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.

Regional and age-specific patterns of pandemic H1N1 influenza virus seroprevalence inferred from vaccine clinical trials, August-October 2009.

The extent of the H1N1 pandemic has been estimated from case counts and deaths but the proportion of exposed populations with inapparent infections has not been described in detail. We analysed haemagglutination-inhibition (HI) antibody titres of pre-vaccination sera from pandemic vaccine trials conducted in six countries on four continents to provide an indication of A/CA/07/2009(H1N1)-like influenza seroprevalence in those populations. Among 7,962 subjects, ranging in age from 12 months to over 60 years, the proportions with HI antibody titres > or =40 to the H1N1pnd virus in the period from August to October 2009 were, by country: Costa Rica 26.4%, United States (US) 22.5%, Switzerland 16.9%, Germany 12.6%, Belgium 10.1%, and Japan 5.9%. Age-specific seropositivity rates in the samples were higher in children and adolescents in Costa Rica and in the US than in Europe and in Japan. The low proportion of seropositive children in Europe and Japan suggests that little local viral transmission had occurred in those regions even as late as September and October 2009, while in the US and Costa Rica, the greater proportion of previously infected children and young adults suggested that a significant number of asymptomatic infections had occurred during the first pandemic wave. Nevertheless, in all locations, the majority of the population remained susceptible to the pandemic virus at the beginning of the influenza season in the northern hemisphere, justifying the implementation of public health interventions.

The missing link--light-induced (280-1,600 nm) free radical formation in human skin.

The recent European recommendation on the efficacy of sunscreen products requests now a minimum ratio of UVA/UVB protection. However, the visible and the infrared (IR) parts of the sun spectrum have received little attention concerning their possible contribution to skin damage. A common biophysical answer for the different wavelengths of the sun spectrum can be found in the creation of excess free radicals - mainly reactive oxygen species (ROS). Thanks to electron spin resonance spectroscopy applied to skin biopsies, we determined for the first time the free radical action spectrum covering UV and visible light (280-700 nm). Convolution of the action spectrum with sunlight spectral irradiance showed that 50% of the total skin oxidative burden was generated by visible light. Creation of ROS by visible light was experimentally confirmed by varying the illuminance of a spotlight. We also evidenced the creation of excess free radicals by near-IR radiation. In that case, free radical generation does not depend exclusively on the dose, but also on the skin temperature increase initiated by near-IR light. Some phenomena which are still unclear, such as the question about the deleterious or beneficial role of sunlight, are reviewed, implying the research on new protection strategies for the prevention of skin cancer.

[UV, visible and infrared light. Which wavelengths produce oxidative stress in human skin?]. / UV, sichtbares Licht, Infrarot. Welche Wellenlängen produzieren oxidativen Stress in menschlicher Haut?

Experimental evidence suggests that the creation of free radicals--mainly reactive oxygen species (ROS)--is the common photobiological answer to the skin-sunlight interaction. The free radical action spectrum (wavelength dependency) for ultraviolet and visible light (280-700 nm) has been determined by quantitative ESR spectroscopy. Visible light produces around 50% of the total oxidative stress caused by sunlight. Reactive species like *O(-)(2), *OH and *CHR are generated by visible light. The amount of ROS correlates with the visible light intensity (illuminance). We demonstrated the creation of excess free radicals by near-infrared light (NIR, 700-1600 nm). Free radical generation does not depend exclusively on the NIR irradiance, but also on the NIR initiated skin temperature increase. The temperature dependence follows the physiological fever curve. Our results indicate that the complex biological system skin creates the same type of free radicals over the entire active solar spectrum. This general response will make it possible to define the beneficial or deleterious action of sunlight on human skin by introduction of a free radical threshold value.

Electron paramagnetic resonance (EPR) imaging in skin: biophysical and biochemical microscopy.

Electron paramagnetic resonance (EPR) is a spectroscopic technique that allows detection of paramagnetic compounds, e.g., free radicals, in skin, and is used for measuring skin membrane fluidity and polarity. EPR imaging is concerned with spatially resolved EPR spectroscopy. We studied EPR images at X-band frequency (9 GHz) in the skin biopsies of hairless mice. Our particular imaging technique utilized a modulated field gradient to obtain cross-sectional images perpendicular to the skin surface. Employing nitroxide free radicals, this approach allows analysis of skin biophysical and biochemical features at the micrometer resolution level. By spin labeling drugs, pharmacokinetic properties of the labeled compound can be monitored in skin. We suggest that EPR imaging has a broad application potential in dermatologic research. In comparison to other spatially visualizing techniques, such as nuclear magnetic resonance (NMR) imaging and ultrasound, EPR imaging has significant advantages, such as high spatial resolution and providing specific biochemical and biophysical information that cannot be obtained by other methods in skin.

Cutaneous tolerance to nitroxide free radicals in human skin.

No data are available on the irritant effect of nitroxide free radicals in human skin. Nitroxides are important biomedical skin probes used in Electron Paramagnetic Resonance spectroscopy and imaging. Our purpose was to study the skin irritation potential of different nitroxide free radical structures in skin of healthy human subjects. We investigated the following nitroxides: Tempo (2,2,6,6-tetramethyl-1-piperidinoxy), Doxo (2,2,5,5-tetramethyl-3-oxazolidinoxy), Proxo (2,2,5,5-tetramethyl- -dihydro-pyrrolinoxy), and Imidazo (2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl). Cutaneous irritation was determined in human skin following a single application and after repetitive applications in comparison to the standardized irritant sodium lauryl sulfate (SLS). The response was evaluated clinically as well as by a bioengineering method analyzing transepidermal water loss (TEWL) and skin hydration (capacitance). The nitroxides were classified clinically from nonirritant (Imidazo, Proxo), to slightly irritant (Doxo, 100 mM), or moderately irritant (Tempo 100 mM) after a single application. The TEWL values were significantly increased by Doxo and Tempo, but capacitance values were not changed significantly. In the cumulative irritation test Tempo was scored as a slight irritant (10 mM). TOLH (2,2,6,6-tetramethyl-1-hydroxypiperidin), the hydroxylamine of Tempo, which is the major skin metabolite, did not cause skin irritation after a single or repetitive applications. This may indicate that a loss of cellular reducing equivalents may be involved in the inflammation process caused by Tempo. The order of nitroxide irritation potency (Tempo > Doxo >> Imidazo = Proxo) is inverse to the order of nitroxide biostability in human skin (Imidazo = Proxo >> Doxo > Tempo). In conclusion, nitroxide free radicals are classified as nonirritant to moderately irritant in human skin. Particularly, the pyrrolidine and imidazoline type nitroxides have a low potential to cause acute or subacute skin toxicity.

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides.

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).

Electron paramagnetic resonance studies on nitroxide radical 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) redox reactions in human skin.

Electron paramagnetic resonance (EPR) is currently being explored for the study of living biological systems. Among biophysical and biochemical applications, the study of nitroxide radical interactions with tissue antioxidants and oxidants is of growing interest. Skin is a target organ of the EPR methodology and is frequently exposed to oxidative stress. We investigated the piperidine-type nitroxide 2,2,5,5-tetramethyl-4-piperidin-1-oxyl (TEMPO) because it is skin permeable and readily accepts electrons in biological systems. TEMPO is readily scavenged on the surface of cultured human skin. Pretreatment of skin cultures with butylhydroperoxide, which decreases intracellular ascorbate and glutathione, causes inhibition of nitroxide scavenging. Exposure of skin cultures to dehydroascorbate, which is internalized and converted to ascorbate, leads to stimulation of nitroxide scavenging. In human keratinocytes and fibroblasts, the TEMPO radical is reversibly reduced to the hydroxylamine depending on the oxygen concentration and the availability of intracellular glutathione and ascorbate. Cell exposure to the glutathione synthetase inhibitor buthionine-sulfoximine depleted intracellular glutathione and inhibited nitroxide reduction; exposure to dehydroascorbate or glutathione-monoethylester increased intracellular ascorbate or glutathione concentration and stimulated nitroxide reduction. Quantitative considerations indicate that the major reduction site of TEMPO in skin and skin cells is the cytosol ascorbate/glutathione redox cycle. We suggest that analysis of TEMPO radical scavenging by the EPR technique is a convenient method for measuring skin ascorbate and thiol-dependent antioxidant activity in vitro and in vivo.

In vitro and in vivo assessment of the irritation potential of different spin traps in human skin.

No clinical data are available on the acute cutaneous toxicity of spin traps which are frequently used in combination with the electron paramagnetic resonance (EPR) technique for detection of free radicals and reactive oxygen/nitrogen species. The purpose of this study was to evaluate the acute dermatotoxicity of the following spin traps in human skin: C-phenyl-N-tert.-butyl nitrone (PBN), C-(4-pyridinyl-N-oxide)-N-tert.-butylnitrone (POBN), 5, 5-dimethyl-l-pyrroline-N-oxide(DMPO), 5 diethoxyphosphoryl-5-methyl-l-pyrroline-N-oxide (DEPMPO), diethyldithiocarbamate (DDC) and N-methyl-D-glucamine dithiocarbamate (MGD). The corrosivity of the test substances was first assessed in human skin in vitro by measurement of transcutaneous electrical resistance (TER). In this assay all spin traps were non-corrosive at 500 mM concentration. Subsequently cutaneous irritation of the spin traps was determined at different concentrations (50, 250 and 500 mM) in human skin according to a routine four h human patch test in comparison to the standardized irritant sodium laurylsulfate (SLS, 20%). The response was evaluated clinically as well as by a biophysical method analyzing transepidermal water loss (TEWL). PBN and DEPMPO caused a transient and weak inflammatory reaction at 500 mM in four of 17 and in two of 17 volunteers, respectively. DMPO, POBN, DDC, MGID, and the iron complexes of DDC and MGD were clinically non-irritant at all concentrations tested and no delayed-acute inflammatory reactions were observed. However, the TEWL values were significantly increased by all spin traps except DMPO at 500 mM, indicating disturbed epidermal barrier function. We conclude that the spin traps investigated have a low potential to cause acute skin toxicity and may be used safely for in vivo EPR studies in human skin.

Cutaneous tolerance to nitroxide free radicals and nitrone spin traps in the guinea pig.

The attempts to use nitroxide free radicals and nitrone spin traps topically in skin requires analysis of their potential cutaneous adverse effects. The objective of this study was to investigate the skin irritation and sensitizing potential of nitroxides and nitrones in the guinea pig. The following unsubstituted nitroxides were investigated: 2,2,6,6-tetramethyl-1-piperidinoxyl (Tempo), 2,2, 5,5-tetramethyl-3-oxazolidinoxyl (Doxo), 2,2,5,5-tetramethyl-1-dihydro-pyrrolinoxyl (Proxo), 2,2,3,4,5,5-hexamethyl-imidazoline-1-yloxyl (Imidazo) and the nitrones: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert.-butyl-phenylnitrone (PBN). Cutaneous irritation was determined following the modified Draize protocol. The response was evaluated clinically as well as by a biophysical method analyzing transepidermal water loss (TEWL). The nitroxides and nitrones were classified clinically from non-irritant (Proxo, Imidazo, DMPO) to slightly irritant (Tempo, Doxo, PBN) according to the Draize protocol. In agreement with the clinical scoring, the TEWL values were significantly increased by Tempo, Doxo and PBN. TOLH, the hydroxylamine of Tempo and its major skin metabolite, did not cause skin irritation. The sensitizing effect was evaluated according to the Magnusson and Kligman test. The results showed no cutaneous hypersensitivity to all nitroxides and nitrones, indicating a weak sensitizing potential. That concludes that the nitroxides and nitrones tested in this study have a low potential of acute skin intolerance.

SURF_ER--surface electron spin resonance (ESR) of the surface domain of large objects.

SURF_ER is a method for spectral and spatial electron spin resonance measurements on the surface of large objects which extension is only restricted by the width of the pole gap of the magnet and the homogeneity of the magnetic field and not by the cavity dimensions. The application of several techniques like SURF_ER for spectroscopic measurements, SURF_ERM for spatial scanning and SURF_ERI for spatial measurements of the depth of the surface region are discussed and represented for the skin of a human being as an example.

Electron paramagnetic resonance for everybody--MICROspec-X--a new class of electron paramagnetic resonance spectrometer.

The electron paramagnetic resonance spectroscopy is the only method for detecting free radicals. Free radicals have an increased importance in our daily life. A small transportable EPR spectrometer is presented for the popularisation of the EPR method. The technical construction and some applications are illustrated which show the usability of the spectrometer.

[Development and validation of a questionnaire measuring hospital consumer satisfaction ]. / Sviluppo e validazione di un questionario di rilevazione della soddisfazione dell'utenza ospedaliera.

This study describes the process of development of a new instrument for measuring patient satisfaction with hospital care in Italy. The self-administered questionnaire included a total of 48 items, -contributing to the construction of 8 scales, each of them describing a specific area of interest such as medical care and nursing, several aspects of organisation, comfort and overall outcome of care. Psychometric characteristics of the questionnaire were in terms of acceptability, validity, reliability and the ability to discriminate different levels of satisfaction in various patient groups. Psychometric analyses resulted in a convincing construct validity and reliability, as described by the Cronbach Alpha coefficient estimates [range 0.73-0.95]. The high compliance obtained (67.3%) can be considered a sign of good acceptability of the questionnaire and of its mode of administration. These analyses demonstrate a good performance of the questionnaire in terms of both validity and reliability, suggesting that this instrument can represent a solid base for future applications.

Influence of dietary carotenoids on radical scavenging capacity of the skin and skin lipids.

Nutrition rich in carotenoids is well known to prevent cell damage, premature skin aging, and skin cancer. Cutaneous carotenoids can be enriched in the skin by nutrition and topically applied antioxidants have shown an increase in radical protection after VIS/NIR irradiation. In this paper, it was investigated whether orally administered carotenoids increase the radical scavenging activity and the radical protection of the skin using in vivo electron paramagnetic resonance spectroscopy and the skin lipid profile was investigated applying HPTLC on skin lipid extracts. Furthermore, in vivo Raman resonance spectroscopy was used to measure the cutaneous carotenoid concentration. A double blind placebo controlled clinical study was performed with 24 healthy volunteers, who have shown a slow but significant and effective increase in cutaneous carotenoids in the verum group. The enhancement in carotenoids increases the radical scavenging activity of the skin and provides a significant protection against stress induced radical formation. Furthermore, the skin lipids in the verum group increased compared to the placebo group but only significantly for ceramide [NS]. These results indicate that a supplementation with dietary products containing carotenoids in physiological concentrations can protect the skin against reactive oxygen species and could avoid premature skin aging and other radical associated skin diseases.

Encapsulated curcumin results in prolonged curcumin activity in vitro and radical scavenging activity ex vivo on skin after UVB-irradiation.

The phytochemical curcumin possesses antioxidant activity; however, it becomes unstable after being exposed to light or heat or loses activity during storage. This is especially important when curcumin is applied to the skin within a cosmetic or pharmaceutical formulation, since sun exposure is unavoidable. This drawback can be directly addressed by encapsulation of curcumin in photo-stable nanospheres. Therefore, curcumin was encapsulated into nanoparticles consisting of ethyl cellulose and/or methyl cellulose. Nanoparticles were subjected to processing conditions commonly used in industry, for example, temperature and pressure and thus retained their morphology. Furthermore, sun exposure resulted in the protection of curcumin by nanoparticles, whereas non-encapsulated curcumin degraded completely. Determination of the radical protection factor resulted in similar antioxidant activity of encapsulated and non-encapsulated curcumin indicating that curcumin maintains its antioxidant activity. Application of lotions containing curcumin or curcumin nanoparticles to the skin and subsequent UVB-irradiation resulted in less radical formation compared to lotion application only. Moreover, radical formation was even less after nanoparticle application compared to free curcumin. Nanoencapsulation protects curcumin from photo degradation and can therefore prolong the antioxidant activity of curcumin.

Associations of the correlates of protection and implication on the statistical power for demonstrating non-inferiority: application of a re-sampling method on a large phase III influenza vaccine clinical trial.

BACKGROUND: In the later stage of the clinical development of new vaccines it is required to demonstrate their efficacy with the immunogenicity measures established as correlates for disease protection. Evaluation of interpandemic trivalent influenza vaccines is commonly assessed by three immunogenicity measures for each strain in different age groups: Seroprotection, Seroconversion and Geometric Mean Titers. US and European guidelines with respect to this topic have been issued for the licensure of new influenza vaccines. The statistical power of comparative trials, which consider these endpoint variables, could be affected to the extent that these measures are correlated. Results from a large non-inferiority trial in the clinical development of a novel cell-derived influenza vaccine have been analyzed with the aim of evaluating how statistical dependency between the above-mentioned three immunogenicity measures might affect the power to demonstrate non-inferiority. METHODS: The statistical non-inferiority criteria, which were met in the trial, were applied to different subsets (n=250, n=370 and n=500) using a re-sampling method from the original dataset (re-samples=10,000). RESULTS: The measures of immunogenicity were highly correlated, and the fulfillment or failure of any of the non-inferiority criteria for a specific measure partially predicted the same outcome for the other measures. Due to this dependency within each strain, the levels of power obtained by re-sampling methods were always higher than those obtained by theoretical calculations, which were based on the assumptions of independency between the three measures of immunogenicity. Seroconversion and Geometric Mean Ratio (GMR) showed a higher correlation. A failure in the fulfillment of the non-inferiority criteria for GMR predicted the failure for Seroconversion in >76% of cases. CONCLUSIONS: The correlation between different measures of immunogenicity should be taken into account when evaluating statistical power for non-inferiority in influenza vaccine trials and in establishing sample sizes. Statistical approaches that include either all three measures of immunogenicity or both Seroconversion and the ratio of GMTs as co-primary non-inferiority endpoints might create redundancy and could increase the probability of not meeting at least one non-inferiority criterion by chance, due to multiplicity.

Safety, tolerability and immunogenicity of a mammalian cell-culture-derived influenza vaccine: a sequential Phase I and Phase II clinical trial.

This sequential, observer-blind, randomised, single-centre, combined Phase I and Phase II clinical trial compared the tolerability and immunogenicity of a single intramuscular dose of a novel cell-culture-derived influenza vaccine (CCIV), produced in Madin-Darby canine kidney cells, with a conventional egg-based vaccine. The immunogenicity of both vaccines was assessed by SRH assay, a well-recognized test by EMEA, in compliance with the requirements of the EU Committee for Medicinal Products for Human Use (CHMP). The Phase I part of the trial comprised 40 healthy adults (18-40 years of age); the subsequent Phase II part involved 200 healthy adult (n=80, 18-60 years of age) and elderly (n=120, > or =61 years of age) subjects. Both vaccines showed similar reactogenicity and any solicited local or systemic reactions were mostly mild or moderate. Regarding immunogenicity, both the CCIV and the control vaccine met all of the EU Committee for Medicinal Products for Human Use criteria for influenza vaccines for each strain and in both age groups. In conclusion, the CCIV produced in mammalian cell-culture is as well tolerated and as immunogenic as the control egg-based vaccine in non-elderly and elderly adults.