Galectin-7 in the stratum corneum: a biomarker of the skin barrier function.

OBJECTIVE: Skin barrier disruption often occurs in diseased and damaged skin conditions such as atopic dermatitis (AD). We focused the galectin-7 protein (Gal-7) as a biomarker of skin condition and assessed whether the content of Gal-7 in stratum corneum (scGal-7) could be used as an indicator of skin barrier disruption and as an index of local skin symptoms in AD patients. METHODS: Alteration of Gal-7 expression levels in keratinocyte and scGal-7 contents after barrier disruption by sodium dodecyl sulphate were evaluated in vitro and in vivo, respectively. Correlation between scGal-7 content and transepidermal water loss (TEWL) was examined in 126 healthy subjects. We performed single measurements of scGal-7 contents in 34 AD patients and serial measurements of 15 inpatients among them. SC samples were collected by the tape-stripping method, and scGal-7 content was determined using enzyme-linked immunosorbent assay. RESULTS: Gal-7 expression in keratinocytes increased after barrier disruption. The scGal-7 content reflected the disruption of the skin barrier. The scGal-7 contents and TEWL values correlated in healthy subjects. The scGal-7 level was higher in AD patients than in healthy subjects. The scGal-7 contents in the cheek and neck of AD patients significantly correlated with the total and local skin lesion severity scores. Serial measurements in the inpatients showed that the scGal-7 contents in the cheek and neck decreased in tandem with local severity scores in response to treatment. CONCLUSION: Measurement of scGal-7 content in tape-stripped samples was useful for the evaluation of the skin barrier function in dry skin conditions such as AD.

Chlamydophila pneumoniae Infection Induced Nodular Vasculitis.

We present the case history of a 48-year-old male patient with Chlamydophila (Chlamydia) pneumoniae who developed a nodular vasculitis. He developed a cutaneous vasculitis with the onset of respiratory symptoms. The diagnosis of Chlamydophila pneumoniae infection was based on serology. Since this infection is very common in our population, although often asymptomatic, it should be systematically considered as a causative agent of nodular vasculitis.

Neuroprotective mechanisms of lidocaine against in vitro ischemic insult of the rat hippocampal CA1 pyramidal neurons.

To compare neuroprotective effects of lidocaine and procaine against ischemic insult, intracellular recordings were made from rat hippocampal CA1 pyramidal neurons in slice preparations. Superfusion of the slices with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization 6 min from the onset. When oxygen and glucose were reintroduced, the membrane depolarized further until it reached 0 mV, and thereafter the membrane showed no functional recovery. Pretreatment with lidocaine (10 microM), but not procaine (50 microM), restored the membrane potential after the reintroduction of oxygen and glucose. Lidocaine, compared to procaine, significantly inhibited the reduction in both tissue ATP content and flavoprotein fluorescence during and after in vitro ischemia. Under electron microscopy, only lidocaine well preserved the structure of mitochondria in the CA1 pyramidal cell body. Extracellular recordings revealed that procaine reduced the field postsynaptic potential whereas lidocaine augmented it. Both drugs reduced the presynaptic volley dose-dependently. Neither lidocaine nor procaine significantly affected a rapid rise of the intracellular Ca2+ level produced by in vitro ischemia in the CA1 region. All the results suggest that the neuroprotective lidocaine action is due to the protection of the mitochondria to maintain the tissue ATP content during and after in vitro ischemia.

Arachidonic acid metabolites contribute to the irreversible depolarization induced by in vitro ischemia.

Intracellular recordings were made from hippocampal CA1 neurons in rat slice preparations. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization approximately 5 min after the onset of the superfusion. Even when oxygen and glucose were reintroduced immediately after rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV (irreversible depolarization) after 5 min from the reintroduction. The pretreatment of the slice preparation with a phospholipase A2 (PLA2) inhibitor, para-bromophenacyl bromide, or a cytochrome p-450 inhibitor, 17-octadecynoic acid, significantly restored the membrane to the preexposure potential level after the reintroduction of oxygen and glucose. The administration of 14,15-epoxyeicosatrienoic acid or 20-hydroxyeicosatetraenoic acid did not change the latency of the rapid depolarization and did not allow the membrane potential to recover after the ischemic exposure. In contrast, after pretreatment with cyclooxygenase or lipoxygenase inhibitors, such as indomethacin, resveratrol, Dup-697, nordihydroguaiaretic acid, and 3,4-dihydrophenyl ethanol, a minority of neurons tested showed postischemic recovery from the persistent depolarization. Improved recovery was also seen after treatment with the free radical scavengers, edaravone and alpha-tocopherol. These results suggest that the activation of the arachidonic acid cascade via PLA2 and the free radicals produced by arachidonic acid metabolism contribute to the irreversible depolarization produced by in vitro ischemia.

Dietary soy oil content and soy-derived phytoestrogen genistein increase resistance to alopecia areata onset in C3H/HeJ mice.

Alopecia areata (AA) is a complex, multi-factorial disease where genes and the environment may affect susceptibility and severity. Diet is an environmental factor with the potential to influence disease susceptibility. We considered dietary soy (soya) oil content and the soy-derived phytoestrogen genistein as potential modifying agents for C3H/HeJ mouse AA. Normal haired C3H/HeJ mice were grafted with skin from spontaneous AA affected mice, a method previously shown to induce AA. Grafted mice were given one of three diets containing 1%, 5% or 20% soy oil and observed for AA development. In a separate study, mice on a 1% soy oil diet were injected with 1 mg of genistein three times per week for 10 weeks or received the vehicle as a control. Of mice on 1%, 5%, and 20% soy oil diets, 43 of 50 mice (86%), 11 of 28 mice (39%), and 2 of 11 mice (18%) developed AA, respectively. Four of 10 mice injected with genistein and 9 of 10 controls developed AA. Mice with AA had hair follicle inflammation consistent with observations for spontaneous mouse AA, but no significant association was observed between the extent of hair loss and diet or genistein injection. Mice that failed to develop AA typically experience white hair regrowth from their skin grafts associated with a moderate macrophage and dendritic cell infiltration. Soy oil and derivatives have previously been reported to modify inflammatory conditions. Hypothetically, soy oil compounds may act on C3H/HeJ mice through modulating estrogen-dependent mechanisms and/or inflammatory activity to modify AA susceptibility.

The presynaptic modulation of glutamate release and the membrane dysfunction induced by in vitro ischemia in rat hippocampal CA1 neurons.

Superfusion with an oxygen and glucose deprived medium (in vitro ischemia) of rat hippocampal CA1 pyramidal neurons in tissue slices produced a rapid depolarization within 5 min and thereafter showed no functional recovery (irreversible membrane dysfunction), even if oxygen and glucose were reintroduced. We previously suggested that such a rapid depolarization is triggered by the accumulation of extracellular glutamate (Glu). As a result, we examined the effects of either the activation or inhibition of presynaptic receptors, which modulate Glu release from the nerve terminal, on the potential change produced by in vitro ischemia. The adenosine A1 receptor antagonist, 8-cyclopenthyl theophylline, A2a receptor antagonist, ZM241385, and A2b receptor antagonist, alloxazine, did not significantly alter either the latency or the maximal slope of the rapid depolarization. In addition, the GABAB receptor antagonist, 2-hydroxysaclofen, or the metabotropic Glu receptor type 4 antagonist, alpha-methylserine-O-phosphate, did not change either the latency or the maximal slope. The adenosine A(1) receptor agonist, 2-chloro-N6-cyclopentyladenosine, A2a receptor agonist, CGS2168, or A2b receptor agonist, 5'-(N-ethylcarboxamido)-adenosine, did not affect these parameters either. None of these drugs restored the membrane potential to the pre-exposure level after the reintroduction of oxygen and glucose. Simultaneous intracellular recordings from CA1 and CA3 pyramidal neurons in the same slices revealed the membrane of the CA3 neurons to be hyperpolarized when a rapid depolarization occurred in the CA1 neurons. These results suggest that presynaptic Glu release does not accelerate during the generation of the rapid depolarization induced by in vitro ischemia.

17alpha-estradiol induces aromatase activity in intact human anagen hair follicles ex vivo.

For topical treatment of androgenetic alopecia (AGA) in women, solutions containing either estradiol benzoate, estradiol valerate, 17beta- or 17alpha-estradiol are commercially available in Europe and some studies show an increased anagen and decreased telogen rate after treatment as compared with placebo. At present it is not precisely known how estrogens mediate their beneficial effect on AGA-affected hair follicles. We have shown recently that 17alpha-estradiol is able to diminish the amount of dihydrotestosterone (DHT) formed by human hair follicles after incubation with testosterone, while increasing the concentration of weaker steroids such as estrogens. Because aromatase is involved in the conversion of testosterone to estrogens and because there is some clinical evidence that aromatase activity may be involved in the pathogenesis of AGA, we addressed the question whether aromatase is expressed in human hair follicles and whether 17alpha-estradiol is able to modify the aromatase activity. Herewith we were able to demonstrate that intact, microdissected hair follicles from female donors express considerably more aromatase activity than hair follicles from male donors. Using immunohistochemistry, we detected the aromatase mainly in the epithelial parts of the hair follicle and not in the dermal papilla. Furthermore, we show that in comparison to the controls, we noticed in 17alpha-estradiol-incubated (1 nM) female hair follicles a concentration- and time-dependent increase of aromatase activity (at 24 h: 1 nM = +18%, 100 nM = +25%, 1 micro M = +57%; 24 h: 1 nM = +18%, 48 h: 1 nM = +25%). In conclusion, our ex vivo experiments suggest that under the influence of 17alpha-estradiol an increased conversion of testosterone to 17beta-estradiol and androstendione to estrone takes place, which might explain the beneficial effects of estrogen treatment of AGA.

Extrusion of intracellular calcium ion after in vitro ischemia in the rat hippocampal CA1 region.

Simultaneous recordings of intracellular Ca(2+) ([Ca(2+)](i)) signal and extracellular DC potential were obtained from the CA1 region in 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid penta-acetoxymethyl ester (Fura-2/AM)-loaded rat hippocampal slices. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) for 5-6 min produced a rapid rise of the [Ca(2+)](i) level in the stratum radiatum (rising phase of the [Ca(2+)](i) signal), which occurred simultaneously with a rapid negative DC potential (rapid negative potential). When oxygen and glucose were reintroduced, the increased [Ca(2+)](i) signal diminished rapidly (falling phase of the [Ca(2+)](i) signal) during the generation of a slow negative DC potential (slow negative potential), which occurred within 1 min from the onset of the reintroduction. Thereafter, the [Ca(2+)](i) signal partially and the slow negative potential completely returned to the preexposure level approximately 6 min after the reintroduction. The changes in [Ca(2+)](i) signal during and after in vitro ischemia were very similar to the changes in the membrane potential of glial cells. The rising and falling phases of [Ca(2+)](i) signal corresponded to the rapid depolarization and a depolarizing hump, respectively, in the repolarizing phase of glial cells. A prolonged application of in vitro ischemia or a reintroduction of either glucose or oxygen suppressed the falling phase after ischemic exposure. The application of ouabain (30 microM) generated both a rapid negative potential and a rapid elevation of [Ca(2+)](i), but no slow negative potential or rapid reduction in [Ca(2+)](i) were observed. When oxygen and glucose were reintroduced to slices in the Na(+)-free or ouabain- or Ni(2+)-containing medium, the falling phase was suppressed. The falling phase was significantly accelerated in Ca(2+)- and Mg(2+)-free with EGTA-containing medium. In contrast, the falling phase was significantly slower in the Ca(2+)-free with high Mg(2+)- and EGTA-containing medium. The falling phase of the [Ca(2+)](i) signal after ischemic exposure is thus considered to be primarily dependent on the reactivation of Na(+), K(+)-ATPases, while the extrusion of cytosolic Ca(2+) via the forward-mode operation of Na(+)/Ca(2+) exchangers in glial cells is thought to be directly involved in the rapid reduction of [Ca(2+)](i) after ischemic exposure.

Mechanisms underlying the depression of evoked fast EPSCs following in vitro ischemia in rat hippocampal CA1 neurons.

The mechanisms underlying the depression of evoked fast excitatory postsynaptic currents (EPSCs) following superfusion with medium deprived of oxygen and glucose (in vitro ischemia) for a 4-min period in hippocampal CA1 neurons were investigated in rat brain slices. The amplitude of evoked fast EPSCs decreased by 85 +/- 7% of the control 4 min after the onset of in vitro ischemia. In contrast, the exogenous glutamate-induced inward currents were augmented, while the spontaneous miniature EPSCs obtained in the presence of tetrodotoxin (TTX, 1 microM) did not change in amplitude during in vitro ischemia. In a normoxic medium, a pair of fast EPSCs was elicited by paired-pulse stimulation (40-ms interval), and the amplitude of the second fast EPSC increased to 156 +/- 24% of the first EPSC amplitude. The ratio of paired-pulse facilitation (PPF ratio) increased during in vitro ischemia. Pretreatment of the slices with adenosine 1 (A1) receptor antagonist, 8-cyclopenthyltheophiline (8-CPT) antagonized the depression of the fast EPSCs, in a concentration-dependent manner: in the presence of 8-CPT (1-10 microM), the amplitude of the fast EPSCs decreased by only 20% of the control during in vitro ischemia. In addition, 8-CPT antagonized the enhancement of the PPF ratio during in vitro ischemia. A pair of presynaptic volleys and excitatory postsynaptic field potentials (fEPSPs) were extracellularly recorded in a proximal part of the stratum radiatum in the CA1 region. The PPF ratio for the fEPSPs also increased during in vitro ischemia. On the other hand, the amplitudes of the first and second presynaptic volley, which were abolished by TTX (0.5 microM), did not change during in vitro ischemia. The maximal slope of the Ca(2+)-dependent action potential of the CA3 neurons, which were evoked in the presence of 8-CPT (1 microM), nifedipine (20 microM), TTX (0.5 microM), and tetraethyl ammonium chloride (20 mM), decreased by 12 +/- 6% of the control 4 min after the onset of in vitro ischemia. These results suggest that in vitro ischemia depresses the evoked fast EPSCs mainly via the presynaptic A1 receptors, and the remaining 8-CPT-resistant depression of the fast EPSCs is probably due to a direct inhibition of the Ca(2+) influx to the axon terminals.

Influence of estrogens on the androgen metabolism in different subunits of human hair follicles.

The molecular pathways involved in estrogen-mediated induction of hair growth in androgenetic alopecia are unknown. Some authors found that estradiol (E) inhibited 5alpha-reductase (5alpha-R) activity and therefore we addressed the question whether 17alpha- or 17beta-E are able to modulate the activity of 5alpha-R, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) or 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in isolated compartments of human hair follicles. For this purpose, scalp biopsies from volunteers were taken and from each biopsy root sheaths, connective tissue sheaths and dermal papillae (DP) were dissected and incubated in the presence of 3H-testosterone (T) and, in addition, either 17alpha-E, 17beta-E, progesterone or finasteride for up to 48 hrs. Thereafter high-performance liquid chromatography analysis of culture supernatants was performed to detect T-metabolites. At the tested concentrations, finasteride was found to be a major inhibitor of dihydrotestosterone (DHT) formation. Even 1 nM finasteride inhibited DHT synthesis in DP by 86% and 1 nM progesterone by 75%. Estrogens were less able to inhibit the synthesis of DHT in DP (e.g. 100 nM 17alpha-E: 20%; 100 nM 17beta-E: 60%). Whether E directly inhibits 5alpha-R in DP's or whether the effect of estrogens might be explained by an increased conversion of T to the weaker androgens such as androstendione (via 17beta-HSD), androstenediol (via 3beta-HSD) or 17beta-E (via aromatase), thereby diminishing the amount of T available for the conversion to DHT, remains to be shown.

Acrodermatitis acidemica secondary to malnutrition in glutaric aciduria type I.

We encountered a patient with glutaric aciduria type I (GA-I) associated with skin lesions resembling acrodermatitis enteropathica (AE). This child was being fed with a low-protein diet when the skin disorder developed. A deficiency in plasma levels of essential amino acids, particularly isoleucine, and zinc was confirmed. Supplementation of a high-caloric, protein-rich diet together with zinc, selenium and vitamins led to a prompt improvement of the skin lesions. We assume that in our patient the skin lesions were the result of malnutrition, rather than being primarily associated with the underlying metabolic disease. To our knowledge, no other report is so far available concerning GA-I complicated by skin eruptions.

The feasibility of quantitative analysis of androgen metabolism by use of single dermal papillae from human hair follicles.

Androgenetic alopecia (AGA) is a dihydrotestosterone-mediated process, characterized by continuous miniaturization of androgen sensitive hair follicles (HF). Although increased 5 alpha-reductase (5aR) activity in affected HF is a key feature in the pathogenesis of AGA, only little is known about the in vivo expression of 5aR within AGA-affected HF. Recent studies have shown that the dermal papilla (DP) is the predominant site of type 2 5aR expression within the human HF, but direct measurements of 5aR activity in intact DP of AGA-affected HF have not been reported so far, mainly because of technical problems. Hence there is a need for a reliable and sensitive method of measuring 5aR activity in fresh tissues. As a novel approach, we used freshly isolated, intact DP and a highly sensitive HPLC-radiomatic flow scintillation system to measure 5aR. In this way we were able to measure 5aR even in small DPs from miniaturized HF. Our results show that DP from the occipital scalp express ex vivo considerable amounts of 5aR activity, but the measurable enzyme activities of individual DP differ considerably. Therefore the use of only one or two DP is at present not a reliable tool to analyze 5aR activity ex vivo.

Steroid sulfatase in the human hair follicle concentrates in the dermal papilla.

5 alpha-dihydrotestosterone is known to play a crucial part in the regulation of hair growth and in the development of androgenetic alopecia. 5 alpha-dihydrotestosterone is formed locally within the hair follicle from the systemic precursor testosterone by cutaneous steroid 5 alpha-reductase. Moreover, adrenal steroids such as dehydroepiandrosterone are converted to 5 alpha-dihydrotestosterone by isolated hair follicles, which may provide an additional source of intrafollicular 5 alpha-dihydrotestosterone levels. Elevated urinary dehydroepiandrosterone and serum dehydroepiandrosterone sulfate have been reported to be present in balding young men. These reports suggest that dehydroepiandrosterone sulfate may act as an important endocrine factor in the development of androgenetic alopecia. Hence the question arises whether the dehydroepiandrosterone sulfate can be metabolized within the hair follicles to yield dehydroepiandrosterone by the microsomal enzyme steroid sulfatase, and where steroid sulfatase might be localized. We therefore performed immunostaining for steroid sulfatase on human scalp biopsies as well as analysis of steroid sulfatase enzyme activity in defined compartments of human beard and occipital hair follicles ex vivo. Using both methods steroid sulfatase was primarily detected in the dermal papilla. Steroid sulfatase activity was inhibited by estrone-3-O-sulfamate, a specific inhibitor of steroid sulfatase, in a concentration-dependent way. Furthermore, we show that dermal papillae are able to utilize dehydroepiandrosterone sulfate to produce 5 alpha-dihydrotestosterone, which lends further support to the hypothesis that dehydroepiandrosterone sulfate contributes to androgenetic alopecia and that steroid sulfatase inhibitors could be novel drugs to treat androgen-dependent disorders of the hair follicle such as androgenetic alopecia or hirsutism.

The novel drug CS-891 inhibits 5alpha-reductase activity in freshly isolated dermal papilla of human hair follicles.

The local conversion of testosterone (T) to the more potent androgen dihydrotestosterone (DHT) by 5alpha-reductase (5aR) is implicated in the pathogenesis of androgenetic alopecia (AGA). Recently, the clinical effectiveness of finasteride, a selective type II 5aR inhibitor, in treating AGA has been documented, and these clinical studies have shown that circulating DHT is lowered by 60-70% in men taking finasteride. The source of the residual circulating DHT is presumed to be due to type I 5aR activity which is not affected by finasteride. Several novel compounds with potent dual inhibitory activity on both isoenzymes have been described and CS-891 is one of them. This compound may be likewise effective in the prevention or treatment of AGA. As a prerequisite for such an action CS-891 should be able to inhibit 5aR activity in its target tissue: the hair follicles (HF). Here we report on the capability of CS-891 to inhibit 5aR activity in dermal papillae (DP) of human HF.