Ciro R. Martins

The Pathogenic Effect of IgG4 Autoantibodies in Endemic Pemphigus Foliaceus (Fogo Selvagem)

Endemic pemphigus foliaceus, or fogo selvagem, is an autoimmune blistering skin disease caused by IgG autoantibodies to a desmosome-associated glycoprotein. We studied the IgG subclasses with autoantibody activity in serum from 29 patients with active disease and in the skin lesions of 18 patients by immunofluorescence, using IgG-subclass-specific monoclonal antibodies. The predominant disease autoantibodies present in all patients were of the IgG4 subclass. IgG1 and IgG2 autoantibodies were detected in low titer in the 29 patients: IgG1 in 23 patients and IgG2 in 9. IgG3 autoantibodies were not detected in the serum of any patient. Direct immunofluorescence testing of skin lesions showed a preferential deposition of IgG4 on the keratinocyte surface. The pathogenic effect of IgG4 was demonstrated by the passive transfer of fractions containing IgG4 autoantibodies from the patients to neonatal BALB/c mice. The disease of the patients was reproduced clinically, histologically, and immunologically in these animals. Only IgG4 autoantibodies were detected by direct immunofluorescence, bound to the epidermis in the lesions of the mice, and by immunoelectron microscopy at the keratinocyte surface. IgG4 has previously been reported to be a blocking or protective antibody because it has poor effector functions in vitro, as compared with the other IgG subclasses. The finding that it is the pathogenic autoantibody in fogo selvagem raises the possibility that it may also be important in other autoimmune disease.

Endemic pemphigus foliaceus (fogo seivagem). I. Clinical features and immunopathology

Endemic pemphigus foliaceus is an autoimmune disease that has remarkable features. Endemic foci are found in characteristic environments within the interior of Brazil. The epidemiologic data strongly suggest that an environmental factor initiates the autoantibody response in the host. As such it is an important disease for in-depth study. A group of interested investigators in both Brazil and the United States has been formed to attempt to do just that. As part of the overall effort, this Cooperative Research Group for the Study of Fogo Selvagem presents a definition of the disease, a proposed clinical classification for various forms of the disease, and an outline of what is currently known of its immunopathologic characteristics.

Clinical and epidemiologic characterization of photosensitivity in HIV-positive individuals

Background: An increased prevalence and severity of cutaneous photosensitivity has been recognized in association with human immunodeficiency virus (HIV) infection. However, this disorder remains poorly characterized in terms of its epidemiology, predisposing factors, clinical, and environmental associations.

DNA Methylation in Anal Intraepithelial Lesions and Anal Squamous Cell Carcinoma

Purpose: Anal intraepithelial neoplasia is associated with human papillomavirus infection and may progress to invasive squamous cell carcinoma (SCC), which is increasing in immunocompromised patients. We hypothesize that anal intraepithelial neoplasia is associated with abnormal DNA methylation and that detection of these events may be used to improve screening programs. Experimental Design: Seventy-six patients were identified who underwent anal cytology screening and subsequent biopsy at our institution between 1999 and 2004. The specimens from these patients included 184 anal biopsies [normal, n = 57; low-grade squamous intraepithelial lesion (LSIL), n = 74; high-grade squamous intraepithelial lesion (HSIL), n = 41; and invasive SCC, n = 12] and 37 residual liquid-based anal cytology specimens (normal, n = 11; LSIL, n = 12; HSIL, n = 14). The methylation status of the following genes was determined for each biopsy and cytology sample using real-time methylation-specific PCR: HIC1, RASSF1, RARB, CDKN2A, p14, TP73, APC, MLH1, MGMT, DAPK1, and IGSF4. Results: Methylation-specific PCR analysis of biopsy samples revealed that DNA methylation was more common in SCC and HSIL than LSIL and normal mucosa. Specifically, methylation of IGSF4 and DAPK1 was prevalent in SCC (75% and 75% of cases, respectively) and HSIL (59% and 71%, respectively) but was absent in LSIL and normal biopsy samples. Methylation profiles of cytologic samples were similar to those found in the biopsy samples. Conclusions: Aberrant DNA methylation is a frequent event in anal HSIL and SCC. Methylation of IGSF4 and DAPK1 is specific for HSIL and SCC, and may serve as a useful molecular biomarker.

The histopathology of folliculitis in HIV-infected patients.

Background:  Cutaneous disease referable to human immunodeficiency virus (HIV) infection has become less common with the advent of widespread administration of antiretroviral medications, particularly the protease inhibitors. Pruritic eruptions that fall into the general categorization of folliculitis continue to be problematic.

Endemic pemphigus foliaceus in western Paraná, Brazil (1976-1988).

Endemic pemphigus foliaceus or fogo selvagem (FS) is a blistering autoimmune disease indigenous to certain states of Brazil. In the state of Parana the disease has been reported in the north‐central regions where a total of 632 cases were documented in the period of 1940–80. The present study describes a new focus of FS in the western region of the state of Parana. This focus includes a total of 213 new cases of FS and only 11 cases of pemphigus vulgaris seen in this region from February 1976 to July 1988. Over 90% of these patients were peasants working in agriculture or involved in other outdoor activities.

Pemphigus foliaceus antigen: Characterization of an immunoreactive tryptic fragment from BALB c mouse epidermis recognized by all patients' sera and major autoantibody subclasses

The pemphigus foliaceus antigen (PF Ag) is a 160-kDa desmosomal core glycoprotein, desmoglein I. A 50-kDa soluble immunoreactive fragment of the PF Ag was recently prepared from trypsinized cornified cell envelope preparations by papain treatment (R.S. Labib et al. 1989, J. Invest. Dermatol. 93, 272-279). This papain fragment (pf-PF) is associated with upper cell layers of the epidermis and appears to be trypsin resistant in situ. The present work describes the preparation of another fragment by trysinization of the viable lower cells of the epidermis of neonatal BALB/c mice. This tryptic fragment (tf-PF) is a 45-kDa glycoprotein that is partially purified by concanavalin A affinity chromatography of the trypsinization medium. The partially purified tf-PF preparation is capable of completely blocking the indirect immunofluorescence of high titer PF sera. The tf-PF is immunoprecipitated by all PF sera tested (n = 19) and by the two major subclasses of PF autoantibodies, IgG1 and IgG4. Autoantibodies of both the predominant IgG4 and the less prevalent IgG1 subclasses precipitate the same tf-PF as demonstrated by a single compact spot of pI 5.5 by two-dimensional polyacrylamide gel electrophoresis. Chemical and immunological comparison of the tf-PF and pf-PF may explain why the acantholytic lesions of PF appear only in the upper epidermis, despite the presence of the PF Ag throughout all layers of the epidermis. The availability of these two soluble immunoreactive fragments of the PF Ag will be of great value for the further immunochemical characterization of the antigenic epitopes and their role in cell-cell adhesion.

Pemphigus vulgaris and pemphigus foliaceus

Abstract The blistering diseases of the skin grouped under the term pemphigus comprise unique organospecific autoimmune disorders in which the epidermis is injured by autoantibodies. The clinical syndromes manifested depend on the specificity of the autoantibodies Present in the patient, i.e., pemphigus vulgaris (PV) autoantibodies produce suprabasilar acantholysis while pemphigus foliaceus autoantibodies cause subcorneal acantholysis. The antigens recognized by these autoantibodies appear to be desmosomal-associated glycoproteins. The etiology of all forms of pemphigus is unknown except for the endemic form of pemphigus foliaceus known as fogo selvagem (PS). FS is thought to be initiated by exposure to environmental factors) present in certain regions of Brazil in which the disease is endemic. This review examines the relevant clinical, histological, and immunologic characteristics of PV and FS. PV is a potentially lethal disease that affects predisposed individuals sharing the HLA-DRw4 and -DRw6 phenotypes and a unique-DQ β-restriction fragment length of the major histocompatibility complex (MHC) polymorphism. Patients develop mucosal erosions and vesicles and bullae in the skin. The majority of patients possess autoantibodies directed against a cell surface antigen of squamous epithelia. The epidermal lesions show suprabasilar acantholysis. Experimental data strongly implicate PV autoantibodies as the cause of the epidermal lesions by mechanisms under current investigation. The IgG fraction from the sera of these patients induces similar lesions when injected into experimental animals. Similarly, F(ab′)2 but not Fab′ fragments from PV IgG are pathogenic. These findings suggest that cross-linking of a keratinocyte surface antigen by the autoantibodies may be the initial step in the development of acantholysis in the epidermis of patients with PV. FS is a superficial erosive dermatosis that affects young adults and children living in remote villages of certain regions of Brazil where there are more than 15,000 registered cases. Epidemiological data strongly implicate an environmental factor(s) as the cause of FS. If this is established, it would represent a major step forward in the understanding of human autoimmunity. The disease involves only the skin and produces subcorneal vesicles that can lead to exfoliated erythroderma. The majority of patients have autoantibodies against a keratinocyte surface antigen, desmoglein I. These autoantibodies induce epidermal subcorneal vesicles when injected intraperitoneally into experimental animals. FS IgG as well as the F(ab′)2 and Fab′ fragments are pathogenic when tested in the experimental animal model. This suggests that the autoantibodies in this disease may trigger subcorneal acantholysis by impairing the function of a cell adhesion molecule of the keratinocyte. The mechanisms by which pemphigus autoantibodies precipitate acantholysis continue under investigation in different laboratories. Current experiments suggest that activation of proteases such as plasminogen activator may be implicated in this process. Experimental data from other investigators suggest that complement activation may be an amplifying mechanism of acantholysis. Finally, it has been postulated that pemphigus autoantibodies may impair the assembly of desmosomes, thereby inducing acantholysis. The advances in the immunopathology of pemphigus have proved important in establishing the rationale for the treatment of these patients. All therapies are focused on lowering and eliminating pemphigus autoantibodies from the circulation of the patient. Thus, these therapies for PV and FS (as a prototype of pemphigus foliaceus) include systemic steroid and immunosuppressive agents. As we advance in the understanding of the etiology and pathogenesis of pemphigus, we will be able to develop new approaches in the management of these patients.

3) Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies

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.