Subject: Broad histopathologic patterns of non-glabrous skin and glabrous skin from patients with a new variant of endemic pemphigus foliaceus-part 1

September 11, 2009 - Endemic pemphigus foliaceus

Comment: Dr Abreu work is of supreme word importance because she discovered a new autoimmune disease with several autoantibodies to cells junctions (that are down regulates in most cancers. Type 1 diabetes, rheumatoid arthritis and lupus are familiar autoimmune diseases; but it’s the rare new variant of endemic pemphigus foliaceus in El Bagre (El Bagre-EPF) which plagues nearly 3 percent of men age 30 to 70 and a smaller percentage of postmenopausal women living on the outskirts of the small municipality of El Bagre, Colombia, South America (SA) providing a rare opportunity to dissect the factors that prompt the immune system to harm instead of protect. Mal nutrition, and environmental pollution associated with ultraviolet radiation and contact with herpes simplex virus exacerbate this disease.

One of the major discoveries of Dr Abreu with extensive world impact was the discovery of a new variant of Endemic pemphigus foliaceus (EPF). EPF is the only known endemic autoimmune disease occurring in some geographic rural regions of S.A., in Central America and in Africa. The occurrence of EPF has been associated with massive destruction of the rain forest and enormous and abrupt colonization. The occurrence of EPF has also been associated with massive mining pollution of mercuric selenides and other trace elements. The uniqueness of this autoimmune model made of it tremendous tools to study the natural course and more relevant aspect of autoimmune diseases. The geographic restriction makes EPF an invaluable natural-model for studying how the environment, genetic background, and other aspects such us life style are capable to induce, maintain, modify, or even cure this disease. Based on this discoveries by Dr Abreu they are more than a 100 world wide web citations of her discoveries and her work have been included as references ins several books through the world

In Brazil there are several foci of EPF where the disease is called fogo selvagem (FS). In Colombia, we have confirmed the endemic nature of El Bagre-EPF. We have performed preliminary studies in characterizing it, and in comparing it with other forms of EPF including FS. Dr Abreu described a 10-year prospective, controlled epidemiologic, humanitarian, and immunologic fieldwork case-control survey was performed in El Bagre. Our work revealed that this disease is endemic in rural areas surrounding El Bagre. The disease appeared in 4.7% of middle-aged and older men and postmenopausal women from these rural areas. This disease differs from previously described forms of EPF. It shares some heterogeneous immunoreactivity with paraneoplastic pemphigus but seems not to be associated with malignant tumors. The disease resembles Senear-Usher syndrome (pemphigus and lupus) but occurs endemically either with a localized stable clinical course or in a possible systemic form. This systemic form may affect organs other than skin and is characterized by episodic relapses and poor prognosis in comparison with the localized form.

Some of the insights and major discoveries of Dr Abreu that had extended world impact included the characterization of several autoantigen profiles for several sera samples obtained from patients with this condition using case controlled molecular epidemiological studies. Dr Abreu used different methods, such us immunofluorescence, (both direct and indirect) (DIF, IIF), various immunoblot (IB) analyses with different antigen sources and detection methods, such as radioactivity, ECL, and immunoprecipitation (IP). By using IIF or DIF with the use of human skin sections as antigen detected IgG autoantibodies against keratinocyte cell surfaces, fibrinogen and other molecules and possible unknown molecules possibly related either to cell junction or cell-matrix junctions. The results of IB and IP analysis indicated that all sera had antibodies reactive with desmoglein 1 (Dsg1). In addition, in various IB assays, many sera reacted with several other proteins with molecular weights of 250 kd, 210 kd, and 190 kd, which later Dr Abreu demonstrated to correspond to desmoplakin I, envoplakin, and periplakin, respectively for these assays we used recombinant proteins of various plakin domains. Molecules of the plakin family ubiquitously detected in other organs such the heart, gastrointestinal tissue, muscle, kidney among other and preliminary studies pointed to possible affection of other internal organs and not only the skin. Of major importance was the discovery of desmoplakin and envoplakin by Dr Abreu.

In addition, Dr Abreu solved the identity of one of the most important antigenic moiety, a 45 kDa tryptic fragment which is recognized by all sera from all the patients with active disease. We were able to identify this conformational epitope using a tryptic cleavage product from cow snouts, and glycosylated peptides partially purified Con-A affinity column. A further immunoaffinity purification using an EPF patient's serum covalently coupled to a Staphylococcus aureus protein A column was incubated with the concanavalin A- (Con-A) eluted products and the immuno-isolated antigen was subjected to amino acid sequence analysis revealing which matched the mature form of the extracellular domain of bovine Dsg1. The identification of this molecule was elusive to the world community with multiple failed attempts performed by scientist worldwide, however Dr Abreu was able to discover the ectodomain of this molecule

Dr Abreu developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these El Bagre-EPF patients to be used for epidemiological studies. We succeeded using the protein extract obtained from trypsin-digested fresh bovine cow snouts and further purified on a Con A matrix was used as antigen. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for EPF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibility’s of 98% (intra-assay) and 95% (inter-assay). We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples and correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.

Dr Abreu latest world-made world breakthrough findings in El Bagre-EPF including the discovery of palms with a polyclonal autoimmune response in patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America. Dr Abreu discovered this when performing skin examination of the palms and soles of El Bagre-EPF patients revealing edematous texture at touch and mild hyperkeratosis, in comparison with their non-glabrous skin. Based on this observation, Dr Abreu tested the palms of 20 El Bagre-EPF cases and 20 controls from the endemic area for any alterations in the samples by direct immunofluorescence (DIF). Dr Abreu findings showed that DIF showed deposits of fibrinogen and albumin, as well as IgG, IgA, IgM, C3C, and IgD, at the epidermal basement membrane zone, around some areas in the epidermis, in the vessels, nerves, peri-neurovascular areas and around the sweat glands. Specific markers for blood vessels [anti-intercellular adhesion molecule 1 (ICAM-1)], [anti-junctional adhesion molecule (JAM-A)] and nerves [anti-glial fibrillary acidic protein (GFAP)], and [anti-human neuron specific enolase (NSE)] co-localized with the patients’ autoantibodies. Dr Abreu is the first person in the world of describe these findings and she concluded that although no blisters, ulcerations, pustules or erosions are clinically observed in the palms of El Bagre-EPF patients, DIF detected distinct immunoreactivity. These alterations may contribute to the edematous texture of the palms and the mild hyperkeratosis found in most of these patients. We may propose that normal glabrous skin and non-glabrous skin seem to be different in types of cell or expression of some molecules that may vary in number, size or structural organization, depending on the anatomical site. We also propose that the immunoreactivity to nerves that we found, may play a role in the recurrent sensation or burning or “being in fire” in patients with endemic pemphigus. Our discoveries may set-up some bases in the discovery of the pathophysiology of the burning sensation that is a hallmark in patients affected by endemic pemphigus foliaceus. Our findings may also explain in part the hyperkeratosis palmaris that has been associated with a chronic phase of fogo selvagem in Brazil.

Dr Abreu just recently made another major breakthrough in the study of autoimmune blistering and autoimmune disease with world-wide impact. Dr Abreu just discovered for the first time in the history that human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in el Bagre, Colombia, South America. Dr Abreu had previously discovered a new variant of endemic pemphigus foliaceus (EPF) in Colombia, South America (El Bagre-EPF). Dr Abreu continued with the characterization of this variant of endemic pemphigus foliceus EPF, and she focused on one of our previously reported clinical findings: the presence of ocular lesions. These ocular lesions are seen in patients having extensive skin involvement, as measured by the Lund and Browder scale, which is generally utilized for patients with skin burns. Dr Abreu specifically evaluated the possibility of several eyelid structures autoreactivity in these patients, and correlated our immunological data with the clinical findings. We performed indirect immunofluorescence studies utilizing normal human eyelid skin from routine blepharoplasties as substrate tissue. We tested sera from a) twelve El Bagre-EPF patients with ocular lesions, b) five patients with sporadic (non-endemic) pemphigus foliaceus, and c) twenty normal control subjects (ten from the El Bagre EPF endemic area and ten from non-endemic areas). We used goat antiserum to human total IgG/IgA/IgM (FITC-conjugated) as a secondary antibody. In addition, we used antibodies to human fibrinogen, albumin, IgG, IgE, C1q and C3, conjugated with either FITC, Texas Red, Alexa Fluor 555 or Alexa Fluor 594. Ki-67 (a cell proliferation marker) was utilized to determine the cell proliferation rate, and nuclear counterstaining was performed with either DAPI or Topro III. Dr Abreu findings revealed autoreactivity to multiple eyelid structures, including meibomian glands and tarsal muscle bundles at different levels, and some areas of the epidermis and the dermis close to the isthmus of the eyelids. Tarsal plate autoreactivity was seen in 10/12 of the El Bagre-EPF sera and in one control with pemphigus erythematosus. Furthermore, immunoprecipitation using an eyelid sample as a substrate with 1 mM sodium orthovanodate showed new autoreactivity to several antigens, including some of possible lipid origin. Dr Abreu was the first person in the world who identified for the first time autoantibodies to meibomian glands and tarsal muscle in El Bagre-EPF. Our findings represent a milestone and suggest that the autoantibodies to the ocular structures cause the clinical and histopathological findings in the ocular lesions in El Bagre-EPF.

One of Dr Abreu recent innovations was to report broad histopathologic patterns of non-glabrous skin and glabrous skin from patients with a new variant of EPF prospective, controlled epidemiologic survey performed in El Bagre, Colombia revealed a new variant of endemic pemphigus disease, occurring in a gold mining region. The disease resembled Senear-Usher syndrome, and occurred in an endemic fashion. The aim of this study is to describe the most frequent histopathologic patterns in non-glabrous skin and in glabrous skin observed in these patients, and their clinical correlation. The study was performed on non-glabrous skin biopsies of 30 patients from the dominantly clinical affected areas (either on the chest, arms or face). Simultaneously, biopsies from the palms were obtained in 10 randomly chosen patients of the 30 total patients. The specimens were examined following hematoxylin and eosin (H&E) staining. The most common blisters observed were subcorneal, although in some cases intraspinous and subepidermal blisters were visualized. Our results showed a very heterogeneous pattern of histopathologic patterns in non-glabrous skin, which seemed to correlate with the clinical features. The most common pattern was typical pemphigus foliaceus-like, with some lupus erythematosus-like features. A non-specific, chronic dermatitis pattern prevailed in the clinically controlled patients taking daily corticosteroids. In the patients who have had the most severe and relapsing pemphigus, early sclerodermatous changes and scleredermoid alterations prevailed in their reticular dermis. In addition to the scleredermoid alterations, the reticular dermis showed a paucity of appendageal structures. On the contrary, in the palms, a similar pattern was seen in all cases, including thickening of the stratum corneum, hypergranulosis, edema in the papillary and reticular dermis and a dermal perivascular lymphocytic infiltrate. The direct immunofluorescence of the glabrous vs. the non-glabrous skin also showed some differences. We conclude that the histopathologic features of this new variant of endemic pemphigus are complex, therefore, classical histopathologic features previously described for superficial, endemic pemphigus cannot be used alone to diagnose this disease.

Dr Abreu just recently made another major break-through in the study of autoimmune blistering and autoimmune disease with world-wide impact. Dr Abreu reported the presence of autoantibodies in sweat glands detected by different methods in serum and in tissue from patients affected by a new variant of endemic pemphigus foliaceus. No one had shown autoantibodies in sweat glands in endemic pemphigus foliaceus in the world. Dr Abreu discovery came after examining the patients with a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, (El Bagre-EPF), Dr Abreu noted several polymorphic clinical lesions around their axillary areas. Based on our clinical findings and on previous histopathological studies on the skin of these patients that showed abnormalities in their sweat glands, and the presence of mercuric selenides and iodines by autometallography assay, Dr Abreu decided to investigate immunoreactivity to the sweat glands in these patients. Dr Abreu tested for autoreactivity utilizing direct and indirect immunofluorescence (DIF, IIF). To be able to distinguish between non-specific immune deposits and real autoimmune response, and knowing that sweat glands have some intrinsic autofluorescence for the presence of lipofuscin granules (that naturally fluoresce under the UV light microscope), as well as by the presence of secretory IgA, we used simultaneously immunohistochemistry (IHC). Dr Abreu tested ten El Bagre-EPF patients, ten healthy controls from the endemic area and ten healthy controls from the United States. We were able to visualize a specific autoreactivity in sweat glands in 8/10 cases of El Bagre-EPF by DIF, IIF and by IHC. In addition when using anti-human monoclonal antibodies to CD3, CD68, and CD20, we confirmed the presence of several specific immune responses in situ, and around the sweat glands. No healthy control cases yielded positive findings. In some chronic cases, a decrease and sometimes a complete absence of sweat glands and other skin appendices was found. In addition to this, sclerodermoid changes or early sclerodermatous changes sometimes extending into the adipose tissue as a membranous lipodystrophy were observed. Autoreactivity to the neurovascular components around the sweat glands were also observed. Dr Abreu data demonstrates for the first time that there is an immunoreactivity toward sweat glands in El Bagre-EPF patients that seems to destroy some of these structures.

The latest scientific advance of Dr Abreu was to discover some degree of genetic association in a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia: using the Hardy-Weinberg-Castle law and linked short tandem repeats. Dr Abreu performed a Complex Segregation Analysis (CSA) and short tandem repeats to discriminate between environmental and/or genetic factors in this disorder. The CSA analysis was carried out according to the unified model, implemented using the transmission probabilities implemented in the computer program POINTER, and evaluated by using a software package for population genetic data analysis (GDA), Arlequin. We performed pedigree analyses by using Cyrillic 2.1 software, with a total of 30 families with 50 probands (47 males and 3 females) tested. In parallel to the CSA, we tested for the presence of short tandem repeats from HLA class II, DQ alpha 1, involving the gene locus D6S291 by using the Hardy-Weinberg- Castle law.

Dr Abreu results indicate that the best model of inheritance in this disease is a mixed model, with multifactorial effects within a recessive genotype. Two types of possible segregation patterns were found; one with strong recessive penetrance in families whose phenotype is more Amerindian-like, and another of possible somatic mutations. Dr Abreu concluded that the penetrance of 10% or less in female patients 60 years of age or older indicates that hormones could protect younger females. The greatest risk factor for men being affected by the disorder was the NN genotype. These findings are only possible due to somatic mutations, and/or strong environmental effects. We also found a protective role for two genetic loci (D6S1019 AND D6S439) in the control group.

Some of the latest publications of Dr Abreu are:

Abreu-Velez, IC Avila, J Segovia, MM Yepes, WB Bollag. Rare clinical form in two patients affected by a new variant of endemic pemphigus in northern Colombia. Skinmed, November 1, 2004; 3(6): 317-21.

Abréu-Vélez AM., Abréu-Vélez CE, Patiño PJ, Montoya-Maya F,

Bollag B W. The tryptic cleavage product of the mature form of the bovine desmoglein 1 ectodomain is one of the antigen moieties immunoprecipitated by all sera from symptomatic patients affected by a new variant of endemic pemphigus. Eur Journal of Dermatol Jul-August, 13; 359-66, 2003.

Hisamatsu Y, Abreu Velez AM, Amagai M, Ogawa MM, Kanzaki T, Hashimoto T. Comparative study of autoantigen profile between Colombian and Brazilian types of endemic pemphigus foliaceus by various biochemical and molecular biological techniques. J Dermatol Sci. 2003 Jun; 32(1):33-41.

Abréu-Vélez AM., Warfvinge G. Leon-Herrera W.,Abréu-Vélez, Montoya-Maya F, Hardy D, Bollag B. W, Hashimoto K. Detection of Mercury and Other Undetermined Materials in Skin Biopsies of Endemic Pemphigus Foliaceus. The American Journal of Dermatopathology, Oct 25(5):384-391, 2003.

Abréu-Vélez, AM, Beutner, E, Montoya F, Bollag WB, Hashimoto T. Analyses of autoantigens in a new form of endemic pemphigus foliaceus in Colombia. J American Academy of Dermatolology. Oct (49), 4:609-614, 2003.

Abréu-Vélez, AM, Hashimoto T, Tobón S, Londoño ML, Montoya F, Bollag, WB, Beutner, E. A unique form of endemic pemphigus in Northern Colombia. J American Academy of Dermatology. Oct (49), 4:609-614, 2003.

Abréu-Vélez AM, Yepes MM, Patiño PJ, Bollag WB, Montoya F. A cost-effective, sensitive and specific enzyme-linked immunosorbent assay useful for detecting a heterogeneous antibody population in sera from people suffering a new variant of endemic pemphigus. Arch Dermatol Res. 2004 Jan 17.

Howard M, Yepes MM, Maldonado JG, Villa E, Jaramillo A, Botero J, Patino PJ, Hashimoto T. Abreu-Velez AM. Broad histopathologic patterns of non-glabrous skin and glabrous skin from patients with a new variant of endemic pemphigus foliaceus (part 1). The Journal of Cutaneous Pathology, 2009 ms published on line.

Abreu-Velez AM, Howard MS, Hashimoto K, Hashimoto. Autoantibodies to sweat glands detected by different methods in serum and in tissue from patients affected by a new variant of endemic pemphigus foliaceus. Archives Dermatol Reserch, 2009 published on line June 23.

Abreu-Velez AM, Howard MS, Loebl A. Autoreactivity to sweat and sebaceous glands and skin homing T cells in lupus profundus.. Clin Immunol. 2009 132, 420-424.

Abreu-Velez AM, Howard MS, Hashimoto T, Grossniklaus. Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America. Ms in press. J American Academy of Dermatology.

Abreu-Velez AM, Howard MS, Pinto FJ Jr. Dyshidrotic eczema: relevance to the immune response in situ. North Am J Med Sci, 2009, August, Vol 1. No 3.

Abreu-Velez AM, Howard MS, Hashimoto T. Palms with a polyclonal autoimmune response in patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America. Ms submitted to European Journal of Dermatology.

Abreu-Velez AM, Billie Jackson B, Howard MS A. Deposition of immunoreactants in a cutaneous allergic drug reaction. North Am J Med Sci 2009; 1: 180-183.

Abreu-Velez AM, Villa-Robles E, Howard MS. A new variant of endemic pemphigus foliaceus in El-Bagre, Colombia: the Hardy-Weinberg-Castle law and linked short tandem repeats. Ms in press. North Am J Med Sci 2009; 1: 169-179

Abreu-Velez AM, Girald J, Howard MS. J. Antigen presenting cells in a patient with hair loss of and systemic lupus erythematosus. North Am J Med Sci 2009; 1: 205-210.

Abreu-Velez AM, Smith G Jr, Howard MS. Neutrophil extracellular traps (NETS), Igd, myeloperoxidase (MPO) and antineutrophil cytoplasmic antibody (ANCA) associated vasculitides. Ms submitted .to North Am J Med Sci.