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J R Stanley

Publications and source records attributed to J R Stanley.

At least 73 records · Page 4Linked to original sources
PubMed1993

Cloning of the 5' mRNA for the 230-kD bullous pemphigoid antigen by rapid amplification of cDNA ends.

The 230-kD bullous pemphigoid antigen (BPAG1), defined by autoantibodies in patient sera, is a hemidesmosomal plaque protein in the same gene family as the intracellular proteins desmoplakin I/II and plectin. We had previously isolated, from a lambda gt11 library, overlapping cDNA clones with 6921 bp of mRNA sequence for BPAG1. The coding sequence encoded by these clones included the 3' stop codon but not the 5' coding and non-coding region of the mRNA. To obtain these sequences we used the polymerase chain reaction (PCR) method called rapid amplification of cDNA ends (RACE). The PCR products were cloned into plasmids and sequenced. With five PCR primers we were able to obtain overlapping clones containing the 5' region of the mRNA. An upstream stop codon in frame with the rest of the coding sequence demonstrates that the full 5' coding sequence is obtained. Four different PCR products from two separate reactions had the same 5' end, suggesting that this 5' end is near, or at, the transcription start site. No alternatively spliced clones were found and no transmembrane site was predicted, confirming that BPAG1 is an intracellular hemidesmosomal plaque protein.

Amino Acid Sequence↗
PubMed1993

Comparative study of bullous pemphigoid antigens among Japanese, British, and U.S. patients indicates similar antigen profiles with the 170-kD antigen present both in the basement membrane and on the keratinocyte cell membrane.

There are a number of controversies relating to studies of bullous pemphigoid (BP) antigens from different institutions, mainly regarding the detection of the 230-kD and 170-kD BP antigens. In this study, in an attempt to resolve the discrepancies, we have examined and compared the reactivity by immunofluorescence, immunoblotting, and immunoprecipitation among the sera from Japanese, British and U.S. BP patients. Both the 230-kD and 170-kD BP antigens were detected by various sera from all populations with immunoblotting, whereas immunoprecipitation detected only the 230-kD BP antigen but not the 170-kD BP antigen. Immunoprecipitation was more sensitive than immunoblotting to detect the 230-kD antigen. These results indicate that both the 230-kD and 170-kD proteins are BP antigens found in all three populations. By immunofluorescence cell surface staining in the lower epidermis in addition to basement membrane zone staining was shown by a considerable number of patients' sera in all populations. Comparison between the results of immunofluorescence and immunoblotting revealed a clear correlation of this cell surface staining with the presence of antibodies against the 170-kD BP antigen. That the affinity-purified antibodies specific to the 170-kD BP antigen showed this cell surface staining further confirmed this correlation. These results may indicate a different nature of the 170-kD BP antigen from that of the 230-kD BP antigen.

Antigens, Surface↗
PubMed1992

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.

Amino Acid Sequence↗
PubMed1992

Immunomorphologic and biochemical identification of the pemphigus foliaceous autoantigen within desmosomes.

Desmosomes are specialized domains of the plasma membrane that play a fundamental role in intercellular adhesion. This adhesive function is mediated at least in part by the cadherin homologous cell adhesion molecule (CAM) desmoglein (dg). Autoantibodies (aab) from patients with pemphigus foliaceous (pf), a blistering disease of the epidermis, have been shown by immunochemical methods to bind to desmoglein. However, the molecular localization of the binding sites of these antibodies, especially as it relates to the ultrastructure of the desmosomes, has not been definitively characterized. We therefore performed pre-embedding direct immunoelectron microscopy (IEM) on perilesional skin of patients with pf and post-embedding indirect IEM using sera from five patients with pf. We first confirmed by immunoprecipitation and immunoblotting that these sera bound dg. Both IEM methods showed that pf-aab exclusively bind to desmosomes. Double-labeling IEM of several other constitutive desmosomal proteins further suggests that most likely pf-aab bind to an extracellular domain of the transmembrane CAM dg. Our studies suggest one possible pathophysiologic mechanism for the clinical manifestations of pf: namely, that the binding of aab to an extracellular epitope of desmoglein might impair the adhesive properties of desmosomes mediated by dg and result in the loss of cell adhesion leading to acantholysis and blister formation.

Autoantigens↗
PubMed1992

Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic.

Complementary DNA cloning of the 130-kD pemphigus vulgaris (PV) autoantigen (PVA) has indicated that it is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules. By homology with typical cadherins, PVA has five extracellular domains (EC1 through EC5). To localize immunogenic domains and to determine whether antibodies against them might be pathogenic, we produced beta-galactosidase fusion proteins with cDNA encoding different portions of the extracellular domains of PVA (EC1-2, EC3-5, and each individual domain). Immunoblot analysis of these fusion proteins with 23 PV patients' sera demonstrated that major immunogenic regions of PVA are located on the EC1, EC2, and EC4 domains. IgG was affinity-purified from PV sera on fusion proteins representing the amino (EC1-2) and carboxy (EC3-5) terminus of the extracellular PVA, and injected into neonatal mice. PV IgG affinity-purified on the EC1-2 fusion protein caused suprabasilar acantholysis, the typical histological finding of PV, but IgG affinity-purified on the EC3-5 fusion protein or beta-galactosidase alone did not. These results indicate that at least one pathogenic epitope, which is sufficient to cause suprabasilar acantholysis in neonatal mice, is located on the amino-terminal region of PVA, an area thought to be important in cadherin homophilic adhesion.

Amino Acid Sequence↗
PubMed1991

Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.

Pemphigus vulgaris (PV) is a life-threatening skin disease in which autoantibodies against a keratinocyte cell surface 130 kd glycoprotein, PV antigen (PVA), cause loss of cell-cell adhesion, with resultant epidermal blisters. We used affinity-purified PV IgG to isolate cDNA, containing the entire coding sequence for PVA, from human keratinocyte expression libraries. Northern blot analysis indicated PV mRNA expression only in stratified squamous epithelia. The deduced amino acid sequence of PVA was unique but showed significant homology with members of the cadherin family of Ca(2+)-dependent cell adhesion molecules, most markedly to desmoglein I. These findings demonstrate that a novel epithelial cadherin is the target of autoantibodies in PV.

Amino Acid Sequence↗
PubMed1991

Comparison of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins.

Bullous pemphigoid is a subepidermal blistering disease in which patients have autoantibodies against the plaque of the hemidesmosome. Starting with a previously isolated 2-kilobase (kb) cDNA for bullous pemphigoid antigen (BPA), we used primer extension of keratinocyte mRNA to isolate overlapping cDNAs with a combined open reading frame of 6.3 kb, encoding most (243 kDa) of the BPA, but lacking the far amino terminus. Analysis of this amino acid sequence revealed a carboxyl-terminal domain containing two regions of 174 and 176 residues with high sequence identity. Most of the amino-terminal two-thirds of BPA is predicted to be in an alpha-helical conformation in which two chains would aggregate into a coiled-coil rod structure. BPA and desmoplakin I, a desmosome plaque protein, show remarkable sequence and structural homology. In its carboxyl-terminal domain, desmoplakin I also has 176 residue repeats with 40% sequence identity to those in BPA. The repeats in both molecules have a regular linear distribution of acidic and basic residues with a period of 9.5, the same as that found in the 1B segment of keratin filaments, suggesting a means of ionic interaction between keratin and these plaque proteins. Also, desmoplakin I, like BPA, is predicted to have a rod domain, which in both proteins has similar regular charge periodicities, suggesting a means of ionic self-aggregation. These findings extend those of Green et al. (Green, K. J., Parry, D. A. D., Steinert, P. S., Virata, L. A., Wagner, R. M., Angst, B. D., and Nilles, L. A. (1990) J. Biol. Chem. 265, 2603-2612) which show that BPA and desmoplakin I represent the first members of a new family of adhesion junction plaque proteins.

Amino Acid Sequence↗
PubMed1991

Drug-induced pemphigus: autoantibodies directed against the pemphigus antigen complexes are present in penicillamine and captopril-induced pemphigus.

Pemphigus is an autoimmune blistering disease characterized by circulating autoantibodies directed against the keratinocyte cell surface. The two variants, pemphigus foliaceus and pemphigus vulgaris, can be distinguished at the molecular level by immunochemical studies. The large majority of patients with pemphigus develop the disease spontaneously; however, there is a small group of patients who develop pemphigus after treatment with certain medications, of which penicillamine and captopril are the best documented. Most patients with drug-induced pemphigus have circulating and/or tissue bound epidermal cell surface autoantibodies; however, the molecular specificity of these autoantibodies has not been studied. We performed immunoprecipitation studies utilizing extracts of 125I-labeled suction blister epidermis and the sera of three patients with drug-induced pemphigus foliaceus (two due to penicillamine and one due to captopril) and one patient with captopril-induced pemphigus vulgaris. We found that the three patients with drug-induced pemphigus foliaceus had circulating autoantibodies that are directed against the pemphigus foliaceus antigen complex and that the one patient with drug-induced pemphigus vulgaris had circulating autoantibodies that are directed against the pemphigus vulgaris antigen complex. This study demonstrates that autoantibodies from drug-induced pemphigus patients have the same antigenic specificity, on a molecular level, as do autoantibodies from other pemphigus patients.

Aged↗
PubMed1991

Southern analysis of the 230-kD bullous pemphigoid antigen gene in normal humans, animals, and patients with junctional epidermolysis bullosa.

To begin to characterize the 230-kD bullous pemphigoid antigen (BPA) gene, we performed Southern analysis on genomic DNA with probes derived from 7 kb of cDNA that spans most of the coding region of this hemidesmosomal plaque protein. When hybridized to a 1-kb fragment of this BPA cDNA, normal human genomic DNA digested with EcoRI, BamHI, PstI, HindIII, or EcoRV showed only a single band, which was unique for each enzyme, indicating a single human gene for BPA. To determine if a related gene exists in animals, we used probes covering the full 7 kb of cDNA for Southern analysis of genomic DNA from various vertebrates. A related gene was detected in other mammals (monkey, cow, dog, rabbit, mouse, and rat) but not in chicken, frog, or fish. Under these same hybridization conditions a probe for human beta-actin could detect an actin gene in all these species. Furthermore, immunofluorescence showed that an antibody raised against portions of the 230-kD BPA bound to the epidermal basement membrane of mammals but not that of a bird or amphibian. Finally, because most patients with junctional epidermolysis bullosa (JEB) have defective hemidesmosomes in ultrastructure, and probably function, we analyzed genomic DNA from these patients. No restriction fragment length polymorphisms (RFLP) were detected when the DNA from 11 normals and 8 JEB patients (representing 16 possible defective genes) was digested with BamHI, EcoRI, or PstI and hybridized to any part of the cDNA. These findings indicate that 1) there is a single BPA gene in humans; 2) a closely related gene exists in other mammals but not birds, amphibia, or fish; and 3) gross abnormalities of the BPA gene are not characteristic of JEB patients.

Animals↗
PubMed1991

Coexistence of pemphigus foliaceus and bullous pemphigoid. Demonstration of autoantibodies that bind to both the pemphigus foliaceus antigen complex and the bullous pemphigoid antigen.

Pemphigus and bullous pemphigoid are autoimmune blistering diseases of the skin characterized by circulating autoantibodies directed against the keratinocyte cell surface and the epidermal basement membrane zone, respectively. The coexistence of pemphigus and bullous pemphigoid is very uncommon. We describe a patient with pemphigus foliaceus who later developed bullous pemphigoid and show, by means of immunoprecipitation studies utilizing both cultured keratinocytes and suction blister epidermis, that our patient had circulating autoantibodies directed against both the pemphigus foliaceus antigen complex and the bullous pemphigoid antigen. This report is the first to demonstrate the coexistence of pemphigus foliaceus and bullous pemphigoid at the molecular level.

Antigen-Antibody Complex↗
PubMed1990

Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.

BACKGROUND AND METHODS: We describe five patients with underlying neoplasms in whom painful mucosal ulcerations and polymorphous skin lesions developed, usually with progression to blistering eruptions on the trunk and extremities. Histologic examination showed vacuolization of epidermal basal cells, keratinocyte necrosis, and acantholysis. Immunofluorescence testing revealed atypical pemphigus-like autoantibodies in perilesional epithelium and serum from all five patients. We studied the antigenic specificities of the autoantibodies by indirect immunofluorescence and immunoprecipitation, using extracts of 14C-labeled human keratinocytes. IgG purified from the serum of one patient was passively transferred to four neonatal mice to test for pathogenicity. RESULTS: Immunofluorescence testing showed that the autoantibodies bound to the surface of tissues containing desmosomes, including complex and simple epithelia, and myocardium. An identical and unique complex of four polypeptides with molecular weights of 250, 230, 210, and 190 was immunoprecipitated by all serum samples. The 250-kd polypeptide comigrated with desmoplakin I (a protein found in the desmosomes of all epithelia), and the 230-kd antigen comigrated with the antigen of bullous pemphigoid. Cutaneous blisters, a positive Nikolsky's sign, and epidermal and esophageal acantholysis developed in all mice into which the autoantibody was injected. Electron microscopy showed epidermal acantholysis similar to lesions of experimentally induced pemphigus vulgaris. CONCLUSION: These five patients with cancer had a novel acantholytic mucocutaneous disease characterized by autoantibodies that were pathogenic after passive transfer. The autoantibodies from these patients reacted with an antigen complex composed of desmoplakin I and the 230-kd antigen of bullous pemphigoid and two as yet unidentified epithelial antigens. We suggest the term "paraneoplastic pemphigus" for this disease.

Acantholysis↗
PubMed1990

IgG antibodies from patients with bullous pemphigoid bind to localized epitopes on synthetic peptides encoded by bullous pemphigoid antigen cDNA.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized in part by the presence of tissue-bound and circulating antibodies specific for basement membrane zone proteins, the BP Ag. The purpose of the present study was to determine seroreactivity of patients with BP to six nonoverlapping synthetic peptides representing sequences in the carboxyl domain of the recently cloned 230-kDa BP Ag. Sera from 40 patients with BP, 57 normal subjects, and 18 patients with other autoimmune blistering skin diseases were examined in an ELISA for binding to six synthetic peptides varying between 17 and 19 amino acids in length. The binding of IgG from patients with BP to three synthetic peptides, P1-2, P1-1, and P3-1, was significantly different from that seen in the normal controls (p less than 0.001, Fisher's exact test). Affinity-purified anti-P1-2 antibody from a patient with BP bound in a characteristic linear band to the epidermal side of 1 M NaCl split skin and immunoprecipitated the native 230-kDa BP Ag. Serum IgG antibodies from a rabbit immunized with a BP fusion protein that contains the sequences for P1-1 and P1-2, bound on ELISA to P1-2 but not to P1-1. These data suggest that multiple epitopes on the 230-kDa BP Ag are recognized by circulating autoantibodies in patients with this disease, and that an epitope encoded within the synthetic peptide P1-2 is expressed on the native molecule and may be relevant in the generation of an immune response both in man and in an animal model.

Amino Acid Sequence↗
PubMed1990

Pemphigus. Skin failure mediated by autoantibodies.

Pemphigus is a group of diseases in which autoantibodies mediate a loss of cell-to-cell adhesion, resulting in blister formation and ultimate failure of the barrier function of the skin. These autoantibodies are directed against complexes that contain polypeptides found in adhering junctions. Exactly how these antibodies mediate loss of cell adhesion, whether through secondary mechanisms such as release of proteases, direct interference of junction formation, or both, remains an area of active and exciting investigation.

Adolescent↗
PubMed1990

Studies of the relationship of the 230-kD and 180-kD bullous pemphigoid antigens.

In bullous pemphigoid (BP), autoantibodies from most patients recognize a high molecular weight 230-kD epidermal antigen (Ag) by immunoprecipitation. By Western immunoblotting, 50-70% of sera recognize the high molecular weight Ag, but 30-50% recognize a low molecular weight, 180-kD epidermal Ag. We examined the specificities of affinity-purified antibodies against these Ag. Antibodies specific for the 230- and 180-kD Ag were prepared by immunoaffinity against Ag immobilized on nitrocellulose and released by acid glycine. IgG eluted from the 230-kD Ag band retained its specific binding to the 230-kD Ag by immunoblotting, and bound to the epidermal basement membrane zone (BMZ) by indirect immunofluorescence (IF) and to hemidesmosomes by indirect immunoelectron microscopy (EM). IgG affinity purified by the 180-kD Ag band bound only the 180-kD Ag in immunoblotting, with no cross reaction to the 230-kD Ag, bound the epidermal BMZ by indirect IF, and also bound to hemidesmosomes in immuno-EM. IgG specific for the 230-kD Ag in immunoblotting immunoprecipitated only the 230-kD Ag, with no apparent precipitation of the 180-kD Ag. Surprisingly, IgG specific for the 180-kD Ag precipitated both the 180- and the 230-kD Ag in immunoprecipitation, and the 230-kD Ag band was much more intense than the 180-kD Ag band. This study shows that apparent cross-reactivity between these Ag by BP autoantibodies can only be detected in native conditions by immunoprecipitation, and cannot be demonstrated using denatured Ag in immunoblotting. The two proteins appear to be distinct Ag, closely associated in the epidermal hemidesmosome, but the exact relationship of these Ag to each other may not be clarified until complete structural data become available.

Antibodies↗
PubMed1990

Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA.

A partial cDNA clone (called BP cDNA) with coding sequences for the carboxy-terminal region of bullous pemphigoid (BP) antigen has been recently isolated and sequenced. In order to determine whether specific peptides encoded by the cDNA could be used to raise antibodies against BP antigen, fusion proteins derived from fragments of the BP cDNA and 17-mer or 19-mer synthetic peptides, corresponding to its deduced amino acid sequence, were used to generate rabbit antibodies. Three restriction enzyme fragments, 1179 bp (5' end), 264 bp (middle), and 546 bp (3' end), of the 1992 open reading frame (ORF) of BP cDNA were subcloned in frame into pEX plasmids to make beta-galactosidase fusion proteins FP1, FP2, and FP3, respectively. Fusion proteins of the predicted molecular weight, and which bound anti-beta-galactosidase antibodies, were produced, confirming the length of the predicted ORF. Rabbits immunized with FP1, but not FP3, produced antibodies, similar to authentic antibodies from BP patients, which: 1) bound the epidermal basement membrane at titers over 10,000, as determined by indirect immunofluorescence; 2) bound the basement membrane on the roof of 1 M NaCl-split skin; 3) immunoprecipitated the 230-kD BP antigen; and 4) bound the hemidesmosome, as determined by immunoelectron microscopy. Rabbits immunized with FP2 also produced lower titer BP-like antibodies. We further showed that short hydrophilic synthetic peptides, contained in FP1, could induce similar BP-like antibodies in rabbits at immunofluorescence titers up to 2560. These rabbit antibodies should prove useful for further studies on the function and structure of particular epitopes of BP antigen as well as on the pathophysiology of disease.

Animals↗