Helmut Näher

Polymerase chain reaction-based detection of dermatophyte DNA with a fungus-specific primer system

Summary. There is significant clinical interest in primers which are specific for fungi and do not hybridize to DNA of other eukaryotes or prokaryotes. Such primers would allow specific amplification of fungal DNA from human tissue samples containing fungi. Fungal identification to the species level could follow by direct sequencing or restriction analysis. Several previously described primer systems cross‐react with DNA of plants and animals.

S-100β-Protein im Serum als Tumormarker beim malignen Melanom Aktueller Kenntnisstand und klinische Erfahrungen

ZusammenfassungS-100 ist ein saures, kalziumbindendes Protein, das als Heterodimer aus 2 isomeren Untereinheiten α und β besteht und erstmalig in Zellen neuroendokrinen Ursprungs beschrieben wurde. Es spielt eine Rolle bei verschiedenen zellulären Prozessen, wie z.B. der Zelldifferenzierung und der Proliferation und interagiert mit dem Tumorsuppressorprotein p53. S-100 ist ebenfalls in Melanomzellen vorhanden, und sein immunhistochemischer Nachweis ist bei der histopathologischen Diagnostik des malignen Melanoms weit verbreitet. Nachdem S-100β im Serum von Patienten mit malignem Melanom nachgewiesen wurde, folgten zahlreiche klinische Studien zur Etablierung dieses Proteins als Tumormarker in verschiedenen Stadien der Erkrankung. Die Resultate zeigen, daß S-100β-Protein im Serum von Patienten mit malignem Melanom ein unabhängiger prognostischer Marker und ein ergänzender klinischer Parameter für die Progression der metastasierten Erkrankung sowie für das Monitoring der Patienten während einer systemischen Therapie sein kann. Bei Lymphknoten- oder Fernmetastasierung gibt es jedoch auch Patienten mit negativen S-100-β-Werten, bei denen eine Korrelation mit dem Krankheitsverlauf nicht hergestellt werden kann. Eigene Ergebnisse bestätigen diese Grundaussage für Patienten im Stadium III/IV. Werden aber wiederholt positive S-100β-Serumwerte bei der statistischen Auswertung nur einmalig berücksichtigt, zeigt sich ein deutlich geringerer Anteil von Patienten im Stadium III/IV mit positiven S-100-Werten, als in der Literatur angegeben. Für das Monitoring im Stadium I und II scheint S-100β nicht geeignet.SummaryS100 is an acidic-calcium-binding protein, composed as a heterodimer of two isomeric subunits α and β and was first described in cells of neuroendocrine origin. It plays an important role in various cellular processes such as cell differentiation and proliferation and interacts with the tumour suppressor gene p53. S100 is also present in melanoma cells and its immunhistochemical detection is widely used in the histopathological diagnosis of malignant melanoma. S100 has been detected in the serum of patients with malignant melanoma and many clinical studies have been performed to establish this protein as a tumor marker in different stages of the disease. The data suggest that S-100β-protein in serum of patients with malignant melanoma could be an independent prognostic marker and an additional clinical parameter for progression of metastatic disease and serological monitoring during systemic therapy. However there are patients in stage of lymph node- or systemic metastasis with negative S-100β-serum levels and no correlation to the course of disease. Our results confirm the findings for patients in stage III/IV. However, the percentage of S-100β-positive patients in stage III/IV is lower than reported in the literature, if repeatedly positive samples are excluded from statistical analysis. For monitoring in stage I and II it seems to be not helpful.

Extracorporeal photophoresis augments function of CD4+CD25+FoxP3+ regulatory T cells by triggering adenosine production.

Background. During extracorporeal photophoresis (ECP), peripheral blood mononuclear cells are treated with DNA-intercalating agents and irradiated with ultraviolet light. This procedure exerts immunosuppressive effects, most likely mediated by regulatory T cells (Treg). However, the underlying mechanisms are not clear yet. In our study, we investigated the effect of ECP on frequency and function of Treg in the peripheral blood of patients suffering from graft-versus-host disease. Methods. Whole blood samples from graft-versus-host disease patients were taken before and after the ECP treatment on 2 consecutive days. Phenotypical analysis of changes in distinct leukocyte subsets within the peripheral blood of patients and healthy controls was performed by means of flow cytometry. Functional analysis of the Treg population after magneto bead isolation was performed using conventional suppression assays, and adenosine was detected by means of high pressure liquid chromatography and Lanzetta assays. Results. We show that the frequency of CD4+/CD25+/FoxP3+ Treg in the peripheral blood increases after each cycle of ECP and also in the course of treatment. The suppressive capacity of Treg after ECP was increased compared with that of Treg before ECP, although not reaching the suppression levels obtained with Treg from healthy controls. Furthermore, we show that ECP stimulates the CD39-mediated production of adenosine by Treg, which substantially reduces the T-cell proliferation in in vitro suppression assays. Conclusion. Our data indicate that ECP stimulates the conversion of ATP to adenosine by the ectonucleotiodase CD39, which acts as a novel soluble immunosuppressive reagent mediating immunosuppression of Treg.

Detection of C trachomatis in urogenital specimens by polymerase chain reaction.

OBJECTIVE--To establish a polymerase chain reaction (PCR) protocol for the detection of urogenital C trachomatis infection and to compare it with the detection in cell culture. SPECIMENS--Urethral specimens were collected from 62 male patients and cervical specimens from 106 female patients. SETTING--Department of Dermatology and Venereology, Ruprecht-Karls-Universität, Heidelberg. METHODS--Urogenital specimens were simply boiled for 15 minutes and subsequently subjected to amplification without prior extraction of nucleic acid. The DNA sequence selected for amplification is located in the third open reading frame of the ubiquitous C trachomatis plasmid pCTT1. The amplified products were demonstrated by agarose gel electrophoresis followed by Southern blot hybridization. In addition, specimens were investigated with cell culture. MAIN OUTCOME MEASURES--Results of PCR and cell culture. RESULTS--PCR detected all C trachomatis serovars relevant for urogenital infections (D-L2). Serial dilution experiments revealed that the PCR procedure was 100 fold more sensitive than cell culture. The investigation of 168 urogenital specimens showed that the PCR confirmed all 30 cell culture positive results, however, out of the 138 cell culture negative specimens 16 were positive using the PCR. CONCLUSIONS--A substantial number of urogenital C trachomatis infections detectable by PCR may be missed by the cell culture technique.

Therapie des metastasierten malignen Uveamelanoms

ZusammenfassungDie Uvea ist nach der Haut der zweithäufigste Entstehungsort maligner Melanome, und das Uveamelanom der häufigste primäre intraokuläre Tumor des Erwachsenenalters. Aufgrund seiner Lokalisation, Biologie, Histologie, Genetik und seiner von kutanen Melanomen differierenden Prognose wird diese maligne Neoplasie zunehmend als eigenständige Entität in der Gruppe der malignen Melanome angesehen. Während Primärtumoren von Ophthalmoonkologen therapiert werden, findet die Behandlung von Patienten im Stadium der Metastasierung häufig in dermatoonkologischen Zentren statt. Die hämatogene Tumoraussaat betrifft häufig primär und überwiegend die Leber und bleibt oftmals über einen lüngeren Zeitraum auf dieses Organ beschränkt. Fernmetastasierte Uveamelanome zeigen eine weitgehende Resistenz gegenüber Zytostatika, die für das kutane maligne Melanom etabliert sind. Neue Therapieansätze mit einer lokalen intraarteriellen Chemotherapie über die A. hepatica, ggf. in Kombination mit einer Embolisation der die Lebermetastasen versorgenden Gefäße, konnten zu einer Prognoseverbesserung beitragen. Bei der lokal-hepatischen und systemischen Chemotherapie scheint das Nitrosoureaderivat Fotemustin anderen Zytostatika überlegen und ist als Therapeutikum der 1. Wahl anzusehen. In dieser Arbeit werden nach einer Charakterisierung des okulären Tumors Uveamelanom die verfügbaren Daten über bisherige Behandlungsergebnisse im Stadium der Metastasierung zusammengefasst und ein Überblick über neue und zukünftige Behandlungsstrategien gegeben.AbstractThe uvea is the most common site for extracutaneous melanoma and uveal melanoma is the most frequent primary intraocular tumour in adults. Because its different location, biology, histology, genetic features and prognosis in comparision to cutaneous melanoma, this tumour is considered as a distinct entity in the group of malignant melanoma. While primary uveal melanoma is usually treated by ophthalmologic oncologists, metastatic diseases is often managed by dermatologic oncologists. Hematogenous spread predominantly involves the liver and is often restricted to this organ for a long period. Metastatic uveal melanoma is usually resistant to chemotherapeutic regimens established for the therapy of cutaneous melanoma. Newer therapeutic modalities, such as local intra-arterial chemotherapy into the hepatic artery, perhaps combined with embolisation of feeder blood vessels of liver metastases, improves the prognosis of metastatic uveal melanoma. Currently the nitrosourea derivate fotemustine is the drug of choice in the local hepatic and systemic treatment and seems to be superior to other chemotherapeutic agents. Following the characterisation of primary uveal melanoma, we summarize the results of different treatment protocols for metastatic disease and give an overview of new strategies.

B-raf Exon 15 Mutations Are Common in Primary Melanoma Resection Specimens but Not Associated with Clinical Outcome

Downstream of Ras, the serine/threonine kinase B-raf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations. With respect to the B-raf protein sequence, the V599E mutation was predicted in 63% of these positive melanomas, followed in frequency by the V599K transition (31%). Detection of B-raf exon 15 mutations or prediction of the activating mutation V599E were not statistically associated with the risk for subsequent metastasis in the follow-up of patients. Altogether, the B-raf oncogene is affected in a substantial subset of melanoma resection specimens. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, activation of this oncogene may contribute to the development of melanoma.

Analysis of Losses of Heterozygosity of the Candidate Tumour Suppressor Gene DMBT1 in Melanoma Resection Specimens

Deleted in malignant brain tumours 1 (DMBT1), a candidate tumour suppressor gene located on chromosome 10q25.3-q26.1, has recently been identified and found to be deleted in several different types of human tumours. In melanomas, the chromosomal region 10q22-qter is commonly affected by losses, hence we screened primary melanoma samples for losses of heterozygosity (LOH), and acquired melanocytic naevi and melanomas for transcription of DMBT1 and protein expression. Of 38 informative melanomas, 1 nodular melanoma and 2 subcutaneous metastases showed LOH of both microsatellites flanking the gene, suggesting loss of 1 DMBT1 allele. Three further melanomas showed LOH at 1 informative locus but were heterozygous for the second marker. Applying reverse-transcription polymerase chain reaction (RT-PCR), DMBT1 transcription was not found in melanomas. However, DMBT1 transcription was also absent from the majority of naevi from which melanomas frequently arise, making down-regulation of gene transcription during transformation from naevus to melanoma unlikely. Immunohistochemistry showed nerves, sweat glands and the stratum spinosum of the epidermis to be DMBT1 protein positive, whereas the naevi and melanoma cells themselves were negative. All considered, the candidate tumour suppressor gene DMBT1 does not appear to be a major inactivation target in the development of melanomas.

Detection of Epstein-Barr virus DNA in anal scrapings from HIV-positive homosexual men

Epstein-Barr virus (EBV) can infect epithelial cells as well as B lymphocytes. Infection of the male and female genital tracts has recently been demonstrated, and it has been suggested that the virus may be sexually transmissible. In our study we investigated whether EBV can be found in the anal region of sexually active homosexual men. Anal scrapings from HIV-positive homosexual men and a heterosexual control population were investigated using the polymerase chain reaction (PCR) to screen for EBV DNA. EBV DNA was detected in 8 of 27 anal samples (29.6%) from the homosexual men and 3 of 34 samples (8.8%) from the heterosexual men. Our study shows that, like the genital tract, the anal region can habour EBV subclinically. This finding suggests that the anal region may be a reservoir for EBV and that sexual transmission of this virus may be possible.

New mutation in the CYLD gene within a family with Brooke‐Spiegler syndrome

Brooke‐Spiegler syndrome is a rare, autosomal dominant disease characterized by multiple skin appendage tumors caused by various mutations in the CYLD gene on chromosome 16q12‐q13. We describe a family, in which we performed a molecular‐genetic examination and found a new mutation in exon 19 in the CYLD gene leading to a frameshift. It is important to be aware of this syndrome and its pathogenesis as its phenotypic features can vary so that apparently different diseases are caused by the same genetic defect. In addition, there may be malignant transformation of the generally benign tumors, so that a timely diagnosis is essential for appropriate monitoring and therapy.

Diagnostik von Dermatomykosen mit der Polymerase-Kettenreaktion

ZusammenfassungEin Nachweissystem für Dermatophyten mittels Polymerase-Kettenreaktion (PCR) wurde entwickelt. Die Primer „TR1” und „TR2” flankieren eine 581 bp lange Sequenz innerhalb des Gens, das für die kleine ribosomale Untereinheit (18S rRNA) von Pilzen kodiert. Mittels PCR ließ sich die DNA von Isolaten der 7 häufigsten Dermatophyten amplifizieren, daneben die von einigen Hefe- und Schimmelpilzen. Durch Hybridisierung mit spezifischen Detektionsoligonukleotiden gelang die Identifizierung von Dermatophyten und Candida albicans. Die Restriktionsfragmentanalyse erlaubte die Abgrenzung der Dermatophyten gegenüber den Hefe- und Schimmelpilzen. Die Spezifität der PCR für Pilze wurde an menschlicher DNA aus 42 Dermis- und Epidermisproben sowie an ausgesuchter tierischer und pflanzlicher DNA überprüft. Um die klinische Relevanz des Verfahrens zu evaluieren, wurden 69 routinemäßig gewonnene Haut- und Nagelproben mit der PCR und der Kultur untersucht. Mit der PCR wurden in 35 und mit der Kultur in 28 der insgesamt 38 positiv bewerteten Patientenproben Dermatophyten nachgewiesen. Die Sensitivität der PCR (92%) war höher als die der Kultur (73%). Das Ergebnis dieser Untersuchung zeigt, daß die PCR Vorteile gegenüber der Kultur beim Nachweis von Dermatophyten hat.SummaryA polymerase chain reaction (PCR) assay for the detection of dermatophytes was established. The primers ”TR1” and ”TR2” amplify a 581 bp fragment within the gene coding for the small ribosomal subunit (18S rRNA) of fungi. PCR allowed the detection of isolates of 7 common dermatophytes and in addition several yeasts and moulds. Hybridisation with specific oligonucleotides results in the identification of dermatophytes and Candida albicans. Restriction analysis of the PCR product allowed us to distinguish between dermatophytes and yeasts or moulds. The specifity of the PCR with respect to fungi was assessed by testing human DNA collected from 42 dermis and epidermis specimens as well as DNA from selected plants and animals. To evaluate the clinical relevance of the PCR assay, 69 routinely collected skin and nail specimens were examined by PCR and culture. PCR detected dermatophytes in 35 and culture in 28 of 38 specimens that were classified as positive. Sensitivity of PCR (92%) was higher than that of culture (73%). These results show that PCR has advantages over culture for the detection of dermatophytes.