Epidermolysis bullosa simplex generalized severe induces a T helper 17 response and is improved by apremilast treatment.

BACKGROUND: Epidermolysis bullosa simplex generalized severe (EBS-gen sev) is a genetic disorder caused by mutation in the KRT5 or KRT14 genes. Although it is usually considered a mechanical disease, recent data argue for additional inflammatory mechanisms. OBJECTIVES: To assess the inflammation in the skin of patients with EBS-gen sev. METHODS: A first immunohistochemical retrospective study was performed on frozen skin samples from 17 patients with EBS-gen sev. A second multicentre prospective study was conducted on 10 patients with severe EBS-gen sev. Blister fluid and epidermis were processed for immunochemical analysis and quantitative real-time polymerase chain reaction. Cytokine expression was analysed in blister fluid and compared with that in controls. RESULTS: Histological analysis showed a constant dermal perivascular CD4+ lymphocyte infiltrate in skin biopsies of both blister (n = 17) and rubbed skin (n = 5), an epidermal infiltration of neutrophils and eosinophils in 70% of cases, and increased immunostaining for CXCL9 and CXCL10 in blistering skin. High levels of T helper 17 cytokines were detected in lesional skin. Three adult patients with EBS-gen sev were treated with apremilast, with a dramatic improvement of skin blistering and good tolerance. CONCLUSIONS: Our study demonstrates the importance of inflammation in patients with EBS-gen sev and underlines the key role for T helper 17 cells in its pathogenesis. In addition, this study provides promising new therapeutic approaches for this disabling disorder.

Interaction of plectin with keratins 5 and 14: dependence on several plectin domains and keratin quaternary structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.

T-lymphocytes are directly involved in the clinical expression of migratory circinate erythema in epidermolysis bullosa simplex patients.

Epidermolysis bullosa simplex with migratory circinate erythema (EBS-MCE) is a rare EBS subtype characterised by migratory blistering lesions that resolve with brownish pigmentation. It is caused by a recurrent readthrough mutation, c.1649delG, in the tail of keratin 5. Here, we report a child with EBS-MCE and investigated the immunologic mechanisms underlying the migratory lesions in this patient. A skin biopsy from the patient from an active border of an erythematous lesion was used for the immunohistochemical characterisation of the inflammatory infiltrate and for TUNEL assay to detect apoptotic cells. We found abundant CD4+ and CD8+ T lymphocytes infiltrating the papillary dermis and lining the dermal-epidermal junction. A number of these cells expressed the activation marker CD69. CD83+ dendritic cells were present both in the epidermis and papillary dermis. Finally, TUNEL staining showed apoptosis of basal and suprabasal keratinocytes. These findings suggest a critical role of the cellular immunity in determining the EBS-MCE phenotype.

Consequences of two different amino-acid substitutions at the same codon in KRT14 indicate definitive roles of structural distortion in epidermolysis bullosa simplex pathogenesis.

Numerous inherited diseases develop due to missense mutations, leading to an amino-acid substitution. Whether an amino-acid change is pathogenic depends on the level of deleterious effects caused by the amino-acid alteration. We show an example of different structural and phenotypic consequences caused by two individual amino-acid changes at the same position. Epidermolysis bullosa simplex (EBS) is a genodermatosis resulting from KRT5 or KRT14 mutations. Mutation analysis of an EBS family revealed that affected individuals were heterozygous for a, to our knowledge, previously unreported mutation of c.1237G>C (p.Ala413Pro) in KRT14. Interestingly, 2 of 100 unrelated normal controls were heterozygous, and 1 of the 100 was homozygous for a different mutation in this position, c.1237G>A (p.Ala413Thr). In silico modeling of the protein demonstrated deleterious structural effects from proline substitution but not from threonine substitution. In vitro transfection studies revealed a significantly larger number of keratin-clumped cells in HaCaT cells transfected with mutant KRT14 complementary DNA (cDNA) harboring p.Ala413Pro than those transfected with wild-type KRT14 cDNA or mutant KRT14 cDNA harboring p.Ala413Thr. These results show that changes in two distinct amino acids at a locus are destined to elicit different phenotypes due to the degree of structural distortion resulting from the amino-acid alterations.

[Immunofluorescence mapping for diagnosis of congenital epidermolysis bullosa]. / Mapeo por inmunofluorescencia para el diagnóstico de epidermólisis ampollosa congénita.

The tools for diagnosis of epidermolysis bullosa have advanced greatly since Hintner's group introduced antigen mapping as a diagnostic test for this family of genodermatoses. Monoclonal or polyclonal antibodies raised against some of the specific proteins found in the epidermis and basement membrane of the epidermis have allowed 4 types of epidermolysis bullosa de be identified and all variants to be classified. When a newborn baby presents with blisters, many conditions are implicated in the differential diagnosis. Examination under an optical microscope can suggest epidermolysis bullosa, but immunofluorescence mapping and electron microscopy are required for confirmation of the diagnosis and further classification of congenital epidermolysis bullosa. This article explains the importance of immunofluorescence antigen mapping and describes the methods employed for classification and subclassification of epidermolysis bullosa.

Altered expression of a new antigen of the dermal-epidermal junction (NU-T2 DEJ Ag) in junctional epidermolysis bullosa.

NU-T2 antigen (Ag) is a new and recently described antigen of the dermal-epidermal junction, recognized by an anti-CD1b monoclonal antibody denominated NU-T2. We studied NU-T2 Ag expression in junctional epidermolysis bullosa (13 patients) and in other forms of hereditary epidermolysis bullosa (23 patients), comparing the results with nicein expression. In junctional epidermolysis bullosa gravis type no differences were found between the expression of NU-T2 and nicein, both being negative in bullous as well as in non-bullous skin. Interestingly, in mitis type junctional epidermolysis bullosa, NU-T2 Ag was found to be absent or reduced in five of six patients both in lesional and in uncleaved skin. When compared with nicein expression, clearcut differences were found, further suggesting that these two antigens are different. These data confirm that NU-T2 Ag is a novel epitope of the dermal-epidermal junction, probably relevant in dermal-epidermal cohesion, and it could be responsible, together with nicein, 19-DEJ-1 and other adhesion molecules, for the different subtypes of junctional epidermolysis bullosa. Finally, NU-T2 monoclonal antibody is a new relevant tool for the diagnosis, classification, and prenatal diagnosis of junctional epidermolysis bullosa.

Immunohistochemical and ultrastructural characterization of tonofilament and hemidesmosome abnormalities in a case of epidermolysis bullosa herpetiformis (Dowling-Meara).

BACKGROUND: A neonate with epidermolysis bullosa herpetiformis (EBH) (Dowling-Meara) had an undescribed ultrastructural and immunohistochemical abnormality. OBJECTIVE: The objective was to clarify the ultrastructural and immunohistochemical abnormalities in EBH to gain further insight into the pathogenesis of this disorder. METHODS: Tissue from the patient was studied with routine histochemistry, electron microscopy, and immunohistochemistry. RESULTS: Excessive clumping of tonofilaments on electron microscopic examination, anomalous hemidesmosomes, and immunohistochemical evidence of aberrant keratin expression by basal epidermal cells was found. CONCLUSION: This case of EBH provides further evidence for primary abnormalities involving cytoskeletal-membrane attachment plaque formation in this rare disorder.

Bullous pemphigoid antigens (BPAGs): identification of RFLPs in human BPAG1 and BPAG2, and exclusion as candidate genes in a large kindred with dominant epidermolysis bullosa simplex.

Bullous pemphigoid antigens (BPAGs) are defined as autoantigens in a blistering skin disease, bullous pemphigoid. Two of the BPAGs, a 230-kD (BPAG1) and a 180-kD (BPAG2) protein, have been localized to hemidesmosomes, attachment structures at the basal keratinocyte-basement membrane interphase. We have recently cloned cDNAs corresponding to human BPAG1 and BPAG2, and mapped the corresponding genes to human chromosomes 6p and 10q, respectively. These cDNAs have now been used in a search for RFLPs in the corresponding genes. Southern hybridizations of genomic DNA from normal unrelated individuals with a BPAG1 cDNA detected an informative MspI RFLP, and similar hybridizations with a BPAG2 cDNA revealed an informative TaqI RFLP. These RFLPs were applied to a large kindred with epidermolysis bullosa simplex (EBS), generalized (Koebner) type, consisting of 14 affected and 12 unaffected individuals in three generations. Linkage analysis excluded the EBS locus in this pedigree approximately 9 cM and approximately 5 cM on either side of the BPAG1 and BPAG2 loci, respectively, when a lod score of -2.0 was taken as the limit of exclusion. This study demonstrates that mutations in the BPAG1 or BPAG2 genes are not the primary genetic defect in this family with EBS.