Subclinical oral involvement in patients with endemic pemphigus foliaceus

Background We have described a variant of endemic pemphigus foliaceus (EPF) in El Bagre area known as pemphigus Abreu-Manu. Our previous study suggested that Colombian EPF seemed to react with various plakin family proteins, such as desmoplakins, envoplakin, periplakin BP230, MYZAP, ARVCF, p0071 as well as desmoglein 1. Objectives To explore whether patients affected by a new variant of endemic pemphigus foliaceus (El Bagre-EPF) demonstrated oral involvement. Materials and Methods A case-control study was done by searching for oral changes in 45 patients affected by El Bagre-EPF, as well as 45 epidemiologically matched controls from the endemic area matched by demographics, oral hygiene habits, comorbidities, smoking habits, place of residence, age, sex, and work activity. Oral biopsies were taken and evaluated via hematoxylin and eosin staining, direct immunofluorescence, indirect immunofluorescence, confocal microscopy, and immunohistochemistry. Results Radicular pieces and loss of teeth were seen in in 43 of the 45 El Bagre-EPF patients and 20 of the 45 controls (P < 0.001) (confidence interval [CI] 98%). Hematoxylin and eosin staining showed 23 of 45 El Bagre-EPF patients had corneal/subcorneal blistering and lymphohistiocytic infiltrates under the basement membrane zone and around the salivary glands, the periodontal ligament, and the neurovascular bundles in all cell junction structures in the oral cavity; these findings were not seen in the controls (P < 0.001) (CI 98%). The direct immunofluorescence, indirect immunofluorescence, confocal microscopy, and microarray staining displayed autoantibodies to the salivary glands, including their serous acini and the excretory duct cell junctions, the periodontal ligament, the neurovascular bundles and their cell junctions, striated muscle and their cell junctions, neuroreceptors, and connective tissue cell junctions. The autoantibodies were polyclonal. IgA autoantibodies were found in neuroreceptors in the glands and were positive in 41 of 45 patients and 3 of 45 controls. Conclusions Patients affected by El Bagre-EPF have some oral anomalies and an immune response, primarily to cell junctions. The intrinsic oral mucosal immune system, including IgA and secretory IgA, play an important role in this autoimmunity. Our data contradict the hypothesis that pemphigus foliaceus does not affect the oral mucosa due to the desmoglein 1-desmoglein 3 compensation.

Furthermore, each patient had to be positive by immunoblotting for reactivity against Dsg1 [2,3], as well as for plakin molecules; each patient's serum immunoprecipitated a concanavalin A affinity-purified bovine tryptic 45 kDa fragment of Dsg1 [4]; and each patient's serum had to yield a positive result using an enzyme-linked immunosorbent assay test when screening for autoantibodies to pemphigus foliaceus antigens [5].
All samples were run with positive and negative controls.
Several years ago, the first [12] discovered new autoantigens to several organs other than the skin. Because of the complexity of the immune response in these patients, we

Introduction
We have described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu) [1][2][3][4][5]. El Bagre-EPF differs from other types of EPF clinically, epidemiologically, and immunologically. Previous studies have shown that patients affected by EPF in Brazil have some oral findings [7,8]. Selected authors have described the presence of autoantibodies using hematoxylin and eosin (H&E) staining, direct and indirect immunofluorescence (DIF, IIF), and electron microscopy studies [9][10][11]. In the current study, our aim was to search for oral clinical lesions and an oral autoimmune response in patients affected by EPF in El Bagre, Colombia (El Bagre-EPF) [1][2][3][4][5] and to compare our findings with those described in the medical literature for Brazilian EPF patients.  [1,2], and have serum displaying intercellular staining (ICS) between epidermal keratinocytes and the basement membrane zone (BMZ) of the skin via either DIF or IIF using fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies Conclusions: Patients affected by El Bagre-EPF have some oral anomalies and an immune response, primarily to cell junctions. The intrinsic oral mucosal immune system, including IgA and secretory IgA, play an important role in this autoimmunity. Our data contradict the hypothesis that pemphigus foliaceus does not affect the oral mucosa due to the desmoglein 1-desmoglein 3 compensation.

ABSTRACT
Large varicosities were found in 10 of 45 patients at the base of the involved lingual renine veins or in vessels of the ventral surface of the tongue or the floor of the mouth, with no control varicosities recorded. Small ulcers were seen in the palatal mucosa in 5 of 45 El Bagre-EPF patients. Dental caries were also found in most participants.

H&E Staining
The H&E staining showed that 23 of 45 El Bagre-EPF patients had corneal and/or subcorneal epidermal blisters and dermal edema and lymphohistiocytic infiltrates under the BMZ and around the salivary glands (including their serous acini and the excretory duct cell junctions and the neurovascular bundles). These findings were not observed in the controls (P < 0.001) (CI 98%) ( Figure 1b).
The controls demonstrated secretory IgA in the salivary glands, including serous acini and the excretory duct cell junctions; these findings were also noted in the El Bagre-EPF patients (P < 0.001) (CI 98%) (Figures 1 and 2). The

Questionnaires on Oral Habits
Deleterious oral habits include bruxism parasomnias, traumatic brain injury, neurological disabilities, nail biting, morphological factors, temporomandibular joint dysfunction, tongue thrusting, mouth breathing, smoking habits, and chewing on plants and/or gum. Other questions included how often toothbrushes were changed, use of dental floss, dental visits, and frequency of brushing teeth.

Imgenex Microarray IIF Using Frozen Normal Oral Organs
Our microarray work was performed as described for our IIF; as our antigen source, we used a commercial human tissue microarray in duplicate from Imgenex Corporation (San Diego, CA, USA).

Statistical Analysis
We used the Fisher exact test to compare 2 nominal variables (eg, positive and negative) of the antibody response. P < 0.01 with a 98% degree of confidence or more was considered statistically significant. We used the software Graph-Pad QuickCalcs (GraphPad Software Inc., La Jolla, CA, USA).

Questionnaires on Oral Habits
Deleterious oral habits did not show any statistical significance between the cases and controls. The oral health habits were poor in all study participants (37/45 patients and 38/45 controls). Most never visited the dentist for economic reasons

Oral Evaluation
The most significant alteration in the El Bagre-EPF patients was the finding of multiple radicular pieces and loss of teeth

IHC Staining
Using metallothionein we observed patchy spot staining at the BMZ, as well as in the neurovascular bundles, the salivary glands including serous acini and the excretory ducts, cell junctions, striated muscle, mesenchymal-endothelial cell junction    revealed FS in the acute and bullous phases of the disease and significant periodontal disease [26].
Other authors studied 15 patients with FS reporting subcorneal acantholysis and no oral blisters or erosions, but DIF demonstrated the presence of tissue-bound autoantibodies in both the epidermis and the oral epithelium of all patients [6].
We observed an autoimmune response to neural receptors in El Bagre-EPF patients' oral mucosa and salivary glands; indeed, we have described autoreactivity to skin neurovascular structures and neural receptors in previous studies [27,28]. The neuronal/transmitter control of salivary glands is performed by both dopaminergic and serotonergic neurons and receptors [29]. Both classes of transmitters elicit saliva secretion. The neurons contain γ-aminobutyric acid (GABA).
GABA-positive fibers form a network around most salivary acinar lobules and a dense plexus in the interior of a minor fraction of acinar lobules.
We conclude that patients affected by El Bagre-EPF have an autoimmune response in the oral mucosa. We suggest that this process results in loss of teeth; IgA, and the mucosal immune system seem to play important roles. Given our observations, the Dsg1-Dsg3 compensation theory offered to explain a "lack" of oral compromise in pemphigus foliaceus (including its endemic variants) may need reassessment.
In this case-control study we found some clinical alterations such as loss of teeth in the El Bagre-EPF group. Prednisone therapy and lack of oral hygiene can explain these findings. However, the controls have similar demographic factors, with the exception of the intake of prednisone. The autoantibodies to the periodontal ligament could contribute to weakness of the patients' teeth. We previously reported autoantibodies to several smooth muscle structures including the arrector pili muscle in most El Bagre-EPF patients [13].
In theory, clinical involvement of the oral mucosa is not typically present in pemphigus foliaceus. Previously published data indicate that in pemphigus foliaceus, desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) are expressed in a pathogenic distribution throughout the squamous mucosal epithelia and the skin [16][17][18]. In our data, we observed something completely different that contradicts this hypothesis, ie, the "theory of Dsg1-Dsg3 compensation." Measuring salivation in the endemic area is also difficult, but with use of multicolor immunofluorescence [19] we were able to observe positivity to neuroreceptors using high magnifications and color contrast. We also previously demonstrated that the El Bagre-EPF patients have autoantibodies to their palms and soles, as well as to their sweat glands with an IgA response (immune-specific to these anatomic sites) [20,21].
Our findings pointed us to an IgA autoimmune response that is part of the mucosal innate immunity, including the saliva containing lysozymes, bacteriocidins, defensins, cationic proteins, and lactoferrin [22]. Our findings brought our attention to the specific immunity that the oral mucosa has in comparison with the skin. Tomasi and his colleagues in the mid-1960s originally documented oral "local immunity" with the presence of IgA antibodies in secretions including saliva [23][24][25].
A group of Brazilian authors performed a study of the oral cavity of 56 patients with fogo selvagem (FS). Histopathological and clinical examination of the gingivae of 8 patients