H. P. Grundmann

Genotypic, phenotypic and functional analysis of CD4+CD7+ and CD4+CD7- T lymphocyte subsets in Sézary syndrome

Abstract  The expansion of CD4 + CD7 – T cells in the peripheral blood of Sézary syndrome (SS) is well known. It remains unclear whether this population contains the dominant T cell clone. Peripheral blood mononuclear cells (PBMC) of five SS patients were sorted by fluorescence-activated cell sorting into CD4 + CD7 – and CD4 + CD7 + populations. These populations were analysed separately for clonality of the T cell receptor γ chain (TCR-γ) by PCR-DGGE. The cytokine profile of both populations was investigated by RT-PCR ELISA for IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-15. In three other patients with known Vβ-usage, the dominant T cell clones were phenotypically characterized by double staining. PCR-DGGE of TCR-γ demonstrated that all patients had a clonal population in their blood and that this population was present in CD4 + CD7 – and CD4 + CD7 + populations. Concerning mRNA cytokine transcription, the two populations did not show any consistent differences. In three patients with identified clones (Vβ 3.1, 5.3 and 6.7), double staining revealed positivity for CD2, CD3, CD4, CD5, CD45RO and CD7 in a significant proportion (at least 35%). We conclude that the CD4 + CD7 – population does not represent the dominant T cell clone in patients with SS. An increase in this population of PBMC in SS might account for deviations in the T cell functions of the patients.

Deficiency of arylsulfatase C in cultured skin fibroblasts of X-linked ichthyosis

In October 1978 it was reported by Shapiro et al. [13] that cultured skin fibroblasts of patients with X-linked recessive inherited ichthyosis had a deficiency of steroidsulfatase. The same could be demonstrated for cultured epidermal cells [6]. Such a steroidsulfatase deficiency is also observed in the placenta of mothers with low urinary estriol excretion [1,2,3, 12]. It had also been reported that amnion fluid cells as well as cultured skin fibroblasts from offsprings of mothers with placental steroidsulfatase deficiency showed this inborn enzym defect. This defect of steroidsulfatase activity in the placenta is also associated with low arylsulfatase C activity as shown by biochemical [2, 3, 12] and histochemical methods [4, 5, 7]. Koppe et al. [5] reported three pregnant women with low estrogen excretion due to low placental arylsulfatase C activity. The three boys born were normal at birth but later developed ichthyosis vulgaris of the X-linked inherited type. Histochemical investigations of the skin demonstrated the same deficiency of arylsulfatase C activity in all three children. In conclusion, a deficiency of the two microsomal enzymes steroidsulfatase [2] and arylsulfatase C [10] seems to be associated with X-linked recessive ichthyosis, tough these two enzymes are not identical as demonstrated electrophoretically [3]. The two lysosomal arylsulfatases A and B are not affected [ 12]. The biochemical demonstrat ion of arylsulfatase C deficiency in cultured skin fibroblasts from patients with X-linked ichthyosis has to our knowledge not yet been reported. Skin biopsies from patients with Xlinked ichthyosis were tr immed free of fat, cut into

Determination of arylsulfatase C in hair follicles.

The use of hair follicles for the determination of genetically defined enzyme defects was described by several authors in the last ten years, especially for the detection of heterozygotes in X-linked disorders [3, 4, 6 8 , 15, 20] or autosomal recessive inherited disease [11]. But in some cases it may be of great interest to detect a certain enzyme defect in the patient itself in order to ensure the clinical diagnosis. Steroid sulfatase and arylsulfatase C deficiencies have recently been shown to be associated with X-linked ichthyosis [12, 13, 17], a skin disorder which is difficult to distinguish clinically and histologically from other forms of ichthyosis [10], whereas marked ultrastructural differences can be detected [1, 2]. Having these facts in mind and in order to have a simple and fast assay in hand it was obvious to try to estimate sulfatase in hair follicles. From each individual 2 0 3 0 scalp hairs were collected and microscopically differentiated. Only anagen hairs proved to be useful for the assay. In most cases hairs were tested immediately after plucking but it was possible to store them for at least 3 4 days at room temperature without loss of activity. All selected anagen hairs were cut about 3 4 mm above the hair bulbs as described by others [3] and placed separately in 50 pl of 0.1 M phosphate buffer pH 8.0. Before we had shown that the use of 0.1 M phosphate prevents the simultaneous estimation of the activities of the arylsulfatases A and B because these enzymes are inhibited by phosphate [5, 14]. The buffer solutions with the single hairs were then subjected to l 0 cycles of freezing and thawing using liquid nitrogen and a waterbath of 37 ~ C to make the cells of the hair follicles penetrable for the substrate. Because the arylsulfatase C is tightly bound to microsomal membranes [5] the substrate has to penetrate into the cell. To the 50 ~1 of phosphate buffer containing the hair follicle 100 pl of 0.4 mM 4-methylumbelliferyl sulfate potassium salt [9, 18, 19] in 0.1 M phosphate buffer were added and this solution together with the hair follicle was incubated for 5 h in a waterbath at 37 ~ C. The reaction was then stopped by the addition of 1 ml of 0.1 M glycine-carbonate buffer pH 10.3. The fluorescence which had developed by the release of free methylumbelliferone was measured with the

Plasma Concentrations of Beta-Carotene and Canthaxanthin during and after Stopping Intake of a Combined Preparation

During 17 days two groups of 6 and 8 volunteers of the Department of Dermatology ingested 4 and 6 capsules per day of carotenoids (10 mg beta-carotene + 15 mg canthaxanthin per capsule; Phenoro). Heparinized blood was collected twice a week until day 21 and then weekly until day 77. Concentrations of beta-carotene and canthaxanthin in plasma were analyzed by high-performance liquid chromatography during 77 days. Plasma levels of carotenoids were higher in group 2 (6 capsules) than in group 1 (4 capsules) but reached maximum concentrations within the same time. Hence, therapeutic plasma levels of beta-carotene were reached earlier in group 2. According to blood levels, group 2 could be clearly divided in low and high responders. In contrast to canthaxanthin, beta-carotene exhibited a biphasic decline in both groups, with a monophasic asymptotic decrease which was expressed clinically by a pronounced orange to yellow pigmentation of the skin.

Onychomycosis (trichophyton mentagrophytes) A scanning electron microscopic observation

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 he 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, “lunnel”‐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.

Fluorometric determination of DNA in epidermis and cultured fibroblasts, using 4′-6-diamidino-2-phenylindole (DAPI)

Summary4′-6-diamidino-phenylindole (DAPI) which is used for the detection of mycoplasmic infections of cell cultures or cytofluorometric investigations proved to be a sensitive agent for the fluorometric assay of alkali extracted DNA in cultured cells and human epidermis. Various experiments to optimize assay conditions led to the following procedure: 1 part of freeze dried tissue (1 mg/ml) and 1 part of 0.2 mol/l NaOH (w/v) were heated at 95° for 40 min. After cooling for 1 h to room temperature, 0.1 ml alkaline extract was mixed with 2.9 ml DAPI-solution (0.2 μg/ml in McIlvaine buffer containing 1 mmol/l MgCl2). After 10 min in the dark the emitted fluorescence was estimated at 453 nm with an excitation at 360 nm. This assay procedure allowed a detection limit of 25 ng DNA.

Inhibitory effect of 8-methoxysporalen plus UVA (PUVA) on the systemic induction of contact sensitivity to dinitrochlorbenzene (DNCB) in guinea pigs

SummaryTo investigate the influence of 8-MOP and UVA on the induction of cell-mediated immunity, guinea pigs, sensitized with a single injection of dinitrofluorbenzene (DNFB) in Freunds complete adjuvant (FCA) in all footpads and the nuchal skin, were treated with 8-MOP, UVA, or 8-MOP+ UVA on days 0, 2, and 5 and tested with dinitrochlorbenzene (DNCB) on day 14 after sensitization. Two control groups, exposed in a covered condition to the PUVA 4000 lamp, to observe the heat effect, showed a slightly enhanced contact sensitivity in comparison to the only sensitized control group. No altered reactivity was observed after irradiation with longwave UV light alone, whereas a statistically significant enhanced resp. reduced contact sensitivity was obtained after the treatment with 8-MOP alone and the combined action of 8-MOP +UVA, respectively.ZusammenfassungUm etwas über den Einfluß der PUVA-Behandlung auf die Induktion der cellulären Immunität zu erfahren, wurden Meerschweinchen, die durch eine einmalige Injektion von Dinitrofluorbenzol (DNFB) in Freunds komplettem Adjuvans (FCA) in die Pfoten und die Nackenhaut sensibilisiert worden waren, an den Tagen 0, 2 und 5 mit 8-MOP, UVA oder 8-MOP+UVA behandelt und 14 Tage nach der Sensibilisierung mit Dinitrochlorbenzol (DNCB) getestet. Zwei Kontrollgruppen, behandelt mit oder ohne 8-MOP wurden, zugedeckt mit chirurgischen Tüchern, unter die PUVA 4000 Lampe gelegt, um den Wärmeeffekt zu prüfen. Beide Gruppen zeigten eine leicht erhöhte Kontaktüberempfindlichkeit. Keine veränderte Reaktivität wurde nach UVA allein beobachtet, während sich eine statistisch signifikant erhöhte, bzw. erniedrigte Kontaktüberempfindlichkeit nach 8-MOP allein, bzw. 8-MOP +UVA ergab.

Properties of acid phosphatase in human stratum corneum.

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.

Steroid Sulfatase = Aryl Sulfatase C? Chromatographic and Electrophoretic Properties in Extracts from Placental Microsomes and Skin Fibroblasts

Although the deficiency of steroid sulfatase (STS) as well as aryl sulfatase C (ASC) activities in patients with X-linked recessive ichthyosis has been confirmed by several groups all over the world, the question whether STS = ASC is not yet completely answered. To obtain more information, Miranol H2M extracts from placental microsomes and cultured skin fibroblasts were subjected to gel permeation chromatography and polyacrylamide gel electrophoresis. STS (3H-dehydroepiandrosterone sulfate) and ASC (4-methylumbelliferone sulfate) activities were estimated in the eluted gel permeation chromatography fractions and within the same gel cylinders in half gel slices. None of these two methods allowed a separation of the two microsomal sulfatase activities. From these results and from different behavior of STS and ASC (not deficient in uncultured skin preparations of X-linked recessive ichthyosis, different kinetic properties between STS and ASC, etc.) we propose microsomal sulfatase activities to be assembled in an enzyme aggregate.

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcus aureus antigens

SummaryThe development of delayed hypersensitivity against staphylococcal antigens in guinea pigs was observed from day 3 to day 50 after sensitization with staphylococcal homogenate in Freunds incomplete (FIA) and Freunds complete adjuvant (FCA). Skin test reactivity, stimulation of lymph node lymphocytes, and peripheral blood lymphocytes and the titre of precipitating antibodies were followed during this period. Maximal skin test reactivity as well as maximal lymphocyte responsiveness occurred at day 21 after sensitization in FCA-sensitized guinea pigs. In FIA-sensitized animals highest skin reactivity was observed at day 14 and maximal lymphocyte stimulation 35 days after sensitization. Precipitating antibodies reached a plateau at day 20 in plasma of animals sensitized with FCA and at day 35 in FIA-sensitized animals.ZusammenfassungDie Entstehung einer Spättypüberempfindlichkeit gegenüber Staphylokokkenantigenen bei Meerschweinchen wurde vom Tag 3 bis zum Tag 50 nach der Sensibilisierung mit Staphylokokkenhomogenat in Freunds inkomplettem (FIA) oder komplettem Adjuvans (FCA) verfolgt. Als Parameter wurden während diesem Zeitabschnitt die Hautreaktionen nach Intracutantestung, die Stimulation von Lymphknoten- und peripheren Lymphocyten und der Verlauf des Titers präzipitierender Antikörper gemessen. Sowohl stärkste Hautreaktionen, als auch maximale Lymphocytenstimulation von Lymphknoten- und peripheren Lymphocyten wurden bei FCA-sensibilisierten Tieren nach 21 gefunden, während bei FIA-sensibilisierten die Maxima für den Hauttest nach 14 und für den Lymphocytentransformationstest nach 35 Tagen auftraten. Der Titer der päzipitierenden Antikörper gegen Staphylokokkenhomogenat erreichte im Plasma von FCA-sensibilisierten konstante Werte bei 20 Tagen und im Plasma von FCA-Tieren bei 35 Tagen.