Martin Laimer

Treatment‐resistant classical epidermolysis bullosa acquisita responding to rituximab

SIR, Epidermolysis bullosa acquisita (EBA) is a chronic severe subepidermal blistering disease of the skin and mucous membranes characterized by marked resistance to topical and systemic therapy. We report a patient with a 6-year history of EBA refractory to any immunosuppressive medication (Fig. 1), who responded well to a treatment with the anti-CD20 monoclonal antibody rituximab. A 71-year-old woman presented with blisters and erosions on mechanically strained areas including hands, elbows and feet, that healed with scarring and milia formation and resulted in nail dystrophy, loss of toenails and scarring alopecia. Involvement of mucous membranes was initially confined to the oral cavity (Fig. 2), and later also occurred on oesophageal and pharyngolaryngeal mucosa. Histological examination of perilesional skin showed subepidermal blister formation and a mild infiltrate composed of lymphocytes and neutrophils. Conventional direct immunofluorescence of perilesional skin detected linear deposition of IgG and C3 at the dermoepidermal junction. No IgA or IgM deposits were found. Indirect immunofluorescence (IIF) with the patient’s serum on salt-split skin revealed an exclusive dermal binding of circulating IgG antibasement membrane zone (BMZ) antibodies at a titre of 1 : 2560. These antibodies recognized a band at 290 kDa, and a 145-kDa band corresponding to the a-chain of collagen VII in immunoblotting on keratinocyte extracts. Thus, the diagnosis of a classical mechanobullous type of EBA was made. Over a 6-year period several immunosuppressive agents, including corticosteroids, azathioprine, ciclosporin, plasmapheresis, mycophenolate mofetil, colchicine, gold preparations, highand low-dose immunoglobulins and daclizumab were administered but none of them resulted in prolonged clinical benefit (Fig. 1). Then in 2004, disease activity increased, mainly due to massive erosions of the pharyngolaryngeal and oesophageal mucosa, which resulted in upper and lower oesophageal stenoses. The anti-BMZ titre (1 : 320) was at this time point lower than in the previous years of the disease. The patient required endoscopic dilatation of oesophageal stenoses, percutaneous endoscopic gastrostomy and tracheotomy because of severe obstruction of the upper airways. We therefore decided to initiate an anti-CD20 monoclonal antibody therapy attempt. Concomitantly, vaccination against influenza A and Streptococcus pneumoniae was carried out. Five rituximab infusions were given intravenously at a frequency of one infusion per week. Because of the poor health condition of our patient and to avoid potential side-effects we used a reduced dose of 144 mg m each time. Concomitant immunosuppressive therapy was azathioprine 2Æ0 mg kg daily. Our patient tolerated the rituximab therapy well with absence of any side-effects or infections. Four weeks after the first rituximab infusion the patient showed marked improvement of the skin and mucous membrane condition (Fig. 2b) and concomitant therapy of azathioprine was discontinued. Transiently, IIF became negative and a repeated immunoblotting study could no longer detect antibodies reactive with the

Rapid, high-quality and epidermal-specific isolation of RNA from human skin

Abstract:  As global transcriptome analyses with a growing demand on layer‐specific applications are widely used in cutaneous biology, we investigated the effect of established and optimized dermo‐epidermal separation methods on the quality of RNA. We compared enzymatic separation with dispase, chemical separation with 1 m sodium chloride and heat separation to a treatment with 3.8% ammonium thioyanate. The impact of freezing as well as the addition of 10 mm aurintricarboxylic acid was considered in the evaluation of the amount and quality of isolated RNA from dermis and epidermis. Using the low abundant gene kallikrein 12 for real‐time PCR analysis, we were able to demonstrate the superior RNA quality after dermo‐epidermal separation using 3.8% ammonium thiocyanate. In addition to the time effectiveness this separation technique promises dermal and epidermal purity and is therefore the method of choice for producing high‐quality RNA for genome‐wide dermal and epidermal transcriptional analysis.

Seborrheic keratosis: Reflectance confocal microscopy features and correlation with dermoscopy

BACKGROUND Differentiation between seborrheic keratosis (SK) and skin cancers may be difficult. Reflectance confocal microscopy (RCM) enables noninvasive assessment of skin neoplasms at cellular-level resolution. OBJECTIVE We sought to describe RCM features of SK and to correlate these RCM findings with dermoscopic structures. METHODS Clinical, dermoscopic, and RCM images of 45 consecutive SK were obtained at a private and university dermatology clinic. Fourteen SK were biopsied because of equivocal clinical or dermoscopic features. RESULTS With RCM, all SK displayed a regular honeycomb pattern of the epidermis and densely packed, round to polymorphous, well-circumscribed dermal papillae at the dermoepidermal junction, features suggestive of a benign neoplasm. RCM features indicating the diagnosis of SK were also observed, including epidermal projections (43/45 SK; 96%) and keratin-filled invaginations (36/45 SK; 80%) at the lesion surface; corneal pseudocysts at epidermal layers (19/45 SK; 42%); and melanophages (21/45 SK; 47%) and dilated round and linear blood vessels (21/45 SK; 47%) in the papillary dermis. Of biopsied SK, 93% (13/14) displayed at least 3 characteristic RCM findings in the absence of RCM features suggestive of malignancy. LIMITATIONS This was a limited study sample and retrospective study design. CONCLUSIONS SK display a distinct set of RCM criteria despite their variable clinical and dermoscopic appearances.

Epidermolysis bullosa nevi.

Epidermolysis bullosa (EB) nevi are large, eruptive, asymmetrical, often irregularly pigmented melanocytic lesions. Such nevi may give rise to small satellite nevi surrounding the primary nevus, and thus frequently manifest clinical features suggestive of melanoma. They usually arise in sites of previous bullae or erosions. At least twice a year all persisting wounds and EB nevi should be evaluated with a low threshold for histopathologic examination if warranted. Our practice is to punch biopsy EB nevi showing dermoscopic features of concern as well as dermoscopically featureless lesions. Given the skin fragility and potentially impaired wound healing in EB patients, we avoid prophylactic total excision of large EB nevi, but rather use the dermoscope to select appropriate sites for punch biopsies within giant EB nevi.

Cytokeratin-related loss of cellular integrity is not a major driving force of human intrinsic skin aging.

The contribution of extracellular matrix components to intrinsic skin aging has been investigated thoroughly, however, there is little information as to the role of the cytoskeletal proteins in this process. Therefore, we compared the expression of the constituents of the cytoskeleton, keratins 1-23 (K1-K23) as well as junction-plakoglobin (JUP), alpha-tubulin (TUBA), and beta-actin (ACTB) in human foreskins of both young (mean 6.4 years) and aged (mean 54.3 years) individuals. By applying RNA expression analysis to intrinsically aged human skin, we demonstrated that the mRNA levels of the genes for K1, K3, K4, K9, K13, K15, K18, K19 and K20 are downregulated in aged skin, K5 and K14 are unchanged, and K2, K16 and K17 are upregulated in aged skin. The mRNA data were confirmed on the protein level. This diverse picture is in contrast to other cytoskeletal proteins including components of the desmosome (JUP), microtubuli (TUBA) and microfilaments (ACTB) - often regarded as house-keeping genes - that were all reduced in aged skin. These cytoskeletal features of intrinsic aging highlight the importance of the cellular compartment in this process and demonstrate that special attention has to be given to RNA as well as protein normalization in aging studies.

Gene therapy of epidermolysis bullosa.

Easy access to the organ and identification of underlying mutations in epidermolysis bullosa (EB) facilitated the first cutaneous gene therapy experiments in vitro in the mid-1990s. The leading technology was transduction of the respective cDNA carried by a retroviral vector. Using this approach, the genotypic and phenotypic hallmark features of the recessive forms of junctional EB, which are caused by loss of function of the structural proteins laminin-5 or bullous pemphigoid antigen 2/type XVII collagen of the dermo–epidermal basement membrane zone, have been corrected in vitro and in vivo using xenograft mouse models. Recently, this approach has also been shown to be feasible for the large COL7A1 gene (mutated in dystrophic EB), applying PhiC31 integrase or lentiviral vectors. Neither of these approaches has made it into a successful Phase I study on EB patients. Therefore, alternative approaches to gene correction, including modulation of splicing, are being investigated for gene therapy in EB.

Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging

Abstract:  Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two‐dimensional (2‐D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun‐protected human foreskin samples pooled from ‘young’ and ‘old’ men. In addition, we analyzed these candidate gene products by 1‐D and 2‐D western blotting to obtain corroborative protein expression data, and by both real‐time PCR (RT‐PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post‐translationally regulated candidates such as phosphatidyl‐ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay

How splicing, the process of intron removal in pre-messenger RNA (mRNA), is carried out with such fidelity in human cells is still not understood, although some general rules are being proposed mainly by in vitro experiments. These rules are currently being redefined by analysis of splicing mechanisms in patients presenting splicing defects. We analysed material of a patient suffering from junctional epidermolysis bullosa, a heritable blistering skin disease. Absence of laminin-5 protein together with hypoplastic hemidesmosomes at the dermo-epidermal junction in the patients skin was shown by immunohistochemical analysis and immunoelectron microscopy. Subsequent DNA analysis revealed heterozygosity for the mutations R635X and 3009C → T in the LAMB3 gene. The latter did not alter codon translation, but introduced an exonic splice site in exon 20. Interestingly, this exonic splice site, which presented a splice score of only 68.6, was preferentially used by the spliceosome over the wild-type splice site at the exon 20–intron 20 border, which showed a splice score of 92.2. LAMB3 mRNA was still detectable in RT-PCR analysis although the aberrantly spliced mRNA leads to a stop codon in exon 21, 5′ of the commonly assumed 3′ border for nonsense-mediated mRNA decay. These results describe an exception to the proposed rules of pre-mRNA splicing and RNA degradation.

Zahnveränderung bei junktionaler Epidermolysis bullosa – Bericht über eine Patientin mit einer Mutation im LAMB3‐Gen

During early odontogenesis the basement membrane is known to be important in epithelio-mesenchymal interactions. Mutations in the gene of one of the major structural proteins of the basement membrane such as laminin 5 might therefore be expected either to seriously compromise ameloblast differentiation and/ or interfere with normal basement-membrane formation and degradation and thus the binding of the ameloblasts to their underlying matrix. Teeth of patients suffering from junctional epidermolysis bullosa (JEB) can be severely affected by abnormal dental development and generalized or focal enamel hypoplasia. Those changes are found in 100% of individuals with JEB but the expression is variable. Beside the quantitative alterations, changes in the prismatic structure and orientation of enamel crystals are described. In addition JEB is associated with an increased risk for dental caries, caused by developmentally compromised enamel and external factors such as difficulties in maintaining oral hygiene because of oral lesions or a softer and more refined high caloric diet. Dental care includes three main strategies: Prevention by consequent oral hygiene and reduction of cariogenic nutrition is of paramount importance to minimize caries development; the restoration of enamel and dentin defects with fillings and stainless steel crowns to guarantee structure and function of teeth; and extractions of most severely affected teeth with osteolytic foci to remove continuous sources of oral infections.

Retiform hemangioendothelioma: presentation of a case expressing D2-40.

Retiform hemangioendothelioma (RH) is a low‐grade angiosarcoma with low metastatic risk, usually occurring as a single lesion on the trunk or extremity in middle‐aged adults. Histopathology shows a distinctive pattern with arborizing blood vessels arranged in a retiform pattern (similar to rete testis tissue) and focal papillae with fibrosclerotic (hyaline) cores. The blood vessels are lined by comparatively monomorphic endothelial cells, frequently presenting a hobnail pattern. We report a case of RH presenting as an indolent brownish plaque on the back of a 17‐year‐old male. Surgical resection and sentinel lymph node biopsy showed no evidence of metastasis. In contrast to the recent literature, this case of RH showed positivity for D2‐40, a marker of lymphatic endothelium. We also report ultrastructural findings for this case of RH.