concanavalin-a and Pemphigus

Glucocorticoids (GC) represent the main treatment for pemphigus; however, some patients show GC resistance. GC sensitivity was evaluated in 19 pemphigus patients and 41 controls by the number of binding sites [B(max) (fmol/mg protein)] and the affinity of GC receptor [Kd (nM)] to dexamethasone (DEX) as well as by the pattern of cytokine by DEX-mediated inhibition of concanavalin-A (Con-A)-stimulated PBMC proliferation. The Kd (15.7 ± 2.8 vs.8.1 ± 1.3) and Bmax (6.5 ± 0.9 vs. 3.9 ± 0.3) were higher in pemphigus than controls (p = 0.002). Considering the values above the 95th percentile of normal group as a cut-off (K(d) > 24.9 nM and B(max) > 8.1 fmol/mg protein), elevated K(d) and B(max) were observed in 9.8% and 2.4% of controls and 15.8% and 36.8% of patients (p = 0.02). PBMC proliferation was stimulated by Con-A and inhibited by DEX (p < 0.001) in both pemphigus and control groups. IL-6 and TNFα (pg/mL) basal production were higher in patients than controls. There was an increment of these cytokines after Con-A stimulation, and they were inhibited by DEX (p = 0.002) in controls and remained elevated in pemphigus (p < 0.02). Patients and controls showed no difference in basal and stimulated production of IL-8 and IL-10. There is an alteration on GC sensitivity in pemphigus patients and a higher production of proinflammatory cytokines. Therefore, in pemphigus patients, proinflammatory cytokines might be involved in the mechanism of GC resistance and/or in its maintenance.

Topics: Adolescent; Adult; Binding Sites; Concanavalin A; Dexamethasone; Female; Glucocorticoids; Humans; Interleukin-10; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Male; Middle Aged; Pemphigus; Protein Binding; Receptors, Glucocorticoid; Tumor Necrosis Factor-alpha; Young Adult

Pemphigus foliaceus (PF) antigen is a transmembrane desmosomal glycoprotein (desmoglein I), part of which is located on the keratinocyte surface. Previous studies have shown that after trypsinization of viable human epidermis, this antigen is no longer detected on the surface of detached keratinocytes. It was not known, however, if this loss of antigenic activity was due to destruction, internalization, or cleavage of the antigen itself. In the present study we investigated the fate of the PF antigen after trypsinization of viable human skin. By using Concanavalin-A agarose affinity chromatography, we could partially purify an antigenic glycoprotein fraction that was released by trypsinization into the medium. This antigenic fraction was radiolabeled and tested by immunoprecipitation using sera from endemic pemphigus foliaceus or fogo selvagem (FS), non-endemic pemphigus foliaceus (NEPF), pemphigus vulgaris (PV), and bullous pemphigoid (BP) patients, and sera from normal subjects as controls. Immunoprecipitated labeled proteins were analyzed by SDS-PAGE and autoradiography. All FS sera (20 of 20 FS and five of five NEPF) and 46% of the PV sera (six of 13) immunoprecipitated a band of 45-kD molecular weight. Sera from FS patients in prolonged clinical and serological remission (seven of 10), sera from BP patients (five of five), and sera from normal donors (nine of nine) did not precipitate this 45-kD band. This study showed that a fragment of the PF antigen is released by trypsinization of human skin as a soluble immunoreactive glycopeptide of 45-kD molecular weight. Additionally, this procedure has generated sufficient quantities of the PF antigen for further biochemical characterization.

Topics: Antigens, Surface; Autoantibodies; Chromatography, Affinity; Concanavalin A; Cytoskeletal Proteins; Desmoglein 1; Desmogleins; Desmoplakins; Electrophoresis, Polyacrylamide Gel; Epidermis; Fluorescent Antibody Technique; Humans; Membrane Glycoproteins; Molecular Weight; Pemphigus; Reference Values; Skin; Trypsin

Three cell surface parameters of epidermal cells have been studied by immunofluorescence, pemphigus antigens, concanavalin-A binding sugars, and beta 2 microglobulin. Biopsy specimens were taken from a total of 59 patients with psoriasis, seborrheic and solar keratosis, squamous cell carcinoma, and basal cell epithelioma. We found a loss of demonstrability of all parameters in dedifferentiated tumors or tumor areas in squamous cell carcinoma, premonitory changes in solar keratosis, and no changes in seborrheic keratosis. In the psoriatic epidermis a granular redistribution of the cell surface parameters was occasionally observed in circumscribed areas of the epidermis. A selective loss of the demonstrability of beta 2 microglobulin was the prominent feature in basal cell epithelioma. Our findings demonstrate that the alterations of the cell surface differ in malignant and premalignant skin tumors, in basal cell epithelioma, and in benign psoriatic hyperplasia.

Topics: Antigens; beta 2-Microglobulin; Beta-Globulins; Binding Sites; Carcinoma, Squamous Cell; Concanavalin A; Fluorescent Antibody Technique; Humans; Keratosis; Pemphigus; Precancerous Conditions; Psoriasis; Skin Neoplasms

Human peripheral blood lymphocytes (PBL) are activated to become suppressor T cells (S-T-C) by incubation with Concanavalin-A (Con-A). This has become the standard method for evaluation of suppressor function in patients. S-T-C function has been found to be impaired in several autoimmune diseases, including systemic lupus erythematosus (SLE). Using this assay, we have investigated suppressor-cell function in 2 autoimmune disorders, bullous pemphigoid (BP) and pemphigus vulgaris (PV), studying 6 patients from each group. Three patients with active SLE (positive controls), and 11 normal donors (negative controls) were also included. None of these patients had received systemic therapy with the exception of 2 patients with PV who were treated with gold in the past. PBL from these patients were incubated with and without 40 micrograms/ml Con-A for 72 hr to generate suppressor cells. Both groups of PBL were then irradiated wih 1500 r cobalt. Co-cultures were set up in sextuplicate using normal PBL as responders. Responder PBL were stimulated with 0.5, 1.0, and 2.0 micrograms/ml of phytohemagglutin (PHA) and 5.0, 10.0, and 20.0 micrograms/ml of Con-A. Cultures were pulsed on day 3 with 3H-thymidine and harvested on day 4. Data were analyzed using Student's t-test. S-T-C function was found to be significantly impaired in SLE vs normal (p = 0.0316). No statistically significant difference was seen in BP (p = 0.5883) and PV (p = 0.0921) as compared with normals. A defect in suppressor cell function may still be present in patients with PV and BP for the defect may be antigen-specific and therefore remain undetected by the Con-A suppressor assay.

Topics: Cobalt; Concanavalin A; Culture Techniques; Fluorescent Antibody Technique; Humans; Interleukin-2; Lupus Erythematosus, Systemic; Lymphocytes; Pemphigoid, Bullous; Pemphigus; Phytohemagglutinins; Skin Diseases, Vesiculobullous; T-Lymphocytes, Regulatory

Monoclonal antibodies recognizing human T cell differentiation antigens were used to study lymphocyte populations in three cutaneous diseases. Neoplastic lymphocytes from patients with varying phases of cutaneous T cell lymphoma (mycosis fungoides, Sezary syndrome and related presentations) were reactive with OKT1 and OKT3 (pan T cell reagents) and OKT4 (an antibody defining the functional "helper" T cell subset). The malignant cells lacked membrane antigens reactive with OKT5 and OKT8 (markers of "suppressor" T cells). The presence of an OKT1+, OKT3+, OKT4+, OKT5-, OKT8- phenotype on the neoplastic T lymphocytes of cutaneous T cell lymphoma (CTCL) supports the clinical impression that all phases of CTCL represent a single disease entity. A patient with pemphigus vulgaris, a disease of autoreactive, antiepidermal antibodies was shown to consistently have a marked expansion of the peripheral blood OKT4 reactive T lymphocyte population. These findings suggest that autoantibodies in pemphigus vulgaris may occur in the context of a profound OKT4/OKT5 immunoregulatory imbalance. Peripheral blood lymphocytes from patients ith extensive psoriasis vulgaris had a normal profile of reactivity with the OKT antibodies. In addition, OKT6 (marker of intrathymic T cells) has been shown to react with Ia+ dendritic cells in the epidermis suggesting that this antibody may recognize Langerhans' cells.

Topics: Adolescent; Adult; Aged; Animals; Antibodies, Monoclonal; Cell Transformation, Neoplastic; Concanavalin A; Humans; Lymph Nodes; Lymphocytes; Lymphoma; Mice; Middle Aged; Pemphigus; Phenotype; Psoriasis; Rabbits; Skin Diseases; T-Lymphocytes

Sixteen psoriatic patients and 11 control subjects were investigated by immunofluorescence for skin immunoglobulins (IG) and complement (c) deposits and for keratinocyte membrane markers with anti beta 2 microglobulin (beta 2 m) antibodies (Ab), Con A, and pemphigus Ab. IG and/or c deposits were almost constant in involved epidermis. Three patterns were associated: (1) parakeratotic nuclear (dots-dashes), (2) stratum corneum (SC) intercellular (lamellar pattern), (3) vascular (granular or linear deposits in papillary dermal vessels). In uninvolved epidermis nuclear or vascular deposits could occasionally be present but intercellular pattern was never found in SC. Control specimens were always negative. The three surface markers investigated (beta 2 m, Con A receptor, Pemphigus antigen) could be demonstrated on psoriatic keratinocytes with only slight differences in distribution when compared with controls. Thus, by this methodology no important abnormality could be found in psoriatic keratinocyte membrane antigens.

Topics: beta 2-Microglobulin; Cell Nucleus; Complement System Proteins; Concanavalin A; Fluorescent Antibody Technique; Humans; Immunoglobulins; Membranes; Pemphigus; Psoriasis

Rabbit anti-guinea pig epidermal cell sera (AES) which were prepared by immunizing rabbits with enzymatically dispersed viable guinea pig epidermal cells were shown to react to the intercellular substances of stratified squamous epithelia of guinea pigs and monkeys in a pattern similar to that seen with concanavalin A (ConA) or pemphigus sera (PS) by immunofluorescence. Reciprocal blocking tests were performed on guinea pig lip mucosa after removal of the non-specific binding substances between preincubated substances and subsequently incubated ones. No definite inhibition was noted in the subsequent reactions upon preincubation with AES or PS. However, preincubation with ConA weakened the subsequent reaction with PS, but did not block the reaction with AES. Effects of solvents on the receptors for AES, ConA and PS were also examined. ConA receptor was resistant to both saline solution and ethanol (95% and 99%). PS receptor was labile to both saline solution and ethanol, while AES receptor was labile to ethanol, but resistant to saline solution. These observations suggest that the receptor for AES differs from that for ConA or PS.

Topics: Aged; Animals; Antibody Specificity; Binding Sites, Antibody; Concanavalin A; Epidermis; Ethanol; Fluorescent Antibody Technique; Goats; Guinea Pigs; Haplorhini; Humans; Lip; Mice; Mucous Membrane; Pemphigus; Rabbits; Rats; Receptors, Concanavalin A; Receptors, Drug; Skin; Sodium Chloride

Epidermal antigens partially purified by either isoelectric focusing (the pH 5.2 peak) or concanavalin A (Con A) affinity chromatography react with Con A in tube precipitation reactions. Bands of identity between crude skin antigens, the Con A affinity antigens eluted with alpha-methyl glucoside and the pH 5.2 peak are formed in Ouchterlony gel with rabbit antisera (Rab) to the pH 5.2 antigen. Absorption of Rab or pemphigus antibodies (Pab) with A+ erythrocytes does not affect complement fixation reactions of Rab with the skin antigen nor abolish the ability of Pab to interact with the intercellular cement. The pH 5.2 epidermal antigens react weakly with Pab in tube precipitation reactions and only weakly, if at all, to inhibit Pab reactions in the region of the intercellular cement. High concentrations of Con A inhibit the Pab, peroxidase-anti-IgG tissue reaction whereas the converse inhibition does not occur. Simultaneous use of both Pab and Con A-perodixase reactions at Con A concentrations which do not inhibit Pab, causes enhanced tissue peroxidase reactions in the region of the intercellular cement. These preliminary data indicate that the Pab and Con A-reacting sites are localized on different molecules or antigenic determinants in the intercellular cement. They exclude the possibility that A-blood substances are involved in either site.

Topics: Animals; Antigens; Chromatography, Affinity; Concanavalin A; Epitopes; Humans; Isoelectric Focusing; Pemphigus; Rabbits; Receptors, Concanavalin A; Skin

Immunoglobulin from pemphigus patients binds to the surface of mouse epidermal cells in culture. Cells incubated with the pemphigus antibody are easily detached from culture plates whereas cells incubated with serum from normal patients remain on the plate. Pemphigus antibody-mediated cell detachment is blocked by the addition of the proteinase inhibitors soybean trypsin inhibitor and alpha2-macroglobulin to the culture media. Detachable cells are viable, and activation of the complement cascade is not necessary for cell detachment. The anti-cell-surface antibody of pemphigus appears to disrupt adhesion between viable epidermal cells by activation of proteinase.

Topics: Antibodies; Antigen-Antibody Reactions; Cell Adhesion; Cell Survival; Cells, Cultured; Concanavalin A; Enzyme Activation; Epidermis; Humans; Immunoglobulin Fab Fragments; Pemphigus; Protease Inhibitors

Differences in the cell surface of malignant cells, as compared with normal cells, are believed to be characteristic of many features of tumor cell behaviour. We have obtained evidence suggesting that solid and superficial basal cell carcinomas lack immuno-reactive beta-2-microglobulin (beta-2-m) on the cell surface, in contrast to normal epidermis and that of various non-malignant dermatoses, including basal cell papillomas.

Topics: Antibodies, Neoplasm; beta 2-Microglobulin; Beta-Globulins; Carcinoma, Basal Cell; Cell Membrane; Concanavalin A; Fluorescent Antibody Technique; Humans; Pemphigus; Skin Neoplasms

Inorder to study the relationship between epidermal surface saccharides and pemphigus antigen(s), fluorescein-labelled Concanavalin A (Con A) and Phytohemagglutinin-P (PHA-P) were used. These phytohemagglutinins were found to bind with the intercellular areas of human epidermis. Alpha-methyl-D-mannoside and alpha-methyl-D-glucoside inhibited the epidermal intercellular staining pattern produced by ConA-FITC, while N-acetyl-D-galactosamine blocked the same staining pattern produced by PHA-P-FITC. Normal human skin reacted with pemphigus antibody and pemphigus skin with the deposition of IgG both gave a positive intercellular staining pattern with fluorescein labelled phytohemagglutinins. Our data indicated the non-identity of the binding of Con A and PHA-P, and pemphigus antigen(s).

Topics: Aged; Antibodies; Antigens; Binding Sites, Antibody; Biopsy; Concanavalin A; Female; Fluorescent Dyes; Galactosamine; Glucose; Humans; Immunoglobulin G; Lectins; Mannose; Monosaccharides; Pemphigus; Skin; Staining and Labeling

The interaction of Concanavalin A (Con A) and the surface membranes of the epithelial cells of human oral mucosa, epidermis and guinea-pig lip was studied. Two methods were used: fluorescent Con A and Con A in combination with horse radish peroxidase. With both methods the surface coat of epithelial cells was stained. The specificity of the staining methods could be demonstrated. The interaction between pemphigus antibodies and the surface membranes of epithelial cells could be blocked by Con A.

Topics: Animals; Antibodies; Binding Sites, Antibody; Cell Membrane; Concanavalin A; Epithelial Cells; Epithelium; Epitopes; Glycoproteins; Guinea Pigs; Humans; In Vitro Techniques; Lip; Mouth Mucosa; Pemphigus; Skin

The binding sites of Concanavalin A (Con A) and pemphigus antibody in the intercellular spaces of the human epidermis have been investigated. Extraction of human skin with phosphate-buffered saline (PBS) and 70% ethanol resulted in complete loss of the binding sites of the pemphigus antibody, whereas the extracted skin still retained its reactivity to Con A. Preincubation with more than 0.25 mg/ml of Con A with normal skin caused a reduction in the binding of pemphigus sera. However, preincubation of pemphigus sera with human skin caused no inhibition of the subsequent binding of fluorescein-labelled Con A. These results indicate that Con A binding sites might be related to, but not identical with those of pemphigus antibody.

Topics: Antibodies; Antigens; Binding Sites; Binding Sites, Antibody; Concanavalin A; Humans; Pemphigus; Protein Binding; Skin

An exfoliating substance elaborated by certain phage Group 2 staphylococci causes toxic epidermal necrolysis. Both in man and in the newborn mouse, intraepidermal cleavage is the predominant histologic feature following exposure to this toxin. Electron microscopic study of sequential biopsy specimens obtained from neonatal mice and from organ cultures of human skin revealed intercellular cleavage and cell separation. The extracellular nature of the exfoliative process was confirmed in several ways: (1) perfused tracers did not penetrate cells during cell separation; (2) cultured cells exposed to high doses of exfoliating fractions demonstrated no signs of injury; and (3) cleaved surfaces examined by scanning electron microscopy and surface replication demonstrated intact plasma membranes. When fractions capable of inducing exfoliation were applied to cultured keratinocytes of fibroblasts, sperm, or lymphocyte suspensions, and to human or mouse skin in vivo, they did not alter the distribution or intensity of concanavalin A binding, ruthenium red staining, pemphigus antibody binding, or HL-A surface antigens. Therefore, while the pathogenesis of staphylococcal toxic epidermal necrolysis involves intercellular cleavage, the molecular cell surface target remains unknown.

Topics: Animals; Binding Sites, Antibody; Cell Membrane; Concanavalin A; Cytotoxicity Tests, Immunologic; HLA Antigens; Humans; Lymphocytes; Mice; Organ Culture Techniques; Pemphigus; Ruthenium Red; Skin; Staphylococcal Infections; Stevens-Johnson Syndrome; Toxins, Biological

Topics: Acantholysis; Antibodies; Basement Membrane; Binding Sites, Antibody; Carbohydrate Metabolism; Cell Membrane; Collagen; Concanavalin A; Extracellular Space; Ferritins; Fluorescent Antibody Technique; Humans; Microscopy, Electron; Mouth Mucosa; Pemphigus; Skin

Topics: Cell Membrane; Cell Nucleus; Concanavalin A; Fluoresceins; Humans; Lectins; Microscopy, Fluorescence; Monosaccharides; Pemphigus; Protein Binding; Skin; Staining and Labeling