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J W Bauer

Publications and source records attributed to J W Bauer.

At least 19 recordsLinked to original sources
PubMed2006

Relative quantitation of protein-protein interaction strength within the yeast two-hybrid system via fluorescence beta-galactosidase activity detection in a high-throughput and low-cost manner.

The yeast two-hybrid (Y2H) method is capable of delivering vast amounts of interacting positive yeast colonies from a single library screen, particularly if a multifunctional protein is used as bait. However, the selection of definitive colonies for further molecular analysis is limited by both technical practicality and high costs. Here we demonstrate a cost-effective and simple method for the rapid selection and ranking of those Y2H-positive interaction clones that are suitable for further analysis. We performed a Y2H screen for the identification of human transforming growth factor beta2- interacting proteins in a human skin keratinocyte library. The identified clones were ranked by the amount of beta-galactosidase enzyme produced, as well as by the interaction strength of the positive colonies. The combination of high-throughput microplate fluorescence readers and specific fluorescence assays can be utilized for relative quantitation of protein-protein interaction strength of Y2H-positive colonies in crude yeast-cell lysates. We demonstrate here that the high sensitivity of the fluorescence approach can bypass cumbersome conventional methods of cell lysis used in beta-galactosidase assays, and still deliver accurate values for analysis of protein interaction data. Finally, we also achieved a better understanding of general aspects of beta-galactosidase measurements in the Y2H system, such as protein normalization, the influence of yeast culture incubation time on optimal beta-galactosidase detection, and the linearity of beta-galactosidase detection in crude cell lysates.

Cost-Benefit Analysis↗
PubMed2006

Introducing a fast and simple PCR-RFLP analysis for the detection of mutant thiopurine S-methyltransferase alleles TPMT*3A and TPMT*3C.

BACKGROUND: Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. METHODS: We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. RESULTS: Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. CONCLUSION: This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Alleles↗
PubMed2005

Epidermolysis bullosa naevi reveal a distinctive dermoscopic pattern.

BACKGROUND: Large, asymmetrical and irregularly pigmented naevi in patients with epidermolysis bullosa (EB) have been reported often to mimic cutaneous melanoma clinically. OBJECTIVES: As the biological course of these peculiar moles is benign, we assessed EB naevi with a dermatoscope to determine whether they could be reliably differentiated from cutaneous melanoma. METHODS: We evaluated digital dermoscopic images of 23 EB naevi from 11 patients with EB and analysed these pigmented lesions according to pattern analysis, ABCD rule of dermoscopy and the seven-point checklist. RESULTS: Melanoma-associated dermoscopic criteria such as multicomponent pattern (20 of 23), atypical pigment network (17 of 23), irregular dots/globules (16 of 23), irregular pigmentation (22 of 23) and an atypical vascular pattern (seven of 23) were frequently seen in EB naevi. In contrast, other criteria frequently associated with melanoma progression, such as irregular streaks, blue-whitish veil, regression structures (blue-whitish areas) or black dots, were rarely seen. Most lesions gave false-positive results when the scores of the dermoscopic diagnostic algorithms were calculated. CONCLUSIONS: Recurring dermoscopic structures in EB naevi reveal a distinctive dermoscopic pattern of this recently defined entity. Although EB naevi represent an exception to dermoscopic diagnostic algorithms, their dermoscopic evaluation most often allows us to estimate their benign nature. Nevertheless, as an unequivocal discrimination from malignant melanoma in vivo is sometimes not possible, regular clinical follow up of EB naevi with histopathological evaluation of highly suspicious lesions is mandatory.

Dermoscopy↗
PubMed2004

Telepathology using immunofluorescence/immunoperoxidase microscopy.

We evaluated low-cost, store-and-forward telepathology interpretation of digital images of skin sections stained immunohistochemically, using immunofluorescence (IF) and immunoperoxidase (IP). The sample comprised 17 patients with skin diseases characterized by cutaneous deposition of immunoglobulins, fibrinogen or complement components. Up to 11 digital IF or IP images (median 3) were transferred via email to centres in Graz, Austria, and Kurume, Japan. Both remote centres had expertise in reading immunohistochemical specimens. Although image files were relatively small (approximately 100 kByte), the IF images were of high quality and they were well suited to static telepathology. There was agreement on the diagnoses made by the local and both remote centres by physicians experienced in IF/IP microscopy in 14 of 17 cases (82%). These results suggest that telepathology evaluation of immunohistochemical specimens may be a useful procedure for the discussion of unusual skin disorders, training purposes and second-opinion consultations on difficult cases from centres of excellence in immunohistochemical diagnosis.

Austria↗
PubMed2003

Characteristic immunohistochemical and ultrastructural findings indicate that Kindler's syndrome is an apoptotic skin disorder.

BACKGROUND: Kindler's syndrome is a rare genodermatosis mainly characterized by the onset of skin blistering in early childhood, web formation of fingers and toes, photosensitivity, and progressive poikiloderma. There is still debate whether this disease represents a distinctive entity in the spectrum of congenital bullous poikilodermas or a variant of dystrophic epidermolysis bullosa. OBJECTIVE: To evaluate the recently proposed and debated characteristic immunohistochemical and ultrastructural features of Kindler's syndrome. PATIENT/METHODS: Immunofluorescence (IF) antigen mapping and transmission electron microscopy (TEM) were performed on a skin specimen from non-sun-exposed inner aspect of the upper arm of a 49-year-old patient with characteristic clinical features of Kindler's syndrome. RESULTS: IF studies revealed focally an extensively broadened, partly reticular staining pattern in the dermoepidermal basement membrane zone (BMZ) with antibodies against laminin-5 and type IV as well as type VII collagen. Anti-alpha6 and beta4 integrin staining revealed small gaps in the linear reactivity in the BMZ. Abundant keratin bodies, as detected by anti-immunoglobulin M (IgM) staining, were focally present in the dermis, indicating prominent epidermal apoptosis. This was verified by a histochemical apoptosis stain [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction]. Transmission electron microscopic examination showed manifold reduplications of the lamina densa (with attached anchoring fibrils) as well as a keratin body surrounded by a fibroblast in the upper dermis. CONCLUSION: We present characteristic immunohistochemical and ultrastructural features of Kindler's syndrome identical to those described by Shimizu et al. and provide evidence that Kindler's syndrome might primarily be an apoptotic disorder of basal keratinocytes.

Apoptosis↗
PubMed2003

Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases.

Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.

Cell Line↗
PubMed2003

Type XVII collagen gene mutations in junctional epidermolysis bullosa and prospects for gene therapy.

Non-Herlitz junctional epidermolysis bullosa (nH-JEB) is caused predominantly by mutations leading to premature stop codons on both alleles of the type XVII collagen gene (COL17A1). The analysis of mutations in this gene has provided a means of correlating genotype with phenotype of nH-JEB patients. The phenotype of nH-JEB is characterized by generalized blistering of skin and mucous membranes with atrophic scarring and nail dystrophy. Atrophic alopecia is a distinct feature of nH-JEB patients, but one that is not associated with the severity of the disease at other sites. Enamel hypoplasia and pitting of the teeth are also characteristic for nH-JEB and can be used to facilitate the correct diagnosis in children with a blistering skin disease. Analysis of the biological consequences of mutations in the COL17A1 gene has shown that most patients lack type XVII collagen mRNA due to nonsense-mediated mRNA decay. Patients with these mutations can therefore be a target for corrective gene therapy using vectors coding for full-length type XVII collagen. Proof of principle for this approach has recently been demonstrated. The analysis of naturally occurring phenomena of gene correction in the COL17A1 gene provides evidence for other mechanisms of gene correction in genetic diseases. For example, exclusion of an exon carrying a mutation can lead to a milder phenotype of nH-JEB than predicted by the original mutation. In addition, we have gained data suggesting that COL17A1 exons harbouring pathogenic mutations can also be repaired by trans-splicing, i.e. aligning corrected RNA sequences to introns in the vicinity of faulty exons in the COL17A1 premtRNA.

Alopecia↗
PubMed2003

A novel homozygous nonsense deletion/insertion mutation in the keratin 14 gene (Y248X; 744delC/insAG) causes recessive epidermolysis bullosa simplex type Köbner.

We report the sixth case of a human keratin 14 'knockout' mutation resulting in recessive epidermolysis bullosa simplex (EBS). A novel, homozygous nonsense mutation resulting from a deletion/insertion mutation (744delC/insAG) leads to a premature termination codon in the KRT14 gene (Y248X). The patient suffers from generalized cutaneous blistering since birth, mild nail dystrophy, involvement of mucous membranes and multiple epidermolysis bullosa naevi. The clinical variability noted in K14-deficient EBS patients suggests phenotypic modulation by additional genetic and/or epigenetic factors.

Child↗
PubMed2002

[Unilateral blepharochalasis with IgA-deposits].

A 36-year-old male patient presented with unilateral periocular skin atrophy. The blepharochalasis developed without any obvious inflammation of the eyelids over the past 10 years. Interestingly, elongated blood vessels and microaneurysmatic vessel changes were found in the tarsal conjunctiva. A punch biopsy revealed a nearly complete loss of elastic fibres in the papillary and superficial reticular dermis. The contralateral side was histopathologically normal. On immunohistology IgA-deposits could be observed especially on perifollicular elastic fibres. Immunoelectronmicroscopy confirmed the diagnosis and suggested fibulin and fibronectin as potential binding sites for the autoantibodies. This further report of elastolysis in association with IgA-autoantibodies defines the autoantibody binding site in more detail and suggests that the immune mechanisms may also play a role in vessel changes of the conjunctiva.

Adult↗
PubMed2002

Photodynamic therapy using topical methyl 5-aminolevulinate compared with cryotherapy for actinic keratosis: A prospective, randomized study.

BACKGROUND: Actinic keratoses (AKs) are the most common premalignant tumors. Without treatment, a significant number of patients with AK will experience squamous cell carcinoma. Photodynamic therapy (PDT) using the new highly selective photosensitizer methyl 5-aminolevulinate is a promising new treatment modality for AK. OBJECTIVE: We investigated the complete response rates, cosmetic outcome, and patient satisfaction after photodynamic therapy (PDT) using methyl 5-aminolevulinate (Metvix) versus cryotherapy in the treatment of AKs. METHODS: Patients were randomized to receive either cryotherapy with liquid nitrogen spray or PDT using methyl 5-aminolevulinate cream 160 mg/g, 3 hours application time, and red light (75 J/cm(2)). RESULTS: Efficacy results from 193 patients with 699 lesions (92% face/scalp and 93% thin/moderately thick) were analyzed. Overall complete response rates after 3 months were 69% for PDT and 75% for cryotherapy. Both treatments gave higher response rates in thin lesions (PDT 75%, cryotherapy 80%). PDT gave better cosmetic results and higher patient satisfaction than cryotherapy. CONCLUSION: PDT using methyl 5-aminolevulinate is an attractive treatment option for patients with AK, with a response rate similar to that of cryotherapy, but with superior cosmetic results and high patient satisfaction.

Administration, Topical↗
PubMed2001

A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency.

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

Base Sequence↗
PubMed2001

A localized variant of paraneoplastic pemphigus: acantholysis associated with malignant melanoma.

We report a 72-year-old male patient with a nodular malignant melanoma that was associated with focal suprabasal acantholysis (FSA). This phenomenon, which is regarded as an incidental finding by dermatopathologists, may be associated with inflammatory and also neoplastic skin diseases. Haematoxylin and eosin stained sections from an erythematous plaque surrounding the patient's tumour showed FSA, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) on normal human skin, monkey oesophagus and rat urinary bladder were negative. On electron microscopy few desmosomes could be detected in the basal cell layer of the acantholytic areas and there was a nearly complete loss of these structures in the spinous cell layer. Only remnants of cytoplasmic plaques and keratin filaments could be observed in those areas. In contrast, adherens junctions appeared to be well preserved. An enzyme-linked immunosorbent assay (ELISA) using recombinant fusion proteins as antigens did not show circulating autoantibodies against desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). In contrast, immunoblotting revealed autoantibodies directed against keratinocyte antigens with a molecular weight of 85 kDa and 250 kDa, the first band corresponding to the molecular weight of comigrating plakoglobin. Immunoprecipitation with patient serum also revealed a 85-kDa band. We conclude that these autoantibodies, probably in conjunction with cofactors produced by the tumour, could play a part in the pathogenesis of this variant of FSA, for which we propose the term 'localized paraneoplastic pemphigus.'

Acantholysis↗
PubMed2001

Large melanocytic nevi in hereditary epidermolysis bullosa.

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.

Adolescent↗
PubMed1999

Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a frame-restoring mutation.

Generalized atrophic benign epidermolysis bullosa is an autosomal recessive subepidermal blistering disease typified by null mutations in COL17A1. In 1 large kindred, affected individuals were homozygous for a 2-bp deletion in COL17A1, 4003delTC, which resulted in a downstream premature termination codon, nonsense-mediated mRNA decay, and abrogation of type XVII collagen synthesis. Interestingly, 1 of these patients, although phenotypically identical to her affected siblings, showed focal expression of type XVII collagen in epidermal basement membrane in a pattern suggestive of revertant mosaicism. When studies of randomly obtained epidermal, oromucosal, and peripheral blood cells failed to identify the genetic basis of this apparent mosaicism, microscopic subpopulations of potentially revertant epidermal cells (i.e., those overlying basement membrane containing type XVII collagen) were selectively isolated using laser capture microdissection. Analysis of DNA and RNA from these cells revealed a second mutation, 4080insGG, on 1 allele of COL17A1. This 2-bp insertion corrected the reading frame just proximal to the premature termination codon, countered nonsense-mediated mRNA decay, and allowed protein production by patient keratinocytes in vivo and in vitro. These studies elucidate the molecular basis of a novel form of revertant mosaicism in humans.

Alleles↗