A. Rübben

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

Allelic loss underlies type 2 segmental Hailey-Hailey disease, providing molecular confirmation of a novel genetic concept

Hailey-Hailey disease (HHD) is an autosomal dominant trait characterized by erythematous and oozing skin lesions preponderantly involving the body folds. In the present unusual case, however, unilateral segmental areas along the lines of Blaschko showing a rather severe involvement were superimposed on the ordinary symmetrical phenotype. Based on this observation and similar forms of mosaicism as reported in other autosomal dominant skin disorders, we postulated that in such cases, 2 different types of segmental involvement can be distinguished. Accordingly, the linear lesions as noted in the present case would exemplify type 2 segmental HHD. In the heterozygous embryo, loss of heterozygosity occurring at an early developmental stage would have given rise to pronounced linear lesions reflecting homozygosity or hemizygosity for the mutation. By analyzing DNA and RNA derived from blood and skin samples as well as keratinocytes of the index patient with various molecular techniques including RT-PCR, real-time PCR, and microsatellite analysis, we found a consistent loss of the paternal wild-type allele in more severely affected segmental skin regions, confirming this hypothesis for the first time, to our knowledge, at the molecular and cellular level.

Routine detection of herpes simplex virus and varicella zoster virus by polymerase chain reaction reveals that initial herpes zoster is frequently misdiagnosed as herpes simplex

The differential diagnosis of herpes simplex and zoster may require virological confirmation, yet virus typing is not regarded as necessary in routine dermatological assessment. In an attempt to evaluate the clinical benefits of the routine detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), we analysed skin swabs from 110 patients who were diagnosed at the first clinical visit as having herpes simplex (n = 45) or zoster (n = 65). Viruses were typed using the polymerase chain reaction (PCR) with the general primer pair GPHV‐RU. PCR analysis showed that at the initial clinical presentation, herpes simplex in these patients was not mistaken for zoster but that zoster was incorrectly diagnosed as herpes simplex in nine cases. Thus these results suggest that initial zoster often mimics herpes simplex, hence routine PCR diagnosis of HSV and VZV or alternative rapid diagnostic approaches may be beneficial in these cases.

Clinical features and age distribution of patients with HPV 2/27/57-induced common warts.

Abstract The morphology of common warts depends on the inducing human papillomavirus (HPV) type. In order to assess the impact of the virus type on wart epidemiology we determined the virus type by PCR and recorded anamnestic data of 238 patients with common warts. Warts induced by the related HPV types 2, 27 and 57 predominated in the study population ( n = 202). These warts mostly occurred as multiple verrucae vulgares, mosaic warts or endophytic warts. Patients aged between 10 and 30 years were most affected and they typically displayed a long disease history (mean duration of warts at the time of first clinical examination, 22 months). A different age distribution was observed in HPV 1-induced warts, most of which occurred in children 6–10 years of age. HPV 2-related warts responded only modestly to treatment, as they persisted in approximately 50% of all patients for more than 6 additional months. No sex preference was detected, but an association with atopic diseases was noted as 39.8% of patients with warts containing HPV 2-related viruses showed a history of atopic eczema, pollinosis or asthma as compared with 20.6% of the control population without a history of warts or with short-duration wart disease. Thus, our results indicate that the epidemiology, as well as morphology, of common warts is closely linked to the virus type.

Multilineage progression of genetically unstable tumor subclones in cutaneous T‐cell lymphoma

Abstract:  Molecular analysis of solid malignant tumors has suggested multilineage progression of genetically unstable subclones during early stages of tumorigenesis as a common mechanism of tumor cell evolution. We have investigated whether multilineage progression is a feature of cutaneous T‐cell lymphoma (CTCL). To identify individual tumor cell subclones, we determined the pattern of mutations within microsatellite DNA obtained from multiple histomorphologically confined tumor cell nests of mycosis fungoides (MF) and lymphomatoid papulosis (LyP) lesions. Tumor cells were isolated by laser microdissection, and allelotypes were determined at microsatellite markers D6S260, D9S162, D9S171, D10S215, TP53.PCR15, and D18S65. Nine cases of MF and one patient with anaplastic large cell lymphoma (ALCL) originating from LyP were analyzed at 277 different microdissected areas obtained from 31 individual lesions. Three specimens of cutaneous lichen planus microdissected at 26 areas served as the control tissue. Microsatellite instability in microdissected tissue [MSI(md‐tissue)] was detected in tumor tissues of all CTCL patients. One hundred and fifty‐seven of 469 analyzed polymerase chain reaction (PCR) amplifications contained mutated microsatellite alleles (34%). In lichen planus, MSI(md‐tissue) was seen in only four of 76 PCR products (5%) (P < 0.0001). The distribution of allelotypes in tumor cells from different disease stages was consistent with multilineage progression in five MF cases, as well as in the LyP/ALCL patient. Our results suggest that CTCL may evolve by multilineage progression and that tumor subclones in MF can be detected in early disease stages by mutation analysis of microsatellite DNA obtained from multiple microdissected areas.

Topical 5% Imiquimod Long-Term Treatment of Cutaneous Warts Resistant to Standard Therapy Modalities

Background: Long-lasting cutaneous warts may represent an unbearable stigma to patients and therefore pose a singular challenge for the physician. Generally, these warts are induced by human papillomavirus (HPV) 2, HPV-27 or HPV-57. Objectives: The present study was conducted to evaluate the efficacy and safety of long-term treatment with imiquimod 5% cream applied to long-lasting (mean duration 6.3 years) common warts, which had been resistant to previous therapeutic interventions. Patients and Methods: Imiquimod cream was self-applied by the patients twice daily. Assessment of response and occurrence of side-effects was performed every 4 weeks until clinical cure or up to a maximum of 24 weeks. A total of 37 patients were recruited. Results: 31 out of 37 patients completed the treatment. 10 out of 37 patients experienced a total clearance of their warts (27%). The mean duration to clearance was 19.2 weeks. 18 patients (49%) showed a reduction of more than 50% and 5 patients (14%) a reduction of less than 50%. Conclusions: Our data demonstrate that the long-term topical application of imiquimod 5% cream is an effective treatment for otherwise therapy-resistant cutaneous warts without causing any meaningful side-effects.

Loss of Heterozygosity and Microsatellite Instability in Acquired Melanocytic Nevi: Towards a Molecular Definition of the Dysplastic Nevus

Acquired melanocytic nevi may show signs of histological dysplasia, and epidemiological studies have demonstrated that dysplastic melanocytic nevi (DMN) are associated with an elevated melanoma risk. Nevertheless, the concept of DMN as precursors of melanoma has remained a concept, in view of the difficulty of establishing unambiguous cytological and histological criteria for DMN. Recent molecular data suggest that genetic instability is more frequent in DMN than in benign acquired melanocytic nevi. We have analyzed 54 benign melanocytic nevi and 6 DMN for loss of heterozygosity (LOH) at microsatellite markers D9S171, IFNA, D9S270, D9S265. LOH at one or more loci was detected in 17 out of 54 benign nevi and in 4 out of 6 DMN. LOH was demonstrated at 26 out of 103 amplified and informative microsatellites in benign nevi and at 6 out of 11 microsatellites in DMN. In addition, 6 benign nevi and 6 DMN were microdissected in 4-15 regions per lesion and analyzed for LOH and microsatellite instability (MSI) at D9S162 and D14S53. Both LOH and MSI were detected more frequently in dysplastic nevi (LOH frequency 0.61 vs 0.18; MSI frequency 0.27 vs 0.05). These results confirm that genetic instability is more prevalent in DMN than in benign acquired melanocytic nevi. Therefore, DMN might be defined as a monoclonal and genetically unstable, but limited, melanocytic proliferation that distinguishes this entity from the benign nevus and from malignant melanoma.

Lymphocyte activation in allergic reactions elicited by small-molecular-weight compounds.

The involvement of cytochrome-P450-dependent metabolism of small-molecular-weight compounds as a prerequisite for an optimal lymphocyte response to those compounds is discussed on the basis of studies with compound such as p-phenylenediamine, sulfamethoxazole, chloramphenicol and fragrances. Subsequently, data on cytochrome P450 activity and CYP gene expression in monocytes and MAC-6 cells for cytochromes P450 2E1 and 3A4 are presented.

HPV 18-induced pigmented bowenoid papulosis of the neck.

We describe the case of a 53-year-old man in whom pigmented bowenoid papulosis developed on the skin of the neck. By polymerase chain reaction with general primers for genital human papillomaviruses (HPV) and subsequent restriction enzyme cleavage we could demonstrate HPV 18-related DNA in two biopsy specimens of the pigmented papules. To our knowledge, this report represents the first case of HPV 18-induced extragenital bowenoid papulosis of the neck.

Somatic deletion of the NF1 gene in a neurofibromatosis type 1-associated malignant melanoma demonstrated by digital PCR

BackgroundNeurofibromatosis type 1 (NF1) is the most common hereditary neurocutaneous disorder and it is associated with an elevated risk for malignant tumors of tissues derived from neural crest cells. The NF1 gene is considered a tumor suppressor gene and inactivation of both copies can be found in NF1-associated benign and malignant tumors. Melanocytes also derive from neural crest cells but melanoma incidence is not markedly elevated in NF1. In this study we could analyze a typical superficial spreading melanoma of a 15-year-old boy with NF1 for loss of heterozygosity (LOH) within the NF1 gene. Neurofibromatosis in this patient was transmitted by the boys farther who carried the mutation NF1 c. 5546 G/A.ResultsMelanoma cells were isolated from formalin-fixed tissue by liquid coverslip laser microdissection. In order to obtain statistically significant LOH data, digital PCR was performed at the intragenic microsatellite IVS27AC28 with DNA of approx. 3500 melanoma cells. Digital PCR detected 23 paternal alleles and one maternal allele. Statistical analysis by SPRT confirmed significance of the maternal allele loss.ConclusionTo our knowledge, this is the first molecular evidence of inactivation of both copies of the NF1 gene in a typical superficial spreading melanoma of a patient with NF1. The classical double-hit inactivation of the NF1 gene suggests that the NF1 genetic background promoted melanoma genesis in this patient.