The practical value of the detection of clonality within the T-cell receptor gamma locus by polymerase chain reaction for the diagnosis of cutaneous T-cell lymphomas is well known. However, studies dealing with this subject so far, with special emphasis on the sensitivity of the technique in comparison to, for example, Southern blotting have used mixtures of DNA in various concentrations instead of using mixtures of the cells involved, which would reflect the in vivo situation in a more realistic scope. The purpose of this study was therefore to determine the sensitivity and the limitations of the PCR assay by dilution experiments, using mixtures of cells. Furthermore we studied its applicability to cutaneous T-cell proliferative disorders. Two clonal T-cell lines (MyLa and Jurkat) served as positive control. Dilutions of MyLa cells, cultured normal human keratinocytes and peripheral blood mononuclear cells from lymphoma negative volunteers were used to assess the sensitivity of the PCR-DGGE assay. Skin samples from 4 patients with cutaneous T-cell lymphoma, 1 lesional lymph node, 2 blood samples from a patient with Sézary syndrome and 4 lymphoma-negative tissue samples were analysed. Two samples were uncertain for diagnosis of lymphoma. The PCR-DGGE assay consisted of a 2-round nested PCR with consensus primers within the TCR-gamma locus followed by electrophoretic separation of the product along a denaturing urea/formamide gradient gel. PCR-DGGE sensitivity was, to our knowledge, for the first time investigated for mixtures of lymphocytes (clonal and polyclonal) and keratinocytes. Clonal T-cells were detected in a concentration between 1-0.1% in keratinocytes, whereas the sensitivity was generally lower upon dilution in peripheral blood mononuclear cells or in a mixture of keratinocytes and peripheral blood mononuclear cells. Nevertheless, T-cell clonality was detected in 2 blood samples of a patient with Sézary syndrome, which were negative by Southern blot analysis. The crucial point of this work was the new approach to establish the sensitivity of the PCR-DGGE, in a way which more closely mimics the condition of clinical specimens. Instead of mixing and amplifying DNA extracted from clonal T-cell lines and polyclonal bone marrow cells, we amplified DNA from clonal and polyclonal cells which had been mixed in various ratios before DNA extraction. Polymerase chain reaction in conjunction with denaturing gradient gel electrophoresis is a sensitive and versatile molecular tool for the assessment of clonality of suspect cutaneous lesions. The determination of sensitivity using DNA extracted from premixed cells more closely corresponds to the actual test situation when testing skin samples.

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This report defines the influence of field soft X-ray irradiation on the integrity of epidermal Langerhans cells (LC) in the guinea pig and mouse systems. Male albino (Rockefeller strain) and piebald F1 (2 ×13 strain) guinea pigs as well as C3H mice (H-2K^k) were exposed to one single shot of different dosages of soft X-rays [80,1,200,1,600, and 3,200 R; 30 kV, 0.5 mm aluminium filter, FSD (focus-skin distance) 12 cm]. 1 and 4 weeks after exposure, skin specimens were taken from the irradiated skin. The demonstration and evaluation of LC was performed basing on their expression of specific histochemical (ATPase) and functional immunologic markers (la antigens). Soft X-irradiation had pronounced effects on number and structure of ATPase- and Ia-antigen-positive cells. In the mouse system, 1 week after exposure to 800 and 1,200 R ATPase-positive cells and, in a more or less parellel manner, Ia-antigen-positive cells () were reduced to 74% (74%) and 70% (68%), respectively, and 4 weeks after exposure to 49% (47%) and 43% (40%), respectively. In the guinea pig system one single shot of 1,600 or 3,200 R produced, respectively, a 35% or a 38% reduction of ATPase-positive cells. Prolonged survival did not result in a further depletion of ATPase-positive cells.

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Nails from four patients, infected with dermatophytes, were investigated with the scanning electron microscope (SEM) to gain insight into the spatial arrangements of the dermatophytes within the nail. Fungal hyphae could be detected in nails of all four patients. A toenail from one patient infected with Trichophyton mentagrophytes was more extensively studied and the results are presented in this paper. Light microscopic observations with bright field illumination and Nomarski interference contrast confirmed the dermatophytic infection. Fungal hyphae found with the SEM distally on the ventral part of the toenail showed typical T. mentagrophytes structures and the comparison with cultured material clearly demonstrated the correspondence in morphology and size. Besides the fine structural morphology of the invasion of fungal hyphae into the nail plate between the horny cells and/or directly into corneocytes, "tunnel"-like holes were observed in paraffin embedded sections after removal of the paraffin. Scanning electron microscopy, with its enormous focal depth, yielded far more information than light microscopic techniques about the three dimensional behavior of dermatophytes in the nail.