Epithelium of the human palatal tonsil used for new test antigen in IIF examination (for pemphigus, pemphigoid antibodies and ANA).

The author presents the results of IIF examinations for pemphigus, pemphigoid antibodies and antinuclear antibodies (ANA) on the epithelium of the human palatal tonsil obtained by tonsillectomy in patients with chronic hypertrophic tonsillitis. Tonsil epithelium is more sensitive to intercellular antibodies (IC Ab), and antibodies of the basal membrane zone (BMZ Ab) than sample tissue of the monkey esophagus, while its reaction to ANA is the same or even weaker than monkey esophagus.     

Characterization of 230-kD bullous pemphigoid antigen associated with the detergent-insoluble fraction of cultured keratinocytes

The 230-kD protein identified by antibodies from patients with bullous pemphigoid (BP) has a dual location in cultured normal human epidermal keratinocytes: part in a high-speed supernatant of homogenized cells and part in a particulate fraction, where it is resistant to extraction by non-ionic detergent or mild base. Antibodies were affinity purified from the particulate 230-kD BP antigen, which can be extracted in the presence of urea. The affinity-purified antibodies bind not only the cytosolic 230-kD protein, showing that it is related, if not identical, to the particulate form, but also produce a discontinuous granular pattern by indirect immunofluorescence in the basement membrane zone of rabbit esophagus. In stratifying epidermal cultures, expression of the 230-kD BP antigen is limited to basal cells. These data are consistent with 230-kD BP antigen involvement in keratinocyte basal cell interaction with extracellular matrix and indicate that the cultured cell may provide a useful model for analysis of 230-kD BP antigen function.     

Bullous pemphigoid antibodies. Human skin as a substrate for indirect immunofluorescence assay

Human skin, the target organ for bullous pemphigoid (BP) antibodies, is thought to be a less sensitive substrate for the indirect immunofluorescence assay of BP antibodies than monkey or guinea pig esophagus. To examine the reasons for this puzzling phenomenon, we compared the titers of BP antibodies obtained when human skin, monkey, and guinea pig esophagus were used as substrates. We found the titers of BP antibodies obtained with human skin from sites commonly involved in BP (flexor arm, flexor thigh, popliteal fossa) were as high and usually higher than those obtained with monkey and guinea pig esophagus. In contrast, much lower titers were obtained with human skin from sites rarely involved in the disease (scalp, face, extensor arm). These findings suggest that human skin as a substrate is at least as sensitive as monkey or guinea pig esophagus for the indirect immunofluorescence assay of BP antibodies when the skin is obtained from regions on the body commonly involved in BP.     

In vitro effects of pemphigus antibodies on skin

Acantholysis occurring in rhesus monkey skin explants cultured on sera of 9 pemphigus patients was found to be largely dependent on the titre of intercellular antibody, and not on the participation of complement. Skin explants cultured on normal human sera and pemphigoid sera failed to give rise to intercullular staining or to develop lesions. Six of eight 'negative' pemphigus sera with intercellular antibody titres of less than 20 on skin (and titres ranging from 20 to 160 on monkey esophagus) reacted with the skin explants as revealed by direct immunofluorescence with an anti-IgG conjugate. The binding of antibodies from 3 of these 6 reactive sera resulted in some pathological changes in the explants. At least two of these 3 'negative' sera came from pemphigus patients with skin lesions.     

Immunopathological examination of esophagus as a useful criterion of cure in pemphigus vulgaris

BACKGROUND: We performed histopathological and immunopathological examinations of the esophagus in patients with pemphigus vulgaris during clinical remission. METHODS: In the group of 14 patients without serum antibodies, five were treated with low doses of steroids and cyclophosphamide (as maintenance treatment), while nine had already completed the therapy. RESULTS: In all five cases under maintenance treatment we found bound pemphigus antibodies in vivo. Acantholysis was present in two of these. In all nine non-treated patients, acantholysis and immunopathological findings were negative. CONCLUSIONS: Our studies reveal that the absence of bound 'pemphigus vulgaris' antibodies in the esophagus by immunological examination could be regarded as proof of complete cure and could be the decisive finding to stop pemphigus treatment.     

Proteins of the cornified envelope.

The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum. It is made by cross-linking precursor proteins by a membrane-associated transglutaminase. Using the cornified envelope of cultured human keratinocytes as the immunogen, we obtained a number of monoclonal antibodies which stained epidermis in a variety of ways. The peripheral staining pattern has been associated with several envelope precursors and this has been confirmed by western blots. A mouse IgM monoclonal antibody directed against epidermal basal cell hemidesmosomes was also discovered. By immunofluorescence, the monoclonal antibody produced a strong linear staining of the basement membrane zone and a polar cap on trypsin-dissociated epidermal basal cells. By immunoelectron microscopy, immunoreactants were present in the attachment plaques of hemidesmosomes on guinea pig esophagus. However, no protein reactive with the antibody was detected. This study suggests that an antigen associated with the basal cell hemidesmosomes may be incorporated in the cornified envelope.     

Concomitant Occurrence of Circulating IgA Anti-intercellular and Anti-basement Membrane Zone Antibodies in Autoimmune Blistering Diseases: Immunofluorescence and Immunoblot Studies

Recently, cases with circulating IgA anti-intercellular antibodies have been described. The objective of this study was to present immunofluorescence and immunoblot findings in three cases of bullous diseases with concomitant circulating IgA anti-intercellular and anti-basement membrane zone antibodies. Direct immunofluorescence, indirect immunofluorescence on intact and 1M NaCl-split skin, immunoblotting of epidermal extracts from dispase- and EDTA-separated (two different procedures) human skin, and immunoblotting of the bovine desmosome preparation were performed. All three cases had IgA anti-intercellular and anti-basement membrane zone antibodies. However, immunoblot results were divergent. Case 1 had antibodies against the 150 kD pemphigus foliaceus antigen (IgG), the 170 kD protein (IgG and IgA), and the 97 kD antigen (IgG and IgA). Case 2 had IgG antibodies reactive with the 230 kD and the 170 kD bullous pemphigoid antigens, while case 3 had IgA antibodies against the 97 kD antigen only. The results of immunofluorescence and immunoblot studies in our patients widen the spectrum of laboratory features in blistering skin diseases mediated, at least in part, by antibodies of the IgA class.     

Immunologic sequelae of thermal injury autoantibodies in acute adult burn patients

Summary Indirect immunofluorescent (IF) studies have demonstrated the presence of antinuclear antibodies (ANA) and antibodies reactive with the intercellular area (IC), basement membrane zone, and basal cells of autologous and allogeneic unburned skin in the sera of acute adult burn patients. Further demonstration in burn patients of the occurrence of peripheral ANA masquerading as IC antibodies is presented. In keeping with previous IF identifications of autoantibodies in burn patients, there was no significant relationship between the frequency and titer of these antibodies according to the extent of total body surface burn area and third degree burn area. The possible role of epithelial antibodies in the maintenance of host resistance, as previously suggested, is considered in brief.Presented in part at the workshop on the Immune Consequences of Thermal Injury, Lake Arrowhead, California, 2–5 December, 1979     

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.     

Pemphigus antibodies fixation and keratinocyte differentiation in organ cultures

Summary The effect of some agents, influencing the cyclic adenosine 3,5-monophosphate (cAMP) content of human cells, on the ability of the keratinocytes of binding pemphigus antibodies was studied by using tissue cultures of rabbit esophagus. As demonstrated by immunofluorescence (IF) for IgG, the bound antibodies appeared markedly decreased on esophagus explants grown under standard conditions, that is without test agents, when compared to ones fixed on fresh esophagus. But the IF reaction was remarkably more intense when methylxanthines or epinephrine were added to the growth medium of the cultures. Following the addition of these agents to the cultures some histologic modifications appeared in the explants, indicating that the keratinization process had probably been stimulated. This temporal relationship of immunofluorescence and histologic findings seems to suggest the hypothesis that keratinocyte differentiation, regulation of cAMP intracellular content, and pemphigus antibodies fixation are related processes.     

Sensitivity of indirect immunofluorescence and immunoblotting for the detection of intercellular antibodies in endemic pemphigus foliaceus (fogo selvagem)

BACKGROUND: Two assays are available to detect anti-skin antibodies in patients with fogo selvagem (FS): indirect immunofluorescence (IIF) and immunoblotting (IB). This study was conducted to compare the sensitivity of these assays in detecting FS antibodies. DESIGN: Eighty-nine serum samples of 48 patients with FS and control serum from 15 normal individuals were tested concurrently for the presence of FS antibodies by IIF and IB. IIF studies were conducted using four different substrates: human skin, monkey and guinea pig esophagus, and bovine tongue. RESULTS: FS antibodies were detected much more commonly by IIF than by IB, i.e. in 71% vs. 28% of serum samples respectively. By IIF, the antibodies reacted most strongly against human skin. CONCLUSIONS: IIF is a more sensitive assay than IB for detecting antibodies associated with FS. The sensitivity of the test is maximized by using human skin as a substrate.     

Pemphigus foliaceus associated with absence of intercellular antigens in lower layers of epidermis.

A patient with pemphigus foliaceus was found to lack normal intercellular (IC) antigens in the lower layers of the epidermis. This was evidenced by the inability of her own IC antibodies, or of those from other patients with pemphigus vulgaris, to bind to the IC substance in the lower layers of her epidermis; whereas these same antibodies reacted to IC antigens in all layers of normal allogeneic skin and monkey and guinea pig esophagus. Lack of IC antigens in the lower layers of the epidermis may account for the subcorneal location of bullae in some patients with pemphigus foliaceus.     

Incidence and diagnostic significance of reactive syphilis antibodies in systemic lupus erythematodes and other immunologic diseases

Serum samples of 78 patients with systemic lupus erythematosus, systemic sclerosis and other immunological diseases were tested for antibodies to syphilis. Reactive or weak reactive results were observed in 10% by means of the treponema pallidum hemagglutination (TPHA) test, in 27% by the FTA-Abs test, in 40% applying the IgM-FTA-Abs test, in 10% by the VDRL test and in 3% of the cases using the cardiolipin CF test. Only in 3 patients (4%) we found an antibody pattern characteristic of syphilitic patients (TPHA and FTA-Abs test simultaneously undoubtedly reactive). Neither the comparative qualitative and quantitative determination of antibodies to ANA, nDNS and ENA (extractable salinesoluble nuclear antigen) nor elimination of nDNS and ENA antibodies, or incubation of the treponemal test antigen with DNase lead to a conclusion whether the reactive results of the TPHA, FTA-Abs, and IgM-FTA-Abs tests specifically indicate a syphilitic infection. The low incidence of reactive syphilis tests in SLE and the presence of syphilitic antibodies in other immunological diseases limitate the significance for the criterium in the diagnosis of SLE.     

Unusual manifestation of scleroderma of the trunk

We present the case of an 81-year-old women with progressive systemic scleroderma. In addition to the typical manifestations of diffuse (truncal) scleroderma, including involvement of the esophagus, lungs and muscles, there were two unusual findings: large, non-sclerosing areas on the trunk simulating the disabling type of circumscribed scleroderma and high titers of anticentromere antibodies, while there were no anti-Scl-70 antibodies. The disease responded to treatment with griseofulvin.     

Circulating IgA anti-basement membrane antibodies in linear dermatitis herpetiformis (Duhring): immunofluorescence and immunoelectronmicroscopic studies.

IgA deposits were observed by direct immunofluorescence in linear distribution along the basement membrane zone in a case of dermatitis herpetiformis (Duhring). In addition, in the serum of the same patient circulating IgA antibasement membrane zone antibodies were detected by indirect immunofluorescence, utilizing normal human skin and monkey esophagus as substrates. The ultrastructural localization of in vivo-bound IgA and circulating IgA antibasement membrane zone antibodies fixed to substrate tissue in vitro was found to be in the uppermost strata of the dermis below the basal lamina.     

Expression of interferon-gamma receptors in normal and psoriatic skin.

Psoriatic keratinocytes have a reduced antiproliferative response to interferon (IFN)-gamma, and HLA-DR expression is usually not observed on keratinocytes in psoriatic plaques despite the presence of activated T cells. We have therefore compared the expression of IFN-gamma receptors in psoriatic skin with that of normal human skin. Using mouse monoclonal antibodies and immunoperoxidase staining on cryostat cut sections, we detected IFN-gamma receptors on keratinocytes throughout the epidermal layers except stratum corneum in normal skin (n = 11). Biopsy specimens from involved psoriatic skin (n = 17) consistently showed a staining pattern that differed from that of normal skin in that only the lower part of epidermis reacted with the antibodies to IFN-gamma receptors, whereas the upper layers showed no or minimal staining. Expression of IFN-gamma receptors in uninvolved psoriatic skin (n = 16) did not differ from that of healthy controls. Forty-five percent of the biopsies from lesional psoriatic skin displayed ICAM-1 positive keratinocytes, and only two specimens had a limited expression of HLA-DR reactive keratinocytes. The decreased binding of antibodies against the IFN-gamma receptors in the upper part of psoriatic epidermis might be secondary to abnormal maturation of psoriatic keratinocytes or a primary defect involving abnormal modulation of IFN-gamma receptors.     

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.     

Frequency and isotype distribution of serum antibodies reactive with dietary proteins in adults with chronic urticaria

Forty-eight adult patients with chronic urticaria have been evaluated for scrum antibodies reactive with bovine milk, chicken ovalbumin or wheat gliadin using a solid-phase enzyme-linked immunosorbent assay (ELISA). Of the chronic urticaria sera, 60.4% contained detectable antibodies reactive with one or more of these dietary antigens, compared with 33.9% of sera from 232 healthy adults (P < 0.001). In particular, antibodies reactive with milk (52%; P < 0.001), ovalbumin (39.6%, P < 0.001) and gliadin (20.8%, P > 0.05) were detected at increased frequencies compared with controls (22.8%, 16.8% and 11.6%, respectively). These serum antibodies were predominantly of the IgG isotype, with an IgG subclass restriction mainly to IgG4 for anti-ovalbumin and anti-gliadin antibodies, and to IgG2 and IgG4 for anti-milk antibodies. The increased frequency of IgG antibodies was not significantly associated with either raised total serum IgE or atopic status.     

Production and characterization of monoclonal antibodies to Treponema pallidum.

This study was attempted to produce the monoclonal antibodies specific for Treponema pallidum and to investigate their characteristics, thereby contributing to identify the antigenic structure and to apply the diagnosis of syphilis. The seven clones (YS 3, YS 75, YS 307, YS 481, YS 343, YS 1 and YS 406) secreting monoclonal antibodies reactive with T. pallidum were produced. The isotypes of the seven monoclonal antibodies produced were defined. Optical densities of the 7 monoclonal antibodies were ranged from 0.59 to 1.48 as measured by ELISA. Monoclonal antibodies from 5 of the 7 clones which could agglutinate sheep RBC sensitized with T. pallidum, showed strong fluorescences. The monoclonal antibody from YS 343 was cross-reactive with non-pathogenic treponemes, but the other 6 monoclonal antibodies reacted with T. pallidum specifically. On immunoblotting, monoclonal antibodies from 5 of the 7 clones reacted with a polypeptide of a molecular weight of 47 kDa, and monoclonal antibodies from YS 343 reacted with a polypeptide of 64 kDa common to both T. pallidum and T. phagedenis. The above results revealed that 7 clones secreting monoclonal antibodies reactive to T. pallidum were produced successfully, and specific antibodies reacting with major antigenic polypeptides of a molecular weight of 47 kDa would be useful in the diagnosis of syphilis in the future.     

IgA antibodies recognizing LABD97 are predominantly IgA1 subclass.

Linear IgA bullous dermatosis is a rare acquired subepidermal blistering disease of the skin. A recognized antigen in linear IgA bullous dermatosis is a 97-kDa basement membrane zone protein termed LABD97. Previous studies, using immunofluorescent techniques, have suggested that the IgA response is restricted to the IgA1 subclass. We studied the IgA antibody subclasses in the sera of 6 patients that contained circulating IgA antibodies reactive with LABD97. The methods used included direct and indirect immunofluorescence and Western immunoblot. All patients tested had IgA1 anti-LABD97 antibodies detected by all 3 methods. Two patients had IgA2 antibodies detected by direct immunofluorescence. Three patients had IgA2 antibodies on indirect immunofluorescence. Two of these also had anti-LABD97 IgA2 antibodies and 1 had secretory component containing anti-LABD IgA antibodies on Western immunoblot. We conclude that the predominant IgA antibody subclass reactive with LABD97 in LABD is IgA1, although the IgA2 subclass may be involved in some cases.