14 results on '"Siegel DL"'
Search Results
2. Proteomic Analysis of Pemphigus Autoantibodies Indicates a Larger, More Diverse, and More Dynamic Repertoire than Determined by B Cell Genetics.
- Author
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Chen J, Zheng Q, Hammers CM, Ellebrecht CT, Mukherjee EM, Tang HY, Lin C, Yuan H, Pan M, Langenhan J, Komorowski L, Siegel DL, Payne AS, and Stanley JR
- Subjects
- Amino Acid Sequence, Cell Surface Display Techniques, Chromatography, Liquid, Clone Cells, Complementarity Determining Regions genetics, Desmogleins metabolism, Humans, Mutation genetics, Pemphigus blood, Single-Chain Antibodies chemistry, Single-Chain Antibodies metabolism, Tandem Mass Spectrometry, Autoantibodies immunology, B-Lymphocytes immunology, Pemphigus genetics, Pemphigus immunology, Proteomics methods
- Abstract
In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity., Competing Interests: JL and LK are employees of the Euroimmun AG, a company that develops, produces and manufactures immunoassays for the detection of disease-associated antibodies. The other authors have nothing to disclose., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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3. Modulation of natural IgM autoantibodies to oxidative stress-related neo-epitopes on apoptotic cells in newborns of mothers with anti-Ro autoimmunity.
- Author
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Grönwall C, Clancy RM, Getu L, Lloyd KA, Siegel DL, Reed JH, Buyon JP, and Silverman GJ
- Subjects
- Adult, Antibodies, Anti-Idiotypic blood, Autoantibodies blood, Autoantigens immunology, Cohort Studies, Enzyme-Linked Immunosorbent Assay, Epitopes chemistry, Female, Fetal Blood immunology, Humans, Immunoglobulin G immunology, Infant, Newborn, Malondialdehyde adverse effects, Malondialdehyde chemistry, Malondialdehyde immunology, Mothers, Phosphorylcholine adverse effects, Phosphorylcholine blood, Pregnancy, Pregnancy Complications, Ribonucleoproteins chemistry, Apoptosis immunology, Autoantibodies immunology, Epitopes immunology, Fetus immunology, Immunoglobulin M immunology, Maternal-Fetal Exchange immunology, Oxidative Stress immunology, Ribonucleoproteins immunology
- Abstract
At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
- Published
- 2016
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4. ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 1. Structural and functional characterization in vitro.
- Author
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Ostertag EM, Kacir S, Thiboutot M, Gulendran G, Zheng XL, Cines DB, and Siegel DL
- Subjects
- Adult, Antibody Specificity, Cell Surface Display Techniques, Child, Cloning, Molecular, Cross Reactions immunology, Epitope Mapping, Humans, Immunoglobulin G blood, Middle Aged, ADAMTS13 Protein immunology, Autoantibodies isolation & purification, Purpura, Thrombotic Thrombocytopenic immunology
- Abstract
Background: Acquired thrombotic thrombocytopenia purpura (TTP) is a life-threatening illness caused by autoantibodies that decrease the activity of ADAMTS13, the von Willebrand factor-cleaving protease. Despite efficacy of plasma exchange, mortality remains high and relapse is common. Improved therapies may come from understanding the diversity of pathogenic autoantibodies on a molecular or genetic level. Cloning comprehensive repertoires of patient autoantibodies can provide the necessary tools for studying immunobiology of disease and developing animal models., Study Design and Methods: Anti-ADAMTS13 antibodies were cloned from four patients with acquired TTP using phage display and characterized with respect to genetic origin, inhibition of ADAMTS13 proteolytic activity, and epitope specificity. Anti-idiotypic antisera raised to a subset of autoantibodies enabled comparison of their relatedness to each other and to polyclonal immunoglobulin (Ig)G in patient plasma., Results: Fifty-one unique antibodies were isolated comprising epitope specificities resembling the diversity found in circulating patient IgG. Antibodies directed both to the amino terminal domains and to those requiring the ADAMTS13 cysteine-rich/spacer region for binding inhibited proteolytic activity, while those solely targeting carboxy-terminal domains were noninhibitory. Anti-idiotypic antisera raised to a subset of antibody clones crossreacted with and reduced the inhibitory activity of polyclonal IgG from a set of unrelated patients., Conclusions: Anti-ADAMTS13 autoantibodies isolated by repertoire cloning display the diversity of epitope specificities found in patient plasma and provide tools for developing animal models of acquired TTP. Shared idiotypes of inhibitory clones with circulating IgG from multiple patients suggest common features of pathogenic autoantibodies that could be exploited for developing more targeted therapies., (© 2016 AABB.)
- Published
- 2016
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5. ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model.
- Author
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Ostertag EM, Bdeir K, Kacir S, Thiboutot M, Gulendran G, Yunk L, Hayes VM, Motto DG, Poncz M, Zheng XL, Cines DB, and Siegel DL
- Subjects
- ADAMTS13 Protein immunology, Animals, Cloning, Molecular, DNA, Complementary administration & dosage, Humans, Mice, Models, Animal, Molecular Targeted Therapy methods, Single-Chain Antibodies genetics, Single-Chain Antibodies toxicity, von Willebrand Factor metabolism, ADAMTS13 Protein antagonists & inhibitors, Autoantibodies genetics, Purpura, Thrombotic Thrombocytopenic immunology, Purpura, Thrombotic Thrombocytopenic therapy
- Abstract
Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA., Study Design and Methods: Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined., Results: Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death., Conclusions: These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches., (© 2016 AABB.)
- Published
- 2016
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6. Persistence of anti-desmoglein 3 IgG(+) B-cell clones in pemphigus patients over years.
- Author
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Hammers CM, Chen J, Lin C, Kacir S, Siegel DL, Payne AS, and Stanley JR
- Subjects
- Adult, Aging immunology, Amino Acid Sequence, Antibodies, Monoclonal, Murine-Derived therapeutic use, Cell Lineage, Dose-Response Relationship, Drug, Female, Humans, Immunologic Factors therapeutic use, Longitudinal Studies, Male, Middle Aged, Molecular Sequence Data, Pemphigus drug therapy, Pemphigus immunology, Remission Induction, Rituximab, Treatment Outcome, Aging pathology, Autoantibodies metabolism, B-Lymphocytes immunology, B-Lymphocytes pathology, Desmoglein 3 immunology, Immunoglobulin G immunology, Pemphigus pathology
- Abstract
Pemphigus vulgaris (PV) is a prototypic tissue-specific autoantibody-mediated disease, in which anti-desmoglein 3 (Dsg3) IgG autoantibodies cause life-threatening blistering. We characterized the autoimmune B-cell response over 14 patient years in two patients with active and relapsing disease, then in one of these patients after long-term remission induced by multiple courses of rituximab (anti-CD20 antibody). Characterization of the anti-Dsg3 IgG(+) repertoire by antibody phage display (APD) and PCR indicated that six clonal lines persisted in patient 1 (PV3) over 5.5 years, with only one new clone detected. Six clonal lines persisted in patient 2 (PV1) for 4 years, of which five persisted for another 4.5 years without any new clones detected. However, after long-term clinical and serologic remission, ∼11 years after initial characterization, we could no longer detect any anti-Dsg3 clones in PV1 by APD. Similarly, in another PV patient, ∼4.5 years after a course of rituximab that induced long-term remission, anti-Dsg3 B-cell clones were undetectable. These data suggest that in PV a given set of non-tolerant B-cell lineages causes autoimmune diseases and that new sets do not frequently or continually escape tolerance. Therapy such as rituximab, aimed at eliminating these aberrant sets of lineages, may be effective for disease because new ones are unlikely to develop.
- Published
- 2015
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7. Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.
- Author
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Yamagami J, Payne AS, Kacir S, Ishii K, Siegel DL, and Stanley JR
- Subjects
- Amino Acid Sequence, Animals, Desmogleins genetics, Desmogleins immunology, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Autoantibodies genetics, Autoantibodies immunology, Complementarity Determining Regions genetics, Complementarity Determining Regions immunology, Pemphigus genetics, Pemphigus immunology, Pemphigus pathology
- Abstract
Pemphigus is a life-threatening autoimmune disease in which antibodies specific for desmogleins (Dsgs) cause loss of keratinocyte cell adhesion and blisters. In order to understand how antibodies cause pathogenicity and whether there are commonalities among antibodies in different patients that could ultimately be used to target specific therapy against these antibodies, we characterized Dsg-specific mAbs cloned by phage display from 3 patients with pemphigus vulgaris and 2 with pemphigus foliaceus. Variable heavy chain gene usage was restricted, but similar genes were used for both pathogenic and nonpathogenic mAbs. However, the heavy chain complementarity-determining region 3 (H-CDR3) of most pathogenic, but not nonpathogenic, mAbs shared an amino acid consensus sequence. Randomization of the H-CDR3 and site-directed mutagenesis indicated that changes in this sequence could block pathogenicity but not necessarily binding. In addition, for 2 antibodies with longer H-CDR3s, a tryptophan was critical for pathogenicity but not binding, a result that is consistent with blocking the tryptophan acceptor site that is thought to be necessary for Dsg-mediated adhesion. These studies indicate that H-CDR3 is critical for pathogenicity of a human autoantibody, that a small region (even 1 amino acid) can mediate pathogenicity, and that pathogenicity can be uncoupled from binding in these antibodies.
- Published
- 2010
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8. Antibodies to the desmoglein 1 precursor proprotein but not to the mature cell surface protein cloned from individuals without pemphigus.
- Author
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Yamagami J, Kacir S, Ishii K, Payne AS, Siegel DL, and Stanley JR
- Subjects
- Antibodies, Monoclonal biosynthesis, Antibodies, Monoclonal genetics, Antibody Specificity, Autoantibodies genetics, Cloning, Molecular, Desmoglein 1 isolation & purification, Humans, Immune Tolerance, Membrane Proteins genetics, Membrane Proteins immunology, Pemphigus pathology, Protein Precursors antagonists & inhibitors, Protein Precursors isolation & purification, Purpura, Thrombotic Thrombocytopenic immunology, Purpura, Thrombotic Thrombocytopenic metabolism, Autoantibodies biosynthesis, Desmoglein 1 immunology, Membrane Proteins isolation & purification, Pemphigus immunology, Pemphigus metabolism, Protein Precursors immunology
- Abstract
In pemphigus foliaceus (PF), autoantibodies against desmoglein 1 (Dsg1) cause blisters. Using Ab phage display, we have cloned mAbs from a PF patient. These mAbs, like those from a previous patient, were directed against mature Dsg1 (matDsg1) on the cell surface of keratinocytes and precursor Dsg1 (preDsg1) in the cytoplasm. To determine whether individuals without pemphigus have B cell tolerance to Dsg1, we cloned mAbs from two patients with thrombotic thrombocytopenic purpura and a healthy person. We found mAbs against preDsg1, but not matDsg1. All but 1 of the 23 anti-preDsg1 mAbs from PF patients and those without PF used the VH3-09 (or closely related VH3-20) H chain gene, whereas no PF anti-matDsg1 used these genes. V(H) cDNA encoding anti-preDsg1 had significantly fewer somatic mutations than did anti-matDsg1 cDNA, consistent with chronic Ag-driven hypermutation of the latter compared with the former. These data indicate that individuals without PF do not have B cell tolerance to preDsg1 and that loss of tolerance to matDsg1 is not due to epitope shifting of anti-preDsg1 B cells (because of different V(H) gene usage). However, presentation of peptides from Dsg1 by preDsg1-specific B cells may be one step in developing autoimmunity in PF.
- Published
- 2009
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9. Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.
- Author
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Ishii K, Lin C, Siegel DL, and Stanley JR
- Subjects
- Aged, Animals, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Autoantibodies genetics, Autoantibodies immunology, Desmoglein 1 analysis, Desmoglein 1 antagonists & inhibitors, Epitope Mapping, Epitopes immunology, Female, Fluorescent Antibody Technique, Indirect, Humans, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains immunology, Immunoglobulin Heavy Chains isolation & purification, Immunoglobulin Variable Region immunology, Immunoglobulin Variable Region isolation & purification, Mice, Skin immunology, Antibodies, Monoclonal isolation & purification, Autoantibodies isolation & purification, Desmoglein 1 immunology, Pemphigus immunology, Peptide Library
- Abstract
Pemphigus foliaceus (PF) is a blistering disease caused by autoantibodies to desmoglein 1 (Dsg1) that cause loss of epidermal cell adhesion. To better understand PF pathophysiology, we used phage display to isolate anti-Dsg1 mAbs as single-chain variable fragments (scFvs) from a PF patient. Initial panning of the library isolated only non-pathogenic scFvs. We then used these scFvs to block non-pathogenic epitopes and were able to isolate two unique scFvs, each of which caused typical PF blisters in mice or human epidermis models, showing that a single mAb can disrupt Dsg1 function to cause disease. Both pathogenic scFvs bound conformational epitopes in the N terminus of Dsg1. Other PF sera showed a major antibody response against the same or nearby epitopes defined by these pathogenic scFvs. Finally, we showed restriction of the heavy-chain gene usage of all anti-Dsg1 clones to only five genes, which determined their immunological properties despite promiscuous light-chain gene usage. These mAbs will be useful for studying Dsg1 function and mechanisms of blister formation in PF and for developing targeted therapies and tools to monitor disease activity.
- Published
- 2008
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10. Targeting pemphigus autoantibodies through their heavy-chain variable region genes.
- Author
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Payne AS, Siegel DL, and Stanley JR
- Subjects
- Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antibodies, Monoclonal therapeutic use, Autoantibodies genetics, Autoantibodies therapeutic use, Cell Line, Cells, Cultured, Desmoglein 1 immunology, Desmoglein 3 immunology, Genetic Therapy methods, Humans, Immunoglobulin Idiotypes genetics, Immunoglobulin Idiotypes immunology, Immunoglobulin Idiotypes therapeutic use, Keratinocytes immunology, Keratinocytes pathology, Pemphigus blood, Pemphigus therapy, Autoantibodies immunology, Genes, Immunoglobulin Heavy Chain immunology, Immunoglobulin Variable Region genetics, Pemphigus immunology
- Abstract
Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against cell surface adhesion proteins desmoglein (Dsg) 3 and Dsg1. Previous studies using phage display to clone Dsg-reactive monoclonal antibodies from a PV patient demonstrated that a limited number of antibody variable region genes encode the autoantibody repertoire, with different genes for pathogenic and non-pathogenic mAbs. Here, we investigated the feasibility of specific autoantibody targeting in pemphigus. We produced rabbit anti-idiotypic antibodies against two pathogenic and two non-pathogenic PV mAbs. Antisera inhibited binding of the immunizing mAb to Dsgs by ELISA as well as pathogenicity against cultured human keratinocytes. Antisera also inhibited other mAbs using the same variable region heavy chain (V(H)) genes, despite different light chains or somatic mutations. Additionally, peptide phage display identified peptide sequences that bound PV mAbs in a V(H)-specific manner. To evaluate the therapeutic potential of V(H) gene-targeted reagents, preimmune sera and antisera were used to adsorb pathogenic antibodies from PV sera. Pooled antisera significantly reduced pathogenic activity from the original PV patient's serum and bound pathogenic antibodies from two other PV sera, suggesting shared autoantibody V(H) gene usage among PV patients. Together, these data suggest novel V(H) gene-targeted approaches toward PV treatment.
- Published
- 2007
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11. Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.
- Author
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Payne AS, Ishii K, Kacir S, Lin C, Li H, Hanakawa Y, Tsunoda K, Amagai M, Stanley JR, and Siegel DL
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn, Cadherins immunology, Cells, Cultured, Complementarity Determining Regions genetics, Desmoglein 3, Epidermal Cells, Epidermis metabolism, Epitope Mapping, Humans, Immunoglobulin Variable Region genetics, Immunoglobulin Variable Region immunology, Keratinocytes cytology, Keratinocytes metabolism, Mice, Molecular Sequence Data, Peptide Library, Random Allocation, Sequence Alignment, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Autoantibodies genetics, Autoantibodies immunology, Pemphigus immunology
- Abstract
Pemphigus is a life-threatening blistering disorder of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein 3 (Dsg3) and Dsg1. Mechanisms of antibody pathogenicity are difficult to characterize using polyclonal patient sera. Using antibody phage display, we have isolated repertoires of human anti-Dsg mAbs as single-chain variable-region fragments (scFvs) from a patient with active mucocutaneous pemphigus vulgaris. ScFv mAbs demonstrated binding to Dsg3 or Dsg1 alone, or both Dsg3 and Dsg1. Inhibition ELISA showed that the epitopes defined by these scFvs are blocked by autoantibodies from multiple pemphigus patients. Injection of scFvs into neonatal mice identified 2 pathogenic scFvs that caused blisters histologically similar to those observed in pemphigus patients. Similarly, these 2 scFvs, but not others, induced cell sheet dissociation of cultured human keratinocytes, indicating that both pathogenic and nonpathogenic antibodies were isolated. Genetic analysis of these mAbs showed restricted patterns of heavy and light chain gene usage, which were distinct for scFvs with different desmoglein-binding specificities. Detailed characterization of these pemphigus mAbs should lead to a better understanding of the immunopathogenesis of disease and to more specifically targeted therapeutic approaches.
- Published
- 2005
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12. Genetic analysis of autoantibodies in idiopathic thrombocytopenic purpura reveals evidence of clonal expansion and somatic mutation.
- Author
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Roark JH, Bussel JB, Cines DB, and Siegel DL
- Subjects
- Adult, Aged, Amino Acid Sequence, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antibody Specificity, Autoantibodies chemistry, Autoantibodies immunology, Cloning, Molecular, Female, Humans, Immunoglobulin Heavy Chains immunology, Immunoglobulin Light Chains immunology, Male, Middle Aged, Molecular Sequence Data, Peptide Library, Platelet Glycoprotein GPIb-IX Complex immunology, Recombinant Proteins, Sequence Alignment, Spleen chemistry, Autoantibodies genetics, Blood Platelets immunology, Mutation, Platelet Membrane Glycoproteins, Purpura, Thrombocytopenic, Idiopathic genetics, Purpura, Thrombocytopenic, Idiopathic immunology
- Abstract
Although idiopathic thrombocytopenic purpura (ITP) is the most common autoimmune hematologic disorder, little is known about the associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig) gene usage, clonality, and antigen specificity. Phage display libraries were constructed from splenocytes from 2 patients with chronic ITP, and competitive cell-surface selection was used to isolate several dozen unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in both patients were associated almost exclusively with rearrangements of a single Ig heavy-chain variable-region gene (V(H)3-30), despite an apparent diversity of antigen specificities. Comparative analysis of platelet-reactive Fab Ig gene rearrangements from each patient suggested that they evolved from a restricted number of B-cell clones through somatic mutation with high replacement-to-silent mutation ratios. Although V(H)3-30-encoded heavy chains were found with light chains encoded by several different Ig genes, molecular repairing experiments showed exquisite restriction on the specific heavy- and light-chain pairings that permitted platelet reactivity. Together, these data suggest that the development of platelet-reactive antibodies associated with ITP is driven by an encounter with diverse platelet antigens through the clonal expansion of B cells using genetically restricted and highly specific combinations of heavy- and light-chain gene products. The extraordinarily high usage of the V(H)3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.
- Published
- 2002
13. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies.
- Author
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Arepally GM, Kamei S, Park KS, Kamei K, Li ZQ, Liu W, Siegel DL, Kisiel W, Cines DB, and Poncz M
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, Antigen-Antibody Reactions, Autoantibodies chemistry, Autoimmune Diseases immunology, Binding, Competitive, CHO Cells, Cells, Cultured, Cricetinae, Cricetulus, Cross Reactions, Endothelium, Vascular immunology, Enzyme-Linked Immunosorbent Assay, Epitopes immunology, Female, Genes, Immunoglobulin, Glycosaminoglycans immunology, Heparan Sulfate Proteoglycans immunology, Heparin pharmacology, Humans, Immunoglobulin G chemistry, Immunoglobulin G genetics, Immunoglobulin kappa-Chains genetics, Macromolecular Substances, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Mutagenesis, Site-Directed, Platelet Activation drug effects, Platelet Factor 4 genetics, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins immunology, Sequence Alignment, Sequence Homology, Amino Acid, Species Specificity, Thrombocytopenia immunology, Thrombophilia chemically induced, Thrombophilia immunology, Umbilical Veins, Antibodies, Monoclonal immunology, Autoantibodies immunology, Autoantigens immunology, Autoimmune Diseases chemically induced, Heparin adverse effects, Immunoglobulin G immunology, Platelet Factor 4 immunology, Thrombocytopenia chemically induced
- Abstract
Antibodies to PF4/heparin can be demonstrated in almost all patients with heparin-induced thrombocytopenia/thrombosis (HIT/HITT) and in some persons exposed to heparin who do not have clinical manifestations. The role of anti-PF4/heparin antibodies in the pathogenesis of HIT/HITT has been difficult to establish because the antibodies found in serum are generally polyclonal and polyspecific. To circumvent this problem, we developed a murine monoclonal antibody (mAb) to human (h) PF4/heparin complexes. A monoclonal IgG(2bkappa )antibody (designated KKO) was identified that bound specifically to hPF4/heparin complexes. Maximal binding of KKO to hPF4/heparin complexes occurred at similar molar ratios of PF4:heparin observed for HIT/HITT antibodies. KKO also bound to hPF4 in association with other glycosaminoglycans. Platelet activation by KKO required heparin and was abrogated by blockade of FcgammaRIIA. In the presence of PF4, KKO bound to endothelial cells, but not to CHO cells lacking heparan sulfate proteoglycans. Variants of PF4 complexed to heparin were recognized equally well by KKO and HIT/HITT sera. KKO competes for binding with a subset of HIT/HITT antibodies that are relatively spared by mutations in the 3rd domain of PF4. The nucleotide and predicted amino acid sequences of KKO and RTO, a murine anti-hPF4 mAb that does not require heparin for binding, revealed no obvious relationship in either the heavy- or the light-chain immunoglobulin variable regions. These studies suggest that KKO recapitulates the antigenic and functional specificity of a subset of HIT/HITT antibodies and may, therefore, provide insight into the pathogenesis of thrombocytopenia and thrombosis in affected persons. (Blood. 2000;95:1533-1540)
- Published
- 2000
14. Isolation of human anti-red blood cell antibodies by repertoire cloning.
- Author
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Siegel DL
- Subjects
- Autoantibodies genetics, Autoantibodies immunology, B-Lymphocyte Subsets immunology, Bacteriophage M13 genetics, Bacteriophage M13 immunology, DNA, Complementary genetics, DNA, Recombinant isolation & purification, Escherichia coli genetics, Gene Rearrangement, B-Lymphocyte, Humans, Immunoglobulin Fab Fragments immunology, Immunoglobulin Fab Fragments isolation & purification, Isoantibodies genetics, Isoantibodies immunology, Polymerase Chain Reaction, RNA, Messenger genetics, RNA, Messenger isolation & purification, Rho(D) Immune Globulin genetics, Rho(D) Immune Globulin isolation & purification, Autoantibodies isolation & purification, Cloning, Molecular methods, Erythrocytes immunology, Gene Library, Genes, Immunoglobulin, Immunosorbent Techniques, Isoantibodies isolation & purification
- Published
- 1995
- Full Text
- View/download PDF
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