Alexa Patzelt

Abstract: It has recently been demonstrated by the utilization of different techniques, such as differential stripping and laser scanning microscopy, that the hair follicles represent an efficient long-term reservoir for topically applied substances. In the present paper, the different in vivo techniques are compared to those of in vitro analysis. It was found that in vitro measurements on pig ear skin are highly superior for the analysis of follicular penetration, as compared to in vitro investigations on excised human skin, mainly due to the fact that the human skin contracts after removal. Restretching of the skin to its original size mainly stretches the interfollicular fibres, whereas the fibres around the hair follicles remain contracted. In contrast to excised human skin, pig ear tissue does not contract when the cartilage is not removed. Moreover, it has an intact barrier on both sides of the ear. Regardless of the fact that the hair follicles on pig ear skin are larger than those of humans, the porcine ear skin represents a more suitable in vitro model for the analysis of the penetration and storage of topically applied substances in the hair follicles than excised human skin.

Abstract: In the past, it was assumed that the intercellular route was the only relevant penetration pathway for topically applied substances. Recent results on follicular penetration emphasize that the hair follicles represent a highly relevant and efficient penetration pathway and reservoir for topically applied substances. This study investigates a selective closure technique of hair follicle orifices in vivo assessing interfollicular and follicular absorption rates of topical minoxidil foam in humans. In delimited skin area, single hair orifices or interfollicular skin were blocked with a microdrop of special varnish-wax-mixture in vivo. Minoxidil foam (5%) was topically applied, and transcutaneous absorption was measured by a new surface ionization mass spectrometry technique in serum. Different settings (open, closed or none of both) enabled to clearly distinguish between interfollicular and follicular penetration of the topically applied minoxidil foam. Five minutes after topical application, minoxidil was detected in blood samples when follicles remained open, whereas with closed follicles 30min were needed. Highest levels were found first when both pathways were open, followed by open follicles and subsequently by closed follicles. These results demonstrate the high importance of the follicular penetration pathway. Hair follicles are surrounded by a dense network of blood capillaries and dendritic cells and have stem cells in their immediate vicinity, making them ideal targets for drug delivery.

Abstract: The Organization for Economic Cooperation and Development (OECD) recommends caffeine as a reference substance for in vitro skin absorption tests using Franz diffusion cells (FDC). However, it has not been possible to investigate the follicular penetration pathway using this method until now. The aim of this study was to develop a technique to allow the examination of the follicular penetration pathway of a substance penetrating into the skin. The OECD standard method was therefore combined with the follicle closing technique (FCT), an established in vivo method. By using test skin of varying follicular densities, different penetration values were obtained for the test substance caffeine. The follicular penetration rate was determined by an indirect calculation after modifying the in vivo FCT for use in the in vitro FDC. This method is the first to allow the differentiation of penetration pathways by combining the OECD standard method (using the FDC) and the FCT. Caffeine showed a surprisingly high rate of penetration through the follicular shunts in vitro.

Abstract: An efficient penetration and long-term storage of topically applied substances is important for drug delivery in medical treatment and cosmetics. It has recently become apparent that the hair follicles represent an efficient and long-term reservoir for topically applied substances. It was found that particles sized 300-600 nm penetrate more efficiently into the hair follicles than smaller or larger particles. In the present paper, the hair surface structure of human and porcine hairs was analyzed by electron microscopy. It could be observed that the thickness of the cuticula corresponds to the optimal size of the nanoparticles for penetration into the hair follicles. Additionally, it could be demonstrated that the cuticula of human vellus and terminal hairs were of similar thickness (approx. 530 nm), while the thickness of the cuticula obtained from porcine ear bristles were slightly thinner (approx. 320 nm).

Abstract: The universal sun protection factor (USPF) characterizing sunscreen efficacy based on spectroscopically determined data, which were obtained using the tape stripping procedure. The USPF takes into account the complete ultraviolet (UV) spectral range in contrast to the classical sun protection factor (SPF). Until now, the USPF determination has been evaluated only in human skin. However, investigating new filters not yet licensed excludes in vivo investigation on human skin but requires the utilization of a suitable skin model. The penetration behavior and the protection efficacy of 10 commercial sunscreens characterized by USPF were investigated, comparing human and porcine skin. The penetration behavior found for typical UV filter substances is nearly identical for both skin types. The comparison of the USPF obtained for human and porcine skin results in a linear relation between both USPF values with a correlation factor R(2)=0.98. The results demonstrate the possibility for the use of porcine skin to determine the protection efficacy of sunscreens.

Abstract: Liposomes in the nanosize range have been recognized as a versatile drug delivery system of both hydrophilic and lipophilic molecules. In order to develop a liposome-based topical vaccination strategy, five different types of liposomes were tested as a putative vaccine delivery system on pig ear skin. The investigated liposomes mainly varied in size, lipid composition, and surface charge. Using hydrophilic and hydrophobic fluorescent dyes as model drugs, penetration behavior was studied by means of confocal laser scanning microscopy of intact skin and histological sections, respectively. Follicular penetration of the liposomes was measured in comparison to a standard, nonliposomal formulation at different time points. Dependent on time but independent of their different characters, the liposomes showed a significantly higher penetration depth into the hair follicles compared to the standard formulation. The standard formulation reached a relative penetration depth of 30% of the full hair follicle length after seven days, whereas amphoteric and cationic liposomes had reached approximately 70%. Penetration depth of negatively charged liposomes did not exceed 50% of the total follicle length. The fluorescence dyes were mainly detected in the hair follicle; only a small amount of dye was found in the upper parts of the epidermis.

Abstract: BACKGROUND: Lateralized processing of speech is a well studied phenomenon in humans. Both anatomical and neurophysiological studies support the view that nonhuman primates and other animal species also reveal hemispheric differences in areas involved in sound processing. In recent years, an increasing number of studies on a range of taxa have employed an orienting paradigm to investigate lateralized acoustic processing. In this paradigm, sounds are played directly from behind and the direction of turn is recorded. This assay rests on the assumption that a hemispheric asymmetry in processing is coupled to an orienting bias towards the contralateral side. To examine this largely untested assumption, speech stimuli as well as artificial sounds were presented to 224 right-handed human subjects shopping in supermarkets in Germany and in the UK. To verify the lateralized processing of the speech stimuli, we additionally assessed the brain activation in response to presentation of the different stimuli using functional magnetic resonance imaging (fMRI). RESULTS: In the naturalistic behavioural experiments, there was no difference in orienting behaviour in relation to the stimulus material (speech, artificial sounds). Contrary to our predictions, subjects revealed a significant left bias, irrespective of the sound category. This left bias was slightly but not significantly stronger in German subjects. The fMRI experiments confirmed that the speech stimuli evoked a significant left lateralized activation in BA44 compared to the artificial sounds. CONCLUSION: These findings suggest that in adult humans, orienting biases are not necessarily coupled with lateralized processing of acoustic stimuli. Our results -- as well as the inconsistent orienting biases found in different animal species -- suggest that the orienting assay should be used with caution. Apparently, attention biases, experience, and experimental conditions may all affect head turning responses. Because of the complexity of the interaction of factors, the use of the orienting assay to determine lateralized processing of sound stimuli is discouraged.

Abstract: The important role of hair follicles as penetration pathways and reservoir structures for topically applied compounds has been validated in numerous animal models as well as in humans. Follicular penetration rates are modulated by regional variations in size and proportions and the functional status. Advances have especially been made in the targeting of hair follicle-associated cell populations including antigen-presenting cells and stem cells. Improved investigative methods based on differential stripping, spectrophotometry and confocal laser scanning microscopy have led to the determination of the penetration profiles and kinetics for a multiplicity of drugs and drug delivery systems. The observation that particulate delivery systems aggregate and remain in hair follicle openings and their penetration along the follicular duct occurs in a size-dependent manner, which has led to advanced concepts of targeted drug delivery of bioactive compounds in the field of solid particles, as well as semi-solid particles, such as liposomes. This review summarizes the recent progress in this field, and underlines the necessity for pilot studies in human volunteers to further the development of clinical applications for follicular targeting.

Abstract: Skin aging is mainly caused by the destructive action of free radicals, produced by the UV light of the sun. The human skin has developed a protection system against these highly reactive molecules in the form of the antioxidative potential. Carotenoids are one of the main components of the antioxidants of the human skin. From former studies, it is known that skin aging is reduced in individuals with high levels of carotenoids. Because most of the antioxidants cannot be produced by the human organism, they must be up taken by nutrition. Using noninvasive Raman spectroscopic measurements it is demonstrated that not only fruits and vegetables but also eggs contain high concentrations of antioxidants including carotenoids, which are even doubled in the case of ecological eggs. After a 1-week diet with ecological eggs performed by six volunteers, it is found that the concentration of the carotenoids in the skin of the volunteers increased by approx. 20%. Our study does not intend to recommend exorbitant egg consumption, as eggs also contain harmful cholesterol. But in the case of egg consumption, ecological eggs from hens kept on pasture should be preferred to also receive a benefit for the skin.

Abstract: Nanoparticles are experiencing an increasing application in dermatology and cosmetics. In both application areas, the requirements of nanoparticles are in most cases widely different. As a component of sunscreens, the nanoparticles are supposed to remain on the skin surface or in the upper most layers of the stratum corneum to protect the skin against UV-radiation of the sun. Whereas, on the other hand, when particulate substances are used as carrier systems for drugs, they have to cross the skin barrier to reach the target sites within the living tissue. We discuss the perspectives and risks of the topical application of nanoparticles.

Abstract: Regardless of the fact that several highly efficient antiseptics are commercially available, the antiseptic treatment of chronic wounds remains a problem. In the past, electrical plasma discharges have been frequently used in biometrical science for disinfection and sterilization of material surfaces. Plasma systems usually have a temperature of several hundred degrees. Recently, it was reported that "cold" plasma can be applied onto living tissue. In in vitro studies on cell culture, it could be demonstrated that this new plasma possesses excellent antiseptic properties. We perform a risk assessment concerning the in vivo application of a "cold" plasma jet on patients and volunteers. Two potential risk factors, UV radiation and temperature, are evaluated. We show that the UV radiation of the plasma in the used system is an order of magnitude lower than the minimal erythema dose, necessary to produce sunburn on the skin in vivo. Additionally, thermal damage of the tissue by the plasma can be excluded. The results of the risk assessment stimulate the in vivo application of the investigated plasma jet in the treatment of chronic wounds.

Abstract: BACKGROUND: Anthracyclines, such as pegylated liposomal doxorubicin (PLD) and epirubicin (EP), are effective for the treatment of malignant tumors. Unfortunately, their implementation in therapy is limited due to severe side-effects such as palmar-plantar erythrodysesthesia (PPE). PATIENTS AND METHODS: As the exact pathogenesis of PPE still remains unclear, laser scanning microscopy was utilized to detect PLD, EP and their metabolites in and on the skin surface of patients. RESULTS: It was shown that PLD was significantly more frequently detectable on the skin than was EP (p<0.05), whereas both substances were most frequently seen in the palms and soles. Additionally, it has been visualized that the substances reach the skin surface via sweat, where they distribute and then penetrate back into the skin. CONCLUSION: It was concluded that a high density of sweat glands and a thick stratum corneum might represent important predestined factors for the development of PPE. These findings will help to develop efficient prevention and therapy strategies for PPE.

Abstract: AIMS: Due to ethical reasons, in vivo penetration studies are not applicable at all stages of development of new substances. Therefore, the development of appropriate in vitro methods is essential, as well as the comparison of the obtained in vivo and in vitro data, in order to identify their transferability. The aim of the present study was to investigate the follicular penetration of caffeine in vitro and to compare the data with the in vivo results determined previously under similar conditions. METHODS: The Follicular Closing Technique (FCT) represents a method to investigate the follicular penetration selectively. In the present study, FCT was combined with the Franz diffusion cell in order to differentiate between follicular and intercellular penetration of caffeine into the receptor medium in vitro. Subsequently, the results were compared with the data obtained in an earlier study investigating follicular and intercellular penetration of caffeine in vivo. RESULTS: The comparison of the data revealed that the in vitro experiments were valuable for the investigation of the follicular penetration pathway, which contributed in vivo as well as in vitro to approximately 50% of the total penetration, whereas the kinetics of caffeine penetration were shown to be significantly different. CONCLUSIONS: The combination of FCT with the Franz diffusion cell represents a valuable method to investigate follicular penetration in vitro. Nevertheless, in vivo experiments should not be abandoned as in vitro, structural changes of skin occur and blood flow and metabolism are absent, probably accounting for reduced penetration rates in vitro.

Abstract: Epidermal wound healing is a complex and dynamic regenerative process necessary to reestablish skin integrity. Fluorescence confocal laser scanning microscopy (FLSM) is a noninvasive imaging technique that has previously been used for evaluation of inflammatory and neoplastic skin disorders in vivo and at high resolution. We employed FLSM to investigate the evolution of epidermal wound healing noninvasively over time and in vivo. Two suction blisters were induced on the volar forearms of the study participants, followed by removal of the epidermis. To study the impact of wound ointment on the process of reepithelization, test sites were divided into two groups, of which one test site was left untreated as a negative control. FLSM was used for serial/consecutive evaluations up to 8 days. FLSM was able to visualize the development of thin keratinocyte layers developing near the wound edge and around hair follicles until the entire epidermis has been reestablished. Wounds treated with the wound ointment were found to heal significantly faster than untreated wounds. This technique allows monitoring of the kinetics of wound healing noninvasively and over time, while offering new insights into the potential effects of topically applied drugs on the process of tissue repair.

Abstract: BACKGROUND: The cutaneous antioxidants form an efficient protection system against the destructive potential of free radicals, produced by environmental factors, such as UV-sun irradiation, hazardous substances and lifestyle habits. Most of the antioxidants cannot be produced by the human organism. Thus, they have to be incorporated by food and beverages. MATERIAL AND METHODS: In the present manuscript, the distribution of carotenoids as a marker for antioxidative potential in human skin was investigated with two different in vivo Raman spectroscopy methods with an excitation wavelength of 785 nm (Skin Analyzer) and at 488 nm (resonance Raman spectroscopy). The carotenoid profile was assessed at three different anatomical locations (palm, forehead and volar forearm) in 12 healthy volunteers. RESULTS: In untreated skin, the major fraction of the carotenoids is located in the upper part of the stratum corneum (SC). The amount of carotenoid is lower in the upper part of the SC on the forearm compared to forehead and palm shown with both methods. Both methods detect similar distinction patterns of carotenoid levels for the three anatomical locations. CONCLUSION: The present study supports the hypothesis that antioxidative substances; here carotenoids, are secreted via eccrine sweat glands and/or sebaceous glands to the skin surface. Raman spectroscopic methods are an efficient tool to analyze the distribution of carotenoids in the human skin over time and with the Skin Analyzer over different layers of the epidermis. Resonance Raman spectroscopy is suited to analyze deeper parts of the skin.

Abstract: The penetration of topically applied substances in and through the human skin is of special interest for the development and optimization of topically applied drugs and cosmetic products. In the present study, the efficacy of barrier emulsions in the prevention of the penetration of pollen allergens into the hair follicles was investigated. Because of the sensitising potential of the used pollen allergens, the study was carried out under in vitro conditions. Therefore, excised human skin and porcine ear skin were used as tissue models. Applying laser scanning microscopy and fluorescent labeled grass pollen allergens, we found that the preventive efficacy of the barrier emulsions could be significantly better investigated on porcine ear skin than on excised human skin. This might be due to the contraction of the elastic fibres around the hair follicles in excised human skin after its removal. In contrast to the excised human skin, the porcine ear skin remains on the cartilage during the experiment. Therefore, contraction of the tissue can be avoided. The results give further indication that in vitro studies based on membranes of excised skin are not suitable for the investigation of the follicular penetration pathway of topically applied substances.

Abstract: The penetration of topically applied substances in and through the human skin is of special interest for the development and optimization of topically applied drugs and cosmetic products. In the present study, the efficacy of barrier emulsions in the prevention of the penetration of pollen allergens into the hair follicles was investigated. Because of the sensitising potential of the used pollen allergens, the study was carried out under in vitro conditions. Therefore, excised human skin and porcine ear skin were used as tissue models. Applying laser-scanning microscopy and fluorescent-labeled grass pollen allergens, we found that the preventive efficacy of the barrier emulsions could be significantly better investigated on porcine ear skin than on excised human skin. This might be due to the contraction of the elastic fibres around the hair follicles in excised human skin after its removal. In contrast to the excised human skin, the porcine ear skin remains on the cartilage during the experiment. Therefore, contraction of the tissue can be avoided. The results give further indication that in vitro studies based on membranes of excised skin are not suitable for the investigation of the follicular penetration pathway of topically applied substances.

Abstract: Antioxidant substances in the skin are expected to slow down photo ageing. We therefore developed the hypothesis that high levels of antioxidant substances may be correlated to lower levels of skin roughness. By utilizing modern optical non-invasive in vivo methods, the structures of the furrows and wrinkles as well as the concentration of lycopene were analyzed quantitatively on the forehead skin of 20 volunteers aged between 40 and 50 years. In a first step, the age of the volunteers was correlated to their skin roughness. Here, no significant correlation was found. In a second step, a significant correlation was obtained between the skin roughness and the lycopene concentration (R=0.843). These findings indicate that higher levels of antioxidants in the skin effectively lead to lower levels of skin roughness, and therefore support our hypothesis.

Abstract: WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * In recent years, it has been suggested that hair follicles represent important shunt routes into the skin for drugs and chemicals [1-3]. * In vitro studies have shown the importance of skin appendages for skin penetration by hydrophilic compounds [4]. Investigation of follicular penetration in vivo has been difficult due to the absence of appropriate analytical methods or suitable animal model systems. * Recently, a new method was described that quantifies follicular penetration in vivo by using selective closure of hair follicles [5]. * Caffeine is frequently used in skin penetration experiments as a model for highly water-soluble compounds. Occlusion [6] and skin thickness [7] seem to have little influence on the penetration of caffeine. However, percutaneous absorption rates for caffeine exhibit regional skin differences in humans in vivo[1]. WHAT THIS STUDY ADDS: * The results of the present study demonstrate that a fast drug delivery of caffeine occurs through shunt routes. Therefore, hair follicles are considerable weak spots in our protective sheath against penetration into the body by hydrophilic substances. * We showed that there is a quantitative distinction between follicular penetration and interfollicular diffusion of caffeine in vivo. * These findings are of importance for the development and optimization of topically applied drugs and cosmetics. In addition, such properties must be considered in the development of skin protection measures. AIMS: The skin and its appendages are our protective shield against the environment and are necessary for the maintenance of homeostasis. Hypotheses concerning the penetration of substances into the skin have assumed diffusion through the lipid domains of the stratum corneum. It is believed that while hair follicles represent a weakness in the shield, they play a subordinate role in the percutaneous penetration processes. Previous investigation of follicular penetration has mostly addressed methodical and technical problems. Our study utilized a selective closure technique of hair follicle orifices in vivo, for the comparison of interfollicular and follicular absorption rates of caffeine in humans. METHODS: Every single hair follicle within a delimited area of skin was blocked with a microdrop of a special varnish-wax-mixture in vivo. Caffeine in solution was topically applied and transcutaneous absorption into the blood was measured by a new surface ionization mass spectrometry (SI/MS) technique, which enabled a clear distinction to be made between interfollicular and follicular penetration of a topically applied substance. RESULTS: Caffeine (3.75 ng ml(-1)) was detected in blood samples, 5 min after topical application, when the follicles remained open. When the follicles were blocked, caffeine was detectable after 20 min (2.45 ng ml(-1)). Highest values (11.75 ng caffeine ml(-1)) were found 1 h after application when the follicles were open. CONCLUSIONS: Our findings demonstrate that hair follicles are considerable weak spots in our protective sheath against certain hydrophilic drugs and may allow a fast delivery of topically applied substances.

Abstract: Although in vitro skin absorption studies often detect small residues of applied test material in the epidermis/dermis, it is uncertain whether the residue is within the living skin. We studied the dermal absorption of a hair dye hydroxyanthraquinone-aminopropyl methyl morpholinium methosulphate (HAM) in human skin in vivo and in vitro. In vivo, skin (back and scalp) received 0.5% HAM in a commercial formulation at 20microg/cm2 After 0.5 or 48h, skin was tape stripped, followed by cyanoacrylate biopsies (CAB). Sebum from scalp sites was collected for 48h. In vitro, skin was treated with 20mg/cm2 dye for 0.5h, penetration determined after 24h. In vivo, at 0.5h, total recovery (back) was 0.67microg/cm2 (tape strips+CAB). Fluorescence microscopy showed HAM in the hair follicle openings (HFO). At 0.5h, scalp tape strips contained 1.80microg/cm2, HFO 0.82microg/cm2. At 48h, HFO contained 0.21microg/cm2, sebum 0.80microg/cm2. In vivo, skin residues were in the non-living skin and eliminated via desquamation and sebum secretion. In vitro, the SC contained 1.50microg/cm2, epidermis/dermis 0.86microg/cm2, receptor fluid<0.04microg/cm2, a total of 0.90microg/cm2 was considered to be bioavailable. In vitro epidermis/dermis residues were nearly identical to those located in non-living skin in vivo. In conclusion, in vitro percutaneous penetration studies may produce seemingly bioavailable material , which raises the need for a Threshold of Skin Absorption (TSA) addressing a negligible dermal absorption in order to avoid unnecessary in vivo toxicity studies on substances that produce no significant human systemic exposure.

Abstract: The analysis of wound healing is important for the therapy control and for the development of drugs stimulating the healing process. Wounds cause damage to the skin barrier. A damaged stratum corneum leads to an increased water loss through the skin barrier. The standard measuring procedure for characterization of wound healing is the measurement of transepidermal water loss (TEWL). The disadvantage of this method is that it can be easily disturbed by the perspiration of the volunteers and by topically applied substances, for instance wound healing creams. In the study presented, in vivo laser scanning microscopy and optical coherent tomography were compared concerning the application for their analysis of wound healing processes. The laser scanning microscopy allows the analysis of the healing process on a cellular level. The course of wound healing determined by laser scanning microscopy was correlated with numerical values, allowing the numerical characterization of the wound healing process.

Abstract: In the past, it was assumed that the intercellular route was the only relevant penetration pathway for topically applied substances. Recent results on follicular penetration obtained at the Center for Experimental and Applied Cutaneous Physiology, Charité - Universitätsmedizin Berlin, Germany, emphasize that the hair follicles represent a highly relevant and efficient penetration pathway and reservoir for topically applied substances.

Abstract: Penetration studies are commonly performed on in vitro models, presumably due to a lack of non-invasive in vivo methods. To date, it is not clear whether in vitro models are suitable to reflect the in vivo conditions for percutaneous penetration. Apart from inter and intraspecies skin differences, the excision of a skin sample may influence the penetration rate inter alia as a result of the contraction of the elastic fibres in the skin during excision. Therefore, the aim of the present study was to investigate the follicular reservoir of the hair follicles of human skin in vivo and in vitro utilizing the method of differential stripping. The results obtained revealed a significantly reduced follicular reservoir in vitro, which was only 9.5+/-10.6% of the in vivo reservoir. These results are important for the interpretation of earlier and future penetration investigations. It can thus be assumed that excised skin models are suitable for penetration studies only to a limited extent, as follicular penetration is greatly diminished due to the contraction of the elastic fibres of the skin.

Abstract: Variation in the level of the carotenoid antioxidant substances beta-carotene and lycopene in the human skin of ten healthy volunteers was measured with resonance Raman spectroscopy in an in vivo experiment over the course of 12 months. Information on the lifestyle of the volunteers concerning dietary supplementation and stress factors was obtained daily by the completion of questionnaires. The results showed individual variations in the levels of carotenoid antioxidant substances in the skin of the volunteers, which strongly correlated to specific lifestyles, such as the intake of dietary supplementations rich in carotenoids, and the influence of stress factors. A carotenoid-rich nutrition, based on large amounts of fruit and vegetables, increased the measured carotenoid levels of skin, while stress factors such as fatigue, illness, smoking, and alcohol consumption gave rise to a decrease in carotenoid levels of the skin. These decreases occurred relatively quickly over the course of one day, while the subsequent increases lasted for up to 3 days. During the summer and autumn months, an increase in the level of carotenoids in the skin was measured for all volunteers. The average "seasonal increase" of the carotenoid content in the skin was determined to be 1.26-fold.

Abstract: Eighty percent of hair follicles are in the growing phase. They grow approximately 0.3 mm/day. The hair follicles are surrounded by a close network of capillaries, which supplies them with nutrients. It is well known that substances which influence the metabolic processes of humans also influence hair growth. Steroids, which are used for doping in sport, are among these substances. In the present paper, optical coherent tomography is used for the analysis of changes in the hair structure during the application of steroids for the treatment of patients suffering from auto-immune diseases. Significant differences in the hair cross section could be detected during treatment, while the shape of the hairs was not influenced. It could be demonstrated that optical coherence tomography is a suitable, non-invasive and low-cost measuring technique that can be applied for doping control and screening. As a result of this screening process, only those athletes who show abnormalities in hair parameters would need to be investigated by classical analytical methods. The results presented in this study are not only important for doping controls, but also for several clinical applications, such as therapy and compliance control in cases where the applied substances induce changes in the hair structure.

Abstract: The uppermost layer of the skin--the stratum corneum--represents a barrier of the human organism to the environment. It prevents the penetration of substances coming into contact with the skin into the human body. In cases of strong exposure, the skin has to be protected additionally by barrier creams. In the present study, the influence of microparticles on the homogeneity of distribution of the protection cream on the skin was investigated by laser scanning microscopy. The protection cream contained antioxidant substances with a high radical protection factor for the prevention of palmar-plantar erythema often occurring during chemotherapy with doxorubicin. In this case, the chemotherapeutic substance comes out with the sweat onto the skin surface, from which it penetrates into the skin like topically applied. It was found that particles increase the homogeneity of distribution and, thus, the protection efficacy is significantly increased. The obtained results are important, not only for the prevention of side effects during chemotherapy, but also for the development and application of sunscreens and all types of topically applied drugs and cosmetics, which should form an efficient protection film on the skin.

Abstract: Carcinogenesis is a dynamic and stepwise process, which is accompanied by a variety of somatic and epigenetic alterations in response to a changing microenvironment. Hypoxic conditions will select for cells that have adjusted their metabolic profile and can maintain proliferation by successfully competing for scarce nutritional and oxygen resources. In the present study we have investigated the effects of energy depletion in the context of HPV (human papillomavirus)-induced pathogenesis. We show that cervical carcinoma cell lines are susceptible to undergoing either growth arrest or cell death under conditions of metabolic stress induced by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), a known activator of the AMPK (AMP-activated protein kinase). Our results reveal that AICAR treatment leads to a reduced binding affinity of the transcription factor AP-1 (activator protein-1) and in turn to a selective suppression of HPV transcription. Moreover, the outcome of AICAR on proliferation and survival was dependent on p53 activation and the presence of LKB1, the major upstream kinase of AMPK. Using non-malignant LKB1-expressing somatic cell hybrids, which lose expression after tumorigenic segregation, as well as small interfering RNA LKB1 knockdown approaches, we could further demonstrate that expression of LKB1 protects cells from cytotoxicity induced by agents which modulate the ATP/AMP ratio. Since simulation of low energy status can selectively eradicate LKB1-negative cervical carcinoma cells, AICAR may represent a novel drug in the treatment of cervical cancer.

Abstract: Atopic dermatitis is a chronic remittent skin disease. In the extrinsic form of atopic dermatitis, type IgE-mediated reactions play an important pathophysiological role. The aim of the present study was to examine whether type I allergens can penetrate into the skin. Therefore, pollen proteins were labeled with fluorescein isothiocyanate (FITC), and their penetration profile was studied qualitatively. Solutions of FITC-labeled pollen proteins were applied in vitro on porcine skin and in vivo on human skin. In vitro, the FITC-labeled proteins were observed within the complete stratum corneum (SC) and inside the hair follicles even 15 min after application. They were also distributed inside the dermis around the hair follicles. In vivo, a similar pattern of distribution within the SC and the hair follicles was observed. These results indicate penetration via the SC lipid layers and a faster penetration via the hair follicles. The FITC-labeled proteins entered the dermis via the follicular pathway. Therefore, the follicular penetration should be considered in the development of skin protection strategies. To evaluate such strategies, the developed method can be used, and further studies in atopic dermatitis patients are necessary to determine whether the penetration of type I allergens is increased.

Abstract: PURPOSE: To examine the retinal blood flow in normal eyes before and during retinal stimulation by flickering light. DESIGN: A prospective cross-sectional study. PARTICIPANTS AND TESTING: Twenty-seven eyes of 27 normal subjects with a mean age +/- SD of 38 +/- 15 years (study I) and 21 eyes of 21 normal subjects with a mean age +/- SD of 46 +/- 17 years (study II) were examined with respect to capillary retinal blood flow and central retinal artery and central retinal vein blood flow velocities during flickering light stimulation. A luminance flicker light with a frequency of 8 Hz increased the neuronal activity of retinal ganglion cells. In study I, the retinal capillary blood flow was measured before and during flickering by scanning laser Doppler flowmetry (670 nm, Heidelberg Retina Flowmeter). In study II, the blood flow velocities in the central retinal artery and central retinal vein were examined by pulsed Doppler sonography. MAIN OUTCOME MEASURES: Change in blood flow velocities in the central retinal artery and vein and in retinal capillary blood flow after full-field flicker stimulation. RESULTS: In study I, measurements of blood flow during retinal flicker stimulation showed a significant increase in the mean value of blood flow +/- SD from 317 +/- 72 arbitrary units to 416 +/- 103 arbitrary units. The change was on average 46 +/- 19%. In study II, the systolic and end-diastolic blood flow velocities in the central retinal artery increased significantly (P < 0.0001): systolic, 9 cm/s to 15 cm/s (+62%); end-diastolic, 2.7 cm/s to 5.3 cm/s (+96%). In the central retinal vein, the systolic and end-diastolic blood flow velocities increased significantly (P < 0.0001): systolic, 4.3 cm/s to 6.7 cm/s (+56%); end-diastolic, 1.8 cm/s to 3.6 cm/s (+100%). The authors found no significant change in blood pressure and heartbeat frequency. CONCLUSIONS: Visual stimulation of the retina by flickering light strongly increased the juxtapapillary retinal capillary blood flow and central retinal artery blood flow velocity in normal eyes.

Abstract: The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Abstract: The glycolytic pathway inhibitor 2-deoxyglucose (2-DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non-tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down-regulation is selective, since other reference genes are not affected or even up-regulated under the same experimental conditions. Moreover, 2-DG appears to restore the normal half-life of the tumor suppressor gene product p53, because the protein is strongly up-regulated after HPV 18 E6/E7 suppression. The observed 2-DG-effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV 18 suppression by 2-DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB-8, indicating that Ca2+, a known intracellular "second messenger", is involved in this process. Elevated c-myc and p53 expression appears to be responsible for the time-dependent accumulation of apoptotic cells after prolonged 2-DG treatment. The finding that 2-DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a peculiarity of growing tumors, but even may be an essential prerequisite for the maintenance of virus-specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2-DG or other glucose derivatives in the treatment of precancerous and malignant HPV-associated lesions.