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Endemic pemphigus foliaceus or Fogo selvagem (FS) is an epidermal organ-specific autoimmune disease mediated by autoantibodies. Individuals at risk are peasants who live and work on farms located in the interior of certain endemic states of Brazil. This case-control study compares a group of 52 FS patients with 52 patients suffering from other dermatoses admitted and followed at the hospital for pemphigus (Hospital do Penfigo) in the city of Goiania, state of Goias. Patients and controls matched 1:1 by age, sex, and occupation were examined by two dermatologists at the time of admission and asked to respond to a prepared questionnaire. This questionnaire concerned current and past (1 and 5 years) exposure to environmental risk factors. The following risk factors were assessed: black fly bites, presence of rodents at home, exposure to cereal dust, exposure to fumes or dust released by tree and shrub removal, and exposure to insecticides. Relative risks were estimated from tabulated data by the odds ratio and tested for significance by the chi-square test. The 95% confidence interval for the odds ratio was also calculated for each of the risk factors. The only risk factor showing an odds ratio significantly different from one was exposure to simuliidae bites (odds ratio 4.7, p less than 0.001). This study reinforces the hypothesis that chronic exposure to black fly antigens may precipitate IgG4 antibody formation in predisposed individuals. These antibodies in turn may cross-react with epidermal antigens and cause acantholysis and the clinical expression of the disease known as FS.

Pemphigus foliaceus (PF) and its endemic form, Fogo Selvagem (FS), are characterized by subcorneal vesicles and pathogenic IgG autoantibodies directed against keratinocyte surface antigens. A major pool of FS antigen(s) remains bound to the insoluble epidermal envelope fraction. In this paper we demonstrate that this antigen(s) can be released from the envelope fraction by sonication. By immune precipitation four components can be detected, having molecular weights (MW) of 260, 80, 62, and 45 kD. The 260-kD component is lost by boiling or extraction with glycine HCl at pH 2.8. The major components appear to be the 80- and 62-kD poly-peptides. They chromatograph as a unit by gel filtration in 0.1% SDS, in the MW range of 115-120 kD. The FS antigen(s) appears to be cationic, forming insoluble complexes at low pH with SDS, and is labile to ammonium sulfate and freezing and thawing. It is unaffected by positive pressure concentration, 50% acetone precipitation, and reduction/alkylation. The FS antigen(s) is precipitated by all FS and nonendemic PF sera except those in complete clinical and serologic remission. The FS antigen(s) is also precipitated by 50% of pemphigus vulgaris but none of the bullous pemphigoid sera tested. All FS antigenic components are immunoprecipitated by IgG4 autoantibodies, but the IgG1 subclass from the same patients appear to immunoprecipitate only the 62-kD polypeptide. The FS antigen(s) is able to adsorb human autoantibodies against human desmoglein 1 (DG1), but not rabbit antisera against bovine DG1 or 2. This paper shows that physical stress, i.e., sonication, may be able to solubilize sufficient FS antigen(s) from the epidermal envelope fractions for further chemical characterization. The relationship of these FS antigen(s) to other reported FS antigens is presently unknown.

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.

Based on epidemiologic data, a current hypothesis states that Fogo selvagem (FS) may be triggered by environmental factors present in endemic areas of Brazil. Because the appearance of new cases is limited to those areas, we wanted to ascertain if the presence of the pemphigus autoantibodies was restricted to the patients. To further delineate the restriction of the autoantibody response in these patients we also investigated the presence of lupus-associated autoantibodies. Using indirect immunofluorescence (IF) we tested the sera of patients with FS (n = 196), their relatives (n = 138), their cohabitants (n = 13), and normal donors from endemic (n = 38) and non-endemic areas (n = 44) for pemphigus autoantibodies. Antinuclear antibodies (ANA) and anti-nDNA antibodies were determined by indirect IF against Hep-2 cells and Crithidia lucilliae, respectively. Autoantibodies against nRNP, Ro/SSA, La/SSB, and Sm were assayed by double immune diffusion in agarose gels. FS autoantibodies were present in the sera of all patients with active disease (n = 196, 100%, titers greater than 40 to 2560), but were not found in any sera from normal individuals in endemic or non-endemic areas. The titer of the FS autoantibody showed a rough correlation with the extent and activity of the disease. Furthermore, lupus-associated autoantibodies were not present in any of the tested samples. We conclude the FS antiepidermal autoantibodies are specific serologic markers of the disease and are not present in unaffected individual from the endemic areas. As such, they provide an important marker that should be useful in ongoing epidemiologic studies aimed at identifying putative etiologic agent(s).

This paper details current and historic epidemiologic features of Fogo Selvagem (Endemic pemphigus foliaceus) in Brazil. The following features are described. a) The disease occurs in endemic fashion in regions of Brazil within the states of Goias, Mato Grosso do Sul, Parana, Sao Paulo, and Minas Gerais. It appears that the disease is spreading toward the northwest and west, involving the states of Mato Grosso, Para, Maranhao, Rondonia, Acre, and Amazonas. b) People at risk are young peasants or children of either sex or any race exposed to the local ecology in rural areas of endemic states. Although the disease has been described in urban centers, these occurrences are rare. c) Fogo Selvagem commonly appears in wild areas being colonized and disappears as these areas become urbanized. d) The majority of patients live in close proximity to rivers and within the 10-15 Km flying range of mosquitos or black flies (such as Simulium). It is hypothesized that a black fly, Simulium pruinosum may be the vector that precipitates the disease. f) There is a significant number of Fogo Selvagem in family units where multiple, genetically related individuals are affected. g) Finally, autoantibodies against lupus-associated antigens are not present in the sera of patients with Fogo Selvagem. Clinical examination of the skin, and serologic screening for pemphigus autoantibodies are specific parameters that can be used in the search for the etiologic agents that lead to autoimmune disease of the skin. To identify and prove an etiologic agent for this well-characterized autoimmune disease would be of tremendous importance to the understanding of autoimmune skin diseases, and potentially other organ-specific autoimmune disorders.

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris-acetate-buffered saline (TAS), TAS with 5 mM CaCl+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.

We have identified a novel IgG antikeratin autoantibody in the serum of a Brazilian pemphigus foliaceus patient (Cascas-42). This antibody is specific for the 59 kD acidic murine keratin and its 56.5 kD human counterpart (Moll's catalogue #10), and is distinct from the pemphigus antibody system. Antikeratin autoantibodies present in the Cascas-42 serum were purified by affinity chromatography with a 59 kD murine keratin-agarose column (IAP-Cascas-42 antibodies). The specificity of the IAP-Cascas-42 antibodies was tested by indirect immunofluorescence and immunoelectron microscopy against epidermal cryosections, trypsin-dissociated keratinocytes, and epidermal cell cultures. The serum was also tested with extracts from unlabeled and surface 125I-labeled keratinocytes (Iodo-Gen method) by immunoblot analysis of one- and two-dimensional polyacrylamide gel electrophoresis. The IAP-Cascas-42 antibodies bind the intercellular spaces of murine epidermis, and the cell surfaces of viable, dissociated murine keratinocytes, as well as murine epidermal cells in culture by immunofluorescence and immunoelectron microscopy. These autoantibodies did not stain cytoplasmic keratins and did not react with parallel human epidermal substrates. The Cascas-42 serum identified the 59 kD murine acidic keratin and its 56.5 kD human counterpart in epidermal extracts by two-dimensional polyacrylamide gel electrophoresis and immunoblot analysis. In addition, surface radioiodination of viable murine keratinocytes selectively labeled the 59 kD keratin suggesting that a domain of this molecule is exposed on the cell surface. The 125I-labeled 59 kD keratin was also recognized by the Cascas-42 serum by immunoblotting and autoradiography. These studies suggest that in murine epidermis, the 59 kD keratin is a transmembrane protein with an extracellular domain recognized by the IAP-Cascas-42 antibodies.

In both the endemic and sporadic forms of pemphigus foliaceus (PF), antiepidermal autoantibodies against desmoglein I are present. Desmoglein I is a highly insoluble 160-kD transmembrane glycoprotein of the desmosomal core. The detailed immunochemical characterization of the epitope(s) recognized by the PF autoantibodies is hampered by its large molecular weight and the insolubility of desmoglein I in nondenaturing buffers. This study was designed to identify alternative methods that could yield soluble immunoreactive PF antigen (Ag) from normal human epidermis. The presence of PF Ag in human epidermis and in its soluble or insoluble fractions was monitored by indirect immunofluorescence, immunoadsorption of PF sera, and immunoprecipitation of radiolabeled fractions. The PF Ag from trypsin-resistant, radiolabeled cell envelope preparations was cleaved by papain and immunoprecipitated by PF sera. A 50-kD peptide, isoelectric at pH 5.5-5.8, was immunoprecipitated by sera from all patients with endemic PF (n = 15) or idiopathic PF (n = 4), and by two of four pemphigus vulgaris sera, but by no control sera (n = 7). This study shows that a significant fraction of the PF Ag is insoluble, trypsin-resistant, and is associated with the cornified cell envelope fraction, but an Ag fragment can be obtained in a small molecular weight, soluble, and immunoreactive form by papain digestion. This 50-kD papain fragment is more amenable to detailed chemical and immunologic characterization than the native molecule.