Differential expression of connective tissue growth factor and extracellular matrix proteins in lichen sclerosus.

BACKGROUND: The histopathology of lichen sclerosus (LS) suggests abnormalities in extracellular matrix (ECM) composition. OBJECTIVES: We aimed to investigate the expression pattern of ECM proteins and related growths factors and Smad signal transducers in LS as compared with healthy skin. METHODS: To assess the expression of decorin, biglycan, versican, perlecan, fibronectin, dermatopontin, extracellular matrix protein 1 (ECM-1), matrix metalloproteinase 1, tissue inhibitor of metalloproteinase 1, connective tissue growth factor (CTGF), transforming growth factor ß1, and Smad-3 protein, real-time RT-PCR and immunohistochemistry were performed on skin specimens obtained from the genital region of healthy subjects (n = 10) as well as LS patients (n = 26). RESULTS: Median mRNA as well as mean protein expression of biglycan, versican, fibronectin, and ECM-1 was significantly higher in LS when compared with healthy controls. Both mRNA and protein CTGF expression observed in LS was significantly higher than in controls. CTGF mRNA expression significantly correlated with mRNA expression of biglycan, versican and fibronectin. CONCLUSIONS: Expression of ECM proteins (e.g. proteoglycans, ECM-1) and CTGF is altered in LS. TGF-ß/Smad-3 independent up-regulation of CTGF may induce accumulation of ECM proteins and maintain fibrosis in chronic LS.

Expression of antimicrobial peptides and proteins in etanercept-treated psoriasis patients.

Recent papers highlight the role of dysregulated expression of antimicrobial peptides and proteins (AMPs) in the pathogenesis of psoriasis. Etanercept, a blocker of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α), is effective in the treatment of psoriasis. We aimed to evaluate the expression profiles of AMPs in psoriatic skin before and after a 6-week course of etanercept therapy. We included 12 psoriasis patients who underwent medium-dose etanercept treatment for 6weeks. At baseline and at the end of therapy immunohistochemistry from lesional skin was performed for psoriasin, LL-37, and human ß-defensin 2 (hBD-2). After 6-week treatment, the modified psoriasis area and severity index significantly decreased from 37.5±5.9 to 14±13.4. Lesional immunoreactivity scores of psoriasin, LL-37, and hBD-2 also significantly decreased after a 6-week course of etanercept. We have demonstrated that etanercept-induced improvement of psoriasic lesions is associated with a significant decline of AMP protein expression.

Primary merkel cell carcinoma clinically presenting as deep oedematous mass of the groin.

Merkel cell carcinoma (MCC) is a relatively rare, polyomavirus associated, primary neuroendocrine carcinoma of the skin which is usually arising from dermal skin layers. However, the origin of MCC in the subcutaneous tissue is debatable. We report a 58-year-old female patient with an oedematous mass on her left groin that was firm in consistency and had no discoloration or other visible abnormality of the overlying skin. On histology and immunohistology the tumour was consistent with the diagnosis of MCC showing a predominant subcutanous growth pattern. Pelvic magnetic resonance tomography revealed a tumour conglomerate reaching from the subcutis of the left groin to the left paraaortal and parailiacal region indicating widespread lymphogenic metastisation. Despite complete medical work-up no other MCC primary could be detected. In conclusion, predominant subcutaneous growth pattern as well as tumour localization in the groin are uncommon features of MCC. MCC showing the aforementioned features may be associated with significant delay of diagnosis and therefore represents an unfavourable prognostic factor.

[Positive strip patch test reactions with coexistent negative patch test reactions. Relevance for the assessment practice of occupational skin disease]. / Positive Testreaktionen im Abrissepikutantest bei negativem Standardepikutantest. Bedeutung für die berufsdermatologische Begutachtung.

The strip patch test is recommended whenever a patch test is presumed to be false negative. The aim of this technique is to increase skin sensitivity to test substances by removing the upper layers of the stratum corneum prior to patch testing. We report on a 57-year-old former heavy-current electrical worker, who suffered from hyperkeratotic fissured hand eczema. Sensitization to nickel sulphate was demonstrated in both 2001 and 2009 with strip patch testing even though conventional patch testing was negative. To our knowledge, this is the first description of an allergy to nickel with a pension entitlement according to no. 5101 of the German ordinance on industrial disease based solely on positive strip patch tests.

Significant upregulation of antimicrobial peptides and proteins in lichen sclerosus.

BACKGROUND: Lichen sclerosus (LS) is a chronic inflammatory T cell-driven sclerotic skin condition in which skin barrier disruption frequently occurs. Inflamed and injured epithelia are a particularly rich source of antimicrobial peptides and proteins (AMPs). OBJECTIVES: We aimed to investigate for the first time the expression pattern of AMPs in lesions of LS as compared with healthy skin. METHODS: Twenty-four women with LS as well as 10 healthy women were included in the study. In order to assess the expression of human beta-defensin (hBD)-1, hBD-2, hBD-3, psoriasin (S100A7), the cathelicidin LL-37 and RNase 7, real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry were performed on skin specimens obtained from lesional and healthy skin of the genital region, respectively. RESULTS: Median hBD-2 mRNA levels observed in LS were significantly higher than in controls (0.15 vs. 0.008; P = 0.0037). Moreover, psoriasin (98.2 vs. 28.1; P = 0.0052) mRNA expression was significantly higher in LS lesions as compared with controls. Significant differences in mRNA expression of hBD-2 and psoriasin were also confirmed by immunohistochemistry. For hBD-1, hBD-3, LL-37 and RNase 7, levels did not differ significantly or were significant only at the gene level but not protein level. CONCLUSIONS: We have demonstrated that hBD-2 and psoriasin expression levels in lesional skin of patients with LS are significantly increased when compared with healthy controls. Whether this observation simply reflects an innate defence response caused by an increased risk of local infection, or whether our data indicate a pathogenetic role of AMPs in LS, will be addressed in future studies.

Effects of chronic prenatal ethanol exposure on mitochondrial glutathione and 8-iso-prostaglandin F2alpha concentrations in the hippocampus of the perinatal guinea pig.

It is hypothesised that oxidative stress is a key mechanism of ethanol neurobehavioural teratogenicity, resulting in altered endogenous antioxidant status and increased membrane lipid peroxidation in the hippocampus of chronic prenatal ethanol exposure (CPEE) offspring. To test this hypothesis, timed pregnant guinea-pigs (term, approximately gestational day (GD) 68) received chronic daily oral administration of (i) 4 g ethanol kg(-1) maternal bodyweight, (ii) isocaloric sucrose with pair feeding, or (iii) water. At GD 65 (term fetus) and postnatal day (PD) 0 (neonate), individual offspring were killed, the brain was excised and the hippocampi were dissected. Glutathione (GSH) concentration was measured in the cytosolic and mitochondrial fractions of hippocampal homogenate. The occurrence of lipid peroxidation was determined by measuring the concentration of 8-iso-prostaglandin F2+/- (8-iso-PGF2+/-). There was CPEE-induced decreased brain weight and hippocampal weight at GD 65 and PD 0, decreased mitochondrial GSH concentration in the hippocampus at PD 0, with no change in mitochondrial GSH concentration at GD 65 or cytosolic GSH concentration at GD 65 or PD 0, and no change in mitochondrial or whole-homogenate 8-iso-PGF2+/- concentration in the hippocampus at GD 65 or PD 0. The data demonstrate that CPEE produces selective mitochondrial dysfunction in the hippocampus of the neonatal guinea-pig, involving GSH depletion.

Chronic prenatal ethanol exposure increases apoptosis in the hippocampus of the term fetal guinea pig.

It is hypothesized that chronic prenatal ethanol exposure (CPEE), via maternal ethanol administration, increases mitochondrial-directed apoptosis in the hippocampus of the term fetus that precedes loss of hippocampal CA1 pyramidal cells. To test this hypothesis, timed pregnant guinea pigs received chronic oral administration of: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding or water throughout gestation. At gestational day 65 (term fetus) and postnatal day 0 (neonate), individual offspring were euthanized, and the brain was excised and dissected. CPEE, compared with the isocaloric-sucrose/pair-fed and water control groups, decreased the brain weight of the term fetus and neonate. CPEE did not alter the density of CA1 pyramidal cells in the hippocampus of the term fetus and neonate. In the term fetus, CPEE increased cytochrome c content in the cytosolic fraction of the hippocampus, altered the mitochondrial localization of cytochrome c in cells of the dorsal hippocampus, and increased the percentage of cells in the dorsal hippocampus containing activated caspase-3 and cleaved poly(ADP-ribose) polymerase. The data indicate that CPEE increases neuroapoptosis in the hippocampus of term fetus, which appears to occur via an intrinsic, mitochondrial-directed mechanism initiated by leakage of pro-apoptotic cytochrome c from mitochondria into the cytoplasm.

Isolation of regioisomers of N-alkylprotoporphyrin IX from chick embryo liver after treatment with porphyrinogenic xenobiotics.

Several porphyrinogenic xenobiotics cause mechanism-based inactivation of cytochrome P450 (P450) isozymes with concomitant formation of a mixture of four N-alkylprotoporphyrin IX (N-alkylPP) regioisomers, which have ferrochelatase inhibitory properties. To isolate the four regioisomers of N-methylprotoporphyrin IX (N-methylPP), 3,5-diethoxycarbonyl, 1-4-dihydro-2,4,6-trimethylpyridine (DDC) was administered to untreated, beta-naphthoflavone-, phenobarbital-, and glutethimide-pretreated 18-day-old chick embryos. Separation of the N-methylPP regioisomers by high pressure liquid chromatography (HPLC) revealed no marked difference in the regioisomer pattern among the different treatments. After administration of griseofulvin, allylisopropylacetamide (AIA), or 1-[4-(3-acetyl-2,4,6-triemethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime (ATMP) to untreated and glutethimide-pretreated 18-day-old chick embryos, an N-alkylPP was isolated after AIA administration only. This finding strengthened previous reports of the species specificity of N-alkylPP formation with griseofulvin and ATMP. A series of dihydropyridines, namely 4-ethylDDC, 4-hexylDDC, and 4-isobutylDDC were administered to untreated and glutethimide-pretreated 18-day-old chick embryos and hepatic N-alkylPPs were isolated and separated by HPLC into regioisomers. The regioisomer patterns obtained did not support a previous proposal of masked regions above both rings B and C in the heme moieties of the P450 isozymes responsible for N-alkylPP formation. However, the data support the hypothesis of a partially masked region above ring B alone. The regioisomer patterns were in agreement with results previously obtained in rats showing that the percentage of Nc and (or) ND regioisomers in the regioisomer mixture increases as the length and bulk of the 4-alkyl substituent of a DDC analogue increase. Differences in the regioselectivity of heme N-alkylation may be due to intrinsic chemical features of DDC analogues themselves or to differences in the P450 isozymes inactivated.

Gender differences in N-alkyl protoporphyrin IX production in rats after the administration of porphyrinogenic xenobiotics.

The porphyrinogenicity of 3-[(arylthio)ethyl]sydnone (TTMS) and 3, 5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethylDDC) in rats is dependent on mechanism-based inactivation of selected isozymes of hepatic cytochrome P450 (P450), namely P4501A1/2, 2C6, 3A, and 2C11, followed by formation of ferrochelatase-inhibitory N-alkyl protoporphyrin IX (N-alkylPP). The objective of this study was to determine which P450 isozymes were sources of the N-alkylPPs. Previously, selective inhibition of male rat P4503A showed that it was the major source of N-vinylprotoporphyrin IX after TTMS administration. In the present study, when TTMS was administered to female rats, which lack P4503A2 and 2C11, N-vinylPP formation was 2.3% of that produced by males, which have both of these isozymes. Therefore, although P4503A2 is a major source, P4502C11 is also a significant source of N-vinylPP in males. Selective inhibition of P4503A and 1A1/2 did not decrease N-ethylPP formation in response to 4-ethylDDC administration to male rats, showing that P4503A and 1A1/2 were not sources of N-ethylPP. Thus P4502C6 and 2C11 were the remaining isozyme candidates to be investigated. When 4-ethylDDC was administered to female rats, N-ethylPP formation was 22% of that produced by males. Because female rat livers contain P4502C6 but lack the male specific P4502C11, the likely origin of N-ethylPP in females is P4502C6. Because males produced markedly more N-ethylPP than females, and males have P4502C11 in addition to P4502C6, we conclude that P4502C11 is the major source of N-ethylPP in males, whereas P4502C6 may also be a significant contributor.

cDNA-expressed human cytochrome P450 isozymes. Inactivation by porphyrinogenic xenobiotics.

A number of xenobiotics are known to exert their porphyrinogenic effects in rodents and chick embryos through mechanism-based inactivation of certain cytochrome P450 (P450) isozymes. To facilitate the extrapolation of results from test animals to humans, we have assessed the ability of three prototype porphyrinogenic compounds-namely, 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (DDEP), 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), and allylisopropylacetamide (AIA)-to cause mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, 2C9-Arg144 (2C9), 2D6-Val374 (2D6), and 3A4 in microsomes from human lymphoblastoid cell lines (Gentest Corp., Woburn, MA). The following catalytic markers of human P450 isozymes were used: ethoxyresorufin O-deethylase (P450s 1A1 and 1A2), diclofenac 4-hydroxylation (P4502C9), dextromethorphan O-demethylase (P4502D6), and testosterone 6 beta-hydroxylation (P4503A4). We found that DDEP and TTMS caused mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, and 3A4, whereas only DDEP was able to cause mechanism-based inactivation of cDNA-expressed human P4502C9; neither xenobiotic caused mechanism-based inactivation of cDNA-expressed human P4502D6. A comparison of the human P450 isozyme data with results previously obtained in rat and chick embryo liver showed a close correspondence between the results obtained with P450s 1A and 3A, but not the P4502C subfamily. Because several rat isozymes (P450s 2A1, 2B1, 2C6, 2C11, and 3A1) undergo inactivation by AIA, it was noteworthy that AIA did not inactivate any of the cDNA-expressed human P450 isozymes. Because mechanism-based inactivation of P450 isozymes is related to the porphyrinogenicity of xenobiotics, our results demonstrate the importance of supplementing studies of mechanism-based inactivation of P450 isozymes in animal models with similar studies on cDNA-expressed human P450 isozymes.

The apoprotein of cytochrome P450 3A2 does not selectively direct alkylation to one of four pyrrole rings of N-vinylprotoporphyrin IX.

A variety of porphyrinogenic xenobiotics cause mechanism-based inactivation of a number of rat hepatic cytochrome P450 isozymes. As a result, a mixture of four N-alkylprotoporphyin IX (N-alkylPP) regioisomers, with ferrochelatase-inhibitory properties, are formed. This study was designed to determine whether all four regioisomers are produced on the active site of a single P450 isozyme, or whether the apoprotein of individual P450 isozymes selectively directs the alkylation to one of four pyrrole nitrogen atoms of N-alkylPP. Administration of 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) to untreated and dexamethasone-pretreated rats results in the formation of four regioisomers of N-vinylprotoporphyrin IX (N-vinylPP). Following coadministration of TTMS with troleandomycin, N-vinylPP formation is reduced to 40 and 43% of control in untreated and dexamethasone-pretreated male rats. Since troleandomycin is a selective inhibitor of rat hepatic P450 3A, the difference in N-vinylPP regioisomer formation between control (TTMS alone) and TTMS plus troleandomycin coadministered rats represents the difference due to P450 3A. This allowed us to calculate that all four regioisomers of N-vinylPP are formed by P450 3A isozymes in both untreated and dexamethasone-pretreated male rats. Since untreated male rats express P450 3A2, but not P450 3A1, we conclude that the apoprotein of rat hepatic P450 3A2 does not selectively direct the alkylation to one of four pyrrole nitrogens of N-vinylPP.

Mechanism of glyceryl trinitrate-induced vasodilation. I. Relationship between drug biotransformation, tissue cyclic GMP elevation and relaxation of rabbit aorta.

This study was conducted to test the hypothesis that biotransformation of glyceryl trinitrate (GTN) is involved in GTN-induced relaxation of vascular smooth muscle. The temporal relationship between GTN biotransformation, elevation of cyclic GMP content and vasodilation in rabbit aortic strips (RAS) was determined. Isolated RAS were contracted submaximally with phenylephrine, and then were incubated with 0.62 microM [3H]GTN in a 30-sec time course study. GTN-induced relaxation (inhibition of phenylephrine-induced tone) of RAS was monitored; tissue cyclic GMP content was measured by radioimmunoassay; and GTN, glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN) concentrations in RAS were measured by thin-layer chromatography and liquid scintillation spectrometry. There was time-dependent biotransformation of GTN to GDN by the RAS and a time-dependent increase in cyclic GMP content in the RAS. Statistically significant (P less than .05) biotransformation of GTN and elevation of cyclic GMP content in the tissue occurred at 10 sec, whereas the onset of GTN-induced relaxation of RAS occurred at 12 sec. During the tissue biotransformation of GTN, there was preferential formation of 1,2-GDN compared with 1,3-GDN, with 1,2-GDN/1,3-GDN ratio of 3.0/1 at 10 sec, 5.3/1 at 20 sec and 5.8/1 at 30 sec. The results of this study are consistent with the hypothesis that GTN is a prodrug, such that biotransformation to an active metabolite is involved in GTN-induced relaxation of vascular smooth muscle. The data also indicate that the mechanisms of GTN biotransformation and GTN-induced activation of guanylate cyclase may be related intimately.

Endothelium-dependent relaxation of rabbit aorta by acetylcholine requires ethylenediaminetetraacetic acid.

Experiments were conducted to determine if ethylenediaminetetraacetic acid (EDTA) was essential for the acetylcholine (ACh)-induced relaxation of blood vessels. Isolated rabbit aortic rings were prepared for recording isometric tension. They were maintained in Krebs bicarbonate solution with various concentrations of EDTA. With EDTA concentrations of 0 or 0.003 mM, no ACh-induced relaxation was observed; only the contractile effect of ACh was seen. With 0.03 and 0.30 mM EDTA, ACh induced relaxation with EC50 values of 0.11 and 0.098 microM, respectively. Under the experimental conditions used, EDTA was essential for demonstration of ACh-induced relaxation.

Requirement for reduced, unliganded hemoprotein for the hemoglobin- and myoglobin-mediated biotransformation of glyceryl trinitrate.

The biotransformation of glyceryl trinitrate (GTN) by hemoglobin (Hb) and myoglobin (Mb) was assessed using solutions of various forms of the hemoproteins, viz., the oxy-, deoxy-, carbonmonoxy- and met-forms. After incubation with these, GTN loss was observed only with the deoxy-forms of Hb and Mb. The stoichiometry and products of [14C]GTN biotransformation by deoxy-Hb and deoxy-Mb were determined by measuring the formation of [14C]glyceryl dinitrate [14C]GDN), met-Hb (or met-Mb) and inorganic nitrite anion. Biotransformation of GTN involved the oxidation of 2 mol of heme iron (II) per mol of GTN biotransformed to GDN and inorganic nitrite anion. In addition to the formation of GDN, 1 to 2.5% of the radioactivity could not be extracted from the incubation samples. The ratio of 1,2-GDN/1,3-GDN formed during incubation of deoxy-Hb with GTN was 11:1, which indicated a high degree of regioselectivity for the denitration of the nitrate ester group in position 1 or position 3 of GTN. The metabolite ratio obtained for deoxy-Mb incubation with GTN (1,2-GDN/1,3-GDN,3:1) was less than that for deoxy-Hb, which indicated less regioselectivity for the deoxy-Mb-mediated denitration reaction. This could reflect differences in the topography of the heme pocket of the two hemoproteins and steric differences in the GTN-hemoprotein interaction.