Patterns of Antinuclear Antibodies in a New Variant of Endemic Pemphigus in El Bagre, Colombia, Colocalizing with Antigens against MIZAP, ARVCF, p0071, and Desmoplakins I and II.

BACKGROUND: A new variant of endemic pemphigus foliaceus (EPF) has been documented, El Bagre-EPF. We aimed to study antinuclear antibodies (ANAs) in these patients. METHODS: We performed a case-control study, testing 57 patients affected by this disease and 57 controls from the endemic area matched by work activity and demographics. The participants were evaluated clinically as well as by detection of ANAs utilizing HEp-2 cells. We utilized Triton-induced partial permeabilization of the cell membranes, allowing for the visualization of intracellular and intranuclear antigens. We also immunoadsorbed the ANAs using synthetic peptides to elucidate the nature of the ANA. RESULTS: We detected the presence of a new pattern of ANAs. The new pattern of ANAs was seen in 24% of the El Bagre-EPF patients, compared to our controls (P < 0.001). The new ANA pattern consisted of a thin nuclear and nucleolar rim, finely speckled nucleolar, nuclear membrane pores stains, and a positive intranuclear stain directed against small nuclear components, as well as cytoplasmic deposits of autoantibodies were also observed. The new ANAs pattern perfectly colocalized with commercial antibodies to miocardium-enriched zonula occlusans-1 associated protein (MIZAP), armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), p0071 and desmoplakins I-II (all from Progen Biotechnik). Additionally in 14% of patients with El Bagre-EPF forme fruste and hyperpigmented clinical presentations, a classic homogeneous ANA pattern was observed with autoantibodies specific for Ro, La, Sm, and double-stranded DNA antigens. Immunoadsorption with peptide-based sequences from MIZAP, ARVCF, p0071 and desmoplakins I-II removed the new ANA pattern. CONCLUSIONS: We describe a new pattern of ANAs in El Bagre-EPF, colocalizing with autoantibodies directed against MIZAP, ARVCF, p0071, and desmoplakins I-II.

Genomic study of dilated cardiomyopathy in a group of Mexican patients using site-directed next generation sequencing.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of nonischemic heart failure and death in young adults. Next generation sequencing (NGS) has become part of the diagnostic workup in idiopathic and familial DCM. More than 50 DCM genes have been identified, revealing great molecular heterogeneity and variable diagnostic yield. Interpretation of variant pathogenicity is challenging particularly in underrepresented populations, as pathogenic variant databases include studies mainly from European/Caucasian populations. To date, no studies on genomic diagnosis of DCM have been conducted in Mexico. METHODS: We recruited 55 unrelated DCM patients, 22 familial (F-DCM), and 33 idiopathic (I-DCM), and performed site-directed NGS seeking causal mutations. Diagnostic yield was defined as the proportion of individuals with at least one pathogenic (P) or likely pathogenic (LP) variant in DCM genes. RESULTS: Overall diagnostic yield was 47.3%, and higher in F-DCM (63.6%) than in I-DCM (36.4%, p = 0.047). Overall, NGS disclosed 41 variants of clinical interest (61.0% novel), 27 were classified as P/LP and 14 of unknown clinical significance. Of P/LP variants, 10 were A-band region TTN truncating variants, five were found in DSP (18.5%), five in MYH7 (18.5%), two in LMNA (7.4%), and one in RBM20, ABCC9, FKTN, ACTA1, and TNNT2. NGS findings suggested autosomal recessive inheritance in three families, two with DSP loss of function mutations in affected individuals. The increasing number of mutation reports in DCM, increasing knowledge on the functional consequences of mutations, mutational hotspots and functional domains of DCM-related proteins, the recent refinement ACMG/ClinGen Guidelines, and co-segregation analysis in DCM families helped increase the diagnostic yield. CONCLUSION: This is the first NGS study performed in a group of Mexican DCM patients, contributing to understand the mutational spectrum and complexity of DCM molecular diagnosis.

Autoantibodies to full body vascular cell junctions colocalize with MYZAP, ARVCF, desmoplakins I and II and p0071 in endemic pemphigus in Colombia, South America.

BACKGROUND: We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF). METHODS: Here we aimed to investigate disease autoreactivity to vessels in all body organs/systems. We compared 57 patients and 57 controls from the endemic area, matched by demographics, age, sex, and work activity. We performed immunofluorescence, immunohistochemistry, confocal microscopy, immunoblotting, indirect immune electron microscopy studies, and autometallographic studies. We performed ultrasonography on large patient arteries, investigating for vascular anomalies. In addition, we reviewed autopsies on seven patients who died affected by El Bagre-EPF. We immunoadsorbed any positive vessel immunofluorescence with desmoglein (Dsg1), investigating for new autoantigens. RESULTS: Overall, 57/57 patients affected by El Bagre-EPF displayed autoantibodies to vessels in all the organs/systems of the body via all methods (P < 0.01). The autoreactivity was polyclonal, and the patient's antibodies colocalized with commercial antibodies to desmoplakins I and II, p0071, ARVCF, and MYZAP (all from Progen Biotechnik, Germany; P < 0.01; all present at cell junctions). Immunoadsorption with Dsg1 on positive vessel immunofluorescence showed that the immune response against the vessels was directed against non-Dsg1 antigen(s). Autometallographic studies showed deposits of metals and metalloids in vessel cell junctions and in erythrocytes of 85% of patients (P < 0.01). CONCLUSIONS: Immune response to these vascular antigens is likely altering endothelial cells and vessel shapes, thus disturbing hemodynamic flow. The flow alterations likely lead to inflammation and may play a role in the atherogenesis often seen in these patients.

Patients with a new variant of endemic pemphigus foliaceus have autoantibodies against arrector pili muscle, colocalizing with MYZAP, p0071, desmoplakins 1 and 2 and ARVCF.

BACKGROUND: We identified a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America, which we term El Bagre-EPF, and observed reactivity to arrector pili muscle (APM), thus we tested for autoimmunity to APM. METHODS: We took skin biopsies from 30 patients with El Bagre-EPF and 30 healthy controls (HCs) matched by age, sex and occupation, who were all from the endemic area, and tested these using direct immunofluorescence (DIF), confocal microscopy, immunohistochemistry and immunoblotting (IB). RESULTS: Of the 30 patients with El Bagre-EPF, 27 had autoantibodies to APM that colocalized with commercial antibodies to myocardium-enriched zonula occludens-1-associated protein (MYZAP), desmoplakin (DP)1 and DP2, plakophilin 4, and Armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) (P < 0.001, Fisher exact test). The positive staining also colocalized with Junctional Adhesion Molecule 1 (JAM-A), a control antibody for gap cell junctions. No HC samples were positive. In 27 of the 30 patients, serum that was APM-positive also displayed IB colocalization of their autoantibody molecular weights with the Progen antibodies (P < 0.001, Fisher exact test). CONCLUSIONS: Patients affected by El Bagre-EPF have autoantibodies to APM, colocalizing with the antibodies MYZAP, ARVCF, p0071, DP1 and DP2, suggesting that these molecules are El Bagre-EPF antigens. Further, all of these antigens represent components of cell junctions, indicating that the immune response is directed, at least partially, against cell junctions. The immune response in patients affected by El Bagre-EPF is polyclonal, and it includes B and T lymphocytes, mast cells, IgG, IgA, IgM, IgD, IgE, fibrinogen, albumin, complement/C1q, C3c and C4.

Protein interaction and microRNA network analysis in osteoarthritis meniscal cells.

Osteoarthritis is the most common form of arthritis among elderly adults. Herein, we performed protein-protein interaction (PPI) and miRNA network analysis to evaluate the global correlation between miRNA regulation and the PPI network in human osteoarthritis. Our results showed that desmoplakin (DSP), cystatin A (CSTA), calmodulin 1, tyrosine kinase endothelial, insulin-like growth factor 1 (IGF-1), IGF-binding protein 7 (IGFBP7), syndecan 1 (SDC1), ephrin type-A receptor 4, and PDZ and LIM domain protein 1 were associated with osteoarthritis. Among these proteins, DSP and CSTA interaction and IGF-1, IGFBP7 and SDC1 interaction were observed in our PPI network. Furthermore, these potential target proteins were also linked with individual miRNA in the network. Our findings shed light on the PPIs and mechanisms by which miRNA may regulate the protein interaction network in osteoarthritis, which might provide theoretical support for further studies aimed at discovering new therapeutic strategies.

Cardiac autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America.

Several patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF) have experienced a sudden death syndrome, including persons below the age of 50. El Bagre-EPF patients share several autoantigens with paraneoplastic pemphigus patients, such as reactivity to plakins. Further, paraneoplastic pemphigus patients have autoantibodies to the heart. Therefore, we tested 15 El Bagre-EPF patients and 15 controls from the endemic area for autoreactivity to heart tissue using direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry, immunoblotting, and immunoelectron microscopy utilizing heart extracts as antigens. We found that 7 of 15 El Bagre patients exhibited a polyclonal immune response to several cell junctions of the heart, often colocalizing with known markers. These colocalizing markers included those for the area composita of the heart, such as anti-desmoplakins I and II; markers for gap junctions, such as connexin 43; markers for tight junctions, such as ezrin and junctional adhesion molecule A; and adherens junctions, such pan-cadherin. We also detected colocalization of the patient antibodies within blood vessels, Purkinje fibers, and cardiac sarcomeres. We conclude that El Bagre-EPF patients display autoreactivity to multiple cardiac epitopes, that this disease may resemble what is found in patients with rheumatic carditis, and further, that the cardiac pathophysiology of this disorder warrants further evaluation.

Antibodies to pilosebaceous units along their neurovascular supply routes in a new variant of endemic pemphigus foliaceus in Colombia, South America.

Senear Usher syndrome is a variant of pemphigus foliaceus, confined to seborrheic sites and considered to be a clinical overlap syndrome, with features of both pemphigus foliaceus and lupus erythematosus. We recently described autoantibodies to skin eyelid meibomian glands in patients with a new variant of endemic pemphigus foliaceus (El Bagre EPF) in South America. We tested for El Bagre EPF patient sera autoreactivity to pilosebaceous units utilizing direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry and immunoelectron microscopy. Hematoxylin and eosin staining of skin biopsies revealed that one third of the patients affected by El Bagre-EPF demonstrated some histologic alteration of the pilosebaceous units. By immunohistochemistry, most El Bagre EPF biopsies demonstrated evidence of an autoimmune response along the neural and vascular supply routes of the pilosebaceous units. An active immune response was seen with antibodies such as anti-human mast cell tryptase, myeloid/histoid antigen, CD8, CD20, CD68, CD117/c-kit, ZAP-70 and vimentin. Immunoelectron microscopy demonstrated autoantibodies within the hair follicle and at the basement membrane area of the sebaceous glands. El Bagre-EPF patients have autoantibodies to pilosebaceous units and to their surrounding neurovascular packages. Our results warrant further characterization and may explain the loss of hair described in severe endemic pemphigus foliaceus before the therapeutic steroid era.

Neural system antigens are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia.

BACKGROUND: Endemic pemphigus foliaceus (EPF), is also known as "fogo selvagem" or "wild fire," reflecting the intense burning sensation of the skin reported by patients with this disease. Based on this finding, we tested for neural autoreactivity in patients affected by a new variant of EPF (El Bagre-EPF). METHODS: We tested 20 El Bagre-EPF patients, 20 normal controls from the endemic area, and 20 age- and sex-matched normal controls from outside the endemic area. We tested for autoreactivity to several immunoglobulins and complement. Both human skin and bovine tail were used as antigens. RESULTS: We detected autoreactivity to neural structures, mechanoreceptors, nerves, perineural cell layers of the arachnoid envelope around the optic nerve, brain structures, and to neuromuscular spindles; these structures colocalized with several neural markers. The patient antibodies also colocalized with desmoplakins 1 and 2, with the armadillo repeat protein deleted in velo-cardio-facial syndrome and with p0071 antibodies. Autoreactivity was also found associated with neurovascular bundles innervating the skin, and immunoelectron microscopy using protein A gold against patient antibodies was positive against the nerve axons. Paucicellularity of the intraepidermal nerve endings and defragmentation of the neural plexus were seen in 70% of the cases and not in the controls from the endemic area (p<0.005). Neuropsychological and/or behavioral symptoms were detected in individuals from the endemic area, including sensorimotor axonal neuropathy. CONCLUSIONS: Our findings may explain for the first time the "pose of pemphigus," representing a dorsiflexural posture seen in EPF patients vis-a-vis the weakness of the extensor nerves, and furthermore, the autoreactivity to nerves in EPF could explain the "burning sensation" encountered in EPF disease.

Novel truncating mutations in PKP1 and DSP cause similar skin phenotypes in two Brazilian families.

Inherited mutations in components of desmosomes result in a spectrum of syndromes characterized by variable abnormalities in the skin and its appendages, including blisters and erosions, palmoplantar hyperkeratosis, woolly hair or hypotrichosis and, in some cases, extracutaneous features such as cardiomyopathy. We investigated the molecular basis of two Brazilian patients presenting with clinical features consistent with ectodermal dysplasia-skin fragility syndrome. In patient 1 we identified a homozygous nonsense mutation, p.R672X, in the PKP1 gene (encoding plakophilin 1). This particular mutation has not been reported previously but is similar to the molecular pathology underlying other cases of this syndrome. In patient 2 we found compound heterozygosity for two frameshift mutations, c.2516del4 and c.3971del4, in the DSP gene (encoding desmoplakin). Although there was considerable clinical overlap in the skin and hair abnormalities in these two cases, patient 2 also had early-onset cardiomyopathy. The mutation c.3971del4 occurs in the longer desmoplakin-I isoform (which is the major cardiac transcript) but not in the more ubiquitous desmoplakin-II. In contrast, PKP1 is not expressed in the heart, which accounts for the lack of cardiomyopathy in patient 1. Collectively, these cases represent the first desmosomal genodermatoses to be reported from Brazil and add to genotype-phenotype correlation in this group of inherited disorders. Loss-of-function mutations in the DSP gene can result in a phenotype similar to ectodermal dysplasia-skin fragility syndrome resulting from PKP1 mutations but only DSP pathology is associated with cardiac disease.

Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.

Desmosomes are major cell adhesion junctions, particularly prominent in the epidermis and cardiac tissue and are important for the rigidity and strength of the cells. The desmosome consists of several proteins, of which desmoplakin is the most abundant. Here, we describe the first recessive human mutation, 7901delG, in the desmoplakin gene which causes a generalized striate keratoderma particularly affecting the palmoplantar epidermis, woolly hair and a dilated left ventricular cardiomyopathy. A number of the patients with this syndromic disorder suffer heart failure in their teenage years, resulting in early morbidity. All tested affected members of three families from Ecuador were homozygous for this mutation which produces a premature stop codon leading to a truncated desmoplakin protein missing the C domain of the tail region. Histology of the skin revealed large intercellular spaces and clustering of desmosomes at the infrequent sites of keratinocyte adhesion. Immunohistochemistry of skin from the patients showed a perinuclear localization of keratin in suprabasal keratinocytes, suggesting a collapsed intermediate filament network. This study demonstrates the importance of desmoplakin in the attachment of intermediate filaments to the desmosome. In contrast to null DESMOPLAKIN: mice which die in early development, the truncated protein due to the homozygous 7901delG mutation in humans is not embryonic lethal. This suggests that the tail domain of desmoplakin is not required for establishing tissue architecture during development.

Eicosatrienoic acid (20:3 n-9) inhibits the expression of E-cadherin and desmoglein in human squamous cell carcinoma in vitro.

Eicosatrienoic acid (ETA 5,8,11, n-9) is abnormally increased by essential fatty acid deficiency (EFAD), a condition associated with alterations of cell proliferation and differentiation. In comparison to certain EFAs, addition of ETA at a low concentration resulted in a reduction in the expression of the cell-cell adhesion molecule, E-cadherin, and to a lesser degree, of desmoglein, along with increased invasion of Matrigel by human squamous cell carcinoma (SCC) cells in vitro. At higher concentrations, ETA stimulated the growth of SCC cells. As previously shown, n-6 EFAs (mainly 18:3 n-6, GLA), up-regulated the expression of E-cadherin and desmoglein. This is the first report showing that the abnormal 20:3 n-9 (Mead's acid) is a down regulator of antimetastatic E-cadherin and desmoglein expression.

[Brazilian pemphigus foliaceus (fogo selvagem)]. / Brasilianischer Pemphigus foliaceus (Fogo selvagem).

Most of the clinical, histological and immunohistological features of fogo selvagem resemble those of idiopathic pemphigus foliaceus (PF). Both diseases are clinically characterized by small flaccid bullae evolving into to scaly and crusted lesions, sometimes with pustules, mainly in seborrheic areas of the skin. Mucosal surfaces are mostly spared. The main histologic feature of endemic pemphigus foliaceus is a subcorneal acantholytic blister. Standard immunofluorescence studies demonstrate intercellular IgG deposits throughout the entire epidermis. These IgG antibodies are mainly of the IgG4-subclass. Almost all patients have circulating IgG-autoantibodies in their serum directed against stratified epithelial desmosomes. The fogo selvagem autoantibodies and the PF antibodies are directed against the 160 kD desmosomal glycoprotein desmoglein 1 which together with plakoglobin (85 kD) forms a complex of adhesion proteins with desmosomes of stratified epithelia. Fogo selvagem occurs in endemic foci in some areas of Brazil and possibly in neighbouring South American countries, very often in children, adolescents and young adults. The etiology of fogo selvagem is still unknown. The frequent association with insect bites has lead to the concept of fogo selvagem being a transmissible disease with acquired immunity in adulthood. However, the infectious agent and possible vectors have not yet been identified.

A simple epithelial cell line (MDCK) shows heterogeneity of desmoglein isoforms, one resembling pemphigus vulgaris antigen.

The epidermal blistering disease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130 x 10(3) M(r) polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.

Distribution of desmoplakin I/II in endometrial cells of mice in the artificially induced decidua.

The decidual reaction is characterized by the redifferentitation of the endometrial connective tissue into a tissue with epithelioid features formed by decidual cells. An ultrastructural study showed a special type of junction formed between differentiating (predecidual) cells of the mouse from day six of pseudopregnancy onward. These contacts share ultrastructural characteristics of both desmosome and adherens junctions. These junctions have usually been classified as desmosome-like. In the present work, besides the ultrastructural analysis, we investigated with light microscopy the presence of desmoplakins I and II, using streptavidin-biotin immunoperoxidase technique. We found a positive punctate staining around predecidual cells while a scarce reaction was observed in the other regions of the uterus. These results suggest that these junctions belong to the desmosome family.

Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus.

Recently, it has been shown that desmoglein, pemphigus foliaceus target antigen, and a 130-kD pemphigus vulgaris antigen belong to the cadherin family of cell adhesion molecules. We tried to determine whether desmocollins I/II, other cadherin-like transmembranous glycoproteins present in desmosomes, are also recognized by pemphigus autoantibodies of the IgG class. We examined 16 pemphigus vulgaris sera, 15 pemphigus foliaceus sera, 15 Brazilian pemphigus foliaceus sera, five bullous pemphigoid sera, and 65 normal sera. Four (25%) pemphigus vulgaris sera, one (7%) pemphigus foliaceus serum, eight (53%) Brazilian pemphigus foliaceus sera, and three (5%) normal sera reacted with desmocollins I/II on immunoblots of bovine desmosome preparation. The affinity-purified desmocollins I/II pemphigus autoantibodies were shown to bind the epidermal cell surface by indirect immunofluorescence. Immunoblot analysis revealed one pemphigus vulgaris serum, one Brazilian pemphigus foliaceus serum, and one normal serum recognizing a recombinant protein produced by a desmocollin cDNA clone. Moreover, immunoblot analysis of reactivity of a Brazilian pemphigus foliaceus serum with recombinant proteins produced by deletion mutants of the desmocollin cDNA clone showed that the extracellular portion of desmocollin is immunogenic in this pemphigus patient. We conclude that desmocollins I/II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus. However, the significance of this reactivity remains to be defined.

[Characterization of the desmoglein in renal cells in culture]. / Caracterización de la desmogleína de células renales en cultivo.

Desmosome junctions are found in epithelial tissues. They both link cells externally and anchor cytoplasmic intermediate filaments to the plasma membrane. Quantitative and qualitative abnormalities in intercellular junctions have been described in a broad spectrum of human and animal cancers. Current efforts are aimed at exploring the possibility that some of these defects may account for the hallmarks of malignancy, namely tumour invasion and metastasis. Desmosomes are constituted by several proteins, one of them is desmoglein-1 (DG-1), a transmembrane glycoprotein who glycosylated portion is major component of the adhesion mediating desmoglia. In order to know the similarity between tissue DG-1 and cultured renal cells DG-1 was used antisera raised against DG-1 to identify cross-reacting components. Anti DG-1 antibodies stained cell-cell boundaries in a punctate fashion in epithelial tissue and on densely grown monolayers of renal cells. Radioimmunoprecipitation and immunoelectrotransference show positive reaction with anti DG-1 antibodies with desmosomes obtained from epithelial tissue and renal cells monolayers, but last one was less positive. Results suggest some minor differences between DG-1 extracted from diverse sources but they have a commun immunodominant epitope.