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PubMed2000

Nonparametric tests for stratum effects in the Cox model.

This paper considers the stratified proportional hazards model with a focus on the assessment of stratum effects. The assessment of such effects is often of interest, for example, in clinical trials. In this case, two relevant tests are the test of stratum interaction with covariates and the test of stratum interaction with baseline hazard functions. For the test of stratum interaction with covariates, one can use the partial likelihood method (Kalbfleisch and Prentice, 1980; Lin, 1994). For the test of stratum interaction with baseline hazard functions, however, there seems to be no formal test available. We consider this problem and propose a class of nonparametric tests. The asymptotic distributions of the tests are derived using the martingale theory. The proposed tests can also be used for survival comparisons which need to be adjusted for covariate effects. The method is illustrated with data from a lung cancer clinical trial.

Humans↗
PubMed2003

Unexpected clobetasol propionate profile in human stratum corneum after topical application in vitro.

PURPOSE: The validity of using drug amount-depth profiles in stratum corneum to predict uptake of clobetasol propionate into stratum corneum and its transport into deeper skin layers was investigated. METHODS: In vitro diffusion experiments through human epidermis were carried out using Franz-type glass diffusion cells. A saturated solution of clobetasol propionate in 20% (V/V) aqueous propylene glycol was topically applied for 48 h. Steady state flux was calculated from the cumulative amount of drug permeated vs. time profile. Epidermal partitioning was conducted by applying a saturated drug solution to both sides of the epidermis and allowing time to equilibrate. The tape stripping technique was used to define drug concentration-depth profiles in stratum corneum for both the diffusion and equilibrium experiments. RESULTS: The concentration-depth profile of clobetasol propionate in stratum corneum for the diffusion experiment is biphasic. A logarithmic decline of the drug concentration over the first four to five tape strips flattens to a relatively constant low concentration level in deeper layers. The drug concentration-depth profile for the equilibrium studies displays a similar shape. CONCLUSIONS: The shape of the concentration-depth profile of clobetasol propionate is mainly because of the variable partitioning coefficient in different stratum corneum layers.

Administration, Topical↗
PubMed2001

Physical and physiological effects of stratum corneum tape stripping.

BACKGROUND/AIMS: Tape stripping of human stratum corneum has been performed to measure stratum corneum mass, barrier function, drug reservoir and percutaneous penetration. However, the technique itself requires further development to facilitate interpretation. METHODS: In this study we quantified stratum comeum (SC) tape stripping and water kinetic parameters utilizing three types of adhesive tapes, in an in vivo randomized clinical trial. Stratum corneum was tape stripped, and the mass of SC removed by each tape was quantified utilizing a protein assay. Transepidermal water loss (TEWL) was measured and barrier disruption and SC water kinetics calculated. Three commonly utilized acrylate adhesive tapes were utilized and a comparison made between them. RESULTS: Each type of tape successfully stripped the stratum corneum, but the rayon tape did not induce SC barrier disruption. Neither the type of tape nor the site stripped significantly influenced the mass of SC removed. Water kinetic parameters did not differ significantly for the tapes that did induce barrier disruption. Individual variation in barrier disruption to water following tape stripping was demonstrated. CONCLUSION: The tapes utilized removed a similar amount of SC. The tapes have a different propensity to cause barrier disruption. Some individuals do not demonstrate increased TEWL despite an equivalent mass of SC being removed compared to those who do show a response.

Adhesives↗
PubMed1998

What's water got to do with it? A nuclear magnetic resonance study of molecular motion in pig stratum corneum.

The 1H and 2H magnetic resonance signals from pig stratum corneum were measured as a function of hydration. The 1H free induction decay contained two components, one motionally restricted and the other isotropically mobile on the timescale of 10(-5) s. From its T2 decay, the mobile signal was further subdivided into two components; one, which was 11% of the signal and 5.5% of the mass of the dehydrated stratum corneum, was assigned to nonaqueous tissue (likely hydrocarbons) and the other to water. As water content increased from 0 to 0.25 gH2O/gSC, the second moment of the motionally restricted signal decreased from 5.4 x 10(9) to 3.6 x 10(9) s(-2), whereas the water T2 time increased from less than 0.3 ms to 3.3 ms. The 2H quadrupolar echo from stratum corneum hydrated in 2H2O had a signal from motionally restricted deuterons, attributed to deuterons exchanged onto O-H and N-H groups, and a mobile signal from 2H2O. The amount of exchange, 9.5% of the hydrogen sites in the motionally restricted fraction, was close to the number of exchangeable sites on keratin, the most abundant protein in stratum corneum. Our results are consistent with a model in which the bulk of the water interacts closely with the corneocytes in stratum corneum.

Animals↗
PubMed2003

The role of stratum corneum and dermal microvascular perfusion in penetration and tissue levels of water-soluble drugs investigated by microdialysis.

BACKGROUND: Hydrophilic drugs are poorly absorbed when applied topically, due to low partitioning through the lipid matrix of the stratum corneum. Cutaneous blood flow rapidly clears the absorbed drug, which may result in low tissue levels. This is of importance for topically applied drugs whose site of action is within the epidermis or dermis. Dermal drug levels can be measured using cutaneous microdialysis, which is a means of continuously sampling substances from the dermal extracellular fluid. OBJECTIVES: To measure the contribution of stratum corneum barrier and microvascular perfusion in determining dermal tissue levels of hydrophilic drugs (aciclovir and penciclovir) in vivo. METHODS: Studies were performed using microdialysis of the volar surface of the forearm of healthy volunteers (n = 55) over a 5-h collection period. Stratum corneum was removed by tape stripping, and barrier disruption quantified by measurement of transepidermal water loss (TEWL); dermal microvascular perfusion was modulated by inclusion of noradrenaline in the microdialysis perfusate. RESULTS: With intact skin and normal cutaneous blood flow the concentration of penciclovir recovered was below assay threshold (0.05 ng x mL(-1). With noradrenaline-induced local vasoconstriction, the area under the curve of drug absorbed through normal skin (+/- SEM) was 13.3 +/- 2.9 ng mL(-1) h(0-5) for penciclovir and 27.6 +/- 10.6 ng mL(-1) h(0-5) for aciclovir. Removal of the stratum corneum (to glistening) by tape stripping increased penciclovir absorption by 1300-fold and aciclovir absorption by 440-fold, confirming the stratum corneum as the major barrier to hydrophilic drug absorption. Sequential barrier disruption by tape stripping gave a close correlation between penciclovir concentration absorbed per hour and barrier disruption measured by TEWL (r2 = 0.9283). There was a 15.6-fold difference in the recovery of penciclovir through barrier-deficient skin with and without cutaneous blood flow. There was no relationship between fibre depth and amount of drug dialysed, which suggests free movement of antiviral drug on reaching the aqueous environment of the dermis. CONCLUSIONS: This study defines for the first time the relationship between the degree of mechanical barrier impairment and drug absorption at the same anatomical site in humans, and the role of blood flow in drug clearance in vivo.

Acyclovir↗
PubMed1998

Stratum radiatum giant cells: a type of principal cell in the rat hippocampus.

Neurons of a distinct type in CA1 area stratum radiatum of the rat hippocampus have been found to express a direct cellular form of long-term potentiation (LTP, Maccaferri & McBain, 1996, J. Neurosci. 16, 5334), but their functional identity, i.e. whether interneuron or principal cell, remained unknown. Whole cell recording from hippocampal slices in vitro was combined with light and electron microscopy to answer this question. LTP was robustly induced by a pairing protocol and physiological properties were measured in radiatum giant cells (RGCs) using biocytin containing pipettes. Reconstruction of the cells' dendritic and axonal arbor revealed morphological properties similar to CA1 pyramidal cells with some characteristic differences. They typically had two large diameter apical dendrites, or when only one dendrite arose, it soon bifurcated. Apical dendrites formed a dendritic tuft in stratum lacunosum-moleculare and the dendrites, but not the somata, were densely covered with conventional spines. The axon arose from the basal pole of the soma, descended to stratum oriens and emitted several axon terminals bearing collaterals that travelled horizontally, remaining in stratum oriens. The main, myelinated axon trunks turned towards the fimbria. In the electron microscope axon terminals were found to form asymmetrical synapses on postsynaptic dendritic shafts and dendritic spines in stratum oriens. The dendrites received asymmetrical synapses, mostly on their spines. The axon initial segments also received several synapses, a feature never observed on interneurons. All the above characteristics support the conclusion that RGCs are excitatory principal neurons.

Animals↗
PubMed1999

Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo.

The stratum corneum is located at the interface between body and environment and thus is constantly exposed to a pro-oxidative environment. Previously, we have demonstrated that stratum corneum lipids are targets of oxidative stress induced by ozone and by ultraviolet A and B exposure. Here, we employed an immunoblotting technique to detect protein oxidation in human stratum corneum obtained by tape stripping. After lysis, protein carbonyl groups were measured by derivatization with dinitrophenylhydrazine, separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against dinitrophenyl groups. Keratin 10, identified by use of specific antibodies and by microsequencing, was demonstrated in vitro to be oxidizable by ultraviolet A irradiation, hypochlorite, and benzoyl peroxide. In vivo, a keratin 10 oxidation gradient with low levels in the lower stratum corneum layers, and about 3-fold higher contents of carbonyl groups towards the outer layers was demonstrated in forehead stratum corneum of healthy volunteers (n = 6). As protein oxidation can be associated with an increased susceptibility to proteases, this finding may be important for better understanding the process of desquamation.

Adult↗
PubMed1999

Normal ultrastructure, but altered stratum corneum lipid and protein composition in a mouse model for epidermolytic hyperkeratosis.

Recently, we established keratin 10-deficient mice, serving as a model for the hyperkeratotic skin disorder epidermolytic hyperkeratosis. The considerable ichthyosis in these mice suggested alterations in terminal differentiation and in the formation of a functional epidermal barrier. Here, we report on the ultrastructural organization and composition of the stratum corneum lipids and on the expression of two major cornified envelope proteins. Electron microscopy of ruthenium tetroxide postfixed skin samples demonstrated a normal extrusion and morphology of lamellar bodies as well as the formation of bona fide lamellar layers in neonatal keratin 10-deficient mice. When we studied the composition of the major stratum corneum lipids, however, we found significant changes. Most importantly, the analysis of ceramide subpopulations revealed that the total amount of ceramide 2 was elevated in keratin 10-deficient mice, whereas ceramides 1, 3, 4, and 5 were decreased among total stratum corneum lipids. The amount of the ceramide precursors sphingomyelin and glucosylceramide was reduced in the stratum corneum without accompanying changes in the mRNA coding for acid sphingomyelinase. Notably, we found an increased mRNA and protein content for involucrin in neonatal keratin 10-deficient mice, whereas the expression of loricrin was not changed. Our data demonstrate that, although the formation of lipid layers in the stratum corneum appeared to be normal, its lipid composition is significantly altered in keratin 10-deficient mice.

Animals↗
PubMed1999

Vitamin C, uric acid, and glutathione gradients in murine stratum corneum and their susceptibility to ozone exposure.

The stratum corneum has been recognized as the main cutaneous oxidation target of atmospheric ozone (O3), a major part of photochemical smog. This study reports the presence and distribution of vitamin C, glutathione, and uric acid in murine stratum corneum, and evaluates their susceptibility to acute environmental exposure to O3. Based on tape stripping and a modified extraction method with high performance liquid chromatography electrochemical analysis, we detected vitamin C (208.0 +/- 82.5 pmol per 10 consecutive pooled tapes), glutathione (283.7 +/-96.3), and uric acid (286.4 +/-47.1) in murine stratum corneum as compared with only 16.5 +/- 1.4 pmol alpha-tocopherol. Vitamin C, glutathione (both p < 00.001), and urate (p < 0.01) were found to exhibit a gradient with the lowest concentrations in the outer layers and a steep increase in the deeper layers. To investigate the effect of O3 exposure on hydrophilic antioxidants, we exposed SKH-1 hairless mice to O3 concentrations of 0, 0.8, 1, and 10 p.p.m., and stratum corneum was analyzed before and after exposure. Whereas mock exposure with 0 p.p. m. for 2 h had no significant effect, O3 doses of 1 p.p.m. for 2 h and above showed depletion of all three antioxidants. Vitamin C was decreased to 80% +/- 15% of its pretreatment content (p < 0.05), GSH to 41% +/- 24% (p < 0.01), and uric acid to 44% +/- 28% (p < 0.01). This report demonstrates the previously unrecognized role of hydrophilic antioxidants in the stratum corneum and provides further evidence that O3 induces oxidative stress in this outer skin layer.

Animals↗
PubMed2000

Structural and functional effects of oleic acid and iontophoresis on hairless mouse stratum corneum.

The aim of this study was to assess the effects of chemical and electrical modes of percutaneous penetration enhancement on the intercellular lipid lamellae of the stratum corneum. Hairless mice were treated with either oleic acid/propylene glycol and iontophoresis separately or together. Permeability barrier function was evaluated by measuring transepidermal water loss and correlated with the structure of stratum corneum intercellular lamellae, as evaluated by electron microscopy, using ruthenium tetroxide postfixation. Transepidermal water loss levels did not change following 1 h iontophoresis alone. In contrast, topical applications of 0.3 M oleic acid in propylene glycol for 1 h increased transepidermal water loss significantly. Moreover, the combined use of iontophoresis plus 0.3 M oleic acid for 1 h further increased transepidermal water loss at equivalent time points. Ultrastructural observations demonstrated both marked disorganization of the intercellular lipid lamellae, as well as the presence of distended lacunae within the stratum corneum in oleic acid/propylene glycol plus or minus iontophoresis-treated stratum corneum. This study provides direct evidence that the oleic acid/propylene glycol system can disrupt the stratum corneum lipid lamellar structures, and that coapplications of oleic acid with iontophoresis further enhance the effects of oleic acid. The synergy between chemical and physical enhancement may afford a new approach to promote transdermal drug delivery.

Animals↗
PubMed2001

Attenuated total reflection-Fourier transform infrared spectroscopy as a possible method to investigate biophysical parameters of stratum corneum in vivo.

We investigated the use of attenuated total reflection-Fourier transform infrared spectroscopy as a method to study differences in the molecular components of human stratum corneum in vivo. These variations as a function of the anatomic site and of the depth into its layered structure are important to understand the biology and physiology of the tissue. In this preliminary study we have investigated spectroscopic changes in 18 healthy individuals. Total reflection-Fourier transform infrared spectroscopy represents a potentially powerful tool to study biophysical properties of surfaces. We observed that, in vivo, biophysical parameters of the stratum corneum (such as hydration, lipid composition, and conformation of the aliphatic chains) are indeed dependent on the anatomic site. As in total reflection-Fourier transform infrared spectroscopy experiments the penetration depth of the evanescent field into the stratum corneum is comparable with the thickness of a layer of corneocytes, this technique can be used to follow the distribution of lipids, water, and proteins as a function of depth into the tissue. We found that, in vivo, these molecular components are non-uniformly distributed, in agreement with the presence of water and lipid reservoirs as observed with ex vivo ultrastructural investigations. Composition and conformational order of lipids are also a function of depth into the stratum corneum. Finally we compared the in vivo superficial hydration measured using the infrared absorption of the OH stretch of water, with the hydration measured using the Skicon hygrometer. Our results indicate that total reflection-Fourier transform infrared spectroscopy might be useful to measure important chemical and biophysical parameters of stratum corneum in vivo.

Adult↗
PubMed2002

Decreased levels of sphingosine, a natural antimicrobial agent, may be associated with vulnerability of the stratum corneum from patients with atopic dermatitis to colonization by Staphylococcus aureus.

The stratum corneum of the skin of patients with atopic dermatitis is highly susceptible to colonization by various bacteria, including Staphylococcus aureus. The defense system of the skin against bacterial invasion appears to be significantly disrupted in atopic dermatitis skin, but little is known about the defense mechanism(s) involved. As one sphingolipid metabolite, sphingosine is known to exert a potent antimicrobial effect on S. aureus at physiologic levels, and it may play a significant role in bacterial defense mechanisms of healthy normal skin. Because of the altered ceramide metabolism in atopic dermatitis, the possible alteration of sphingosine metabolism might be associated with the acquired vulnerability to colonization by S. aureus in patients with atopic dermatitis. In this study, we measured the levels of sphingosine in the upper stratum corneum from patients with atopic dermatitis, and then compared that with the colonization levels of bacteria in the same subjects. Levels of sphingosine were significantly downregulated in uninvolved and in involved stratum corneum of patients with atopic dermatitis compared with healthy controls. This decreased level of sphingosine was relevant to the increased numbers of bacteria including S. aureus present in the upper stratum corneum from the same subjects. This suggests the possibility that the increased colonization of bacteria found in patients with atopic dermatitis may result from a deficiency of sphingosine as a natural antimicrobial agent. As for the mechanism involved in the decreased production of sphingosine in atopic dermatitis, analysis of the activities of ceramidases, major sphingosine-producing enzymes, revealed that, whereas the activity of alkaline ceramidase did not differ between patients with atopic dermatitis and healthy controls, the activity of acid ceramidase was significantly reduced in patients with atopic dermatitis and this had obvious relevance to the increased colonization of bacteria in those subjects. Further, there was a close correlation between the level of sphingosines and acid ceramidase (r = 0.65, p < 0.01) or ceramides (r = 0.70, p < 0.01) in the upper stratum corneum from the same patients with atopic dermatitis. Collectively, our results suggest the possibility that vulnerability to bacterial colonization in the skin of patients with atopic dermatitis is associated with reduced levels of a natural antimicrobial agent, sphingosine, which results from decreased levels of ceramides as a substrate and from diminished activities of its metabolic enzyme, acid ceramidase.

Adolescent↗
PubMed1999

[100 MHz sonography in the visualization of the palmar stratum corneum after application of various creams and ointments].

To visualise the effects of different topically applied substances on the stratum corneum in vivo by means of sonography, it is necessary to have an ultrasound unit with a much higher resolution than provided for by the 20 MHz scanners currently used in dermatology. We developed a workstation with a highly focussed transducer with a large bandwidth, providing a resolution of 9 microns axially and 27 microns laterally. Using this ultrasound unit, we investigated the effects of different creams and ointments in 10 persons on the palmar side of the distal finger segments (vaseline, a water-in-oil-emulsion, an oil-in-water-emulsion, 10% urea in an oil-in-water-emulsion and water). Sonograms were taken after an occlusion period of 0, 30, 60, 120, 180 and 240 min. In 100 MHz sonograms of palmar skin, the stratum corneum is represented as an echo-poor band below the echo-rich entry echo--the correlate of the interface between the coupling water and the horny layer. The echo-poor band is traversed by echo-rich coils, representing eccrine sweat gland ducts. It is separated by an echo-rich line from the viable epidermis, which is echo-poor as well. The topically applied substances differ in the time course of the swelling of the stratum corneum. After longer application all externals cause a significant thickening of the stratum corneum (p < 0.005) and a reduction of the echogenicity of the skin entry echo due to altered impedance. 100 MHz sonography is well suited to visualise and quantify the effects of externals on the stratum corneum in vivo.

Administration, Topical↗
PubMed2001

Human stratum corneum penetration by nickel. In vivo study of depth distribution after occlusive application of the metal as powder.

Sequential tape stripping was implemented on three healthy volunteers to examine the surface distribution of nickel through human stratum corneum in vivo following occlusive application of the metal as powder on the volar forearm. Exposure sites were stripped 20 times at intervals from 5 min to 96 h post-dosing and the strips analyzed for metal content by Inductively Coupled Plasma-Mass Spectroscopy with a detection limit for nickel of 0.5 ppb. The gradients of nickel distribution profiles increased proportionally with occlusion time, but after the 10th strip to the 20th strip continued at constant levels. Total nickel removed with 20 stratum corneum strips to the level of the glistening layer after maximum occlusion of 96 h was 41.6 micrograms/cm2 (+/- 12.2; average n = 3). In order to normalize the nickel depth distribution profiles, stratum corneum removed by stripping of untreated skin after occlusion was determined by weighing. Following application of nickel dust over 24 h, analysis of the 20th strip still indicated nickel present at 1.42 micrograms/cm2 (+/- 0.68; average n = 3). These data indicate that, in contact with skin, nickel metal is oxidized to form soluble, stratum corneum-diffusible compounds which may penetrate the intact stratum corneum, presumably by the intercellular route, and have the potential to elicit allergic reactions.

Adult↗
PubMed1998

Colorimetric method for quantifying human Stratum corneum removed by adhesive-tape stripping.

Tape-stripping of the skin is a useful method for removing the stratum corneum and obtaining more information about the function of this skill layer as the main barrier for skin penetration. The amount of stratum corneum removed is of relevance in establishing the concentration profile of chemicals within the stratum corneum after topical application. Weighing is the preferred method for measuring the amount stripped, but because it is often subject to artifacts, alternative methods are sought. We present a simple, colorimetric method for determining the amount stratum corneum removed by sequential adhesive-tape-stripping of human skin in vivo. The method is based on quantification of the sodium hydroxide soluble protein fraction using a commercially available protein assay similar to the Lowry assay. The method is shown to be an accurate and reproducible alternative to weighing, also demonstrating uniform removal of stratum corneum layers following the very initial strips.

Adhesives↗
PubMed2002

Fine structure and Ca-ATPase activity of the stratum intermedium cells during odontogenesis in gars, Lepisosteus, Actinopterygii.

This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

Animals↗
PubMed1984

The diffusion of water across the stratum corneum as a function of its water content.

The flux of tritiated water (HTO) through pieces of stratum corneum at four different levels of hydration has been measured. The concentration of water in the stratum corneum, the concentration of HTO in the presenting solution, and the thickness, density, and weight per unit area of the dry stratum corneum are known. The thickness of the hydrated stratum corneum and the permeability and diffusion constants of HTO were calculated. From these in vitro data it is possible to calculate the in vivo thickness of the stratum corneum, its water concentration profile, and the flux of water (transepidermal water loss) at environments of different relative humidities. Both the transepidermal water loss and the water concentration profile change very little as the environmental relative humidity increases from 0 to 80%. The small decrease in the water concentration of the surface layers of cells as the relative humidity becomes very low, however, may cause an observable alteration in the physical characteristics of the surface layers.

Body Water↗
PubMed1985

A possible function of structural lipids in the water-holding properties of the stratum corneum.

In order to clarify the possible role of lipids in the water-holding property of stratum corneum, the forearm skin of 6 healthy male volunteers was treated with acetone/ether (1/1) for 1, 5, 10, and 20 min. A prolonged treatment period of 5-20 min produced a chapped and scaly appearance of the stratum corneum without any inflammatory reactions. Under these conditions, there was a marked decrease in the water-holding capacity of the stratum corneum accompanied by a considerable and selective loss of intercellular lipids such as cholesterol, cholesterol esters, and phospholipids. These impairments persisted until day 4 after treatment. Electron microscopic observation of the altered stratum corneum revealed that naturally occurring intercellular materials were absent, leaving the area with the appearance of a vacant space. These findings suggest an additional and essential role of the specific structural lipids for the water-holding properties of the stratum corneum.

Acetone↗