25 results on '"Ishii, Ken"'
Search Results
2. Pemphigus Autoantibodies to Desmocollin 3 but Not to Desmocollin 1 Directly Block Heterophilic Desmoglein/Desmocollin Transinteraction.
- Author
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Ishii K, Ishii N, Ishiko A, and Hashimoto T
- Subjects
- Humans, Desmogleins immunology, Desmogleins metabolism, Desmoglein 1 immunology, Desmoglein 1 metabolism, Desmocollins immunology, Desmocollins metabolism, Pemphigus immunology, Pemphigus pathology, Autoantibodies immunology
- Published
- 2024
- Full Text
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3. Pemphigus Vulgaris and Foliaceus IgG Autoantibodies Directly Block Heterophilic Transinteraction between Desmoglein and Desmocollin.
- Author
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Ishii K, Yoshida K, Stanley JR, Yamagami J, Amagai M, and Ishiko A
- Subjects
- Cell Adhesion, Desmocollins antagonists & inhibitors, Desmocollins physiology, Desmoglein 1 antagonists & inhibitors, Desmoglein 1 physiology, Desmoglein 3 antagonists & inhibitors, Desmoglein 3 physiology, Epitope Mapping, Humans, Autoantibodies immunology, Desmocollins immunology, Desmoglein 1 immunology, Desmoglein 3 immunology, Immunoglobulin G immunology, Pemphigus immunology
- Abstract
Anti-desmoglein (Dsg) 1 and Dsg3 IgG autoantibodies in pemphigus foliaceus and pemphigus vulgaris cause blisters through loss of desmosomal adhesion. It is controversial whether blister formation is due to direct inhibition of Dsg, intracellular signaling events causing desmosome destabilization, or both. Recent studies show that heterophilic binding between Dsg and desmocollin (Dsc) is the fundamental adhesive unit of desmosomes. To eliminate cellular contributions to potential pathogenicity of pemphigus antibodies, bead assays coated with recombinant Dsg1, Dsc1, Dsg3, or Dsc3 ectodomains were developed. A mixture of Dsg beads and Dsc beads formed large aggregates, confirming that the heterophilic binding is dominant. The pathogenic anti-Dsg1 and anti-Dsg3 mAbs, which bind the transadhesive interface, blocked the aggregation of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively, whereas nonpathogenic mAbs did not. All sera tested from eight patients with pemphigus foliaceus and eight patients with mucosal pemphigus vulgaris with active disease inhibited the adhesion of Dsg1/Dsc1 and Dsg3/Dsc3 beads, respectively. When paired sera obtained from seven patients with pemphigus foliaceus and six patients with pemphigus vulgaris in active disease and remission were compared, the former inhibited aggregation better than the latter. These findings strongly suggest that steric hindrance of heterophilic transinteraction between Dsg and Dsc is important for disease pathology in both pemphigus foliaceus and pemphigus vulgaris., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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4. Two cases of pemphigus vulgaris in remission showing high titer of anti-desmoglein 3 antibodies.
- Author
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Inaoki M, Oishi K, Nishijima C, Takehara K, and Ishii K
- Subjects
- Autoantibodies immunology, Azathioprine therapeutic use, Enzyme-Linked Immunosorbent Assay, Female, Glucocorticoids therapeutic use, Humans, Immunosuppressive Agents therapeutic use, Middle Aged, Mouth Mucosa immunology, Mouth Mucosa pathology, Pemphigus drug therapy, Pemphigus immunology, Prednisolone therapeutic use, Remission Induction methods, Ribonucleosides therapeutic use, Skin immunology, Skin pathology, Autoantibodies analysis, Desmoglein 3 immunology, Pemphigus pathology
- Published
- 2019
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5. Alopecia developed in a transitional case from pemphigus foliaceus to pemphigus vulgaris.
- Author
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Yoshida K, Ishii K, and Ishiko A
- Subjects
- Alopecia pathology, Female, Humans, Middle Aged, Pemphigus pathology, Scalp pathology, Skin pathology, Alopecia immunology, Desmogleins immunology, Pemphigus complications
- Published
- 2017
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6. Non-pathogenic pemphigus foliaceus (PF) IgG acts synergistically with a directly pathogenic PF IgG to increase blistering by p38MAPK-dependent desmoglein 1 clustering.
- Author
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Yoshida K, Ishii K, Shimizu A, Yokouchi M, Amagai M, Shiraishi K, Shirakata Y, Stanley JR, and Ishiko A
- Subjects
- Cell Adhesion, Desmoglein 1 metabolism, Desmosomes ultrastructure, Fluorescent Antibody Technique, Humans, Imidazoles pharmacology, Keratinocytes immunology, Microscopy, Electron, Organ Culture Techniques, Pemphigus blood, Primary Cell Culture, Pyridines pharmacology, p38 Mitogen-Activated Protein Kinases antagonists & inhibitors, Autoantibodies immunology, Desmoglein 1 immunology, Immunoglobulin G immunology, Keratinocytes physiology, Pemphigus immunology, Single-Chain Antibodies immunology, Skin immunology, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
Background: Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs)., Objective: To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF., Methods: Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay., Results: 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor., Conclusion: These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)
- Published
- 2017
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7. Importance of serological tests in diagnosis of autoimmune blistering diseases.
- Author
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Ishii K
- Subjects
- Humans, Pemphigus blood, Pemphigus immunology, Serologic Tests, Pemphigus diagnosis
- Abstract
Autoimmune blistering diseases are organ-specific autoimmune diseases characterized by autoantibodies against structural proteins that maintain cell-cell (pemphigus diseases) and cell-matrix adhesions (pemphigoid diseases) in the skin and mucous membranes. Over the last few decades, identification of autoantigens and extensive characterization of autoantibodies have improved understanding of the pathogenesis of these diseases. In addition, the development of new sensitive and specific immunoassays enabled accurate diagnosis and proper evaluation of disease activity in autoimmune blistering diseases. In this review, we describe practical updates for molecular diagnostic tests for autoimmune blistering diseases and the basis for interpreting the results of the assays., (© 2015 Japanese Dermatological Association.)
- Published
- 2015
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8. In vitro pathogenicity assay for anti-desmoglein autoantibodies in pemphigus.
- Author
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Ishii K and Amagai M
- Subjects
- Blister complications, Blister immunology, Cells, Cultured, Desmosomes immunology, Desmosomes pathology, Humans, Keratinocytes immunology, Keratinocytes pathology, Organ Culture Techniques methods, Pemphigus complications, Pemphigus immunology, Skin immunology, Autoantibodies immunology, Blister pathology, Desmoglein 1 immunology, Desmoglein 3 immunology, Pemphigus pathology, Skin pathology
- Abstract
Patients with pemphigus have circulating anti-desmoglein (Dsg) 1 and/or Dsg3 autoantibodies that induce blister formation on the skin and mucous membrane. We describe here two assays that measure the pathogenic strength of autoantibodies in blister formation: an in vitro dissociation assay using primary human epidermal keratinocytes to assess pathogenicity of anti-Dsg3 autoantibodies, and an alternative method whereby anti-Dsg3 and Dsg1 autoantibodies are injected into organ-cultured human skin specimen.
- Published
- 2013
- Full Text
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9. Pathogenic anti-desmoglein 3 mAbs cloned from a paraneoplastic pemphigus patient by phage display.
- Author
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Saleh MA, Ishii K, Yamagami J, Shirakata Y, Hashimoto K, and Amagai M
- Subjects
- Aged, Antibodies, Monoclonal analysis, Blister immunology, Desmoglein 3 chemistry, Epitope Mapping, Humans, In Vitro Techniques, Keratinocytes immunology, Male, Paraneoplastic Syndromes blood, Pemphigus blood, Antibodies, Monoclonal blood, Desmoglein 3 immunology, Paraneoplastic Syndromes immunology, Pemphigus immunology, Peptide Library
- Abstract
Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca(2+)-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP.
- Published
- 2012
- Full Text
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10. 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
- Full Text
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11. 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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12. Update on the cloning of monoclonal anti-desmoglein antibodies from human pemphigus patients: implications for targeted therapy.
- Author
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Stanley JR, Ishii K, Siegel DL, and Payne AS
- Subjects
- Desmogleins metabolism, Humans, Immunoglobulin Heavy Chains genetics, Immunoglobulin Heavy Chains metabolism, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains metabolism, Immunoglobulin Variable Region, Peptide Library, Protein Binding, Antibodies, Monoclonal immunology, Cloning, Molecular methods, Pemphigus immunology
- Abstract
Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.
- Published
- 2009
- Full Text
- View/download PDF
13. Pathogenic anti-desmoglein MAbs show variable ELISA activity because of preferential binding of mature versus proprotein isoforms of desmoglein 3.
- Author
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Sharma PM, Choi EJ, Kuroda K, Hachiya T, Ishii K, and Payne AS
- Subjects
- Animals, Epitopes, Humans, Mice, Protein Isoforms, Antibodies, Monoclonal immunology, Desmoglein 3 immunology, Enzyme-Linked Immunosorbent Assay, Pemphigus immunology, Protein Precursors immunology
- Published
- 2009
- Full Text
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14. Pathogenic epitopes of autoantibodies in pemphigus reside in the amino-terminal adhesive region of desmogleins which are unmasked by proteolytic processing of prosequence.
- Author
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Yokouchi M, Saleh MA, Kuroda K, Hachiya T, Stanley JR, Amagai M, and Ishii K
- Subjects
- Antibodies, Monoclonal immunology, Desmoglein 1 immunology, Desmoglein 3 immunology, Desmogleins chemistry, Desmogleins metabolism, Enzyme-Linked Immunosorbent Assay, Humans, Immunoglobulin Fragments immunology, Autoantibodies immunology, Desmogleins immunology, Epitope Mapping, Pemphigus immunology
- Abstract
Pemphigus targets desmogleins (Dsgs), which are thought to be synthesized as inactive precursor proteins with prosequences that are cleaved by substilisin-like proprotein convertases, such as furin, to yield mature adhesive molecules. We hypothesized that some pemphigus pathogenic antibodies (Abs), which presumably interfere with adhesion, only bind the mature form. A pathogenic and three non-pathogenic anti-Dsg1 monoclonal Abs (mAbs) isolated from a pemphigus foliaceus (PF) patient, were used for immunoprecipitation and ELISA of recombinant precursor and mature Dsg1. The pathogenic Ab binds mature Dsg1, whereas non-pathogenic Abs bind either only the precursor or both the precursor and mature Dsg1. Competition ELISA showed that the majority of PF sera target the same or nearby epitopes defined by the pathogenic anti-Dsg1 mAb that blocked >20% binding of 29 out of 40 PF sera. Furthermore, the immunoreactivity of 45 PF sera against the mature Dsg1 was 3.2 fold stronger than that against the precursor Dsg1 by ELISA. Similar results were observed in anti-Dsg3 Abs in 47 pemphigus vulgaris sera, suggesting that most pemphigus sera target epitopes that are unmasked by proteolytic processing. These findings support the idea that at least some pathogenic pemphigus autoantibodies induce the loss of cell adhesion by directly binding the trans-interaction site of Dsgs.
- Published
- 2009
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15. 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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16. IgG autoantibodies directed against desmoglein 3 cause dissociation of keratinocytes in canine pemphigus vulgaris and paraneoplastic pemphigus.
- Author
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Nishifuji K, Olivry T, Ishii K, Iwasaki T, and Amagai M
- Subjects
- Animals, Autoantibodies metabolism, Cell Adhesion, Cell Line, Desmoglein 1 immunology, Desmoglein 1 metabolism, Desmoglein 3 immunology, Desmoglein 3 metabolism, Dog Diseases physiopathology, Dogs, Immunoblotting, Immunohistochemistry, Keratinocytes drug effects, Paraneoplastic Syndromes immunology, Paraneoplastic Syndromes physiopathology, Pemphigus etiology, Pemphigus immunology, Pemphigus physiopathology, Recombinant Proteins, Autoantibodies immunology, Dog Diseases immunology, Keratinocytes immunology, Paraneoplastic Syndromes veterinary, Pemphigus veterinary
- Abstract
Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes. In human patients with pemphigus vulgaris (PV) and paraneoplastic pemphigus (PNP), IgG autoantibodies against desmoglein (Dsg) 3 and Dsg1 play pathogenic roles in blister formation. In contrast, the target for IgG autoantibodies that induce keratinocyte dissociation has not been elucidated in canine pemphigus. The aim of the present study was to determine whether anti-Dsg IgG autoantibodies are present and disrupt the cell-cell adhesion of keratinocytes in canine PV and PNP. The extracellular domains of canine Dsg3 were recognized by IgG in 3/5 (60%) canine PV sera tested. IgG against the extracellular domains of canine Dsg1 was detected exclusively in two dogs that had PV with the mucocutaneous phenotype. In addition, anti-Dsg3 IgG was identified in canine PNP serum. Furthermore, incubation of normal human keratinocytes (NHK) with mucocutaneous canine PV serum and canine PNP serum resulted in dissociation of the NHK sheets, whereas the removal of anti-Dsg3 IgG from these canine sera blocked this dissociation. The present study indicates for the first time that circulating anti-Dsg3 IgG antibodies capable of dissociating keratinocytes are present in dogs with PV and PNP.
- Published
- 2007
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17. Synergistic pathogenic effects of combined mouse monoclonal anti-desmoglein 3 IgG antibodies on pemphigus vulgaris blister formation.
- Author
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Kawasaki H, Tsunoda K, Hata T, Ishii K, Yamada T, and Amagai M
- Subjects
- Aging, Animals, Animals, Newborn, Ascites immunology, Drug Combinations, Drug Synergism, Epitopes, Immunization, Passive, Immunologic Techniques, In Vitro Techniques, Mice, Mice, Knockout, Phenotype, Antibodies, Monoclonal immunology, Blister immunology, Desmoglein 3 immunology, Immunoglobulin G immunology, Pemphigus immunology
- Abstract
Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies. Previously, we generated an active mouse model for PV by adoptive transfer of splenocytes from immunized or naive Dsg3(-/-) mice. In this study, we isolated 10 anti-Dsg3 IgG mAbs (NAK-series) from PV model mice generated by transfer of naive Dsg3(-/-) splenocytes. We characterized their epitopes using domain-swapped and point-mutated Dsg1/Dsg3 molecules and examined their pathogenic activities in blister formation in three different assays. In a passive transfer model using neonatal mice, eight of 10 NAK mAbs showed pathogenic activity when injected together with half the minimum pathogenic dose of anti-Dsg1 IgG autoantibodies from pemphigus foliaceus (PF) patients. None of the mAbs could induce the PV phenotype when individual hybridoma clones were inoculated by peritoneal injection into adult Rag2(-/-) mice. NAK mAbs displayed a range of potency in an in vitro dissociation assay using primary cultured mouse keratinocytes. Interestingly, when multiple hybridoma clones recognizing different epitopes were inoculated in combination, recipient mice developed the PV phenotype. In vitro dissociation assays confirmed that combined NAK mAbs had synergistic pathogenic effects. These findings indicate that although an individual anti-Dsg3 IgG is not sufficient to cause blistering in adult mice, several together can induce the PV phenotype. These mAbs will provide a valuable tool to investigate the molecular mechanisms of blister formation, mimicking the effects of the polyclonal IgG antibodies found in patients.
- Published
- 2006
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18. In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.
- Author
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Ishii K, Harada R, Matsuo I, Shirakata Y, Hashimoto K, and Amagai M
- Subjects
- Adult, Cell Adhesion, Cells, Cultured, Desmoglein 1, Desmoglein 2, Desmogleins, Desmoplakins, Enzyme-Linked Immunosorbent Assay, Exfoliatins pharmacology, Female, Humans, Keratinocytes metabolism, Male, Middle Aged, Autoantibodies toxicity, Cytoskeletal Proteins immunology, Immunoglobulin G toxicity, Keratinocytes cytology, Pemphigus immunology
- Abstract
Patients with pemphigus vulgaris (PV) have circulating anti-desmoglein (Dsg) 3 immunoglobulin G (IgG) autoantibodies that induce blister formation. We developed an in vitro quantitative assay to evaluate the pathogenic strength of anti-Dsg3 IgG autoantibodies in blister formation. To obtain intercellular adhesion mediated dominantly by Dsg3, we used primary cultured normal human keratinocytes expressing low level of Dsg2 in the presence of exfoliative toxin A that specifically digests Dsg1. After incubation with various antibodies, monolayers released by dispase were subjected to mechanical stress by pipetting, and the number of cell fragments were counted. When anti-Dsg3 monoclonal antibodies (mAb) obtained from pemphigus model mice were tested, pathogenic AK23 mAb yielded significantly higher number of cell fragments than AK7 or AK20 non-pathogenic mAb. Dissociation scores, defined with AK23 mAb as the positive control, were significantly higher with active stage PV sera (n=10, 77.4+/-21.4) than controls (n=11, 16.0+/-9.6; p=0.003). When pair sera obtained from 6 PV patients in active stage and in remission were compared, the dissociation scores reflected well the disease activity as those in active stage were four to 17 times higher than those in remission. When sera from different patients showing similar ELISA scores but different clinical severity were tested (n=6), the dissociation scores with sera from severe disease activity were significantly higher than those with sera in remission. These findings indicate that this dissociation assay will provide a simple and objective biological method to measure the pathogenic strength of pemphigus autoantibodies.
- Published
- 2005
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19. 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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20. Update on the cloning of monoclonal anti-desmoglein antibodies from human pemphigus patients: implications for targeted therapy
- Author
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Stanley, John R., Ishii, Ken, Siegel, Don L., and Payne, Aimee S.
- Subjects
Peptide Library ,Immunoglobulin Variable Region ,Antibodies, Monoclonal ,Humans ,Immunoglobulin Light Chains ,Cloning, Molecular ,Desmogleins ,Immunoglobulin Heavy Chains ,Article ,Pemphigus ,Protein Binding - Abstract
Autoantibodies in pemphigus foliaceus (PF) and vulgaris (PV) bind to desmoglein (Dsg) 1 and 3, respectively, and cause loss of keratinocyte adhesion. To characterize the pathogenicity and genetics of such antibodies we have used phage display to isolate monoclonal antibodies (mAbs) from patients. PCR is used to clone the heavy and light chain variable region of the peripheral B cells into a vector that creates a phage particle with the antibody expressed on its surface and the cDNA encoding that antibody inside. The library of phage produced from a PF or PV patient are then panned on a plate containing Dsg1 or Dsg3 to isolate clones. The cDNA of each clone is sequenced to characterize the genetics of the expressed mAb. The mAb from each unique clone is tested for pathogenicity either by injecting into normal human skin organ culture or into neonatal mice. Pathogenic antibodies cause typical pemphigus blisters. In both PV and PF patients the heavy chain (VH) genes used for Dsg-binding antibodies are severely restricted. PV and PF patients have both pathogenic and non-pathogenic mAbs. The immunochemical characteristics of the antibodies (including pathogenicity) sort with the VH, not the VL, gene. These monoclonal pathogenic antibodies can be used to screen peptide libraries to find short peptides that block antibody binding. In summary, the antibody response is restricted and, therefore, it may be feasible to target the specific pathogenic antibodies for therapy.
- Published
- 2009
21. Development of NC1 and NC2 domains of Type VII collagen ELISA for the diagnosis and analysis of the time course of epidermolysis bullosa acquisita patients
- Author
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Saleh, Marwah Adly, Ishii, Ken, Kim, Yool-Ja, Murakami, Akihiro, Ishii, Norito, Hashimoto, Takashi, Schmidt, Enno, Zillikens, Detlef, Shirakata, Yuji, Hashimoto, Koji, Kitajima, Yasuo, and Amagai, Masayuki
- Subjects
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EPIDERMOLYSIS bullosa , *ENZYME-linked immunosorbent assay , *COLLAGEN , *AUTOANTIBODIES , *EPITOPES , *GENE expression , *PEMPHIGUS , *RECOMBINANT proteins , *DIAGNOSIS - Abstract
Abstract: Background: Epidermolysis bullosa acquisita (EBA) is an acquired autoimmune mechanobullous disease. EBA patients possess autoantibodies against type VII collagen which is composed of a collagenous domain flanked by non-collagenous NC1 and NC2 domains. It was reported that major epitopes reside within the NC1 domain and minor epitopes reside within NC2 domain. Objective: The aim of this study is to develop a sensitive and specific ELISA to facilitate the diagnosis of EBA. Methods: We developed ELISAs using recombinant NC1 domain produced by mammalian expression system and recombinant NC2 domain produced by mammalian or bacterial expression system to characterize autoantibodies in EBA. Next, we developed an ELISA using a combination of the NC1 (mammalian expression) and NC2 (bacterial expression). We tested the ELISAs with 49 EBA sera, 55 normal control sera, 20 pemphigus vulgaris and 20 bullous pemphigoid sera. Results: When we evaluated the 49 EBA sera using the NC1 and NC2 ELISAs, 38 (77.5%) reacted with NC1 domain only, 7 sera (14.2%) reacted with both NC1 and NC2 domains, and one serum (2%) reacted with NC2 domain only. Therefore, to increase the sensitivity of the assay, we developed an ELISA coated with a mixture of recombinant NC1 and NC2 domains, resulting in 93.8% sensitivity and 98.1% specificity. By analyzing the time course of two EBA patients, ELISA scores fluctuated in parallel with their disease activity. Conclusion: We conclude that the NC1+NC2 ELISA can be a practical assay for the diagnosis and follow up of the antibody titers of EBA patients. [Copyright &y& Elsevier]
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- 2011
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22. Desmoglein 1 and Desmoglein 3 Are the Target Autoantigens in Herpetiform Pemphigus.
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Ishii, Ken, Amagai, Masayuki, Komai, Ayako, Ebihara, Tamotsu, Chorzelski, Tadeusz P., Jablonska, Stefania, Ohya, Kazuhiko, Nishikawa, Takeji, and Hashimoto, Takashi
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PEMPHIGUS ,AUTOIMMUNE diseases ,CELL membranes - Abstract
Objective: To determine the cell surface autoimmune target of herpetiform pemphigus (HP). Design: Serum samples of HP were examined by immunoblot studies with human epidermal extracts, enzyme-linked immunosorbent assay with baculovirusexpressed recombinant desmoglein (rDsg) 1 and rDsg3, and immunoadsorption assay with rDsg. Patients: Twenty serum samples were obtained from patients with HP who have typical clinical and histological features. All serum samples showed positive staining against keratinocyte cell surfaces by indirect immunofluorescence studies with healthy human skin. Results: Immunoblot results showed that of 17 HP serum samples, only 5 reacted with a 160-kd band and 1 reacted with a 130-kd band. Results of enzyme-linked immunosorbent assays with rDsgl and rDsg3 demonstrated that of 20 HP serum samples, 16 were positive against Dsg1 and 4 were positive against Dsg3. No serum samples reacted with both. Furthermore, in 19 of 20 HP serum samples, immunoreactivity against keratinocyte cell surfaces was completely removed by preincubation with rDsg1 and rDsg3 as shown by indirect immunofiuorescence, excluding a possibility that these HP sera contain autoantibodies against other cell surface molecules. Conclusions: Dsg1 and Dsg3 are the major cell surface target molecules of HP, suggesting that most cases of HP are clinical variants of pemphigus foliaceus and that the rest might be variants of pemphigus vulgaris. Objective: To determine the cell surface autoimmune target of herpetiform pemphigus (HP). Design: Serum samples of HP were examined by immunoblot studies with human epidermal extracts, enzyme-linked immunosorbent assay with baculovirusexpressed recombinant desmoglein (rDsg) 1 and rDsg3, and immunoadsorption assay with rDsg. Patients: Twenty serum samples were obtained from patients with HP who have typical clinical and histological features. All serum samples showed positive staining against keratinocyte cell surfaces by indirect... [ABSTRACT FROM AUTHOR]
- Published
- 1999
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23. Conformational Epitopes of Pemphigus Antigens (Dsg1 and Dsg3) Are Calcium Dependent and Glycosylation Independent.
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Amagai, Masayuki, Ishii, Ken, Hashimoto, Takashi, Gamou, Shinobu, Shimizu, Nobuyoshi, and Nishikawa, Takeji
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PEMPHIGUS , *EPITOPES , *GLYCOSYLATION , *ANTIGENS , *CALCIUM , *ESTERIFICATION - Abstract
The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the antigenicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(β-aminoethyl ether)-N,N,N' ,N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculoproteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens. [ABSTRACT FROM AUTHOR]
- Published
- 1995
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24. p38MAPK contributes to loss of cell adhesion through clustering of desmoglein 1 but is not required for blistering in pemphigus foliaceus.
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Yoshida, Kenji, Ishii, Ken, Shimizu, Atsushi, Yokouchi, Mariko, Amagai, Masayuki, Stanley, John R., and Ishiko, Akira
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PEMPHIGUS , *MITOGEN-activated protein kinases , *CELL adhesion , *DESMOGLEINS , *BLISTERS - Published
- 2017
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25. Recognition of Desmoglein 3 by Autoreactive T Cells in Pemphigus Vulgaris Patients and Normals.
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Hertl, Michael, Amagai, Masayuki, Sundaram, Hema, Stanley, John, Ishii, Ken, and Katz, Stephen
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PEMPHIGUS , *KERATINOCYTES , *AUTOANTIBODIES - Abstract
Pemphigus vulgaris (PV) is an autoimmune blistering disease of the skin and is caused by autoantibodies against desmoglein 3 (Dsg3) on epidermal keratinocytes. Because the production of autoantibodies is presumably T cell dependent, Dsg3-specific T cell reactivity was investigated in 14 PV patients and 12 healthy donors. Peripheral blood mononuclear cells from seven PV patients with active disease showed a primary in vitro response to a recombinant protein containing the extracellular portion (EC1–5) of Dsg3, whereas two of seven PV patients in remission or under immunosuppressive treatment exhibited only secondary (2°) or tertiary (3°) T cell responses to Dsg3. T cell responses to Dsg3 were also observed in four of 12 healthy individuals upon 2° or 3° stimulation with Dsg3. Both PV patients and healthy responders were either positive for DRβ1*0402 – which is highly prevalent in PV – or positive for DR11 alleles homologous to DRβ1*0402. Two CD4+ Dsg3-specific T cell lines and 12 T cell clones from two PV patients and two CD4+ T cell lines and eight T cell clones from two normals were also stimulated by a Dsg3 protein devoid of the EC2–3 (ΔN1), suggesting that epitopes were located in the EC1, EC4, and/or EC5. Using Dsg3 peptides, one immunodominant peptide (residues 161–177) was also identified in the EC2. These observations demonstrate that T cell responses to Dsg3 can be detected in PV patients and in healthy donors carrying major histocompatibility class II alleles identical or similar to those highly prevalent in PV. [ABSTRACT FROM AUTHOR]
- Published
- 1998
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