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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.

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.

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.

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.

Epidermolytic hyperkeratosis is a dominantly inherited ichthyosis, frequently associated with mutations in keratin 1 or 10 that result in disruption of the keratin filament cytoskeleton leading to keratinocyte fragility. In addition to blistering and a severe disorder of cornification, patients typically display an abnormality in permeability barrier function. The nature and pathogenesis of the barrier abnormality in epidermolytic hyperkeratosis are unknown, however. We assessed here, first, baseline transepidermal water loss and barrier recovery kinetics in patients with epidermolytic hyperkeratosis. Whereas baseline transepidermal water loss rates were elevated by approximately 3-fold, recovery rates were faster in epidermolytic hyperkeratosis than in age-matched controls. Electron microscopy showed no defect in either the cornified envelope or the adjacent cornified-bound lipid envelope, i.e., a corneocyte scaffold abnormality does not explain the barrier abnormality. Using the water-soluble tracer, colloidal lanthanum, there was no evidence of tracer accumulation in corneocytes, despite the fragility of nucleated keratinocytes. Instead, tracer, which was excluded in normal skin, moved through the extracellular stratum corneum domains. Increasing intercellular permeability correlated with decreased quantities and defective organization of extracellular lamellar bilayers. The decreased lamellar material, in turn, could be attributed to incompletely secreted lamellar bodies within granular cells, demonstrable not only by several morphologic findings, but also by decreased delivery of a lamellar body content marker, acid lipase, to the stratum corneum interstices. Yet, after acute barrier disruption a rapid release of preformed lamellar body contents was observed together with increased organelle contents in the extracellular spaces, accounting for the accelerated recovery kinetics in epidermolytic hyperkeratosis. Accelerated recovery, in turn, correlated with a restoration in calcium in outer stratum granulosum cells in epidermolytic hyperkeratosis after barrier disruption. Thus, the baseline permeability barrier abnormality in epidermolytic hyperkeratosis can be attributed to abnormal lamellar body secretion, rather than to corneocyte fragility or an abnormal cornified envelope/cornified-bound lipid envelope scaffold, a defect that can be overcome by external applications of stimuli for barrier repair.

Large melanocytic nevi occurring in areas of former blistering in patients with hereditary epidermolysis bullosa (EB) pose a problem to the clinician with regard to prognosis and therapy because they may show clinical and histopathologic features strikingly resembling malignant melanoma. To investigate clinical and histologic criteria as well as the biologic behavior of these nevi, pigmented lesions of 12 patients (EB simplex, n = 1; junctional EB, n = 7; dystrophic EB, n = 4) of the Austrian EB registry were analyzed. Clinically, the nevi are up to palm sized, are initially very dark, and may exhibit stippled pigmentation and irregular borders that outline areas of former blisters. Over time they usually lose pigment, the surface gets papillomatous, and finally they acquire a shagreen-like appearance. Histopathologically, the nevi frequently exhibit a compound congenital or persisting nevus/pseudomelanoma pattern. Despite this combination of features, no malignant transformation of the nevi has been seen by us even after 20 years of prospective surveillance. Because nevi with these criteria do not fit in any of the known categories, we suggest the term EB nevi.

The diagnostic value of antinuclear antibody (ANA) testing must be defined by its correlation to the clinical situation of each patient. Repeatedly we have been faced by a lack of correlation between clinical features and laboratory results. Three case reports are presented, where either a low antibody titer could not predict the rapidly fatal outcome or high positive titers of ANA were observed in patients with a definite mild disease course. Increasing demands for screening as well as for subtyping ANA have led us to establish different-testing algorithms depending on screening results, suspected autoimmune disease and age. The COBAS CORE HEp2 ANA Enzyme Immuno Assay (CC-ANA-EIA) was compared to Indirect Immunofluorescence (IIF). The automated CC-ANA-EIA test system has the advantage of high throughput, lower costs, independency of a trained observer and of the quality of the IF microscope. The CC-beads are coated with a purified HEp2 nuclear extract in addition to recombinant auto-antigens (SSA/Ro 52 and 60, SSB/La, Jo1, SCL70, CENP-B as well as dsDNA). Having the advantage of a greater sensitivity over other ANA-EIAs, which present only a mixture of recombinant antigens, the CC-ANA-EIA serves as a reliable screening test. Neither the CC-ANA-EIA nor the IIF guarantee a 100% sensitivity in detecting ANA. We present our algorithm which helps us to reduce misleading results as well as reduce unnecessary costs. Both test systems are reliable and comparable for testing patients who have the diagnosis or are suspected to have a systemic autoimmune disease. Nevertheless, it is the nature of these diseases that—as shown in our patients—even objective and repeatable results have to be interpreted by a trained clinician to establish a proper assessment of the distinct clinical course of each patient.

Patients with generalized atrophic benign epidermolysis bullosa, a usually nonlethal form of junctional epidermolysis bullosa, have generalized blistering, nail dystrophy, patchy alopecia, and dental abnormalities. Skin fragility in most cases is due to mutations in the gene encoding type XVII collagen (COL17A1). Recently, we reported five Austrian families with generalized atrophic benign epidermolysis bullosa who share the same COL17A1 mutation. Affected individuals in three families are homozygous for 4003delTC, whereas those in two others are compound heterozygotes. To determine if the occurrence of 4003delTC in these unrelated families signifies propagation of an ancestral allele or a mutational hot spot, haplotypes were determined for polymorphisms both within and flanking COL17A1. Five intragenic polymorphisms were chosen based on their informativeness. One of these, not previously reported, was 2988 A or C that introduces a new restriction site for Eco0109 I. All the 4003delTC alleles showed the same haplotype for these five polymorphic markers. Fourteen microsatellite polymorphisms were selected based on their high heterozygosity and their location within 10q23–q25 near COL17A1. Three families shared microsatellite polymorphisms covering at most 19 cM, whereas the others shared smaller regions consistent with cross-over events during passage of this mutation through several generations. These results indicate that 4003delTC occurs on a single ancestral allele.

Patients with generalized atrophic benign epidermolysis bullosa often show decreased expression of type XVII collagen, a transmembrane hemidesmosomal protein encoded by COL17A1. This report documents a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa, and applies a new methodology to define and characterize the resulting mRNA splice variants. Mutational analysis of COL17A1 identified a maternally inherited G-to-T transversion at the -1 position of exon 32. This acceptor splice-site mutation led to the formation of aberrant transcripts present at extremely low levels. Based on our recent finding that cycloheximide stabilized mutant COL17A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effects of the splice-site mutation on splicing of COL17A1 transcripts were determined using reverse transcriptase polymerase chain reaction of total RNA from keratinocytes incubated for 2.5 h in the presence or absence of 10 microg cycloheximide per ml. Using this approach, an abnormally spliced transcript was identified that contains an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splice variants, including one derived from the skipping of exon 32, were also identified. These results indicate the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice-site mutations, because: (i) aberrant splicing often generates a premature termination codon, (ii) transcripts with premature termination codons can occur at low or undetectable levels due to nonsense-mediated mRNA decay, and (iii) the levels of these transcripts can be increased by cycloheximide.

Patients with generalized atrophic benign epidermolysis bullosa (GABEB), an inherited subepidermaI blistering disease, often have no immunologically detectable bullous pemphigoid antigen 2 (BPAG2) in theft epidermal basement membrane. Recently, we analyzed the BPAG2 gene (GenBank no. M91669) in an Austrian family with GABEB and identified a homozygous deletion mutation, 4003delTC, that results in a downstream premature termination codon (PTC). This mutation has now been identified in additional descendants, suggesting transmission of this mutant allele through at least six generations. Screening of four other Austrian GABEB families revealed that affected members were homozygous for 4003delTC in two cases and heterozygous in two others. In the latter, mutational analysis identified two novel nonsense mutations, Q1403X and G8O3X, that were confirmed by restriction endonuclease digestions. Thus, PTCs on both alleles of BPAG2 are present in all of these GABEB families. Immunopre-cipitation and northern blot studies of cultured keratinocytes from homozygous GABEB patients show that 4003delTC results in undetectable levels of BPAG2 protein and mRNA—flndings consistent with the process of nonsense-mediated nRNA decay. Incubating keratinocytes with cycloheximide increased BPAG2 mRNA to a level detectable by northern analysis. When the latter was used in reverse transcription-PCR studies, the mutation was demonstrated, suggesting that cyclohexhnide may allow mutational analysis in cases where low transcript levels have previously thwarted RT-PCR studies. These findings account for the absence of BPAG2 in GABEB patients and attest to the importance of this protein in adhesion of epidermis to epidermal basement membrane.

The 180-kDa bullous pemphigoid antigen (BPAG2) is a candidate gene/protein for mutations in some forms of junctional epidermolysis bullosa. In this study, we searched for mutations in BPAG2 in a large Austrian pedigree with generalized atrophic benign epidermolysis bullosa, a distinct nonlethal form of junctional epidermolysis bullosa, using polymerase chain reaction amplification of genomic DNA, heteroduplex analysis of the polymerase chain reaction products, and direct nucleotide sequencing. We identified a homozygous 2-bp deletion within the coding region of BPAG2 in the affected individuals. This mutation results in a frame-shift and downstream stop codons on both alleles, predicting an absence of functional protein. These findings illustrate the molecular basis of the skin fragility in this family and attest to the importance of the 180-kDa bullous pemphigoid antigen in the attachment of the epidermis to the underlying dermoepidermal basement membrane.

Sneddon's syndrome is a potentially fatal arterio-occlusive disorder characterized by generalized livedo racemosa and cerebrovascular lesions. Skin biopsies often fail to yield diagnostic arterial lesions. In the present series, affected vessels were found in skin biopsies from 12 of 15 patients with Sneddon's syndrome. Selection of the correct biopsy site (seemingly uninvolved skin at the center of a livedo racemosa area), adequate biopsy size (1 to 2 cm), and serial sections are essential for the detection of relevant vascular pathology. Only small to medium-sized arteries of the dermis-subcutis boundary were found to be involved. Lesions follow a distinct course. The initial stage displays partial detachment of endothelial cells, adhesion of mononuclear cells interspersed with fibrin ("endothelitis"), a marked edema of the surrounding connective tissue with numerous dilated capillaries, and a predominantly lymphohistiocytic infiltrate with polymorphonuclear leukocytes. In the early phase a sponge-like plug is formed from mononuclear cells, fibrin, and red blood cells, leading to partial to complete obstruction. A perivascular inflammatory infiltrate, devoid of polymorphonuclear leukocytes, is located around affected arteries; in the adventitia of the occluded vessel, a very regular corona of dilated capillaries appears that is continuous with more peripheral dilated and branching vessels. Organization of the occluding plug ensues in the intermediate stage by "subendothelial cell proliferation," most likely due to immigrating smooth muscle cells. In the final stage, the occluding artery undergoes fibrosis, shrinkage, and atrophy.

We report the second case of amyloid elastosis. Our patient had an underlying primary systemic amyloidosis with lambda light chain paraproteinemia. Salient clinical features included a sclerodermatous facial appearance, cordlike thickening of superficial blood vessels, neck skin resembling that in pseudoxanthoma elasticum, livedo reticularis-like changes on the trunk, Raynaud's phenomenon, arterial and venous thromboses, and the nephrotic syndrome. Amyloid deposits were present in the dermis, around appendages, in blood vessel walls, and in a striking distribution surrounding individual elastic fibers, that appeared shortened and fragmented. Immunofluorescence, electron microscopic, and immunoultrastructural studies with antibodies to lambda light chain, localized the amyloid deposits to the region of the elastic fiber microfibrils, with which amyloid P component (AP) is invariably associated in normal tissues. Because AP binds amyloid fibrils, codistribution of amyloid deposits and AP in amyloid elastosis strongly supports the theory that elastic fiber-associated AP may act as a nidus for amyloid deposition.

Approximately one third of all mutations occurring in the 10 genes causing epidermolysis bullosa (EB), a heritable blistering skin disease, are premature termination codon (PTC) mutations leading to loss of the respective mRNA presumably via nonsense mediated mRNA decay. These mutations might be sensitive to aminoglycoside read-through of the PTC. We tested this hypothesis in immortalized cells from a patient suffering of junctional EB treated with geneticin. These cells harbour the homozygous 4003delTC mutation in the COL17A1 gene, consequently, leading to a PTC further downstream. RT-PCR of the region carrying the mutation revealed detectable levels of COL17A1 mRNA in the vicinity of the mutated exon 52 in treated cells. Immunofluorescence analysis of cultured cells showed intense staining on the cell surface of treated patients cells using a type XVII collagen specific antibody. Western blot revealed a newly synthesized approx. 70kD protein reacting with this antibody. These results suggest that specific COL17A1 mRNA is transcribed to parts of the extracellular domain of type XVII collagen. Functional analysis will determine if this protein can substitute for the wild type protein.

Three patients from two families with complete hereditary deficiency of the fourth component of complement (C4) and systemic lupus erythematosus are described. The syndrome presented by these patients is characterized by early onset in life; exquisite sensitivity to sunlight and to cold exposure, the latter resulting in Raynaud's phenomenon; and skin lesions involving not only exposed areas of the body but also palms and soles and presenting as butterfly rashes, maculopapular eruptions, and lesions similar to those of chronic discoid lupus erythematosus, with marked scaling, atrophy, and scarring. Lupus erythematosus (LE) cell tests were negative and antinuclear antibody (ANA) titers low or negative. The male patient of our series died at the age of 3½ years from septicemia, whereas the two girls, aged 18 and 11 years, respectively, were alive at the time of writing. The C4-deficient gene is associated with HLA-Aw32, Bw38, and Bf S in one family and with HLA-A30, B18, DR7, and Bf S 1 in the other family; the latter is the second family in which this HLA haplotype has been found to be associated with hereditary C4 deficiency.

Stratum corneum (SC) antibodies are directed against antigens in the SC of the epidermis and are known to occur in all normal human sera. They have been shown by indirect immunofluorescence to be frequently associated with upper cytoplasmic (U-Cyt) antibodies. We have recently identified keratin intermediate filaments (KIF) as antigens for U-Cyt antibodies. In this study we investigated whether KIF also bear antigenic sites for SC antibodies. Normal human sera that contained SC and/or U-Cyt antibodies by indirect immunofluorescence were studied. Using immunoblot techniques 3 selected sera were shown to bind to high-molecular-weight (HMW) KIF proteins which had been extracted from 2 different epidermal cell preparations, that is, human callus or epidermis from which the SC had been removed by tapestripping. The 3 test sera were absorbed on KIF which had been reconstituted in vitro from urea extracts from both epidermal substrates. As shown by indirect immunofluorescence, the SC and U-Cyt antibodies of all 3 sera were absorbed out with KIF from callus and with KIF from epidermis without SC. Immunoblot experiments, which are more sensitive than indirect immunofluorescence, demonstrated the absorption of anti-KIF protein antibodies of the 3 test sera on callus KIF and 2 of the sera on KIF obtained from epidermis without SC. This was shown by the lack of staining of the respective HMW KIF proteins with the postabsorption sera. With the third serum a marked reduction of antibody binding was found after absorption on KIF from epidermis without SC. These data indicate that KIF bear antigenic sites for SC antibodies.

The presence of numerous keratin bodies in the upper dermis is a characteristic finding in skin lesions of patients with various dermatoses such as cutaneous graft-versus-host disease, lichen planus, or chronic discoid lupus erythematosus. These keratin bodies are generated by apoptotic keratinocyte death, consist largely of keratin intermediate filaments (KIF), and are constantly covered with immunoglobulins, mainly IgM. Apoptosis is also thought to occur under physiologic conditions in the skin as it does in other organs, but keratin bodies are not frequently reported as being found in nonlesional skin. In order to assess the frequency of keratin bodies in normal skin, we examined serial sections of 10 normal human skin specimens and 5 dermal sheets prepared from normal human skin for the presence of keratin bodies. They were visualized by direct immunofluorescence using a fluorescein isothiocyanate (FITC) rabbit antihuman IgM conjugate. In addition the KIF origin of keratin bodies was demonstrated by a double-staining immunofluorescence procedure using a FITC-conjugated rabbit antihuman IgM followed by a mouse monoclonal antibody against keratin and a sheep antimouse immunoglobulin conjugated with Texas Red. One specimen was also examined for keratin bodies at the ultrastructural level. In serial sections, all 10 normal human skin specimens had numerous keratin bodies as assessed by visualization of globular IgM deposits. Evaluated on dermal sheets, the number of keratin bodies ranged from 39–262 per mm 2 . Nearly all keratin bodies also stained with the antikeratin antibodies. Ultrastructurally the remarkable number of keratin bodies, which consist of filaments measuring approximately 10 nm in diameter or of more granular material, in normal human skin was confirmed. In order to investigate the capacity of KIF material in keratin bodies to function as autoantigen, we examined the sera of the 10 skin donors and, in addition, of 30 normal healthy individuals and 10 patients with rheumatoid arthritis for the occurrence and specificity of IgM-anti-KIF autoantibodies by an enzyme-linked immunosorbent assay and by immunoblot. IgM-anti-KIF autoantibodies were found in all 50 test sera. In the majority of the sera the specificity of these autoantibodies included the 51 kD and the 58 kD KIF protein, which are constituents of KIF in keratin bodies and basal keratinocytes. Quantitatively, the antibody activity of the IgM-anti-KIF autoantibodies varied from serum to serum, being highest in the sera of patients with rheumatoid arthritis. The observation of a large number of keratin bodies in normal human skin and the presence of respective IgM- anti-KIF autoantibodies in normal human sera suggest that the physiologic apoptotic keratinocyte death may provide a significant source of KIF autoantigens and that the in vivo IgM deposition on keratin bodies may represent a specific immune response. These findings more generally suggest that the ubiquitous physiologic apoptotic cell death may cause the liberation of autoantigenic material normally inaccessible to the immune system by its intracellular location, which is followed by the production of autoantibodies in normal healthy individuals.