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Supplementary Figure 2 from Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

Supplementary Figure Legends from Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

Supplementary Figure 1 from Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

Die Versorgung von Patienten mit Epidermolysis bullosa (EB) stellt angesichts der Seltenheit, der Heterogenitat und Komplexitat der Erkrankungsgruppe eine besondere Herausforderung dar. Die primare und sekundare Beteiligung zahlreicher extrakutaner Organsysteme fuhrt zudem zu erheblicher Morbiditat und Mortalitat. Um diese besser zu bewaltigen, sind spezialisierte Behandlungszentren von Noten, die eine individuelle, interdisziplinar akkordierte Betreuung nach aktuellem medizinischen Standard sowie Zugang zu innovativen Therapiemoglichkeiten gewahrleisten. Mit diesem Ziel wurde 2005 das EB-Haus Austria gegrundet, das 2017 zum ersten osterreichischen Expertisezentrum fur Genodermatosen mit Schwerpunkt auf EB designiert wurde und auch als Mitglied in das Europaische Referenznetzwerk fur seltene Hauterkrankungen (ERN Skin) aufgenommen wurde. Die Saulen dieser Institution (Ambulanz, Forschungseinheit, Akademie, Klinisches Studienzentrum) betreiben einen regen Austausch untereinander, mit zahlreichen nationalen sowie internationalen klinischen und wissenschaftlichen Partnern, wie auch insbesondere mit Patienten und deren Angehorige uber die Selbsthilfegruppe DEBRA Austria. Die Entwicklung des EB-Hauses Austria als Referenzzentrum war uber weite Strecken von Pionierarbeit gekennzeichnet, die wegbereitend fur die Optimierung der Betreuung vergleichbarer Erkrankungen sowie allgemeiner Versorgungsstrukturen sein kann.

The care of patients with epidermolysis bullosa (EB) poses a major challenge due to the rarity, heterogeneity and complexity of the disease as well as the occurrence of numerous primary and secondary extracutaneous manifestations, causing a significant morbidity and mortality. Specialized treatment centers are essential for offering these patients adequate care, including individual, interdisciplinary coordinated treatments according to current medical standards, and access to innovative therapeutic options. Against this background, the EB House Austria was founded in 2005 and designated the first national center of expertise for genodermatoses with a focus on EB in 2017. In the same year, it became a member of the European Reference Network for Rare Skin Diseases (ERN Skin). The pillars of this institution (outpatient clinic, research unit, academy, clinical study center) interact closely with each other, with numerous national and international clinical and scientific partners, as well as with patients and their relatives via the DEBRA Austria patient group. The development of the EB House Austria as a reference center is characterized by a long-term pioneering work, which in turn could pave the way for the optimization of care for comparable diseases as well as general care structures.

Accurately addressing the diverse and complex issues of rare diseases (RD) in terms of prevention, recognition, diagnosis, treatment, care and research along key RD specificities, such as great heterogeneity, a limited number of patients, scarcity of relevant knowledge and expertise as well as enormous costs for patient care is a challenging task for healthcare providers and authorities that makes a supranational approach particularly feasible. The European Union has acknowledged RD matters by several initiatives, including efforts to implement national centres of expertise and European reference networks as well as a cross-border referral mechanism to foster access to expert services and to boost dissemination of clinical expertise and research activities. Exemplified by the EB House Austria, a centre of expertise for epidermolysis bullosa cross-linked with international reference partner institutions, this strategy proves its potential to be translated into optimized patient care and to meet the major medical, scientific, social and health-economic impact of RD.

This retrospective multicentre analysis from the Psoriasis Registry Austria (PsoRA) was conducted to determine drug effectiveness and survival of anti-tumour necrosis factor alpha (anti-TNF-α) agents in patients with moderate-to-severe chronic plaque psoriasis over a 9-year period. Data on 1,019 treatment cycles with adalimumab (n = 460), etanercept (n = 501), and/or infliximab (n = 58) administered to 827 patients (272 women, 555 men) were available for analysis. Compared with etanercept, adalimumab and infliximab showed superior short-term effectiveness. Intention-to-treat-calculated median drug survivals for adalimumab (1,264 days) and etanercept (1,438 days) were similar to each other (p = 0.74), but significantly superior to that of infliximab (477 days) (p = 7.0e-07 vs. adalimumab and p=2.2e-07 vs. etanercept, respectively). Their drug survival rates at 36 months were 51.6%, 56.0%, and 22.6%, respectively. Survival rates correlated significantly with effectiveness for adalimumab and etanercept, but not for infliximab.

O1 The European Social Preferences Measurement (ESPM) study project: social cost value analysis, budget impact, commercial life cycle revenue management, and the economics of biopharmaceutical Research & Development (R&D) Michael Schlander, Soren Holm, Erik Nord, Jeff Richardson, Silvio Garattini, Peter Kolominsky-Rabas, Deborah Marshall, Ulf Persson, Maarten Postma, Steven Simoens, Oriol de Sola Morales, Keith Tolley, Mondher Toumi, Harry Telser O2 Newborn Screening: the potential and the challenges James R Bonham O3 Untreatable disease outcomes - how would we measure them? Helmut Hintner, Anja Diem, Martin Laimer O4 Taking Integrated Care Forward: Experiences from Canada to inspire service provision for people living with rare disease in Europe Rejean Hebert O5 Listening to the patient’s voice: social media listening for safety and benefits in rare diseases Nabarun Dasgupta, Carrie E. Pierce, Melissa Jordan O6 Via Opta: Mobile apps making visually impaired patients’ lives easier Barbara Bori, Mohanad Fors, Emilie Prazakova O7 A report of the IRDiRC “Small Population Clinical Trial” Task Force Simon Day O8 HAE patient identification and diagnosis: An innovative, ‘game changing’ collaboration Thomas J. Croce Jr. O9 Co-creating with the community: primary packaging & administration for people with haemophilia Jonas Fransson, Philip Wood O10 Go with Gaucher, taking forward the next generation. How to involve young people to create a new generation of patient advocates Anne-Grethe Lauridsen, Joanne Higgs, Vesna Stojmirova Aleksovska P1 ODAK – Orphan Drug for Acanthamoeba Keratitis Christina Olsen, Ritchie Head, Antonio Asero, Vincenzo Papa, Christa van Kan, Loic Favennec, Silvana Venturella, Michela Salvador, Alan Krol P5 Rare Navigators help people living with rare diseases to manage the social – and healthcare systems Stephanie J. Nielsen, Birthe B. Holm P6 The eAcademy for Tay-Sachs & Sandhoff disease app Daniel Lewi, Patricia Durao P10 The role of a patient organisation in driving the research agenda in a rare disease Heather Band, Andrea West P13 Expertise for rare diseases mapped Marinda J.A. Hammann, Marije C. Effing-Boele, Hanka K. Dekker P14 The hidden costs of rare diseases: a feasibility study Amy Hunter, Amy Simpson P15 FDA’s new natural history grant program: support to build a solid foundation for development of products for rare diseases Gumei Liu, Katherine Needleman, Debra Lewis, Gayatri Rao P17 Understanding the wider impact of adrenal insufficiency: patient organisation involvement in the TAIN project Amy Simpson, Amy Hunter, Martin J Whitaker P20 Bridging the gaps between medical and social care for people living with a rare disease Raquel Castro

The yeast two-hybrid (Y2H) system is one of the most technically straightforward, effective, and widely used tools for the discovery of the binary peptide or protein interactions. However, its exceptional detection sensitivity poses a serious challenge for affinity ranking and hence prioritizing the resultant large number of putative interactors for follow-up analyses. To overcome this apparent bottleneck, we describe here a novel yeast growth curve-based interaction-monitoring approach that permits semiautomatic quantification, comparison, and statistically ascertained scoring of a large collection of Y2H interactions under real-time conditions. Initially, we conducted a proof-of-concept test of five literature-validated peptide-protein interactions with known affinities in the low μM range, and subsequently used the method to classify 88 novel vitamin D receptor-binding peptides derived from high-throughput screening of a highly diverse artificial peptide aptamer library. Based on our in-depth data evaluation, we conclude that real-time monitoring of clone growth as a measure of relative binding strength offers a facile, cost-effective, accurate, reproducible, and further adaptable complement to standard Y2H-derived clone management.

Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries.

Many functional proteomic experiments make use of high-throughput technologies such as mass spectrometry combined with two-dimensional polyacrylamide gel electrophoresis and the yeast two-hybrid (Y2H) system. Currently there are even automated versions of the Y2H system available that can be used for proteome-wide research. The Y2H system has the capacity to deliver a profusion of Y2H positive colonies from a single library screen. However, subsequent analysis of these numerous primary candidates with complementary methods can be overwhelming. Therefore, a method to select the most promising candidates with strong interaction properties might be useful to reduce the number of candidates requiring further analysis. The method described here offers a new way of quantifying and rating the performance of positive Y2H candidates. The novelty lies in the detection and measurement of mRNA expression instead of proteins or conventional Y2H genetic reporters. This method correlates well with the direct genetic reporter readouts usually used in the Y2H system, and has greater sensitivity for detecting and quantifying protein-protein interactions (PPIs) than the conventional Y2H system, as demonstrated by detection of the Y2H false-negative PPI of RXR/PPARG. Approximately 20% of all proteins are not suitable for the Y2H system, the so-called autoactivators. A further advantage of this method is the possibility to evaluate molecules that usually cannot be analyzed in the Y2H system, exemplified by a VDR-LXXLL motif peptide interaction.

Immune recognition and rejection of tissues expressing transfected genes is a major complication of gene replacement therapy for inherited genetic disorders. Owing to the high immunogenicity of human bullous pemphigoid antigen 2 (hBPAG2), the induction and maintenance of tolerance to this neo-antigen is essential to deliver the gene product to patients with epidermolysis bullosa junctionalis. In a skin grafting mouse model, we used gene gun transfection with a construct encoding hNC16A, the immunodominant domain of hBPAG2, to induce antigen-specific immune tolerance. Eighty percent of wild-type mice transfected with hNC16A showed long-term survival of skin grafts expressing hBPAG2. Tolerance was stable and transferable by T cells but not by B cells of tolerant mice to naive hosts. A dense Foxp3(+) regulatory T-cell (T(reg)) infiltrate was noticed in grafts of tolerant mice and depletion of these cells resulted in a loss of tolerance. Taken together, we show that long-lasting hBPAG2-specific tolerance was induced with gene gun delivery of hNC16A through a T(reg)-dependent mechanism. This is of relevance to patients undergoing gene therapy and has broader implications for the treatment of antigen-specific autoimmune diseases.

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

An intact keratin 5/keratin 14 intermediate filament cytoskeleton is vital for the integrity of basal keratinocytes and for the development and maintenance of epidermal structures. In patients with epidermolysis bullosa simplex Dowling-Meara (EBS-DM), heterozygous mutations in the keratin 14 gene in keratinocytes cause a cytoskeletal collapse leading to fragile cells susceptible to cellular stress. The primary aim of this work was to extend analysis of differentially expressed genes in an EBS-DM model cell line to obtain insights into the molecular consequences resulting from the keratin 14 mutation. In a first step, suppression subtractive hybridization (SSH), a powerful technology to enrich for differentially expressed genes, was used to identify genes whose up-regulation may be a direct or indirect result of the keratin 14 mutation, R125P. We discovered 55 candidate genes (SSH genes) that were further analysed by RTq-PCR. Of the 55 SSH genes, 14 (25.45%) were found to be congruently up-regulated. Bioinformatic analysis revealed significant enrichment of genes regulating epidermal development, migration, apoptosis and wound healing.

Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th–40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3′ pre–trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors. Mol Cancer Ther; 10(2); 233–41. ©2011 AACR.

Staphylococcus aureusis a human pathogen of growing clinical significance, owing to its increasing levels of resistance to most antibiotics. Infections range from mild wound infections to severe infections such as endocarditis, osteomyelitis and septic shock. Adherence ofS. aureusto human host cells is an important step, leading to colonization and infection. Adherence is mediated by a multiplicity of proteins expressed on the bacterial surface, including clumping factor B. In this study, we aimed to identify new targets of clumping factor B in human keratinocytes by undertaking a genome-wide yeast two-hybrid screen of a human keratinocyte cDNA library. We show that clumping factor B is capable of binding cytokeratin 8 (CK8), a type II cytokeratin. Using a domain-mapping strategy we identified amino acids 437–464 as necessary for this interaction. Recombinantly expressed fragments of both proteins were used in pull-down experiments and confirmed the yeast two-hybrid studies. Analysis withS. aureusstrain Newman deficient in clumping factor B showed the clumping factor B-dependence of the interaction with CK8. We postulate that the clumping factor B–CK8 interaction is a novel factor inS. aureusinfections.

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele. In epidermolysis bullosa simplex-type Dowling-Meara (EBS-DM), a single amino acid exchange in exon 1 of the keratin 14 gene (K14) triggers a severe skin phenotype, characterized by blistering of the skin and mucous membranes after minor trauma. We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1-7 of the K14 gene. In this approach, the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM) that incorporates a binding domain (BD) and the wild-type sequence of K14. Since the BD is crucial for the trans-splicing functionality, we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs. Co-transfection of the library with a target molecule enabled us to identify highly functional RTMs. The best RTMs were adapted for endogenous trans-splicing in an EBS-DM patient cell line. In this cell line, we were able to detect functional, efficient and correct trans-splicing on RNA and protein levels. Scratch assays confirmed phenotypic reversion in vitro. Owing to concomitant knock-down and repair of the mutated allele, we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease.

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).

Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments. In keratinocytes, type II keratin 6 (K6) interacts with type I keratin 16 (K16). We previously showed that the intermediate filament protein K16 is up-regulated in aged human skin. Here, we report that there is an obvious imbalance of K16 to K6 mRNA in in vivo and in vitro aging, which possibly leads to cellular effects. To unveil a possible biological function of K16 overexpression we investigated the migration potential of keratinocytes having up-regulated K16 expression in vitro. Two cell lines were established by transfection of human keratinocytes (HaCaT cells) with K16 or control vectors and subsequent fluorescence-activated cell sorting. By performing migration assays we were able to show a 90% reduction in the migration ability of the K16-overexpressing keratinocytes. In addition, a delay in wound closure associated with K16-overexpressing cells was shown by scratch assays. Transient overexpression of K6A in K16-overexpressing keratinocytes partially corrected the cell-migration defect. By real-time PCR we excluded co-regulation of the annotated interaction partner, K6, in the K16 cell line. Finally, we observed a decreased level of tyrosine phosphorylation in K16-overexpressing cells. Taken together, these data highlight the possibility of a physiological role for K6/K16 heterodimers in keratinocyte cell migration, in addition to the heterodimer's known functions in cell differentiation and mechanical resilience.

Immunofluorescence mapping is based on the detection of structural proteins of keratinocytes or of the dermo-epidermal junction using specific poly- or monoclonal antibodies. Through this method, the level of split formation can be determined by investigating the location of a given antigen in a natural or induced blister. This method also allows testing for the normal expression, reduction or absence of various structural proteins depending on the antibodies used. It has widely replaced transmission electron microscopy and is used as the initial laboratory test to prove the clinical diagnosis of epidermolysis bullosa.

Please cite this paper as: K16 is a further new candidate for homotypic intermediate filament protein interactions. Experimental Dermatology 2010; 19: e241–e250. Abstract: Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments (IF). These filaments mediate resilience and mechanical strength to epithelial cells and maintain tissue integrity. Specific type I/type II pairs are co-expressed in vivo and serve as markers for distinct tissue layers and cell differentiation states. Heterodimerization has been regarded the undisrupted hallmark of IF. We show now that recombinantly expressed cytokeratin 16 (K16) interacts with itself and forms homodimers even in denaturating SDS–PAGE analysis. Detailed FRET experiments in HaCaT keratinocytes were in accordance with our in vitro observations and showed clearly that K16 is able to form strong homodimers. Homotypic keratin interactions has been previously shown for keratin 17 (K17) and keratin 18 (K18) by Schnabel et al. (Biochim Biophys Acta, 1998: 1403: 158), and we now proved K16 to be the third type I keratin that is able to form homodimers.

Chronic involvement of orogenital and conjunctival mucosa in the course of either genetically based (epidermolysis bullosa hereditaria) or auto-immunologically mediated (as for example pemphigus vulgaris, mucous membrane pemphigoid or epidermolysis bullosa acquisita) blistering diseases can cause significant morbidity. To provide accurate care, recognition of clinical, pathogenic and diagnostic features as well as awareness of recent advances in the development of new therapeutic modalities are mandatory and thus will be discussed in this review.

Scleromyxoedema is a rare disease of unknown aetiology that is characterized by progressive cutaneous mucinosis and paraproteinaemia. A variety of systemic (e.g. gastro intestinal, neurological, pulmonary, cardiac and renal) complications may lead to significant morbidity and mortality necessitating therapeutic intervention. The latter remains challenging. Numerous treatment modalities have been reported in the literature, often, however, with inconsistent responses, frequent relapses and potentially serious side-effects. Moreover, the rarity of scleromyxoedema has prevented the execution of controlled therapeutic trials. This paper discusses current proposed therapeutic strategies and reports the case of a 64-year-old male patient with progressive scleromyxoedema associated with IgG-lambda paraproteinaemia in whom monthly administrations of vincristine, idarubicin and dexamethasone in addition to daily oral thalidomide led to clinical and laboratory remission within 12 weeks.

A 74-year-old woman presented with a four-week history of locally expanding, highly pruritic urticarial plaques, bullae and erosions on her left breast. She had undergone surgery for an infiltrating ductal carcinoma of the same breast six months before and received intra- and postoperative radiotherapy (RT) followed by adjuvant hormonal anticancer treatment. Histopathological, immunological and serological data confirmed the diagnosis of localized bullous pemphigoid (BP) and treatment with systemic and local corticosteroids led to a sustained remission. After excluding other factors, we concluded that RT was the most likely trigger of her localized BP.

Mutations in the gene coding for the transmembrane receptor protein Patched (PTCH) are implicated in the autosomal dominant disorder Gorlin syndrome (also known as naevoid basal cell carcinoma syndrome), characterized by congenital abnormalities and cancer predisposition. Tumour promotion is thought to be associated with aberrant function of PTCH, leading to misregulation of the hedgehog signalling network. However, the transcriptional events that underlie the reduced tumour suppression effects of PTCH have not been studied in detail. We describe a patient with Gorlin syndrome who had three molecular aberrations resulting in biallelic disruption of the PTCH gene, leading to abnormal protein expression and development of basal cell carcinoma. Remarkably, within tumour cells, the somatic nonsense mutation G1019X was associated with activation of a cryptic splice donor site, in which an in-frame deletion of the exon sequence containing the nonsense mutation occurred. However, the function of the resulting PTCH protein variant was still compromised. The pathogenetic alterations described give insights into the sequence of events leading to cellular transformation and underscore the importance of the PTCH protein in skin homeostasis.

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5' pre-trans-splicing molecule encoding wild-type exons 2-9 led to specific replacement of the mutated 5' portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.

Systemic sclerosis is a generalized autoimmune connective tissue disease of unknown aetiology. Profound vascular and immunological dysregulations result in tissue fibrosis affecting the skin and internal organs. Currently, two main clinical subtypes are distinguished, i.e. limited and diffuse cutaneous systemic sclerosis, which differ significantly in the clinical course and prognosis. Autoantibodies against topoisomerase (Scl-70), centromere-associated proteins, and nucleolar antigens are important for the diagnosis of the disease and give clues for its clinical manifestations and prognosis (prognostic autoantibodies). For most of these antibodies, however, the role in pathogenesis is not established. Anti-centromere antibodies are associated with limited cutaneous involvement and risk for pulmonary hypertension, whereas anti-topoisomerase I is associated with diffuse progressive disease and severe interstitial lung disease. Anti-Th/To positivity is associated with limited skin involvement but a high risk for severe internal organ involvement (Kidneys, PAH, Lung fibrosis). Anti-RNA polymerase I/III antibodies are associated with a high risk for renal involvement. Autoantibodies against the PDGF receptor and fibrillin-1 seem to play important roles in the pathogenetic process of systemic sclerosis.

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.

The generalized severe subtype of junctional EB (formerly junctional EB Herlitz type) is the usually most severe lethal variant of the EB group of mechanobullous genodermatoses. Extreme blistering of the skin and mucous membranes and impaired wound healing lead to chronic erosions and formation of exuberant granulation tissue which, in addition to pigmentary changes, nail anomalies, and dental hypoplasia, are clinical hallmarks of the disease. Complications arising from numerous extracutaneous manifestations involving, for instance, the eye and gastrointestinal, laryngeal, and genitourinary tract also account for the devastatingly high morbidity and mortality in early childhood.

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.

An important characteristic of epidermolysis bullosa simplex Dowling-Meara (EBS-DM) keratinocytes is the increased level of Jun N-terminal kinase (JNK) stress signalling, which is thought to contribute to the disease phenotype. In this work, we report on the dramatic up-regulation of cytokeratin 14 (K14) in the EBS-DM model cell line KEB7 at both the transcriptional and translational levels, which is noteworthy because KEB7 patient cells are heterozygous for a missense mutation (R125P) in K14. By performing functional assays, we show a direct link between overexpressed wild-type K14 and increased JNK signalling in healthy, immortalized keratinocytes. This observation led us to hypothesize a positive feedback model in which mutant (R125P) K14 triggers JNK signalling, leading to increased AP1-dependent expression of K14, which in turn amplifies JNK signalling further. We therefore suggest that an imbalance of cytoplasmic K14 monomers and K14 incorporated into the intermediate filament (IF) network leads to elevated stress signalling, potentially altering IF dynamics by phosphorylation, which as a side effect, weakens EBS-DM keratinocytes.

1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) imposes cell cycle block in late G1 phase in cultured human keratinocytes. We wanted to identify early vitamin D target genes using a subtractive screening approach. Human foreskin keratinocytes were grown to about 70% confluence, treated with 2×10 −7 M 1α,25(OH) 2 D 3 or left untreated and RNA from both populations were isolated after 22 h of incubation. cDNA was synthesised and cloned into plasmid vectors. For screening of the libraries, cDNA was amplified in vitro using T7 RNA polymerase and then the amplified RNA (driver, control population) and single stranded cDNA (tester) were used for subtractive hybridisation. Heterohybrids were then separated from single stranded nucleotides using a hydroxyapatite column. The radiolabeled single stranded cDNA was used for screening a colony blot. Positive clones were rescreened, plasmid DNA was isolated and used for verifying the results by Northern blot analysis, using RNA isolated from untreated keratinocytes, as well as RNA isolated after 6 h, 12 h and 24 h of vitamin D treatment.

Seasonal variations can be found in almost any parameter of an organism's biochemistry, physiology, endocrinology, and behaviour. This phenomenon, generally called photoperiodism, results from one of the major functions of the circadian system, i.e. the translation of environmental information into rhythmic intraorganismic signals, which then regulate or influence physiology and pathology. We induced melanoma in three groups of syngeneic C57BL/6 mice synchronised to different photoperiods (8, 12, or 18 h of light within 24-h days) by subcutaneous injections of HFH18 melanoma cell suspensions. All animals from all three photoperiodic groups developed exponentially growing tumors. The average tumor volume on day 31 post injection was significantly smaller in animals exposed to light/dark conditions (LD) 8 : 16 h as compared with animals held in LD 18 : 6 h and intermediate in animals from the equinox group. These results indicate that C57BL/6 mice react to photoperiod, which can exert a significant effect on tumor growth.