Linkage of pemphigus vulgaris antibody to the major histocompatibility complex in healthy relatives of patients.

Pemphigus vulgaris (PV) is an autoimmune disease caused by high concentrations of antibody to an epidermal cadherin. The disease is associated with two kinds of HLA-DR4, DQ8 haplotypes dominantly distributed among Jewish patients, and these plus DR6, DQ5 haplotypes in non-Jewish patients. Low levels of the PV antibody were found in 48% of a total of 120 asymptomatic parents, children, and siblings of 31 patients, thus exhibiting dominant inheritance. The inheritance of these low levels of antibody in asymptomatic relatives was linked to the major histocompatibility complex with a highly significant logarithm of the odds score of 9.07, almost always to a DR4 or DR6 haplotype of the patient. Disease appears to occur in susceptible individuals with low levels of antibody when a second factor, either environmental or genetic, induces high levels, sufficient to produce blisters.

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.

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.

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.

A distinct stratum corneum antigen in psoriasis and its reactions with stratum corneum autoantibodies.

Stratum corneum antibodies are ubiquitous and can be detected by various immunological methods. Of these, the ones detected by hemagglutination undergo changes in antibody titers and have been implicated in psoriasis. The purpose of our study was to examine if differences exist in the activities of the antigens isolated from psoriatic scales in comparison to normal callus. Stratum corneum antigens were prepared by trypsin-phenol-water extraction. The water phase, which contains the stratum corneum antigen, was used to sensitize the red blood cells in the hemagglutination assay. The antibody activity in human sera was determined before and after absorption with antigens isolated from callus, psoriatic scales, and cell envelopes. We found notable differences in the antigens obtained from callus and psoriatic scales. These include higher antibody titers to the antigens of the scales, the presence of unique antigenic determinants on psoriatic scales and the localization of the antigen on cell envelopes. These immunological differences were corroborated by the marked biochemical differences of certain amino acids, most notably glycine and proline, and these differences were unique to psoriatic scales as they were not shared with other hyperproliferative disorders.

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.

Developments in immunofluorescence: the need for standardization.

DIFFICULTIES EXPERIENCED BY THE NEWCOMER TO THE FLUORESCENT ANTIBODY STAINING METHOD LARGELY ARISE FROM TWO CAUSES: (1) the many modifications in the procedure which have been suggested over the last decade; (2) the variability of the basic reagent, fluorescent conjugate, in terms of potency and specificity. The ensuing problem of non-reproducibility is clearly an important factor for clinical laboratories now faced with increasing demands for routine tests involving immunofluorescence. In this paper a simple approach to standardization is described which, if adopted, would lead to a more efficient use of the method.

Studies on defined immunofluorescence in clinical immunopathology. III. Past progress, some new methods, and prospects for the future.

Experience with chessboard titrations over the past 9 years has shown that essentially all indirect IF staining systems that employ anti-IgG conjugates can be defined with the aid of assays for F/P ratios and/or antibody concentrations. Some improvements in the radial diffusion assays for antibody protein have been effected by refinements in methodology. Studies with a form of the Dass system showed that over a 16-fold range of light intensities, relatively little change occurred in "titers" of IgG (comparably to titers of patient sera) or in PEP conjugates as seen in DASS chessborad titrations. An analysis of 233 PEP values included in 12 literature reports on indirect IF tests indicate that 88% fall within one doubling dilution of 1/16 unit/ml or about 12.5 mug labeled Ab/ml. The DASS reactions also are within this range. Factors responsible for variation beyond these limits in indirect IF tests were analyzed. The most important factor appears to be experimental errors. Other factors include low titers of primary antibodies, some aberrant conjugates, and a recent shift toward greater sensitivity of chessboard titrations. The latter appears to be due primarily to modes of reading and should be controlled by the constant use of reference sera. It now seems possibe that future IF studies may be defined with the aid of this DASS system. To achieve this goal, it will be necessary to not only elaborate microanalytic techniques of the type that have been considered at this Conference but also to generate macroanalytic methods that can be used in parallel with them as a basis for interpreting microscopic observations both in the DASS system and in IF studies of sera and tissues.

Studies on defined immunofluorescence in clinical immunopathology. II. Relationship of F/P ratios pf conjugates and staining properties in indirect immunofluorescence.

The effect of conjugate F/P ratios on titers of patient antibodies was evaluated with two model systems in IIF chessboard titrations, notably, antinuclear antibodies and the intercellular antibodies of pemphigus. The plateau titers of both antibodies and F/P conjugate ratios were analyzed and proved to have a high degree of linear relationship statistically. Single titrations of other tissue antibodies, including skin basement membrane antibodies of bullous pemphigoid, MTA, GPA, SMA, and ThA with the same group of conjugates yielded similar information. A quantitative relationship between the two parameters was established.

Complement-fixing intercellular antibodies in a case of cicatricial pemphigoid.

A patient with ocular and oral lesions, as well as cicatricial pemphigoid, had intercellular antibodies to a titer of 80 by indirect immunofluorescence (IF) microscopy staining tests with her serum. These were identified as pemphigus-like antibodies, since direct IF microscopy indicated no intercellular IgG deposits. Complement IF microscopy tests of the serum gave a closely comparable titer (40) of intercellular antibodies. Since serum samples of patients with true pemphigus either fail to fix complement or have much lower complement fixation titers, the complement IF microscopy tests of patients' serum samples may be of value in differentiating pemphigus-like antibodies from pemphigus antibodies.

Action of topically applied arachidonic acid on the skin of patients with psoriasis.

Concentrations of arachidonic acid ranging from 0.1% to 2% were applied under occlusive dressings to psoriatic plaques in 45 patients. Alleviation of the clinical symptoms of psoriasis including complete clearing in some cases was obtained with the use of 0.5% to 2% arachidonic acid applied under occlusion every 24 to 48 hours five to seven times. Histologic examination showed polymorphonuclear leukocytes penetrating into the stratum corneum and formation of microabscesses or wide-spread accumulations of polymorphonuclear leukocytes in the stratum corneum, with its eventual destruction. The parakeratotic horny layer became detached; this was followed by restoration of the granular layer and an apparently normal stratum corneum. While arachidonic acid metabolites can be proinflammatory and proproliferative, they may also be important in the healing process for psoriasis.

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.

Reticulin and endomysial antibodies in bullous diseases. Comparison of specificity and sensitivity.

Reticulin antibodies of IgG- and IgA-class and endomysial antibodies, which are of the IgA class, were studied by indirect immunofluorescence in 30 normal subjects, and in 45 patients with dermatitis herpetiformis (DH), 31 with pemphigoid, and 30 with pemphigus. Endomysial antibodies were present in 65% of patients with DH maintained on a normal diet and were absent in those on a gluten-free diet and in those with other diseases and in normal controls. Reticulin antibodies of the IgA class were disease specific for DH and occurred in only 25% of such patients. IgG-class reticulin antibodies, on the other hand, were not specific for DH, as they occurred in similar frequencies in patients with pemphigoid and pemphigus and in normal subjects. IgA-class reticulin antibodies occurred primarily in those patients with DH who had high titers of endomysial antibodies. None of the 20 such patients who were negative for endomysial antibodies had IgA-class reticulin antibodies. These studies indicate the high degree of specificity and sensitivity of endomysial antibodies.

Coexistence of bullous pemphigoid and systemic lupus erythematosus.

A vesiculobullous eruption with clinical and histological features of bullous pemphigoid developed in a 28-year-old woman with proven systemic lupus erythematosus (SLE). Serum of this patient contained elevated titers of antinuclear antibodies but basement membrane antibodies could not be detected at first, though they did appear in blister fluid. Normal monkey skin explants cultured on this patient's sera gave positive direct immunofluorescence (IF) at the basement membrane zone (BMZ) for IgG deposits. The use of tissue culture methods may be helpful because of the capacity of this test system to reveal the presence of the antibodies to the BMZ despite the presence of the antinuclear antibodies that appear to interfere with their demonstration in standard indirect IF tests.

Stripping of the stratum corneum in patients with psoriasis: production of prepinpoint papules and psoriatic lesions.

In patients with psoriasis, partial stripping of the stratum corneum induced minute erythematous and edematous, nonscaling papules six hours to seven days later. These "prepinpoint papules" PPPs) comparable to spontaneous PPPs, which we described earlier, appeared in 75 of 159 patients with active psoriasis but in none of 27 controls; 73% to 91% of these PPPs, depending on the activity of the disease, transformed into pinpoint papules. Histological and histochemical examinations of the PPPs showed infiltrates containing numerous polymorphonuclear leukocytes around the vessels and penetrating into the epidermis, partly destroying it. The earliest change in the epidermis was thinning of the granular layer without other features of psoriasis. Immunoglobulin G and complement deposits on stratum corneum (SC) antigen sites were found in the PPPs initially in about one third, and later in two thirds of the papules, whereas they were present in virtually all of the psoriatic pinpoint papules. Papules induced by stripping, similar to spontaneous PPPs, play a central role in the etiology of at least some forms of psoriasis. Polymorphonuclear leukocyte infiltrates and SC antibody binding are key features of the conversion of PPP to pinpoint psoriatic lesions.

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.

Dermatitis herpetiformis and bullous pemphigoid. Intermediate and mixed forms.

Nine patients had clinical and histological features suggestive of both dermatitis herpetiformis (DH) and bullous pemphigold (BP). Five patients responded to treatment with sulfapyridine or sulfones: in two the response was inconsistent, and the disease was controlled by combined treatment with prednisone; in one patient, there was no response to sulfapyridine or sulfones. Immunofluorescence studies showed IgA deposits in a linear homogeneous pattern at the basement membrane zone in all patients, and IgG was present in five. No circulating anti-basement membrane antibodies were detected by repeated immunofluorescence examinations. The authors conclude that the occasional overlapping of BP and DH should not lead to dropping the distinction between the two entities. For overlap cases that cannot be classified as BP or DH, the term "intermediate or mixed form of DH and BP" seems to be most suitable.

Herpes gestations with identical lesions in the newborn. Passive transfer of the disease?

Cutaneous lesions of herpes gestation is developed in a newborn on the fifth day of life and disappeared spontaneously after three weeks. The immunologic phenomena were analogous in the mother and child. No circulating or in vivo-bound immunoglobulins were demonstrated, but components 3 and 4 of complement were present at the basement membrane zone. We discuss a possible passive transfer of the disease to the newborn, and a supposedly pathogenetic role of immunologic phenomena in herpes gestationis.

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.

Immunologic phenomena in herpes gestationis.

Immunofluorescence studies were performed in 12 cases of herpes gestations. Direct IF staining (performed in 10 of them) revealed a highly characteristic pattern, notably 1) Constantly BMZ deposits of complement component C3, 2) In most biopsy specimens also C4 and C5, 3) Deposits of C4, C5, properdin and IgG variable in different specimens of given patients. About half of the specimens examined contained properdin and about one third stained for IgG, 4) Findings of IgM, IgA, IgD and IgE were consistently negative, 5) Serum findings of BMZ antibodies in given cases were variable. In two cases circulating BMZ antibodies were detected in one of the samples examined. On the base of these findings it seems that HG is related to BP.