Volker Steinkraus

Kinetics of DNA strand breaks and protection by antioxidants in UVA- or UVB-irradiated HaCaT keratinocytes using the single cell gel electrophoresis assay

The aim of this study was to characterize the genotoxic action of UVA and UVB in human keratinocytes by application of the single cell gel electrophoresis assay (SCGE assay). Dose dependence of DNA damage, the time course of its repair, and the influence of cellular antioxidant status were assessed. Irradiation with UVA or UVB both resulted in a dose-dependent increase in the level of DNA damage. A time course study to evaluate the repair kinetics in keratinocytes irradiated with 5 J/cm2 UVA revealed an immediate occurrence of DNA effects which subsequently disappeared within about 1 h, indicating removal of DNA lesions. This rapid repair of DNA damage is consistent with the observation that 5 J/cm2 UVA did not impair cellular viability. In contrast, exposure to 15 mJ/cm2 UVB resulted in a prolonged repair of DNA damage which lasted about 25 h. Thus, the repair kinetics of UVA- and UVB-induced DNA damage clearly differed from each other, implicating the induction of different types of DNA lesions by UVA and UVB. Neither a pretreatment with Mg-ascorbyl phosphate or D,L-alpha-tocopherol, nor depletion of endogenous glutathione altered cellular sensitivity to UVB. In contrast, the DNA damaging effects of UVA could be counteracted by a pretreatment with these antioxidants. These observations confirm that the UVA-induced effects on DNA are related to radical mediated strand breaks and DNA lesions forming alkali-labile sites. The UVB-induced effects mainly occur as a consequence of excision repair-related strand breaks. The observed repair kinetics of DNA lesions and the influence of cellular antioxidant status may help to elucidate protective mechanisms against the carcinogenic effects of UV radiation present in sunlight.

Increased in vitro expression of beta2-adrenoceptors in differentiating lesional keratinocytes of vitiligo patients

Keratinocytes were established in serum-free culture medium from lesional and nonlesional skin of a patient with vitiligo (skin type III) and from an age-matched healthy control subject. Both differentiating and undifferentiated cells were examined for the presence of beta2-adrenoceptors in culture medium containing either low (0.1×10−3M) or high (1.5×10−3M) calcium concentrations. Binding experiments were performed with saturating levels of radiolabeled (—)-[3H] CGP 12177, a nonselective beta-adrenergic antagonist. Controls for nonspecific binding were determined by the addition of the beta-adrenergic antagonist, propranolol (5 Μmol), before the introduction of (—)-[3H] CGP 12177 to cell cultures. Undifferentiated keratinocytes yielded the highest expression of beta2-adrenoceptors, whereas differentiating keratinocytes grown in medium with a low calcium concentration (0.1×10−3M) had a significantly lower expression of receptors with the exception of vitiliginous cells, which retained high densities of receptors, similar to undifferentiated cells. In addition, these vitiliginous keratinocytes showed a defect in 45calcium uptake. In contrast, differentiated keratinocytes from all three cell strains, grown in medium containing a high calcium concentration (1.5×10−3M) revealed a significantly lower receptor density compared to undifferentiated cells. This finding identified the importance of the extracellular calcium concentration in the expression of beta2-adrenoceptors. Our results: (1) confirmed the importance of beta2-adrenoceptors in the regulation of intracellular calcium concentrations in keratinocytes; (2) showed an increase in catecholamine receptors in differentiating vitiliginous keratinocytes compared to cells established from nonlesional and control skin cultured in medium with a low calcium concentration (0.1×10−3M); and (3) demonstrated the importance of the extracellular calcium concentration in the down-regulation of the beta2-adrenoceptors in vitiliginous keratinocytes. These new observations further suggest an association between the sympathetic nervous system and the pathogenesis of vitiligo.

Unusual histopathological features of cutaneous leishmaniasis identified by polymerase chain reaction specific for Leishmania on paraffin-embedded skin biopsies

Background  Cutaneous leishmaniasis (CL) is rare in Northern Europe and may be overlooked because colleagues have little experience with it.

Autoradiographic mapping of beta-adrenoceptors in human skin

A high density of β2-adrenoceptors has been found in human skin. Using autoradiographic mapping we investigated the distribution of β1- β2-receptors in normal and diseased human skin. Cryostat sections of human skin obtained at biopsy were incubated with [125I]-iodocyanopindolol and nonspecific binding was identified by incubation of adjacent sections with 200 μM (-)-isoproterenol; β2-receptors were visualized using CGP 20712A and β1-receptors using ICI 118,551 as competing agents. The epidermis was densely labelled with an even distribution throughout all layers. Most of the β-receptors were of the β2-subtype with practically no β1-receptors. β-Receptors were also localized to eccrine sweat glands, dermal blood vessels, and perivascular inflammatory cells, but there was no labelling of sebaceous glands. Topical glucocorticoids caused an increase in the density of epidermal β-receptors. We conclude that keratinocytes and eccrine sweat glands express high densities of β2-receptors suggesting that they may have a physiological role in the regulation of these cells.

Β-Adrenergic receptors in psoriasis: evidence for down-regulation in lesional skin

Lesional psoriatic skin displays reduced responsiveness to Β-adrenergic stimulating agents. To elucidate whether the receptor protein itself is responsible for this, lesional and non-lesional psoritatic skin was investigated ex vivo for maximal Β-adrenergic binding density (Bmax) and Β-adrenergic binding affinity (KD). Epidermal crude membrane homogenates (ECMH) were prepared from split-thickness skin biopsies and saturated with the lipophilic Β-adrenergic antagonist (—)-125I-iodocyanopindolol (ICYP) as radioligand. Specific binding was saturable and Scatchard transformation of the binding data revealed a homogeneous class of Β-adrenergic receptors in all nine experiments. The maximal Β-adrenergic binding density was significantly less in lesional than in non-lesional psoriatic skin (Bmax=49.7 ± 7.2 fmol/mg protein vs. 67.1 ± 2.2 fmol/mg protein, n=9, P<0.05). The binding affinity was similar in lesional and in non-lesional skin (KD=9.0 ± 1.5 pmol/l vs. 8.0 ± 0.9 pmol/l). These results could at least partially explain the reduced responsiveness of the Β-adrenergic system in lesional psoriatic skin seen after stimulation with Β-adrenergic agonists.

High density of beta2-adrenoceptors in a human keratinocyte cell line with complete epidermal differentiation capacity (HaCaT)

SummaryA non-tumorigenic keratinocyte cell line with complete epidermal differentiation capacity (HaCaT) was used in radioligand binding experiments to determine the number of beta-adrenoceptors. Intact cells were saturated with 3H-labelled (−)CGP-12177 (CGP), a hydrophilic non-selective beta-adrenergic antagonist as radioligand. In order to investigate the beta-adrenergic subtype selectivity, displacement experiments were performed with different antagonists and agonists. Binding of CGP to keratinocytes has been shown to be reversible and saturable and to have high affintiy (Bmax=114.0±8.8 fmol/107 cells with 6866 receptors/cell, KD=0.095±0.017 nmol/l; n=11). Betaadrenergic antagonists inhibited binding yielding monophasic displacement curves. IC50-values (nmol/l) were: propranolol (non-selective) 1.68; CGP-12177 (non-selective) 1.08; ICI 118,551 (beta2-selective) 2.92; bisoprolol (beta1-selective) 1230; and CGP-20712 (beta1-selective) 24980. Agonists displaced CGP in the order isoprenaline> adrenaline>noradrenaline. We conclude that HaCaT cells express a high density of beta2-adrenoceptors providing a good model system to study adrenergic receptor mechanisms under reproducible experimental conditions in keratinocytes.

Time course and extent of α1-adrenoceptor density changes in rat heart after β-adrenoceptor blockade

1 It has been suggested that impaired β‐adrenoceptor stimulation is a condition under which the functional role of cardiac α1‐adrenoceptors is enhanced. We therefore investigated the extent and time course of changes in α1‐adrenoceptor characteristics after chronic treatment with the β‐adrenoceptor blocker propranolol in rat heart. For comparison β‐adrenoceptors were also studied. The mechanism of the changes in adrenoceptor density was investigated with cycloheximide, an inhibitor of protein synthesis. The functional significance of an increased α1‐adrenoceptor density was tested by measuring isometric force of contraction in the presence of phenylephrine or isoprenaline in right ventricular papillary muscles. 2 Rats were treated with propranolol (9.9 mg kg−1 daily) or 0.9% NaCl, applied with osmotic minipumps for 1, 2, 3 or 7 days. Propranolol treatment resulted in a maximally 28% increase of α1‐adrenoceptor density after 3 days (NaCl 95.9 ± 3.5 vs. propranolol 123.0 ± 1.6 fmol mg−1 protein, n = 6, P < 0.01). This up regulation reached significant levels after 2 days of treatment and was reversible after cessation of treatment within two days. KD‐values were the same for NaCl‐ and propranolol‐treated rats. Changes of Bmax and KD in β‐adrenoceptor binding assays did not reach significant levels. 3 Cycloheximide (1.5 mg kg−1 i.p. daily for 3 days) inhibited the propranolol‐induced increase in Bmax of α1‐adrenoceptors completely. In addition, cycloheximide also decreased the density of α1‐ and β‐adrenoceptors also under control conditions. 4 pD2‐values for the positive inotropic effect of phenylephrine and isoprenaline in isolated electrically driven papillary muscle were similar in NaCl‐ and propranolol‐treated rats (phenylephrine: 5.41 ± 0.11 vs. 5.41 ± 0.19, n = 7; isoprenaline: 6.31 ± 0.18 vs. 6.65 ± 0.19, n = 7). The observed increase in α1‐adrenoceptor density in healthy rat heart may therefore not be high enough to enhance the phenylephrine‐induced increase in force of contraction. 5 In conclusion, time course and effects of cycloheximide indicate that the increase in Bmax of myocardial α1‐adrenoceptors is due to de novo synthesis of receptors. However, at least for the rat heart model, a functional significance of this increase could not be demonstrated.

Cutaneous metastases from carcinoma of the prostate

Cutaneous or subcutaneous metastases occur in 2% to 9% of visceral malignancies.lm4 Their occurrence is associated with advanced disease and poor prognosis. Skin metastases are seen most commonly from carcinomas of the breast, lungs, kidneys, stomach, uterus, and colon.5a6 Carcinomas of the prostate are common7 but account for less than 1% of all skin metastases.6 We describe a patient with carcinoma of the prostate and skin metastases.

Radioligand binding characteristics of β2–adrenoceptors of cultured melanoma cells

The existence of β‐adrenoceptors on intact cells of the human malignant melanoma cell line A‐ 375 was investigated using the binding properties of the tritiated radioligand (‐)‐[3H]CGP‐ 12177, a hydrophilic non‐selective β‐adrenoceptor antagonist. Displacement experiments of the radioligand from its binding site were performed with antagonists and agonists to determine the β‐adrenoceptor subtype selectivity. The binding of (‐)‐[3H]CGP‐12177 was saturable, of high affinity (Kd= 0.025 nmol/1, n= 12) and was rapid and readily reversible. The maximal number of binding sites (Bmax) was 33.5 ± 1.9 fmol/107 cells or 2018 ± 114 receptors per cell. β‐adrenoceptor antagonists inhibited binding of the radioligand with monophasic displacement curves. IC50 values were (nmol/l): propranolol (non‐selective) 2.82, alprenolol (non‐selective) 2.0, ICI 118,551 (β2‐selective) 3.5 and bisoprolol (β1‐selective) 2200. Agonists inhibited binding in the order of potency of isoprenaline > adrenaline > noradrenaline. It is concluded that cells of the melanoma cell line A‐375 contain a homogeneous population of β2‐ adrenoceptors.

Anaphylactic reactions to food items with latex allergy

Zusammenfassung. Durch Latex-haltige Gummiprodukte bedingte kontakturtikarielle und anaphylaktische Reaktionen stellen ein zunehmendes Problem in zahlreichen medizinischen Disziplinen dar. In jüngster Zeit mehren sich die Berichte über Nahrungsmittelanaphylaxie bei bestehender Latexallergie. Anhand der Kasuistiken 3er Patientinnen, die sowohl auf Latex als auch auf Nahrungsmittel anaphylaktisch reagierten, wird auf die Problematik und Bedeutung möglicher Kreuzreaktionen hingewiesen. Die zu anaphylaktischen Reaktionen führenden Nahrungsmittel waren Banane und Avocado, Banane, Avocado und Buchweizen sowie Avocado, Banane und Tomate, wobei bislang Berichte über Buchweizen und Tomate in diesem Zusammenhang fehlten.Abstract. Contact urticaria and anaphylactic reactions to latex-containing rubber products are being recognized with increasing frequency in all kinds of medical disciplines. Recently a number of reports have been published describing anaphylactic reactions to food items in patients with latex allergy. The cases of three patients who developed anaphylactic reactions to both latex and food items are presented, and the importance of the association of latex and cross-reactivity with food items is stressed. The food items that led to anaphylactic reactions were banana and avocado; banana, avocado and buckwheat; and banana, avocado and tomato. The cross-reactivity of latex to buckwheat and tomato has not been reported before.