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J R Stanley

Publications and source records attributed to J R Stanley.

At least 91 records · Page 5Linked to original sources
PubMed1989

Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris.

Pemphigus foliaceus and pemphigus vulgaris are skin diseases in which antibodies against the cell surface of keratinocytes destroy the adhesion between epidermal cells, producing blisters. Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides of 260, 160, and 85 kd (the foliaceus complex), whereas patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd polypeptides (the vulgaris complex). The 160-kd polypeptide of the foliaceus complex has been identified as desmoglein, a desmosomal glycoprotein. We suspected that the 85-kd component in both these antigenic complexes might be plakoglobin, another molecule in the adhering junctions of cells. To characterize these antigenic complexes, we used the serum of five patients with pemphigus foliaceus, that of four patients with pemphigus vulgaris, and monoclonal antiplakoglobin antibodies. We found that monoclonal antibodies to plakoglobin immunoprecipitated the 85-kd polypeptide from the dissociated foliaceus and vulgaris complexes and precipitated both complexes from epidermal extracts. Serum from patients with pemphigus foliaceus or pemphigus vulgaris (but not from four normal controls) bound desmoglein and the 130-kd polypeptide, respectively, showing that these peptides (and not plakoglobin) are the antigenic binding sites in these disorders. We conclude that plakoglobin, a protein of the adhering junctions of epidermal cells, is the 85-kd molecule in the antigenic complexes found in both pemphigus foliaceus and pemphigus vulgaris, although it is not the binding site in either disorder.

Autoantibodies↗
PubMed1989

Epidermal autoantibodies in erythema multiforme.

We report a case of refractory erythema multiforme associated with atypical epidermal autoantibodies. The patient was a 63-year-old woman with clinical and histologic features of erythema multiforme and a large mediastinal T cell lymphoma. Immunofluorescence studies disclosed high serum titers of cell surface antibodies against epidermal keratinocytes in a pattern typical of pemphigus vulgaris. The epidermal antigen reacting with these circulating antibodies was characterized by incubating 14C-labeled keratinocytes with serum. The patient's serum pattern precipitated multiple proteins keratinocytes antigens), but not the 130,000- and 85,000-dalton polypeptides characteristic of pemphigus vulgaris. Thus autoantibodies reacting selectively with non-pemphigus vulgaris epidermal keratinocyte antigens may be the cause of a false-positive immunofluorescence test for pemphigus vulgaris.

Aged↗
PubMed1989

A 230-kD basic protein is the major bullous pemphigoid antigen.

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of BP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP sera by both immunoprecipitation of extracts of radiolabeled cultured human keratinocytes and immunoblotting of extracts of normal human epidermis. Thirty-six of 37 BP sera immunoprecipitated the 230-kD polypeptide, and 11 of 13 BP sera identified the 230-kD polypeptide by immunoblotting. Eighty-six disease and normal control sera did not bind the 230-kD BP antigen. Immunoprecipitation was a more sensitive assay than was immunoblotting to detect the 230-kD antigen; BP sera that were weak or negative by immunoblotting were strongly positive by immunoprecipitation. To demonstrate that this 230-kD polypeptide shared epitopes, defined by BP sera, with the epidermal basement membrane zone, we showed that BP IgG affinity purified on this polypeptide bound the epidermal basement membrane zone by immunofluorescence. To further characterize the BP antigen and to compare the antigen synthesized by cultured human keratinocytes to the antigen extracted from epidermis, we analyzed the 230-kD BP antigen by two-dimensional gel electrophoresis. BP antigen immunoprecipitated from culture extracts or BP antigen identified by immunoblots of epidermal extracts was a single spot on two-dimensional gels, with a pI of about 8. These data indicate that, although other polypeptides are sometimes identified (e.g., a 166 kD band by immunoprecipitation or a 180 kD band on immunoblots), the major BP antigen identified in cultured human keratinocytes is similar to the antigen found in normal human epidermis and is a basic 230-kD polypeptide.

Autoantigens↗
PubMed1989

Biochemical differentiation of types of pemphigus.

There are two major types of pemphigus as determined by clinical and histologic criteria: PV, in which the blister forms at the suprabasilar layer of the epidermis, and PF, in which the blister forms in the more superficial epidermis. The autoantibodies from both types of pemphigus bind in an identical fashion to the cell surface of keratinocytes. Blocking studies suggest that PF and PV sera recognize different epidermal antigens. Absorption studies have suggested that a variety of antigens react with pemphigus autoantibodies, but the conditions of these studies may have absorbed or inactivated the antibodies nonspecifically. By immunoblotting it has been shown that a subset of PF and FS patients' sera bind to a 160-kD glycoprotein that is identical to DG I, a desmosomal core glycoprotein. Also by immunoblotting, a few PV sera recognize a 33-kD protein from human epidermis and a 140-kD protein from bovine tongue. All PV sera tested by immunoprecipitation bind to a 210-kD glycoprotein that is synthesized by cultured human keratinocytes. This glycoprotein consists of two disulfide-linked polypeptides of 130 and 80 kD. PF sera do not recognize these polypeptides. Thus, even though the antigen specificities at a molecular level of all PF and PV sera are not fully known, it is clear that PV and PF autoantibodies bind different molecules. These findings of disease specific antigens may account for the different clinical forms of these diseases.

Antigens, Surface↗
PubMed1988

Human dermal fibroblasts synthesize laminin.

Laminin, a glycoprotein of approximately 900,000 daltons, is a major component of the basement membrane that separates the epidermis from dermis in human skin. Previous studies have shown that keratinocytes and other epithelial cells synthesize laminin and utilize it for attachment to other extracellular matrices such as heparan sulfate proteoglycan and basement membrane collagen. The relationships between phenotypically normal mesenchymal cells and laminin have been much less emphasized in the literature. In this study, we have used antibodies that specifically label the A and B chains of laminin (but not fibronectin or other unrelated proteins) by Western blot analysis to immunoprecipitate biosynthetically derived laminin from [35S] methionine labeled cultures of neonatal and adult human skin fibroblasts. To be sure that the precipitated bands were laminin and not fibronectin, which has a molecular size very close to that of the laminin B chains, experiments were performed in which fibronectin was removed from the radiolabeled proteins by first immunoprecipitating with antifibronectin antibody and then sequentially immunoprecipitating laminin from the fibronectin-depleted supernates with antilaminin antibody. These experiments definitively demonstrate that human dermal fibroblasts synthesize and secrete laminin.

Adult↗
PubMed1988

Epidermal antigens in pemphigus vegetans. Report of a case.

A patient is described who had typical pemphigus vegetans, according to clinical, histopathological and immunofluorescence criteria. To determine the specific molecules bound by antibodies in the serum of this patient, an immunoprecipitation analysis was performed with extracts of [14C]-amino acid labelled cultured normal human keratinocytes. The patient's serum precipitated the 130 kd and 85 kd polypeptides of the pemphigus vulgaris antigen, but also precipitated other polypeptides, the significance of which is not clear.

Antibody Specificity↗
PubMed1988

Identification of pemphigus vulgaris antigen extracted from normal human epidermis and comparison with pemphigus foliaceus antigen.

Immunoprecipitations of cultured keratinocyte extracts have shown that pemphigus vulgaris (PV) sera bind a polypeptide of 210,000 mol wt with disulfide-linked chains of 130,000 and 85,000 mol wt. To identify proteins in normal human skin recognized by PV antibodies, we performed immunoprecipitations of normal human epidermal extracts. All 22 PV sera tested immunoprecipitated a complex of polypeptides (PV complex) of 210,000, 130,000, and 85,000 mol wt, after reduction. One- and two-dimensional gel electrophoresis showed that the 130,000- and 85,000-mol-wt polypeptides of the PV antigen from both cultured keratinocytes and epidermis have identical charges and sizes. In addition to precipitating the PV complex, 14 of 22 PV sera also have antibodies to a calcium-sensitive epitope on a different complex of polypeptides (PF complex) which has previously been shown to be precipitated by all pemphigus foliaceus (PF) sera. The PF complex consists of polypeptides of 260,000, 160,000, 110,000, and 85,000 mol wt. Although the majority of PV sera also precipitate the PF complex, no PF sera precipitate the PV complex. Thus, PV and PF can be absolutely distinguished on a molecular level using the patients' autoantibodies. The PV and PF complexes, although distinct, have certain similarities. The 85,000-mol-wt polypeptide of each is identical. The 160,000-mol wt-peptide of the PF complex and the 130,000-mol-wt peptide of the PV complex have the same isoelectric point and both are capable of disulfide linkage to the 85,000-mol-wt polypeptide. The PV and PF complexes are closely related and may prove important in cell adhesion.

Autoantigens↗
PubMed1988

Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.

Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.

Amino Acid Sequence↗
PubMed1987

Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.

Pemphigus foliaceus (PF) patients have antibodies against a tightly, but noncovalently bound complex of polypeptides, which consists of desmoglein I (DGI) and other, possibly desmosomal, proteins. Most PF antibodies bind a calcium-sensitive epitope on this complex and chelation of calcium destroys the reactivity of these sera with the complex, but not the complex itself. The PF sera that do bind the desmosomal complex in the absence of calcium are those sera capable of binding denatured DGI on immunoblotting, and these same sera also immunoprecipitate only DGI when the desmosomal complex is dissociated with SDS. These findings demonstrate that autoantibodies against a complex of desmosome-associated proteins are characteristic of PF and define a calcium-sensitive conformational epitope on this complex.

Animals↗
PubMed1987

Pemphigus foliaceus antibodies and a monoclonal antibody to desmoglein I demonstrate stratified squamous epithelial-specific epitopes of desmosomes.

Pemphigus foliaceus (PF) is a human autoimmune disease in which autoantibodies are directed against the cell surface of epidermal cells. Using an immunoblotting technique, we recently demonstrated that a subgroup of PF patients have autoantibodies to the desmosomal core glycoprotein, desmoglein I (DGI). There are desmosomes in all epithelia and in heart, yet PF affects only stratified squamous epithelia. One explanation for this finding might be that there are tissue-specific differences in desmosomes. Thus, to determine whether certain epitopes of DGI are tissue-restricted, we performed immunofluorescence studies on various monkey tissues with the following antibodies: a rabbit polyclonal antiserum against whole desmosomes, which demonstrated the desmosomes in all tissues tested; a mouse monoclonal antibody against DGI, MmDGI-1; and PF sera that bound DGI on immunoblotting (PF IB+). In addition, we tested the tissues with PF sera that did not bind DGI by immunoblotting (PF IB-) to determine if these sera were different from PF IB+ sera in their tissue specificity. PF IB+, PF IB-, and MmDGI-1 antibodies stained all stratified squamous epithelia tested, including skin, tongue, upper esophagus, conjunctiva, and cornea; however, they did not stain heart or any nonstratified squamous epithelia, including gall bladder, small intestine, liver, ureter, and bladder. These results indicate that there is tissue heterogeneity of desmosomes, and that epitopes on DGI defined by both PF antibodies and a monoclonal antibody are present only in stratified squamous epithelia. In addition, PF IB- sera had the same tissue specificity as PF IB+ sera. These results may partially explain why PF involves only stratified squamous epithelia.

Antibodies, Monoclonal↗
PubMed1986

A monoclonal antibody to the desmosomal glycoprotein desmoglein I binds the same polypeptide as human autoantibodies in pemphigus foliaceus.

Recent studies have demonstrated that antibodies from about half of patients with pemphigus foliaceus (PF) bind to a 160 kd polypeptide ("PF antigen") in sodium dodecyl sulfate (SDS) extracts of normal human epidermis. Desmoglein (DG) I, a glycoprotein enriched in desmosomal cores, is approximately the same m.w. as PF antigen. To demonstrate that PF autoantibodies bind to DG I, we used a monoclonal IgG antibody (MmDGI-1) that was raised against bovine muzzle desmosomal cores, and that specifically binds DG I. Double immunofluorescence labeling was performed on the same section of normal human skin with PF antibodies, detected by fluorescein-conjugated goat anti-human IgG, and MmDGI-1, detected by rhodamine-conjugated goat anti-mouse IgG. The pattern of reactivity with both antibodies was identical. Immunoblotting studies on proteins extracted from normal human epidermis and separated by SDS-polyacrylamide gel electrophoresis demonstrated that PF antibodies and MmDGI-1 bound co-migrating polypeptide bands of approximate m.w. 160,000. To confirm that these were identical polypeptides, we performed immunoblots of these epidermal extracts that were separated by two-dimensional gel electrophoreses (isoelectric focusing followed by SDS-PAGE). PF antibodies and MmDGI-1 bound identical spots with pI approximately 5.4 to 5.7 and m.w. approximately 160,000. These studies demonstrate that autoantibodies from certain patients with PF, a disorder of cell adhesion, bind to DG I, a desmosomal core glycoprotein.

Antibodies, Monoclonal↗
PubMed1986

Antigenic specificity of fogo selvagem autoantibodies is similar to North American pemphigus foliaceus and distinct from pemphigus vulgaris autoantibodies.

Fogo selvagem (FS) is clinically, histologically, and immunopathologically similar to sporadic pemphigus foliaceus (PF, as seen in North America and Europe), although the epidemiology of these 2 diseases differs markedly. It has been proposed that FS is identical to PF but, for some reason, occurs in an endemic focus in central Brazil. If this hypothesis is correct, the autoantibodies in FS and PF should have similar antigenic specificities. We studied sera from 13 patients with FS from central Brazil, and compared them with 20 sera from patients with PF from the United States. All these sera had circulating antibodies that bound the cell surface of epithelial cells in identical patterns by indirect immunofluorescence on monkey esophagus or normal human skin. In immunoprecipitation studies none of the 13 FS sera precipitated the pemphigus vulgaris (PV) antigen from radiolabeled extracts of cultured human keratinocytes. This is similar to the findings with PF sera of which 19 of 20 sera did not react with PV antigen, but in sharp contrast to the results with PV sera which, as previously reported, all immunoprecipitate the PV antigen. Immunoblotting performed on extracts of normal human epidermis demonstrated that 7 of 20 PF sera specifically and intensely bound an approximately 160 kD polypeptide, previously identified as desmoglein I, a desmosomal glycoprotein. Similarly, 3 of 13 FS sera specifically bound a 160 kD polypeptide. Thirteen normal sera from North America, 8 normal and disease control sera from central Brazil, 11 PV sera, and 12 bullous pemphigoid sera did not specifically bind this polypeptide. Two-dimensional gel electrophoresis confirmed that the 160 kD polypeptides identified by the subgroup of PF and FS sera were identical. These studies demonstrate that, although the exact molecular specificities of the majority of PF and FS sera remain to be determined, FS autoantibodies do not bind the PV antigen and a subgroup of FS autoantibodies have molecular specificity identical to that of a subgroup of PF autoantibodies.

Antibody Specificity↗
PubMed1986

Severe infantile epidermolysis bullosa simplex. Dowling-Meara type.

We encountered eight patients with epidermolysis bullosa (EB) simplex of the Dowling-Meara type, who presented in infancy with severe blistering and were originally clinically thought to have recessive dystrophic EB. One infant died in the neonatal period, and the others have had reduced blistering with advancing age. However, in two of the three older patients, the development of severe disabling hyperkeratosis of the palms and soles has been a prominent feature. The correct diagnosis of EB simplex was initially not made in five patients, because, on routine histologic examination, the blister was apparently subepidermal. Electron microscopy confirmed the correct diagnosis of EB simplex by demonstration of basal cell cytolysis. There was clumping of tonofilaments in seven patients. Immunofluorescence demonstrated a cleft above the basal layer in three cases. The findings of severe extensive blistering at birth that improves with age, milia formation, acral distribution with herpetiform groups of blisters in older children, intraoral lesions, absence of scarring, and intraepidermal clefting due to basal cell cytolysis and clumping of tonofilaments within these basal cells as seen on electron microscopic examination present a subtype of EB simplex similar to that described by Dowling and Meara. This has been recognized in the European but not in the American literature and is probably more frequent than has been previously reported.

Adolescent↗
PubMed1985

Monoclonal antibodies in dermatologic research. Studies with a unique cell-surface antigen defined specifically for keratinocytes by a monoclonal antibody.

Basic principles of production of monoclonal antibodies are discussed in this paper. In order to illustrate the usefulness of such production, studies of a new monoclonal antibody designated KF-2 which defines a unique cell-surface antigen found exclusively in the stratum spinosum of man and lower primates are recounted.

Acantholysis↗