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Biomedical subjects

M Amagai

Publications and source records attributed to M Amagai.

At least 109 records · Page 6Linked to original sources
PubMed1997

Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1, respectively. Routine immunofluorescence testing of skin and serum from patients cannot distinguish between these two severe diseases since both have IgG Abs directed against keratinocyte cell surfaces. In this study, recombinant Dsg3 and Dsg1, produced as secreted proteins by baculovirus expression, have been utilized to develop ELISAs for the specific characterization of their autoantibodies. Of 49 PV sera, 46 were positive in the Dsg3 ELISA and 44 of 46 PF sera were positive in the Dsg1 ELISA, compared with only 3 of 23 sera of bullous pemphigoid, and none of 53 normal control sera in both ELISAs. Both the Dsg3 and Dsg1 ELISAs were more specific and sensitive than conventional immunofluorescence staining. These Ag-specific ELISAs revealed that more than one-half of PV sera (26 of 49) had anti-Dsg1 Abs in addition to anti-Dsg3 Abs. PV patients who had not only oral mucous lesions but also significant skin involvement tended to have higher titers of anti-Dsg1 Abs. Furthermore, the ELISA reactivity correlated well with clinical disease activity in 5 of 6 PV and 5 of 5 PF patients. This ELISA provides a sensitive and highly specific assay for the diagnosis of patients with PV and PF, the correlation of disease activity with serum Ab levels, and a novel tool for investigating the immunopathogenesis of pemphigus.

Autoantibodies↗
PubMed1997

Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus.

IgA pemphigus showing IgA anti-keratinocyte cell surface autoantibodies is divided into subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN) types. We previously showed by immunoblotting that IgA from some IgA pemphigus patients reacted with bovine desmocollins (Dsc), but not human Dsc. To determine the antigen for IgA pemphigus, we focused on conformation-dependent epitopes of Dsc, because sera of patients with classical pemphigus recognize conformation-sensitive epitopes of desmogleins. We constructed mammalian expression vectors containing the entire coding sequences of human Dsc1, Dsc2, and Dsc3 and transiently transfected them into COS7 cells by lipofection. Immunofluorescence of COS7 cells transfected with single human Dscs showed that IgA antibodies of all six SPD-type IgA pemphigus cases reacted with the surface of cells expressing Dsc1, but not with cells expressing Dsc2 or Dsc3. In contrast, none of seven IEN-type IgA pemphigus cases reacted with cells transfected with any Dscs. These results convincingly indicate that human Dsc1 is an autoantigen for SPD-type IgA pemphigus, suggesting the possibility of an important role for Dsc1 in the pathogenesis of this disease. This study shows that a Dsc can be an autoimmune target in human skin disease.

Animals↗
PubMed1997

Pemphigus foliaceus: preferential binding of IgG1 and C3 at the upper epidermis.

The immunopathological mechanism of pemphigus foliaceus acantholysis remains controversial. Previous workers have found that IgG autoantibodies are reactive with the epidermal surface and capable of blister induction. The presence of complement components in lesions also suggests their potential role in this disease. Complement participation, however, has not been clearly demonstrated. We describe a case of pemphigus foliaceus (PF) in a 58-year old woman which was confirmed by histopathological and immunopathological studies, and by ELISA assay on recombinant PF antigen. Unusual features of this case include the predominance of IgG1 anti-epithelial antibody in both the deposits and circulation, and the preferential binding of IgG and complement component 3 at the upper epidermis. We believe that this pattern of co-localized deposition suggests a location-specific, non-random complement activation.

Autoantibodies↗
PubMed1997

Herpetiform pemphigus showing reactivity with pemphigus vulgaris antigen (desmoglein 3)

We report a patient with herpetiform pemphigus (HP) who showed reactivity only with pemphigus vulgaris (PV) antigen but not with pemphigus foliaceus (PF) antigen. Direct and indirect immunofluorescence revealed keratinocyte cell surface staining in the lower layers of the epidermis, where desmoglein 3 (Dsg3) is expressed. Immunoblot analysis, using ethylenediamine tetra-acetic acid-separated human epidermal extracts, revealed that the patient's serum recognized only a 130-kDa polypeptide which co-migrated with Dsg3. By antigen-specific immunoadsorption studies, using desmoglein 1 (Dsg1) and Dsg3 recombinant protein produced by baculovirus expression system, immunoreactivity of the patient's serum was completely adsorbed by Dsg3 alone, but not by Dsg1. These results indicate that this HP patient produced only anti-Dsg3 autoantibodies and no other autoantibodies against components of the keratinocyte cell surface. HP could be a variant of PV, in addition to PF, with unique clinical and histological features.

Autoantigens↗
PubMed1996

The majority of bullous pemphigoid and herpes gestationis serum samples react with the NC16a domain of the 180-kDa bullous pemphigoid antigen.

A series of previous studies have indicated that the NC16a domain of the 180-kDa bullous pemphigoid antigen (BP180) is the most immunogenic and probably pathogenic region in both bullous pemphigoid and herpes gestationis. To confirm these previous results by a large-scale study, we examined serum from 154 bullous pemphigoid and 43 herpes gestationis patients using an immunoblot technique with the BP180 NC16a domain fusion protein, which had been prepared using cDNA obtained from a human keratinocyte cDNA library by polymerase chain reaction amplification. This fusion protein was recognized by 90% of bullous pemphigoid and 79% of herpes gestationis serum samples, but not by any other disease or normal control serum samples. These results indicate that this system is very sensitive and specific for the detection of anti-BP180 antibodies and should be useful for the diagnosis of various subepidermal bullous diseases.

Autoantigens↗
PubMed1996

Pemphigus: from immunofluorescence to molecular biology.

Since the discovery of autoantibodies in patients with pemphigus, pemphigus has been intensively studied by dermatologists and cutaneous or cellular biologists by means of various techniques including immunofluorescence, immunoelectron microscopy, immunoprecipitation, immunoblotting, and molecular biology. In this article, up-dated topics on pemphigus obtained by each individual technique are reviewed. In the course of immunofluorescence studies on unusual cases of blistering diseases, a new entity characterized by immunoglobulin A (IgA)-type autoantibodies directed against keratinocyte cell surfaces has been discovered. Immunoelectron microscopy using low temperature post-embedding gold labeling enabled us to quantitate binding sites of pemphigus autoantibodies within desmosomes at different levels of epidermis. Immunoprecipitation and immunoblot analyses allowed us to characterize antigen complexes in paraneoplastic pemphigus. Finally, approaches using molecular biology not only have given us a fundamental insight that pemphigus autoantigen is a cadherin-type cell adhesion molecule both in pemphigus vulgaris and pemphigus foliaceus, but also provided tools to develop novel diagnostic and therapeutic strategies.

Autoantibodies↗
PubMed1996

Analysis of antigens recognized by autoantibodies in herpes gestationis. Usefulness of immunoblotting using a fusion protein representing an extracellular domain of the 180 kD bullous pemphigoid antigen.

Herpes gestationis (HG) is a rare pregnancy-associated disease. The aim of this study was to compare various immunohistochemical and immunobiochemical techniques with respect to their diagnostic sensitivity for HG. We studied 43 HG sera; only half of these reacted with the basement membrane zone (BMZ) with both indirect immunofluorescence (IF) and complement IF of normal human skin. 81% of the sera reacted with the epidermal side of 1 M NaCl-split skin. In general, titers of anti-BMZ antibodies in HG sera were lower than those in bullous pemphigoid (BP) sera. Immunoblot analysis of human epidermal extracts showed that 51% of HG sera recognized the 180 kD BP antigen (BP180) and 26% recognized the 230 kD BP antigen (BP230). We also studied the reactivity of HG sera with fusion proteins representing either the NC16a domain of human BP180 or the C-terminal region of mouse BP230. Whereas 79% of HG sera reacted with the BP180 fusion protein, only 5% recognized the BP230 fusion protein. Our results suggest that indirect IF of 1 M NaCl-split skin and immunoblotting of a fusion protein representing the BP180 NC16a domain are more sensitive techniques for the diagnosis of HG than conventional and complement IF or immunoblotting of crude epidermal extracts.

Animals↗
PubMed1996

Pemphigus vulgaris antigen (desmoglein 3) is localized in the lower epidermis, the site of blister formation in patients.

In Patients with pemphigus vulgaris, autoantibodies against the desmosomal glycoprotein desmoglein 3 (Dsg3) cause blisters due to loss of keratinocyte cell-cell adhesion in the basal and immediate suprabasal layer of the deeper epidermis, leaving the superficial epidermis intact. Autoantibodies from these patients, however, usually bind to the cell surface of keratinocytes throughout the entire epidermis, as determined by indirect immunofluorescence. To explain this apparent paradox, we immunoadsorbed pemphigus vulgaris sera with the extracellular domains of Dsg3 and desmoglein 1 (Dsg1) produced by insect cells infected with recombinant baculovirus. When adsorbed with extracellular domains of both Dsg3 and Dsg1, these sera no longer stained epidermis, demonstrating that most, if not all, of their cell surface reactivity can be attributed to antibodies against the extracellular domains of these desmogleins. Adsorption with only the Dsg1 extracellular domain left antibodies that stained only the basal and immediate suprabasal layers of the epidermis and immunoprecipitated only Dsg3, not Dsg1, from extracts of cultured cells synthesizing these molecules. In contrast, adsorption with only the Dsg3 extracellular domain left antibodies that stained only the more superficial epidermis and immunoprecipitated only Dsg1. These data localize Dsg3 exactly to the area in the epidermis where blisters occur in pemphigus vulgaris.

Autoantibodies↗
PubMed1996

Transport to endoplasmic reticulum by signal peptide, but not proteolytic processing, is required for formation of conformational epitopes of pemphigus vulgaris antigen (Dsg3).

Desmoglein 3 is the autoimmune target of pemphigus vulgaris. Most, if not all, pathogenic autoantibodies are raised against conformational epitopes on desmoglein 3. In this study, we examined whether posttranslational modification in endoplasmic reticulum is required for the proper three-dimensional structure formation of recombinant pemphigus vulgaris antigen. Previously, we have produced by baculovirus expression a secreted form of desmoglein 3, PVIg, which represents equivalent conformational epitopes of the native antigen and showed that PVIg is able to immunoadsorb heterogeneous autoantibodies from pemphigus vulgaris patients' sera. To elucidate the role of proteolytic processing, we constructed a mutant PVIg molecule, PVIg-fXa, whose putative endoproteolytic cleavage site was replaced by the recognition sequence of serum coagulation factor Xa. PVIg-fXa was produced without proteolytic processing; however, exogenous treatment of PVIg-fXa with factor Xa resulted in cleavage of the prosequence. Interestingly, not only the processed PVIg-fXa, but also the unprocessed form, showed the immunoadsorptive activity. Furthermore, to elucidate the role of endoplasmic reticulum signal peptide at the amino terminus, we constructed another mutant, PVIg-delta sig, which lacks the signal peptide and prosequence. PVIg-delta sig was not secreted and accumulated in the cytosol. PVIg-delta sig failed to show the immunoadsorption. Together with our previous finding on the role of glycosylation, these observations indicate that the conformational epitopes of the recombinant pemphigus antigen are not affected either by glycosylation or proteolytic processing, although they need to be formed in the endoplasmic reticulum, and emphasize the importance of conformation of the antigen in pathogenic autoantibody binding.

Adsorption↗
PubMed1996

Specific detection of anti-cell surface antibodies in herpes gestationis sera.

We examined 42 herpes gestationis sera with immunofluorescence of normal human skin sections, and found that anti-keratinocyte cell surface antibodies were detected specifically in 10 herpes gestationis sera. The diagnosis of these herpes gestationis cases was confirmed by detecting antibodies against the 180 kD bullous pemphigoid antigen with immunoblotting of its fusion protein. The results of immunoadsorption assay using baculoproteins of both pemphigus vulgaris and pemphigus foliaceus antigens indicated that the herpes gestationis sera did not recognize common pemphigus antigens. Immunoblotting of human epidermal extracts and immunofluorescence of various tissues also suggested that the sera did not recognize any other desmosomal components or paraneoplastic pemphigus antigens. The significance of this reactivity is unclear. However, because no control bullous pemphigoid sera showed this reactivity, it may suggest a different pathophysiology between herpes gestationis and bullous pemphigoid.

Antigens, Surface↗
PubMed1996

A case of herpetiform pemphigus associated with autoimmune hemolytic anemia: detection of autoantibodies against multiple epidermal antigens.

We report a case who was clinically and histopathologically diagnosed as herpetiform pemphigus (HP) and associated with autoimmune hemolytic anemia (AIHA). However, immunofluorescence studies demonstrated concurrent anti-cell-surface and anti-basement-membrane-zone antibodies in the patient's serum. Immunochemical studies showed that the patient's serum reacted with both the pemphigus foliaceus antigen and the two bullous pemphigoid antigens. Subsequently, the patient developed AIHA. Both anemia and skin lesions were successfully treated with oral prednisolone. We believe that this is the first case with HP in association with AIHA. The presence of autoantibodies against multiple antigens suggests an abnormal immunologic tolerance in the antibody production system in this patient.

Anemia, Hemolytic, Autoimmune↗
PubMed1995

Induction of autoantibodies in normal mice by injection of nucleobindin and natural occurrence of antibodies against nucleobindin in autoimmune MRL/lpr/lpr mice.

Our previous works have shown that nucleobindin (Nuc) or recombinant (r) Nuc not only augments anti-DNA antibody production in vitro but also accelerates autoimmune response in vivo in MRL/+/+ (MRL/n) mice which are the substrain of autoimmune MRL/lpr/lpr (MRL/l) mice. To investigate whether rNuc can induce autoimmune response similarly in naive mice, we carried out intraperitoneal (i.p.) injection of rNuc (5 micrograms) without adjuvant into 8-week-old female BALB/c mice and continued injection twice a week for 12 weeks. About 5 weeks after the first injection, all the mice began to show IgG hypergammaglobulinemia (HG) followed by elevation of a number of autoantibodies of the IgG class such as anti-double-stranded (ds) DNA, anti-U1 ribonuclear protein (RNP), anti-ssB(La) and anti-Fc antibodies (RF), but not by anti-Sm antibodies. However, the IgG anti-dsDNA antibody response and histopathological changes in the kidney of these BALB/c mice were not so noticeable as those in MRL/n mice induced by rNuc in our previous experiment. In contrast, the IgG anti-rNuc antibody response of normal BALB/c mice induced by rNuc was stronger than that of MRL/n mice induced by rNuc. Since the titers of each autoantibody of BALB/c mice induced by rNuc were not always associated with the level of IgG HG, and either IgG HG or IgG autoantibodies could not be induced by control administration of extracts (5 micrograms) of Escherichia coli with or without harboring plasmid alone, polyclonal B cell activation (PBA) appeared not to be the mechanism of this autoimmunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.

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 anti-genicity 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(beta-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 baculo-proteins 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.

Adsorption↗
PubMed1995

A case of pemphigus vulgaris showing reactivity with pemphigus antigens (Dsg1 and Dsg3) and desmocollins.

Both pemphigus vulgaris antigen (PVA; Dsg3) and pemphigus foliaceus antigen (PFA; Dsg1) are members of the desmoglein subfamily of the cadherin supergene family. Another desmosomal cadherin, desmocollin, is occasionally recognized by certain pemphigus sera. We present a 38-year-old Japanese male who showed clinically and histopathologically typical features of pemphigus vulgaris, whose sera reacted with all PVA, PFA, and desmocollins using immunoblotting of both human epidermis and bovine snout epidermis. Studies using domain-specific fusion proteins of PFA and PVA suggested that this patient's serum reacted with the intracellular domain of PFA and the extracellular domain of PVA, the latter of which seems to be responsible for initiating the skin lesion. The patient's serum showed reactivity with human desmocollin and was shown to react with bovine Dsc2 fusion protein, further suggesting the significance of anti-desmocollin autoantibodies in pemphigus. These results indicate that certain pemphigus cases may produce antibodies against multiple antigen molecules, although the complex mechanism of the production of autoantibodies remains to be elucidated.

Adult↗
PubMed1995

Antigen-specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus.

Patients with the autoimmune blistering disease pemphigus foliaceus (PF) have circulating autoantibodies directed against the desmosomal cadherin desmoglein 1 (Dsg1). Based on the fact that purified IgG fractions from PF patients induce loss of cell adhesion in organ culture and in a neonatal mouse model, it has been proposed that these anti-Dsg1 antibodies play a pathogenic role in blister formation. To directly address whether antibodies in PF sera specific for the Dsg1 extracellular domain are indeed pathogenic in the disease, PFIg, a chimeric protein containing the entire extracellular domain of human Dsg1 and the constant region of human IgG1, was produced by baculovirus expression. Incubation of PF patients' sera with the PFIg baculoprotein removed the immunoreactivity of autoantibodies against keratinocyte cell surfaces in all 20 PF and eight Brazilian PF patients' sera tested. This adsorption was conformation dependent, because PFIg protein denatured by low pH or heat was no longer able to adsorb the immunoreactivity of PF sera. Furthermore, the incubation with the PFIg baculoprotein eliminated the pathogenic activity of PF patients' sera and prevented gross blister formation in a neonatal mouse model of pemphigus. Anti-Dsg1 antibodies eluted from the PFIg protein column were pathogenic as they resulted in the appearance of gross blisters in neonatal mice with typical histologic findings of PF. These observations indicate that the extracellular domain of Dsg1 expressed by baculovirus is capable of specifically immunoadsorbing pathogenic autoantibodies from PF patients' sera and provide direct evidence that the anti-Dsg1 autoantibodies in PF sera are indeed pathogenic. The availability of this Dsg1 recombinant protein may facilitate the development of antigen-specific plasmapheresis as a novel therapeutic strategy in pemphigus.

Animals↗
PubMed1995

Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.

We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.

Autoantigens↗