in nicotine binding.

Structural determinants of L-[3H]nicotine binding to the sequence flanking Cys 192 and Cys 193 of the Torpedo acetylcholine receptor alpha1 subunit were investigated using synthetic peptides (residues 181-200) and fusion proteins (residues 166-211). Nicotine binding at a single concentration (30 nM) was compared with 71 peptides and fusion proteins in which individual amino acids at positions 181-200 were substituted. Substitution of Lys 185, Tyr 190, Cys 192, Cys 193, Thr 196, and Tyr 198 resulted in the greatest reduction in nicotine binding. Equilibrium binding of [3H]nicotine to peptide 181-200 revealed a binding component with an apparent KD of 1.2 microM. Substitution of Lys 185 (with Glu), His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 resulted in a significant reduction in affinity. Affinity was not affected significantly by substitution of Arg 182, Lys 185 (with Gly or Arg), Val 188, Tyr 189, Pro 194, Asp 195, Thr 196, and Asp 200. It is concluded that Lys 185, His 186, Tyr 190, Cys 192, Cys 193, and Tyr 198 play the greatest role in nicotine binding to residues 181-200 of the alpha1 subunit. Previous studies have implicated Tyr 190, Cys 192, Cys 193, and Tyr 198 in agonist binding to the acetylcholine receptor. These results confirm a role for these residues and also demonstrate a function for Lys 185 and His 186 in nicotine binding.

Amino Acid Sequence↗
PubMed1998

Binding of single substituted promiscuous and designer peptides to purified DRB1*0101.

MHC class II molecules present antigenic peptides to T cells. The sequence characteristics of peptides associated with various class II alleles have been examined by analysis of peptide mixtures extracted from purified class II molecules as well by direct binding assays with substituted synthetic peptides and purified class II molecules. Here, in vitro binding assays with purified DRB1*0101 and glycine substituted analogues of H gamma 321-340 and alanine substituted analogues of TT948-967, universal CD4+ epitopes of the gamma subunit of the human nicotinic acetylcholine receptor and tetanus toxin, respectively, were able to compete for binding to an extent similar to that of the unsubstituted peptides. Testing whether this is a property of promiscuous, but not allele-specific peptide epitopes, a designer peptide containing the proposed anchor residues for binding DRB1*0101 was used in similar binding assays. As expected, the binding capacity of this designer peptide was much higher than that of the universal epitope peptides. However, substitution of the anchor residues for alanine in the context of this designer peptide did not abrogate binding to DRB1*0101 and, in fact, enhanced it. Therefore, it appears that for both class II allele-specific and universal epitope peptides, binding is a result of the combinatory effects of a few residues. The individual replacement of these residues with the sterically and electrostatically neutral residue alanine does not negatively affect binding in the continued presence of other anchor residues.

Binding, Competitive↗
PubMed1998

Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.

We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans). A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool. We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209. The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206. The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans. In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209. The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans. These results indicate that even purified constructs from E. coli contain bacterial contaminants recognized by CD4+ cells. They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens. Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations. Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells. Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells.

Amino Acid Sequence↗
PubMed1998

TCR-Vbeta usage in the thymus and blood of myasthenia gravis patients.

In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vbeta families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vbeta usage was found in the thymi of MG patients and controls, except Vbeta2, which showed a small increase in MG patients' thymi. Blood T cells of MG patients used Vbeta4, Vbeta6, Vbeta15, Vbeta16 and Vbeta24 significantly more than those of the controls. Vbeta4 and Vbeta6 are the gene families most frequently used by anti-AChR CD4(+) cells in MG patients. Blood T cells from MG patients used Vbeta12, Vbeta14, Vbeta17 and Vbeta18 significantly less than controls. MG patients used Vbeta4 and Vbeta6 significantly more in the blood than in the thymus, while the opposite occurred for Vbeta7, Vbeta12 and Vbeta14. Controls used Vbeta17 more and Vbeta24 less in the blood than in the thymus. The preferential expansion of Vbeta4 and Vbeta6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vbeta usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vbeta usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vbeta usage in blood and thymi of MG patients are likely to be due to preferential Vbeta usage by the anti-AChR T cells in the blood.

Adolescent↗
PubMed1998

Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors.

We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.

Animals↗
PubMed1998

T-cell recognition of muscle acetylcholine receptor subunits in generalized and ocular myasthenia gravis.

OBJECTIVES: Our purpose was to identify the muscle acetylcholine receptor (AChR) subunits recognized by autoimmune CD4+ T cells in myasthenia gravis (MG) and determine whether they differ in generalized (gMG) and ocular MG (oMG), and as gMG progresses. METHODS: We tested the proliferative response of blood CD4+ cells from 25 patients with gMG and four patients with oMG to synthetic peptides spanning the sequence of each subunit of human muscle AChR. We also investigated the antisubunit response of Th1 cells (a CD4+ subset frequently involved in autoimmune phenomena) using an enzyme-linked immunospot (ELISPOT) assay of antigen-induced secretion of interferon-gamma by individual CD4 cells. RESULTS: In gMG patients both the total CD4+ population and the Th1 subset recognized all AChR subunits to comparable extents. oMG patients recognized the AChR epsilon subunit minimally, and other subunits consistently and more strongly. gMG patients whose disease had lasted less than 5 years had lower antisubunit responses, and several of them did not recognize some AChR subunits; patients whose disease had lasted for 5 or more years had higher antisubunit responses and always responded to all AChR subunits. CONCLUSIONS: CD4+ and Thl responses in MG involve the entire AChR molecule. This likely results from spreading of the CD4+ sensitization to increasingly larger parts of the AChR as the disease progresses. The differential recognition of AChR subunits in oMG might be related to the preferential involvement of extrinsic ocular muscles, which express AChR containing the gamma subunit.

Adult↗
PubMed1998

Human vascular endothelial cells express functional nicotinic acetylcholine receptors.

ACh receptors sensitive to nicotine (nAChR) are present in human skin keratinocytes and in bronchial epithelial cells. They are stimulated by ACh secreted by the same cells that express them, and they modulate cell motility and shape. A variety of non-neuronal tissues, including endothelial cells, synthesize ACh, which raises the possibility that they are sensitive to nicotine. We demonstrate here that endothelial cells that line blood vessels express functional nAChRs. Their structure and ion-gating properties are similar to those of the nAChRs expressed by ganglionic neurons and by skin keratinocytes and bronchial epithelial cells. In situ hybridization experiments using primary cultures of endothelial cells from human aorta demonstrated the presence in these cells of the subunits believed to contribute to ganglionic ACh receptors (AChRs) of the alpha3 subtype: alpha3, alpha5, beta2 and beta4. Binding of radiolabeled epibatidine-a high-affinity specific ligand of certain neuronal AChRs, including the alpha3 subtypes-revealed the presence of approximately 900 specific binding sites per cell. We assessed the presence of functional AChRs by patch-clamp experiments. Cultured human endothelial cells express ion channels that are opened by (+)-anatoxin-a and are blocked by dihydro-beta-erythroidine. These are specific agonist and antagonist, respectively, of neuronal AChRs of the alpha3 subtype. The ion-gating properties of the endothelial AChRs were similar to those of neuronal ganglionic AChRs. The presence of AChRs sensitive to nicotine in endothelial cells may be related to the toxic effects of nicotine on the vascular system.

Animals↗
PubMed1997

Prevention of experimental myasthenia gravis by nasal administration of synthetic acetylcholine receptor T epitope sequences.

T cell tolerization prevents and improves T cell-mediated experimental autoimmune diseases. We investigated here whether similar approaches could be used for antibody (Ab)-mediated autoimmune diseases. Myasthenia gravis, caused by IgG Ab against muscle acetylcholine receptor (AChR), is perhaps the best characterized of them. We used an animal model, experimental myasthenia gravis induced in C57Bl/6 mice by immunization with Torpedo acetylcholine receptor (TAChR), to demonstrate that nasal administration of synthetic sequences of the TAChR alpha-subunit- forming epitopes recognized by anti-TAChR CD4+ T helper cells (residues alpha150-169, alpha181-200, and alpha360-378), given before and during immunization with TAChR, causes decreased CD4+ responsiveness to those epitopes and to TAChR, reduced synthesis of anti-TAChR Ab, and prevented experimental myasthenia gravis. These effects were not induced by nasal administration of synthetic epitopes of diphtheria toxin. Secretion of IL-2, IL-4, and IL-10 by spleen T cells from TAChR immunized mice, in response to challenge with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to TAChR, while peptide-treated mice had also an AChR-specific Th2 response. The TAChR peptide treatment induced also in vitro anergy to the TAChR of the spleen T cells, which was reversed by IL-2.

Administration, Intranasal↗