Organ culture studies of pemphigus antibodies. II. Ultrastructural comparison between acantholytic changes in vitro and human pemphigus lesions.

The ultrastructural and light microscopic features of acantholysis produced in organ culture were compared with those of human pemphigus lesions. In both, an intraepidermal split was seen an typical suprabasal acantholytic cells were present. These cells contained small bundles of tonofilaments, usually located away from the cell periphery. Desmonsomal plaques with inserted tonofilaments frequently remained along the periphery of acantholytic cells and along the upper portion of the periphery of basal cells. The ultrastructural similarity between in vitro and in vivo lesions provides additional evidence to suggest that organ cultures may provide a valid model for studying the dynamics of pemphigus lesion formation.

Juvenile dermatitis herpetiformis versus "benign chronic bullous dermatosis of childhood." Are these immunologic diseases?

Seven cases of juvenile dermatitis herpetiformis have been investigated. Immunofluorescence and histologic studies were made in all and jejunal biopsies in three. Immunopathologic results were positive in all cases including one that had previously been reported to be negative. Two groups could be distinguished according to clinical and histologic criteria, response to sulfapyridine, and character of the immunoglobulin deposits. The first corresponded to dermatitis herpetiformis (DH) of adults, with characteristic lesions of the jejunal mucosa; the second corresponded either to bullous pemphigoid (BP), although in the majority of the cases without circulating antibasement-membrane antibodies, or to a mixed type with the combined features of DH and BP. Repeated biopsies with serial sections are essential for demonstrating immune deposits. The question arises whether any immunologically negative cases of "benign chronic bullous dermatosis of childhood" actually exist.

Immunogold localization of the 97-kD antigen of linear IgA bullous dermatosis (LABD) detected with patients' sera.

The classification of linear IgA bullous dermatosis in the group of subepidermal blistering diseases is still a matter of controversy. This situation is due partly to the considerable clinical heterogeneity of the disease but also results from the difficulties in characterization and localization of the specific basement membrane zone antigen(s) recognized by immunoglobulin (Ig)A antibodies. In the present study, we have combined the Western blot detection of circulating autoantibodies with an ultrastructural immunogold labeling of human skin antigens using the same patients' sera. Our results, obtained with a short series of sera showing exclusive IgA class reactivity with the epidermal portion of salt-split skin, indicate that the antibodies recognizing the 97-kD antigen on immunoblot bind to the hemidesmosomal plaques of basal keratinocytes and the adjacent lamina lucida. These homogeneous laboratory results remain in striking contrast to the heterogeneity of clinical pictures in the patients studied, suggesting a participation of complementary, possibly not humoral, phenomena in the pathogenesis of linear IgA bullous dermatosis.

The ultrastructural localization of IgA deposits in chronic bullous disease of childhood (CBDC).

A case of bullous disease in a child with linear IgA immune deposits at the basement membrane zone and with some clinical, histological, and electron microscopic characteristics both of dermatitis herpetiformis and bullous pemphigoid, is described. The bulla formed between the basal lamina and basal cell membranes as in bullous pemphigoid, but at the same time there were numerous inflammatory cells in the dermis just below the partly destroyed basal lamina and also abundant fibrin deposits in very recent bulla and in the skin, all of which is rather characteristic of dermatitis herpetiformis. Ultrastructurally, the IgA deposits were located chiefly below the lamina basalis (the dermal type) but also, though less abundantly, in the lamina lucida, very much as we have seen them to be in adult cases with linear IgA immune deposits at the basement membrane zone. The investigations have supplied further evidence showing the chronic bullous disease of childhood to be actually a counterpart of the form in adults with the same linear localization of IgA deposits.

Detection of DNA-psoralen photoadducts in mammalian skin.

An immunofluorescence (IF) method for the detection of 8-methoxypsoralen (8-MOP) photoadducts to DNA has been developed to assess nuclear damage in keratinocytes and melanocytes after psoralen plus UVA (PUVA) treatment, both under in vitro and in vivo conditions. Cryostat sections of the albino and pigmented guinea pig and human skin were used for in vitro studies to establish minimal and maximal drug concentration and UVA dosimetry for the detection of DNA-8-MOP photoadducts. Limits of detection were as low as 10 ng/cm2 8-MOP and 1 J/cm2 UVA for skin sections and sodium bromide-split epidermal sheets. Guinea pigs treated with topical PUVA revealed positive IF stain in epidermal cell nuclei at a threshold dose of 100 micrograms/cm2 8-MOP and 13 J/cm2 UVA. Pretreatments of cryostat cuts with ethanol and alkali before IF test enhanced the sensitivity of detection in vivo about 10-fold and enabled us to follow the repair of DNA damage after treating normal guinea pig skin with a dose of 50 micrograms/cm2 8-MOP plus 6 J/cm2 UVA. The most interesting findings were as follows: A sensitive method to detect PUVA-induced nuclear damage in epidermal and dermal cells was developed. PUVA treatment induced nuclear DNA damage to melanocytes as well as to adjacent keratinocytes, and melanocytes appeared to be 10 times less vulnerable to photo-damage than keratinocytes. There was a greater propensity for the proliferative cells to be damaged by PUVA. PUVA induced nuclear damage up to 700 micron depth in the dermis. The usefulness of the IF test in detecting DNA damage in microgram and ng amounts in vivo and in following the repair of damaged DNA induced by PUVA.

The ultrastructural localization of IgA in skin of a patient with mixed form of dermatitis herpetiformis and bullous pemphigoid.

A case with mixed features of dermatitis herpetiformis and bullous pemphigoid was investigated by immuno-electron microscopy. There were clinical, histological, and ultrastructural characteristics of both diseases, the response to sulfapyridine was dramatic at the beginning, but intestinal lesions were absent. Direct immunofluorescence tests were made 6 times in the 4 year period and demonstrated in all biopsies exclusively linear IgA deposits. The IgA deposits were shown to occupy the entire lamina lucida and to adhere to the basal cell membranes and lamina densa, very much like IgG deposits in bullous pemphigoid. Antibodies against the basement membrane zone could not be demonstrated in the serum.

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.

Substrate specificity of anti-epithelial antibodies of pemphigus vulgaris and pemphigus foliaceus sera in immunofluorescence tests on monkey and guinea pig esophagus sections.

The indirect immunofluorescent (IF) reactivity of the pemphigus antibodies in sera of 21 cases of pemphigus vulgaris (PV), 15 cases of pemphigus foliaceus (PF), and 14 cases of Brazilian PF (BPF) was compared on 2 substrates, notably monkey esophagus (ME) sections and guinea pig esophagus (GPE) sections. The IF reactions of the pemphigus antibodies of PV could be distinguished from those of PF or BPF by differences in their reactivity on ME and GPE sections in 98% of the cases examined in this study. In most cases, the pemphigus antibodies of PV cases gave higher titers and stronger IF staining reactions on ME sections, while those of PF and BPF cases gave stronger reactions on GPE sections. In addition, most (13 of 21) PV sera react with the lowest 3-4 cell layers of ME sections, while most (13 of 15) PF sera failed to do so but did react with the upper layers of the sections. Importantly, in 8 of the 50 cases examined by IF, the choice of substrate affected the detectability of the pemphigus antibodies, i.e., 4 of 15 PF and 2 of 14 BPF sera reacted only with GPE and 2 of 21 PV sera reacted only on ME. These research findings point to the need for an evaluation of the combined use of ME and GPE in routine diagnostic studies of pemphigus antibodies.

IgA antibodies in chronic bullous disease of childhood react with 97 kDa basement membrane zone protein.

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease occurring in prepubertal children. Both CBDC and its adult counter-part, linear IgA bullous dermatosis (LABD), are characterized by linear deposition of IgA along the cutaneous basement membrane zone (BMZ). Circulating IgA antibody in LABD has been found to bind to a 97-kDa BMZ antigen, whereas the antigen in CBDC has not been well characterized. The purpose of this study was to evaluate the immunoreactivity of BMZ IgA antibodies in a series of CBDC patients. We evaluated 12 sera from patients with CBDC with circulating IgA anti-BMZ antibodies on indirect immunofluorescence (IIF), which stained the epidermal side of split skin with titers ranging from 1:20 to 1:640. Immunoblotting was performed against two preparations of BMZ proteins: one enriched with the two bullous pemphigoid antigens (BP230, BP180) and one enriched with the LABD antigen (LABD97). Eight of the twelve sera reacted with a 97-kDa protein that co-migrated with the protein detected in many LABD sera. The intensity of the reaction on immunoblot correlated with serum antibody titers. There was no consistent pattern of reactivity of the IgA anti-BMZ antibodies with either the BP230 or BP180 antigens, although two sera reacted with several higher molecular mass proteins (160-200 kDa). The significance of this reactivity was examined with immunoblotting using BMZ-affinity-purified antibodies, and ELF using nitrocellulose-eluted antibodies. One serum also contained anti-BMZ IgA antibodies that reacted with a 180-kDa protein, corresponding to BP180. We conclude that IgA antibodies in CBDC sera recognize a 97-kDa BMZ antigen present on the epidermal side of BMZ split skin that co-migrates with the antigen previously identified in LABD. These findings suggest that CBDC and LABD are the immunologically related disorders occurring in different age groups.

Substrate specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.

Characterization of an autoantigen associated with chronic ulcerative stomatitis: the CUSP autoantigen is a member of the p53 family.

A unique clinical syndrome has been described in which patients have chronic oral ulceration and autoantibodies to nuclei of stratified squamous epithelium. We have characterized the autoantibodies from patients sera and found that the major autoantigen is a 70 kDa epithelial nuclear protein. Sequencing of the cDNA for this protein, chronic ulcerative stomatitis protein, revealed it to be homologous to the p53 tumor suppressor and to the p73 putative tumor suppressor, and to be a splicing variant of the KET gene. The p53-like genes, p73 and the several KET splicing variants, are recently described genes of uncertain biologic and pathologic significance. This study provides the first clear association of a p53-like protein with a disease process.

Novel non-radioisotope immunoprecipitation studies indicate involvement of pemphigus vulgaris antigen in paraneoplastic pemphigus.

We have developed two different novel immunoprecipitation assays in which radioisotopes are not used, and have examined antigens for four cases of paraneoplastic pemphigus (PNP) including three new patients. The PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder by immunofluorescence, and reacted with a characteristic doublet of the 210 and 190 kD proteins by immunoblotting of normal human epidermal extract, confirming the diagnosis of PNP. In addition, by immunoprecipitation using silver-stain to detect immunoprecipitated proteins, the PNP sera detected the 250, 210 and 190 kD proteins, while control bullous pemphigoid sera detected only the 230 kD bullous pemphigoid antigen. Furthermore, with another immunoprecipitation using cell surface biotinylation, three of the four PNP sera specifically reacted with the 130 kD pemphigus vulgaris antigen (Dsg3), indicating that pemphigus vulgaris antigen may be involved in PNP. This reactivity was further suggested by the immunoblot analysis using recombinant pemphigus vulgaris antigen. In future, these non-radioisotope immunoprecipitation assays should become a useful tool not only to unravel the complex situation for the PNP antigens, but also to study antigens in other autoimmune bullous skin diseases.

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.

Comparison of commercial kits for the detection of anti-nDNA antibodies using Crithidia luciliae.

The reactions of sera from 15 selected patients with connective tissue diseases and 4 selected control people were compared with the use of five commercial kits detecting anti-nDNA antibodies by indirect immunofluorescence on Crithidia luciliae. The cases with systemic lupus erythematosus (SLE) and a related condition reacted with the kinetoplasts of the C. luciliae in each kit tested with one exception, notably a case of drug-induced LE. The four control cases selected for a trace of staining of the nuclei of C. luciliae gave negative reactions with the kinetoplasts. The titer for each individual positive serum varied from 1.36 to 2.67 (mean SG 2.04) geometric standard deviation units. The staining pattern of sera positive for anti-nDNA antibodies on C. luciliae included reactivity of the kinetoplast with or without nuclear staining. The drug-induced LE serum produced only nuclear staining with no significant kinetoplast staining, i.e., a negative test for anti-nDNA antibodies. Patient control sera stained only the nucleus when any reactivity on C. luciliae was present. Generally, there were no major differences in titers and patterns of sera when comparisons were made between manufacturers. The sera were also tested by the Farr radioimmunoassay and the latex nucleoprotein test. The results of both of these assays correlated in most cases with the C. luciliae reactions.

Immunofluorescence of biopsy specimens: comparison of methods of transportation.

Immunofluorescence findings for IgG, IgA, IgM, and C4 on biopsy specimens quick frozen and transported frozen were compared with findings on portions of the same biopsy specimens placed in holding solution and transported at ambient temperatures. A total of 52 biopsy specimens were examined from normal individuals and patients with systemic lupus erythematosus (SLE), discoid lupus erythematosus (DLE), pemphigus, pemphigoid, and dermatitis herpetiformis. Overall agreement of results was 90%: 88% in SLE; 50% to 66% in DLE, 96% in pemphigus; 92% in bullous pemphigoid; and 87% in dermatitis herpetiformis. Except for two of the 42 biopsy specimens, the combined IgG, IgA, IgM, and C4 findings were the same. In one DLE case, only the frozen biopsy specimen was positive. In one case of dermatitis herpetiformis, only the ambient temperature biopsy specimen was positive. Results indicate the holding solution at ambient temperature can be used in place of the frozen method.

Pemphigus antibody action on skin explants: kinetics of acantholytic changes and stability of antigens in tissue cultures of normal monkey skin explants.

The pathogenic effect of the intercellular antibodies of pemphigus was studied by using organ culture of monkey skin. Skin explants that were grown on sera with intercellular antibody titers of 320 or greater fixed these antibodies within one day as demonstrated by direct immunofluorescence for IgG. None of the control sera gave such staining patterns. Following the binding of intercellular antibodies, characteristic histologic changes appeared, notably separation of individual epidermal cells and acantholysis. These histologic changes became more marked in two to five days. During this period, the bound antibodies and the intercellular antigens decreased and disappeared. These temporal relationships of immunofluorescence and histologic findings suggest that pemphigus antibodies play a role in the induction of acantholysis.

Cicatricial pemphigoid of Brunsting-Perry. Immunofluorescent studies.

Six patients with a vesiculobullous eruption of the type described by Brunsting and Perry as benign pemphigoid were studied by direct and indirect immunofluorescence. All six showed linear deposits of IgG but not IgM or IgA at the epidermal-dermal junction. One case also showed C3 deposition and one patient had circulating antibasement membrane zone antibodies in a titer of 1280. These data provide strong evidence that this condition belongs to the cicatricial pemphigoid-bullous pemphigoid spectrum of disease.

Annular atrophic plaques of the face. A variety of atrophic discoid lupus erythematosus?

In a patient whose condition was diagnosed clinically and pathologically as annular atrophic plaques, the lesions bore some resemblance to morphea, lichen sclerosus et atrophicus, and discoid lupus erythematosus (DLE). In a lesion that appeared recently, which was not DLE clinically, the histological characteristics were consistent with DLE, and immunofluorescence examination disclosed an immunofluorescent band at the epidermal-dermal zone. We suggest that annular atrophic plaques may be an atypical variant of DLE, with a tendency to sclerosing.

Anti-endomysial antibodies. A serologic marker of dermatitis herpetiformis.

Direct immunofluorescence (IF) studies of skin biopsies are of value in the diagnosis of most, but not all, cases of dermatitis herpetiformis (DH). Similarly, histologic studies are of help but may be questionable or completely nonspecific. Serologic studies for the presence of IgA-class anti-endomysial antibodies are very specific and are found in 70% of patients with DH and in all untreated patients with celiac disease. The titers of these antibodies are directly associated with the degree of gut disease in these patients. Thus, the presence of these antibodies even in the absence of classic direct IF and histologic findings are diagnostically important. We encountered three cases in which both direct IF and histologic studies were equivocal toward confirming the clinical diagnosis of DH. Serologic studies for the presence of IgA-class anti-endomysial antibodies provided evidence for the diagnosis of DH, and, in each case, results were confirmed by further direct IF studies. Since these antibodies are disease specific for DH and celiac disease and are found in most active cases of DH, they may be considered an adjunct to the direct IF and histologic studies of the skin.

Diagnosis of systemic lupus erythematosus. Importance of antinuclear antibody titers and peripheral staining patterns.

Titers and patterns of antinuclear antibodies (ANA) in sera from 134 normal blood donors, 20 patients with rheumatoid arthritis, 15 patients with systemic scleroderma, and 32 patients with diagnosed or suspected systemic lupus erythematosus (SLE) were studied. The difference between the findings with sera of patients with SLE and normal subjects in terms of high (greater than 160) titers of ANA was greater than in terms of peripheral staining patterns. However, in comparing sera from patients with SLE with sera from patients with other connective tissue diseases, greater differences were found in the incidence of peripheral patterns of ANA compared to differences in the frequency of high ANA titers. Maximum specificity in the diagnosis of SLE was achieved when both titers and patterns of ANA were considered.