Hauke Schumann

The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases

Collagen XVII/BP180, an epidermal adhesion molecule, exists as a full-length transmembrane protein and as a soluble 120-kd ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. Despite a number of studies on autoantibody targets in blistering skin diseases, it has remained unclear whether the physiologically shed ectodomain of collagen XVII plays a role as an autoantigen. Here we isolated the authentic, soluble form of human collagen XVII and showed that it is an autoantigen recognized by IgG and IgA autoantibodies in different blistering skin diseases and is, in some cases, the preferential target. The ectodomain was isolated from the epidermis, keratinocyte media, amniotic fluid, and pemphigoid blister fluid, and autoantibodies affinity-purified with this ectodomain bound to the proximal surface of the epidermis in normal skin but not in collagen XVII-deficient skin. The antibody reactivity was not dependent on the native conformation or the N-glycosylation of the soluble ectodomain, but was abolished by collagenase treatment. Sera of 81 patients with a clinically active blistering skin disease were reacted with full-length collagen XVII, the authentic soluble ectodomain, and recombinant fragments. In bullous and cicatricial pemphigoid, IgG reactive with full-length collagen XVII also recognized the soluble ectodomain. In linear IgA dermatosis and chronic bullous dermatosis of childhood, IgA targeted the soluble ectodomain more efficiently than the full-length protein. The use of recombinant fragments demonstrated that epitopes were present in several noncollagenous and collagenous subdomains of the molecule, and that a significant portion of the sera targeted Col15 domain, a hitherto unrecognized epitope region.

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.

Two Forms of Collagen XVII in Keratinocytes A FULL-LENGTH TRANSMEMBRANE PROTEIN AND A SOLUBLE ECTODOMAIN

The cDNA sequence of human collagen XVII predicts an unusual type II transmembrane protein, but a biochemical characterization of this structure has not been accomplished yet. Using domain-specific antibodies against recombinant collagen XVII fragments, we identified two molecular forms of the collagen in human skin and epithelial cells. Full-length collagen XVII appeared as a homotrimeric transmembrane molecule of three 180-kDa α1(XVII) chains. The globular intracellular domain was disulfide-linked, and theN-glycosylated extracellular domain of three 120-kDa polypeptides was triple-helical at physiological temperatures. A second, soluble form of collagen XVII in keratinocyte culture media was recognized with antibodies to the ectodomain, but not the endodomain. The soluble form exhibited molecular properties of the collagen XVII ectodomain: a triple-helical, N-glycosylated molecule of three 120-kDa polypeptides. Northern blot analysis with probes spanning either the distal 5′or the distal 3′ end of the collagen XVII cDNA revealed an identical 6-kb mRNA, suggesting that both the 180- and 120-kDa polypeptides were translated from the same mRNA, and that the 120-kDa polypeptide was generated post-translationally. In concert, keratinocytes harboring a homozygous nonsense mutation in theCOL17A1 gene synthesized neither the 180-kDa α1(XVII) chain nor the 120-kDa polypeptide. Finally, treatment of normal keratinocytes with a synthetic inhibitor of furin proprotein convertases, decanoyl-RVKR-chloromethyl ketone, prevented the generation of the 120-kDa polypeptide. These data strongly suggest that the soluble 120-kDa polypeptide represents a specifically cleaved ectodomain of collagen XVII, generated through furin-mediated proteolytic processing. Thus, collagen XVII is not only an unusual type II transmembrane collagen, but the first collagen with a specifically processed, soluble triple-helical ectodomain.

Three Novel Homozygous Point Mutations and a New Polymorphism in the COL17A1 Gene: Relation to Biological and Clinical Phenotypes of Junctional Epidermolysis Bullosa

Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.

Molecular diagnostics facilitate distinction between lethal and non-lethal subtypes of junctional epidermolysis bullosa: a case report and review of the literature.

Abstract. The term epidermolysis bullosa (EB) encompasses a heterogeneous group of genodermatoses, characterised by fragility and blistering of the skin, often associated with extracutaneous manifestations. The clinical picture comprises severe subtypes with lethal outcome in the first years of life as well as milder subtypes with localised blistering or minimal symptoms confined exclusively to nail or teeth abnormalities. We present the case of a male infant, who was born with a few bullae and rapidly developed extensive blistering of the skin. The disease was complicated by painful erosions of the oral mucosa, refused ingestion, and recurrent infections. The child died at the age of 4 months because of cardiac failure due to severe sepsis. Antigen mapping of a skin biopsy showed a split within the lamina lucida of the epidermal basement membrane zone and junctional epidermolysis bullosa (JEB) was diagnosed within the first 3 weeks of life. Markedly reduced staining for laminin 5 indicated the Herlitz type of JEB (OMIM 226700), which could be confirmed by mutation analysis in the LAMB3 gene, showing homozygous nonsense mutations. Conclusion: early antigen mapping using antibodies against the proteins affected in epidermolysis bullosa, is a useful tool providing early mutation analysis and valuable prognostic information needed for adequate therapeutic strategies. The recently published literature on current diagnostic procedures and the revised classification system for inherited epidermolysis bullosa aim towards a better understanding of the disease.

Moderne Wundtherapie – praktische Aspekte der lokalen, nicht‐interventionellen Behandlung von Patienten mit chronischen Wunden

Die Behandlung von Patienten mit chronischen Wunden gestaltet sich zunehmend komplexer. Es war daher das Ziel der Arbeitsgemeinschaft Wundheilung (AGW) der DDG die aktuell relevanten Aspekte der nicht‐interventionellen, lokalen Wundbehandlung für den Praxisalltag in einem Übersichtsartikel darzustellen. Neben den notwendigen Maßnahmen wie Wundsäuberung und Debridement werden die gängigen Wundauflagen, deren Indikation sowie die praktische Anwendung beschrieben. Moderne Antiseptika, die heute in der Wundtherapie eingesetzt werden, beinhalten meist Polihexanid oder Octenidin. Es können für verschiedene weitere Aspekte der Wundbehandlung physikalische Verfahren wie beispielsweise Unterdrucktherapien eingesetzt werden. Wichtig ist es zudem, die oft relevante Schmerzsymptomatik der Patienten zu erfassen und adäquat zu behandeln. Die moderne feuchte Wundtherapie kann in der Behandlung von Patienten mit chronischen Wunden die Heilung unterstützen, sie kann Komplikationen vermindern und die Lebensqualität verbessern. Gemeinsam mit einer Therapie der zugrundeliegenden Ursachen ist die moderne Wundtherapie damit ein wichtiger Aspekt in dem therapeutischen Gesamtkonzept bei diesen Patienten.

Betulin-Based Oleogel to Improve Wound Healing in Dystrophic Epidermolysis Bullosa: A Prospective Controlled Proof-of-Concept Study

Introduction Skin fragility and recurrent wounds are hallmarks of hereditary epidermolysis bullosa (EB). Treatment options to accelerate wound healing are urgently needed. Oleogel-S10 contains a betulin-rich triterpene extract from birch bark. In this study, we tested the wound healing properties of topical Oleogel-S10 in patients with dystrophic EB. Methods We conducted an open, blindly evaluated, controlled, prospective phase II pilot trial in patients with dystrophic EB (EudraCT number 2010-019945-24). Healing of wounds treated with and without topical Oleogel-S10 was compared. Primary efficacy variable was faster reepithelialization as determined by 2 blinded experts. The main secondary outcome variable of the study was percentage of wound epithelialization. Results Twelve wound pairs of 10 patients with dystrophic EB were evaluated. In 5 of 12 cases, both blinded reviewers considered epithelialization of the intervention wounds as superior. In 3 cases, only one reviewer considered Oleogel-S10 as superior and the other one as equal to control. Measurements of wound size showed a trend towards accelerated wound healing with the intervention but without reaching statistical significance. Conclusion Our results indicate a potential for faster reepithelialization of wounds in patients with dystrophic EB when treated with Oleogel-S10 but larger studies are needed to confirm significance.

Dermatologische Gentherapie: Utopie oder Realität?

Die Identifizierung ursachlicher Mutationen, das rasant gewachsene Verstandnis der Pathophysiologie von Hautkrankheiten und die schnelle Entwicklung der molekularen Technologien eroffnen fur die Zukunft interessante kausale Therapiemoglichkeiten bei genetischen und erworbenen Erkrankungen. Ein Beispiel ist die somatische Gentherapie, bei der defekte Gene durch funktionsfahige Kopien in der Haut ersetzt werden konnen.

Molekulare Diagnostik hereditärer Dermatosen

Die genetischen Zusammenhange fliesen zunehmend in den arztlichen Alltag ein, und die molekulargenetische Diagnostik gewinnt damit immer mehr an Bedeutung. Wir werden uns im Folgenden deshalb vor allem auf die moderne DNS-Diagnostik konzentrieren. Es handelt sich dabei um die Untersuchung des Erbmaterials, der Desoxyribonukleinsaure (DNS). Alle hereditaren Dermatosen sind durch DNS-Anderungen, Mutationen, in den Genen verursacht, und daher wird die Diagnostik dieser Krankheiten mittels DNS-Analyse durchgefuhrt. Dies bietet den erheblichen Vorteil, dass die gleiche Methodologie fur die Diagnostik verschiedenster Erkrankungen eingesetzt werden kann.