Salivary desmoglein enzyme-linked immunosorbent assay for diagnosis of pemphigus vulgaris: a noninvasive alternative test to serum assessment.

BACKGROUND: Serum desmoglein enzyme-linked immunosorbent assay (ELISA) is used for the diagnosis and monitoring of pemphigus diseases. OBJECTIVES: To compare the diagnostic accuracy of salivary antidesmoglein (Dsg) 1 and 3 ELISA in the diagnosis of pemphigus vulgaris (PV) patients with that of serum desmogleins ELISA. METHODS: Eighty-six untreated PV patients and 180 age- and sex-matched PV-free controls were recruited in this case-control study. PV was diagnosed based on clinical, histopathological, and direct immunofluorescence findings. After processing, serum and salivary anti-Dsg 1 and 3 were measured by the ELISA method using Euroimmun kit (Lübeck, Germany). RESULTS: Using the cut-off point of 20 relative units (RU)/mL, the serum anti-Dsg 1 and 3 ELISA were positive in 62 (72.1%) and 83 (96.5%) patients, respectively, and the salivary anti-Dsg 1 and 3 ELISA were positive in 31 (36.1%) and 63 (73.3%) patients, respectively. The specificity of salivary anti-Dsg 1 and anti-Dsg 3 were both 98.9%. Optimal cut-off values of 7.7 and 13.4 RU/mL were determined for the salivary anti-Dsg 1 and anti-Dsg 3 ELISA, respectively. CONCLUSION: Salivary anti-Dsg 1 and 3 ELISA with high specificities (98.9%) could be suggested as safe and noninvasive methods for the diagnosis of PV when obtaining a blood sample is difficult.

Pemphigus disease activity measurements: pemphigus disease area index, autoimmune bullous skin disorder intensity score, and pemphigus vulgaris activity score.

IMPORTANCE: Recently, the clinical pemphigus disease activity indexes of Pemphigus Disease Area Index (PDAI), Autoimmune Bullous Skin Disorder Intensity Score (ABSIS), and Pemphigus Vulgaris Activity Score (PVAS) were validated to correlate with physician global assessment. The antidesmoglein (anti-Dsg) autoantibodies are known to correlate mostly with pemphigus disease activity. The correlation between these indexes and anti-Dsg1 and anti-Dsg3 enzyme-linked immunosorbent assay values has not been previously evaluated. OBJECTIVES: To evaluate the PDAI, ABSIS, and PVAS in a large number of patients with pemphigus vulgaris and to compare the interrater reliability of these indexes and the convergent validity according to anti-Dsg values. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study was performed in 2012 in a referral university center for autoimmune bullous diseases. One hundred patients with confirmed diagnoses of pemphigus vulgaris and clinical pemphigus lesions (mean [SD] age, 43.3 [1.7] years; age range, 14-77 years; female-male ratio, 1:3) were studied. Three dermatologists familiar with immunobullous diseases and the indexes rated the patients. INTERVENTIONS: All 100 patients were evaluated with the PDAI, ABSIS, and PVAS. Three dermatologists independently rated all 3 indexes for each of the patients on the same day. Serum anti-Dsg1 and anti-Dsg3 enzyme-linked immunosorbent assay values were measured simultaneously. MAIN OUTCOMES AND MEASURES: Analyses of interrater reliabilities, convergent validities according to anti-Dsg titers, correlation between the distribution and types of lesions with disease activity, predictors of higher titers of antibody (multiple regression analysis), and cutoff values of measures for 2 titers of anti-Dsg with optimal area under the curve, sensitivity, and specificity were performed. RESULTS: The interrater reliabilities were highest for the PDAI, followed by the ABSIS and the PVAS (intraclass correlation coefficients of 0.98 [95% CI, 0.97-0.98], 0.97 [95% CI, 0.96-0.98], and 0.93 [95% CI, 0.90-0.95], respectively). The convergent validity was highest for the PDAI, followed by the PVAS and the ABSIS (Spearman ρ = 0.67, 0.52, and 0.33, respectively). Head, neck, and trunk involvement were predictors of higher titers of anti-Dsg1. CONCLUSIONS AND RELEVANCE: Among the 3 studied indexes, the PDAI had the highest validity and is recommended for use in multicenter studies for rare diseases, such as pemphigus vulgaris.

Ultrastructural localization of desmoglein and plakophilin in the human hair suggests that the cell membrane complex is a long desmosomal remnant.

Unlike the superficial part of the corneous layer of the epidermis (Stratum corneum) where desmosomes are degraded and corneocytes flake away, the trichocytes in the hair remain attached to each other after cornification. The permanence and fine localization of cell junctions, in particular of desmosomal proteins in the cornifying and mature human hair, is not known. The present electron microscope immunolocalization study indicates that two protein markers for desmosomes such as desmoglein 4 and plakophilins 1 and 3 are still present in mature cortical and cuticle cells. These proteins remain mainly localized in the cornified cytoplasmic side of desmosomal remnants of cortical cells, but also in the delta layer of the extracellular region of the membrane complex. This suggests that the delta layer represents an extensive desmosomal remnant formed between mature cortical cells and in cuticle cells. The endocuticle appears to be the site of accumulation of desmosomal proteins and degraded nuclear material. The cornification of desmosomal junctions in both cortical and cuticle cells likely contributes to stabilize the integrity of the hair shaft.

[Development of an engraftable skin equivalent based on matriderm with human keratinocytes and fibroblasts]. / Entwicklung eines transplantierbaren Hautäquivalentes auf Basis von Matriderm mit menschlichen Keratinozyten und Fibroblasten.

A cell-based wound coverage with keratinocytes and fibroblasts on the basis of a commercially available dermal substitute (Matriderm ((R)), Kollagen/Elastin matrix) was generated, in order to treat wide burn wounds. First the expansion of keratinocytes was optimised and the culturing time was minimised. Raw material was 1-2 cm (2) split skin. Dermis and epidermis were separated by enzymatic treatment with thermolysin. After treatment of both compartments with trypsin and collagenase I, keratinocytes and fibroblasts were isolated and expanded in collagen I coated dishes. After 10 days fibroblasts were seeded on Matriderm ((R)). After cultivation of the fibroblasts-containing matrix for one week keratinocytes were seeded on top. After an additional week of submersed cultivation the matrix was lifted up to the air-liquid interface to initiate epidermal cell differentiation. After 16 days in the air-liquid interphase the matrix was fixed and underwent immunohistochemical and electron microscopic analysis. Histological analysis showed a regularly stratification of the epidermal part. We observed collagen IV, a marker for the basement membrane, between epidermis and dermis. Desmoglein and the differentiation markers involucrine and cytokeratin 10 were found in the suprabasal layers of the epidermis. Electron microscopic analysis showed the basement membrane in the epidermal junction zone as well as cell-cell connections in the form of desmosomes. Late differentiation characteristics, like granular structures and the cornified layer, were found in the stratum granulosum and stratum corneum. Our results demonstrate that a skin equivalent can be generated by using a collagen/elastin matrix, with an expansion rate of 50-100-fold. This skin equivalent may be useful for covering deep wounds.

Desmosomes are unaltered during infections by attaching and effacing pathogens.

The human attaching and effacing (A/E) intestinal pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and the murine A/E pathogen Citrobacter rodentium cause serious diarrhea in their hosts. These bacteria alter numerous host cell components, including organelles, the host cell cytoskeleton, and tight junctions during the infectious process. One of the proteins that contribute to the intermediate filament network in host cells, cytokeratin-18, is extensively altered during EPEC infections. Based on this, we tested the hypothesis that desmosomes, the only intercellular junctions that interact with intermediate filaments, are also influenced by A/E pathogen infections. We found that the desmosomal transmembrane proteins desmoglein and desmocollin, as well as the desmosome plaque protein desmoplakin, all remain unchanged during EPEC infection in vitro. This evidence is corroborated by the unaltered localization of desmoglein and desmoplakin in vivo in mice infected with C. rodentium for 7 days. Electron microscopic analysis of 7-day C. rodentium-infected murine colonocytes also show no observable differences in the desmosomes when compared to uninfected controls. Our data suggest that, unlike tight junctions, the desmosome protein levels and localization, as well as desmosome morphology, are unaltered during A/E pathogenesis.

Mutations in the desmoglein 4 gene underlie localized autosomal recessive hypotrichosis with monilethrix hairs and congenital scalp erosions.

Localized autosomal recessive hypotrichosis (LAH) is a recently defined disorder characterized by fragile, short, sparse hairs on the scalp, trunk, and extremities. Mutations in desmoglein 4 (DSG4), a novel member of the desmosomal cadherin family that is expressed in the hair follicle as well as the suprabasal epidermis, have been found to underlie LAH. Thus far, the allelic series includes a recurrent intragenic deletion identified in affected Pakastani kindreds and a missense mutation detected in an Iraqi family. We report three siblings of Iraqi and Iranian origin with LAH that presented with congenital scalp erosions and monilethrix-like hairs, features that have not been previously described in this disorder. Follicular hyperkeratotic papules and marked pruritus were also prominent clinical findings. Novel compound heterozygous DSG4 mutations, including a splice-site mutation and a missense mutation that disrupts a conserved calcium-binding site in the extracellular (EC)2-EC3 interface, were found to underlie the disease in this family. These observations broaden the phenotypic and genotypic spectrum of LAH, further illustrating the consequences of DSG4 dysfunction on epidermal and hair shaft integrity.

Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle.

Desmosomes are critical for the tissue integrity of stratified epithelia and their appendages. Desmogleins (DSGs) and desmocollins (DSCs) are transmembrane desmosomal cadherins that interact extracellularly to link neighboring epithelial cells. We recently identified a new member of the DSG family, designated desmoglein 4, whose mutations cause hypotrichosis in human, mouse and rat. In this study, we analyzed in detail the expression domains of human desmoglein 4 protein (DSG4) in human skin relative to differentiation markers and other DSGs. Our results show that DSG4 protein is expressed in the more highly differentiated layers of the epidermis. This expression pattern in vivo is recapitulated in highly differentiated HaCaT human keratinocytes and normal human keratinocytes in vitro. In the human hair follicle, DSG4 is expressed specifically in the hair shaft cortex, the lower hair cuticle, and the upper inner root sheath (IRS) cuticle. Using a green fluorescent protein-tagged version of mouse or rat desmoglein 4 protein (Dsg4) and immuno-electron microscopy, we demonstrate that Dsg4 localizes to desmosomes both in vitro and in vivo. The highly specific expression pattern of DSG4 in the human hair follicle, combined with the phenotype of rodent models and human patients with desmoglein 4 mutations, underscores the importance of this adhesion molecule in the integrity of the hair shaft.