Ultraviolet radiation and nanoparticle induced intracellular free radicals generation measured in human keratinocytes by electron paramagnetic resonance spectroscopy.

BACKGROUND: Several nanoparticle-based formulations used in cosmetics and dermatology are exposed to sunlight once applied to the skin. Therefore, it is important to study possible synergistic effects of nanoparticles and ultraviolet radiation. METHODS: Electron paramagnetic resonance spectroscopy (EPR) was used to detect intracellular free radicals induced by ultraviolet B (UVB) radiation and amorphous silica nanoparticle and to evaluate the influence of nanoparticle surface chemistry on particle cytotoxicity toward HaCaT cells. Uncoated titanium dioxide nanoparticles served as positive control. In addition, particle intracellular uptake, viability, and induction of interleukin-6 were measured. RESULTS: We found that photo-activated titanium dioxide particles induced a significant amount of intracellular free radicals. On the contrary, no intracellular free radicals were generated by the investigated silica nanoparticles in the dark as well as under UVB radiation. However, under UVB exposure, the non-functionalized silica nanoparticles altered the release of IL-6. At the same concentrations, the amino-functionalized silica nanoparticles had no influence on UVB-induced IL-6 release. CONCLUSION: EPR spectroscopy is a useful technique to measure nanoparticle-induced intracellular free radicals. Non-toxic concentrations of silica particles enhanced the toxicity of UVB radiation. This synergistic effect was not mediated by particle-generated free radicals and correlated with particle surface charge and intracellular distribution.

CycleGAN-Based Image to Image Translation for Realistic Surgical Training Phantoms.

Training in surgery is essential for surgeons to develop skill and dexterity. Physical training phantoms provide excellent haptic feedback and tissue properties for stitching and operating with authentic instruments and are easily available. However, they lack realistic traits and fail to reflect the complex environment of a surgical scene. Generative Adversarial Networks can be used for image-to-image translation, addressing the lack of realism in physical phantoms, by mapping patterns from the intraoperative domain onto the video stream captured during training with these surgical simulators. This work aims to achieve a successful I2I translation, from intra-operatory mitral valve surgery images onto a surgical simulator, using the CycleGAN model. Different experiments are performed - comparing the Mean Square Error Loss with the Binary Cross Entropy Loss; validating the Fréchet Inception Distance as a training and image quality metric; and studying the impact of input variability on the model performance. Differences between MSE and BCE are modest, with MSE being marginally more robust. The FID score proves to be very useful in identifying the best training epochs for the CycleGAN I2I translation architecture. Carefully selecting the input images can have a great impact in the end results. Using less style variability and input images with good feature details and clearly defined characteristics enables the network to achieve better results.Clinical Relevance- This work further contributes for the domain of realistic surgical training, successfully generating fake intra operatory images from a surgical simulator of the cardiac mitral valve.

In vivo methods for the analysis of the penetration of topically applied substances in and through the skin barrier.

The efficacy of a drug is characterized by its action mechanism and its ability to pass the skin barrier. In this article, different methods are discussed, which permit this penetration process to be analysed non-invasively. Providing qualitative and quantitative information, tape stripping is one of the oldest procedures for penetration studies. Although single cell layers of corneocytes are removed from the skin surface, this procedure is considered as non-invasive and is applicable exclusively to the stratum corneum. Recently, optical and spectroscopic methods have been used to investigate the penetration process. Fluorescence-labelled drugs can be easily detected in the skin by laser scanning microscopy. This method has the disadvantage that the dye labelling changes the molecular structures of the drug and consequently might influence the penetration properties. The penetration process of non-fluorescent substances can be analysed by Raman spectroscopy, electron paramagnetic resonance, CARS and multiphoton microscopic measurements. Using these methods, the concentration of the topically applied formulations in different depths of the stratum corneum can be detected by moving the laser focus from the skin surface deeper into the stratum corneum. The advantages and disadvantages of these methods will be discussed in this article.

Stabilization of reactive nitroxides using invasomes to allow prolonged electron paramagnetic resonance measurements.

The detection of the antioxidative capacity of the skin is of great practical relevance since free radicals are involved in many skin damaging processes, including aging and inflammation. The nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl) in combination with electron paramagnetic resonance spectroscopy was found suitable for measuring the antioxidative capacity since its reaction with reducing agents is considerably fast. Yet, in order to achieve longer measurement times, e.g. in inflammatory skin diseases, the stabilizing effect of an invasome (ultraflexible vesicle/liposome) suspension with TEMPO was investigated ex vivo on porcine skin and in vivo on human skin. Invasomes increased the measurement time ex vivo 2-fold and the reduction was significantly slowed down in vivo, which is due to membrane-associated and therefore protected TEMPO. Furthermore, TEMPO accumulation in the membrane phase as well as the decreasing polarity of the ultimate surroundings of TEMPO during skin penetration explains the stabilizing effect. Thus, an invasome suspension with TEMPO exhibits stabilizing effects ex vivo and in vivo.

Radical production by infrared A irradiation in human tissue.

The influence of the ultraviolet (UV) irradiation of the sun on the formation of free radicals in human skin is well investigated. Up to now, only small amounts of data are available stating that infrared (IR) irradiation can produce free radicals in the skin. In the present study, the formation of free radicals in human skin, subsequent to IRA irradiation (600-1,500 nm), has been demonstrated by means of two different methods. Firstly, the radical formation was detected indirectly by the degradation of the cutaneous carotenoid antioxidants beta-carotene and lycopene, which was investigated in vivo by resonance Raman spectroscopic measurements. Secondly, the direct observation of produced radicals subsequent to IRA irradiation of the skin was performed in vitro by electron paramagnetic resonance spectroscopy. Taking into account the results of the present study and previous UV light studies, it can be expected that also solar irradiation in the visible spectral range will produce free radicals in the human skin. Therefore, the current sun protection strategies should be reconsidered. Furthermore, it was shown in the present study that the side effect in the form of radical formation could be significantly reduced by increasing the protection system of the human organism in form of the antioxidant network.

Comparative study of carotenoids, catalase and radical formation in human and animal skin.

Animal skin is widely used in dermatological free radical research. Porcine ear skin is a well-studied substitute for human skin. The use of bovine udder skin is rare but its high carotenoid content makes it particularly appropriate for studying the redox state of the skin. Yet, information on the suitability of animal skin for the study of external hazard effects on the redox state of human skin has been lacking. In this study, we investigated the activity of the antioxidant enzyme catalase and the carotenoid content defining the redox status as well as UV-induced radical formation of human, porcine ear and bovine udder skin ex vivo. In human skin only low levels of radical formation were detected following UV irradiation, whereas bovine skin contains the highest amount of carotenoids but the lowest amount of catalase. Porcine ear skin does not exhibit a carotenoid signal but its catalase activity is close to human skin. Therefore, radical formation can neither be correlated to the amount of catalase nor to the amount of carotenoids in the skin. All skin types can be used for electron paramagnetic resonance-based detection of radicals, but porcine skin was found to be the most suitable type.

Muscarinic receptors mediate stimulation of collagen synthesis in human lung fibroblasts.

Clinical observations indicate that in chronic obstructive pulmonary disease patients, the long-acting muscarinic antagonist tiotropium delays decline in airway function, suggesting that cholinergic mechanisms contribute to long-term structural changes. Human lung fibroblasts express muscarinic receptors and the present study aimed to explore their role in controlling collagen synthesis. MRC-5, HEL-299 and primary human lung fibroblasts (phLFb) were cultured. Incorporation of [(3)H]-proline into cellular proteins was determined as measure of collagen synthesis. In MRC-5 cells, the muscarinic agonist carbachol enhanced [(3)H]-proline incorporation in a concentration-dependent manner (effective concentration of 50%: 220 nM, increase at 10 microM by 40-55%, in a different series of experiments). Likewise, 10 microM oxotremorine caused an increase of approximately 65%. For comparison, transforming growth factor-beta1 (5 ng x mL(-1)) caused an increase of approximately 80%. Effects of carbachol on total [(3)H]-proline incorporation and collagenase-sensitive [(3)H]-proline fraction were similar. The effect of 10 microM carbachol was inhibited by tiotropium (inhibitory concentration of 50%: 110 pM), prevented by pertussis toxin and the mitogen-activated protein kinase inhibitor, PD 98059. Muscarinic agonists also enhanced [(3)H]-proline incorporation in a tiotropium-sensitive manner in HEL-299 cells and phLFb. In human lung fibroblasts, muscarinic receptors exert stimulatory effects on collagen synthesis. Prolonged blockade of muscarinic-induced collagen synthesis may contribute to reported beneficial long-term effects of anticholinergics in chronic obstructive pulmonary disease.

Role of Epac1 in mediating anti-proliferative effects of prostanoid EP(2) receptors and cAMP in human lung fibroblasts.

In lung fibroblasts, proliferation is inhibited by activation of EP(2) prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP(2) receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [(3)H]-thymidine incorporation. The selective EP(2) receptor agonist butaprost inhibited [(3)H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [(3)H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [(3)H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP(2) receptor activation.

Disturbed in vitro adrenergic modulation of cytokine production in inflammatory bowel diseases in remission.

OBJECTIVE: Psychological stress has been implicated in the pathophysiology of both inflammatory and functional gastrointestinal (GI) diseases. The goal of this study was to address neuroendocrine modulation of cytokine production by peripheral blood cells in GI diseases. METHODS: We analyzed the in vitro effects of the beta-adrenergic agonist terbutaline and the glucocorticoid agonist dexamethasone on TNF-alpha and IL-10 production by LPS-stimulated monocytes in whole cell blood cultures in patients with inflammatory bowel diseases in remission (N=10), diarrhoea-predominant irritable bowel syndrome (IBS, N=12), patients with a recent gastroenteritis (post-infectious group, N=10), and healthy controls (N=15). RESULTS: In response to terbutaline, there was a significant increase in IL-10 production (concentration effect: p<0.05), which was diminished in IBD (group effect: p<0.01), comparable in IBS and controls, but enhanced in the post-infectious group (group x concentration effect: p<0.05). In contrast, terbutaline resulted in a concentration-dependent suppression of TNF-alpha production, which was comparable in all groups. Dexamethasone suppressed TNF-alpha production in a dose-dependent manner in all groups, but this effect was significantly more pronounced in post-infectious subjects (group effect: p<0.05). CONCLUSIONS: In IBD, disturbed adrenergic regulation of IL-10 could be part of the mechanism(s) underlying the modulation of disease activity by psychological stress. Diarrhoea-predominant IBS was not associated with altered adrenergic or glucocorticoid regulation of cytokine production by peripheral blood cells, whereas a recent history of gastroenteritis was associated with disturbed neuroendocrine modulation of cytokine production, which may play role in the pathophysiology of post-infectious IBS.

Is there a benefit from intensified medical and psychological interventions in patients with functional dyspepsia not responding to conventional therapy?

AIM: In a prospective randomized, controlled trial, to compare the long-term outcome of intensive medical therapy (with or without cognitive-behavioural or muscle relaxation therapy) vs. standard medical therapy in patients with refractory functional dyspepsia (FD), referred to a tertiary referral medical center. METHODS: A total of 100 consecutive FD patients were allocated to a standardized symptom-oriented 4 month therapy (SMT, n = 24), intensive medical therapy (IMT, medical therapy with testing-for and targeting-of abnormalities of motor-and-sensory function, n = 28) or IMT plus psychological interventions (either progressive-muscle relaxation (IMT-MR, n = 20) or cognitive-behavioural therapy (IMT-CBT, n = 28). The symptom intensity (SI) and health-related quality-of-life (HRQoL) after 12 months were prespecified primary outcome parameters. RESULTS: After 12 months, significantly greater improvement of SI occurred in patients with IMT-all (with or without psychological interventions) compared with SMT (P < 0.025 vs. IMT-all). IMT, IMT-MR and IMT-CBT alone also resulted in significantly better improvement of the primary outcome parameters (P all < 0.025 vs. SMT). HRQoL significantly improved in all groups with intensive medical therapy but not standard medical therapy. Differences between intensive medical therapy-all and standard medical therapy were not significant. Concomitant anxiety and depression was improved significantly by IMT-CBT (vs. SMT) but not other treatments. CONCLUSIONS: In FD patients with refractory symptoms, intensified medical management involving function testing and psychological intervention yields superior long-term-outcomes. Additional CBT may be effective for the control of concomitant anxiety and depression.

Neuroendocrine and blood pressure responses to rectal distensions in individuals with high and low visceral pain sensitivity.

BACKGROUND: The mechanisms of interindividual variations in visceral pain sensitivity remain poorly understood. We characterized the neuroendocrine responses to rectal distensions in healthy individuals with high vs. low rectal pain sensitivity. METHODS: Rectal sensory and pain thresholds were determined, and a series of random painful distensions was carried out. Eighteen subjects were stratified into groups with a low rectal pain threshold ("High Sensitivity" group) vs. a high rectal pain threshold ("Low Sensitivity" group) by median split, and were compared with regard to adrenocorticotropic hormone (ACTH) and cortisol, cardiovascular, and emotional responses. RESULTS: Distensions led to an anticipatory stress response, reflected by elevated baseline anxiety, and increased baseline ACTH and cortisol in both groups. In response to distensions, the "Low Sensitivity" group showed significantly greater ACTH and cortisol concentrations analysis of variance (ANOVA time x group for ACTH: p<.05; for cortisol: p<.01), and elevated diastolic blood pressures (BP) (ANOVA group: p<.01) when compared to the "High Sensitivity" group. CONCLUSIONS: Painful rectal distensions are associated with a pronounced anticipatory stress response, reflected by elevated anxiety and elevated stress hormones. Individuals with high rectal pain sensitivity differ from those with low pain sensitivity in distension-induced hormonal and blood pressure responses, suggesting that neuroendocrine responses may be relevant to the pathophysiology of visceral hyperalgesia.

Investigation of cutaneous penetration properties of stearic acid loaded to dendritic core-multi-shell (CMS) nanocarriers.

Dendritic core-multi shell (CMS) particles are polymer based systems consisting of a dendritic polar polyglycerol polymer core surrounded by a two-layer shell of nonpolar C18 alkyl chains and hydrophilic polyethylene glycol. Belonging to nanotransport systems (NTS) they allow the transport and storage of molecules with different chemical characters. Their amphipihilic character CMS-NTS permits good solubility in aqueous and organic solutions. We showed by multifrequency electron paramagnetic resonance (EPR) spectroscopy that spin-labeled 5-doxyl stearic acid (5DSA) can be loaded into the CMS-NTS. Furthermore, the release of 5DSA from the carrier into the stratum corneum of porcine skin was monitored ex vivo by EPR spectroscopy. Additionally, the penetration of the CMS-NTS into the skin was analyzed by fluorescence microscopy using indocarbocyanine (ICC) covalently bound to the nanocarrier. Thereby, no transport into the viable skin was observed, whereas the CMS-NTS had penetrated into the hair follicles down to a depth of 340 µm ± 82 µm. Thus, it could be shown that the combined application of fluorescence microscopy and multi-frequency EPR spectroscopy can be an efficient tool for investigating the loading of spin labeled drugs to nanocarrier systems, drug release and penetration into the skin as well as the localization of the NTS in the skin.

Depolarization of the liver cell membrane by metformin.

Metformin (1,1-dimethylbiguanide; MET) is used in the treatment of type 2 diabetes mellitus. MET's antihyperglycemic action depends at least in part on its inhibitory effect on hepatic gluconeogenesis. As to gluconeogenesis from amino acids (e.g. L-alanine), this is associated with an inhibition of L-alanine uptake into hepatocytes. Since this uptake is mediated by an electrogenic transport mechanism, the aim of the present study was to investigate whether MET has an influence on the liver cell membrane potential which might explain its inhibitory effect on L-alanine uptake. The experiments were performed in vivo in anesthetized rats and in vitro using superfused mouse liver slices with the conventional microelectrode technique. In vivo, MET (160 mg/kg intraperitoneally (i.p.)) significantly depolarized (dV) the liver cell membrane by 6 mV. MET (1 mmol/l) also depolarized the liver cell membrane in vitro (e.g. 15 min after start of superfusion: dV=8 mV). MET's effect was at least partly reversible. Glucagon (10(-7) mol/l), which hyperpolarized the liver cell membrane, abolished MET's effect. Further, the MET-induced depolarization was completely absent during superfusion with low Cl(-) ([Cl(-)]=27 mmol/l) medium, and significantly attenuated by the Cl(-) channel blocker NPPB (25 micromol/l). While MET's effect was only somewhat attenuated by blockade of the Na(+)/K(+)/2Cl(-) cotransporter or by superfusion with (HCO(-)(3)-free) HEPES buffer, the carboanhydrase blocker acetazolamide (1 mmol/l) or blockade of the HCO(-)(3)/Cl(-) exchanger by DIDS (100 micromol/l), which, however, also blocks Cl(-) channels, abolished its effect. The depolarization of the liver cell membrane by MET was unaffected by a blockade of K(+) channels with Ba(2+), a blockade of the Na(+)/K(+) pump or superfusion with low Na(+) medium ([Na(+)]=26 mmol/l). According to these results, the MET-induced depolarization of the liver cell membrane could be due to an activation of the Cl(-)/HCO(-)(3) exchanger and thus depend on intracellular HCO(-)(3) formation. This activation could then lead to a disturbance of the equilibrium between intra- and extracellular Cl(-) and therefore to an enhanced Cl(-) efflux via Cl(-) channels. It is plausible that the depolarizing effect induced by MET is associated with its inhibitory effect on gluconeogenesis by inhibiting uptake of L-alanine and other amino acids into hepatocytes.

[Assessment among German urologists of various conservative treatment modalities for Peyronie's disease. Results of a survey]. / Einschätzung der verschiedenen konservativen Therapieverfahren der Induratio penis plastica unter deutschen Urologen. Ergebnisse einer Umfrage.

BACKGROUND: The aim of this study was to evaluate the frequency and the assessment of the different conservative modalities of treatment in Peyronie's disease. A representative survey among 3187 German urologists was performed using a standardized questionnaire comprising currently used concepts of therapy, their efficacy, and their tolerability. MATERIAL AND METHODS: A total of 636 urologists participated in the study. Altogether they had treated 6019 patients with Peyronie's disease in 2003. The majority of urologists treated between 3 and 15 patients per year. The most frequent treatment modality was the administration of potassium paraaminobenzoate, followed by vitamin E and extracorporeal shock wave therapy. Other oral drugs and intralesional drug administrations were used considerably less frequently. RESULTS: The most commonly used treatment modalities were assessed for the best results in terms of efficacy and tolerability. However, this outcome is contradictory to the few controlled studies regarding the efficacy of the different drugs. CONCLUSIONS: The large number of patients treated demonstrates the importance of conservative therapy for Peyronie's disease. Therefore, it is unfortunate that no conservative treatment modality is currently available that cures the symptoms of this disorder in all patients affected.

Efficacy of artichoke leaf extract in the treatment of patients with functional dyspepsia: a six-week placebo-controlled, double-blind, multicentre trial.

BACKGROUND: This study aimed to assess the efficacy of artichoke leaf extract (ALE) in the treatment of patients with functional dyspepsia (FD). METHODS: In a double-blind, randomized controlled trial (RCT), 247 patients with functional dyspepsia were recruited and treated with either a commercial ALE preparation (2 x 320 mg plant extract t.d.s.) or a placebo. The primary efficacy variable was the sum score of the patient's weekly rating of the overall change in dyspeptic symptoms (four-point scale). Secondary variables were the scores of each dyspeptic symptom and the quality of life (QOL) as assessed by the Nepean Dyspepsia Index (NDI). RESULTS: Two hundred and forty-seven patients were enrolled, and data from 244 patients (129 active treatment, 115 placebo) were suitable for inclusion in the statistical analysis (intention-to-treat). The overall symptom improvement over the 6 weeks of treatment was significantly greater with ALE than with the placebo (8.3 +/- 4.6, vs. 6.7 +/- 4.8, P < 0.01). Similarly, patients treated with ALE showed significantly greater improvement in the global quality-of-life scores (NDI) compared with the placebo-treated patients (- 41.1 +/- 47.6 vs. - 24.8 +/- 35.6, P < 0.01). CONCLUSION: The ALE preparation tested was significantly better than the placebo in alleviating symptoms and improving the disease-specific quality of life in patients with functional dyspepsia.

Differential effects of acetone or Aroclor 1254 pretreatment on the microsomal activation of dimethylnitrosamine to a mutagen.

Pretreatment of mice with acetone enhances the microsomal N-demethylation of dimethylnitrosamine (DMN) at low substrate concentrations (< 5 mM), while pretreatment with Aroclor 1254 represses this activity at low, but enhances it at high (> 35 mM) DMN concentrations. To relate the activity of DMN demethylase with the mutagenicity of DMN, liver microsomes were isolated aseptically from mice 18 h after acetone (3 ml/kg, ip), 5 days after Aroclor 1254 treatment (500 mg/kg, ip), or after treatment with the appropriate injection vehicles, and incubated with S. typhimurium (TA 1535), NADPH and DMN (1, 3 or 70 mM) for 5 to 60 min. After a 48-h incubation on minimal media, revertants per plate were determined. Microsomes from acetone pretreated mice bioactivated DMN to a mutagen at significantly higher (p < 0.001) levels when incubations were performed at 1 mM DMN. Aroclor-1254 microsomes exhibited a decreased ability to convert DMN to a mutagen at both 1 and 3 mM DMN (p < 0.05) and a significantly higher (p < 0.05) ability at 70 mM DMN. These data and published reports suggest multiple microsomal enzymes for DMN bioactivation and that acetone may enhance the enzyme that operates at environmentally important levels of DMN.

[New Insights into the Etiological Pathogenesis of Peyronie's Disease]. / Neue Aspekte zur Atiopathogenese der Induratio penis plastica.

This paper reviews the current knowledge of the etiological pathogenesis of Peyronie's disease. De la Peyronie himself supposed a connection with venereal diseases. Herein, infectious, traumatic, autoimmune and genetic causes are discussed. An important hypothesis is that the recurrent microtraumatisation of the tunica albuginea during sexual intercourse leads to small lesions that activate processes of wound healing and development of fibrotic plaque. According to recent data, an association with the antigens of the HLA-system could be ruled out. Transforming growth factor beta (TGF-beta) seems to have an important impact due to its increased expression in the plaque. Furthermore it is possible to induce a condition similar to Peyronie's disease by intratunical injection of cytomodulin - a substance with TGF-beta-like activity - in an animal model. As in other fibrotic diseases, an imbalance between nitric oxide (NO) and reactive oxygen species (ROS) seems to be of importance. The most important new insights were gained from cell-culture models in which increased expression of basic fibroblast growth factor (bFGF), as well as a change in cell cycle regulation (p53) and cytogenetic instability were observed.