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Correlation between stratum corneum/water-partition coefficient and amounts of flufenamic acid penetrated into the stratum corneum.

The stratum corneum of various donors differs in particular in the composition of the lipoidal phase. Considering the drug amounts penetrating into the stratum corneum a simple methodology to correlate these differences in the stratum corneum composition with the drug amounts detectable within the stratum corneum is desirable. Penetration experiments investigating several incubation times were carried out with three different skin flaps using the Saarbruecken penetration model and the lipophilic model drug flufenamic acid. The drug amounts within the stratum corneum were obtained with the tape-stripping technique, while the drug amounts present in the deeper skin layers were achieved by cryosectioning. The stratum corneum/water-partition coefficient was determined with the same three skin flaps to characterize the lipoidal stratum corneum phase in general, and the differences were attributed to the different amounts of ceramides and sterols. In addition, for the lipophilic drug flufenamic acid, a direct linear correlation was found between the stratum corneum/water-partition coefficients and the drug amounts penetrated into the stratum corneum for all investigated time intervals (correlation coefficients of r(30 min) = 0.998, r(60 min) = 0.998 and r(180 min) = 0.987). In contrast to the stratum corneum/water-partition coefficients, the determination of a corresponding relationship for the stratum corneum and the deeper skin layers failed due to the reason that steady-state conditions could not be achieved for the deeper skin layers during the investigated time intervals. In summary, the stratum corneum/water-partition coefficients offer the possibility to predict drug amounts within the stratum corneum of different donor skin flaps without a time consuming determination of the lipid composition of the stratum corneum.

Anti-Inflammatory Agents, Non-Steroidal↗

Studies in immunodermatology. VI. IF studies of autoantibodies to the stratum corneum and of in vivo fixed IgG in stratum corneum of psoriatic lesions.

Indirect immunofluorescent (IF) tests on sections of normal human skin reveal the presence of antibodies to the stratum corneum in most normal human sera. Sera absorbed with hyperkeratotic scales gave negative reactions. These reactions appeared to correspond to those of the stratum corneum antibodies which were first detected by Krough and Tonder by immune adherence, though this remains to be documented since the conjugate alone stained the stratum granulosum in a pattern comparable to that of the IF staining reported by Krough. The staining was caused by conjugates with high but not by those with low fluorescein to protein ratios. In indirect IF tests, comparable titers of stratum corneum antibodies were found in sera of psoriasis patients and control subjects. Since they reacted with the stratum corneum of the antibody producer they are referred to as autoantibodies. Direct IF tests of psoriatic lesions revealed the presence of in vivo bound IgG as well as other immunoglobulins and complement in the stratum corneum. To differentiate staining with conjugated protein from direct and indirect IF staining of the stratum corneum a four-compartment test was devised. Skin sections were treated with saline or with stratum corneum antibodies and then with conjugates without (compartments 1 or 2) or with (compartments 3 or 4) anti-IgG antibodies. Sections were read by the method of Kawamura and his associates with both UV and BV illumination. Compartment 3 is a direct IF test and compartment 4 is an indirect IF test. In four-compartment tests performed on both biopsies of psoriatic lesions or scales the difference between compartments 1 or 2 and 3 affords a measure of in vivo bound IgG and the difference between compartments 3 and 4 provides information on the presence of free stratum corneum antigen. With this four-compartment test system it was found that in vivo fixation of IgG occurs in the intercellular areas of the stratum corneum of psoriatic lesions or scales and that this corresponds to the site of binding of the stratum corneum autoantibodies. This in vivo binding of IgG in the stratum corneum of psoriatic lesions resembles somewhat the intercellular fixation of IgG in the stratum spinosum in pemphigus. In the materials examined in these preliminary studies most, if not all of the stratum corneum antigen appeared to be covered with in vivo bound IgG.

Animals↗

Analysis of proteins with caseinolytic activity in a human stratum corneum extract revealed a yet unidentified cysteine protease and identified the so-called "stratum corneum thiol protease" as cathepsin l2.

Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was to investigate if human stratum corneum contains proteases different from the above, exhibiting similar properties. After gel filtration, we identified four distinct proteolytic activities in a human stratum corneum extract, a cathepsin-E-like activity (80 kDa), a cathepsin-D activity (40 kDa), a yet unknown cathepsin-L-like form (28 kDa) exhibiting the highest caseinolytic activity, and a chymotrypsin-like protein (24 kDa) containing the acidic activity of the well described stratum corneum chymotryptic enzyme. We named the new 28 kDa protease stratum corneum cathepsin-L-like enzyme. Characterization of stratum corneum cathepsin-L-like enzyme provided clear evidence that this new protease, despite its membership to the cathepsin-L-like family, is distinct from cathepsin L and from the recently described stratum corneum thiol protease. Its ability to hydrolyze corneodesmosin, a marker of corneocyte cohesion, was in favor of a role of stratum corneum cathepsin-L-like enzyme in the desquamation process. A more detailed analysis did not allow us to identify stratum corneum cathepsin-L-like enzyme at the molecular level but revealed that stratum corneum thiol protease is identical with the recently described cathepsin L2 protease. Reverse transcription polymerase chain reaction studies and the use of a specific antibody revealed that, in contrast to earlier reports, expression of stratum corneum thiol protease in human epidermis is not related to keratinocyte differentiation. Our results indicate that the stratum corneum thiol protease is probably expressed as a pro-enzyme in the lower layers of the epidermis and in part activated by a yet unidentified mechanism in the upper layers during keratinocyte differentiation.

Amino Acid Sequence↗

Effect of lactic acid isomers on keratinocyte ceramide synthesis, stratum corneum lipid levels and stratum corneum barrier function.

Alpha-hydroxy acids are effective agents for the treatment of skin xerosis and it is known that, following treatment with lotions containing D,L-lactic acid, the stratum corneum prevents xerosis more effectively. To date, the relative efficacy of the different isomers of lactic acid has not been evaluated and the mode of action of lactic acid in improving stratum corneum resilience is not known. The objective of the present studies was to determine the effects of lactic acid isomers on keratinocyte ceramide biosynthesis, stratum corneum barrier function and the resistance of the stratum corneum to the appearance of skin xerosis. In vitro, lactic acid enhanced the production of ceramides by keratinocytes. L-Lactic acid was more effective than the D isomer (300% increase vs 100% increase). Carbon label from lactic acid was incorporated into all keratinocyte lipid species and a greater incorporation of label into ceramides was achieved with L-lactate than with D-lactate. In vivo, lactic acid increased the levels of stratum corneum ceramides. Whereas, lotions containing L-lactic acid resulted in the greatest increase (48% increase) followed by D,L-lactic acid (25% increase), D-lactic acid had no effect on stratum corneum ceramide levels. The increases in stratum corneum ceramide levels following lactic acid treatment also led to improvements in stratum corneum barrier function, measured by transepidermal water loss following a challenge to the skin with SLS and in the regression phase of a moisturization efficacy study. Significant improvements in barrier function and resistance to the appearance of skin xerosis were observed following L-lactic acid and D,L-lactic acid, but not following D-lactic acid treatment. From these results we believe that lactic acid, particularly the L isomer, stimulates ceramide biosynthesis leading to increased stratum corneum ceramide levels which results in superior lipid barrier and a more effective resistance against xerosis.

Adult↗

Studies in immunodermatology. VII. Four-compartment system studies of IgG in stratum corneum and of stratum corneum antigen in biopsies of psoriasis and control dermatoses.

Biopsies of skin lesions from 50 cases of psoriasis and from 42 cases with other dermatoses were studied by the methods described in the foregoing report, that is by the four-compartment by the methods described in the foregoing report, that is by the four-compartment immunofluorescent (IF) system for detecting stratum corneum autoantibodies and in vivo binding of IgG in psoriatic scales at the site of the stratum corneum antigen. They were also studied histologically. Early and fully developed psoriatic lesions tended to have more widespread deposits of IgG in the stratum corneum than receding lesions. In biopsies with widespread IgG deposits the stratum corneum antigen usually could not be demonstrated because of blocking by in vivo bound IgG. Of the 42 control biopsies of skin lesions from patients with other dermatoses which were examined with this system two gave the IF staining patterns which were seen most commonly in psoriatic lesions. In these two cases strong widespread IgG deposits occurred in compartment 3, and 4 was very similar to compartment 3. One case was diagnosed as lichen planus and one as cornu cutaneum. Comparisons of the IF findings in psoriatic and control lesion biopsies indicate that significantly more of the former have: (1) IgG deposits at the combining sites of stratum corneum antibodies (92 vs. 50%) and (2) no demonstrable stratum corneum antigen because of 'blocking' by in vivo bound IgG, i.e. compartment 3 like 4 (56 vs. 7%). A highly significant inverse relation between the in vivo deposits of IgG and demonstrability of stratum corneum antigen appeared regardless of the clinical condition. This observation lends support to the view that the IgG which is bound to the sites of the stratum corneum antigen in ski; lesions may in fact be in vivo bound stratum corneum antoantibody.

Antigens↗

Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies.

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold-labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation.

Adult↗

Stratum corneum chymotryptic enzyme: a proteinase which may be generally present in the stratum corneum and with a possible involvement in desquamation.

A chymotrypsin-like proteinase that may be involved in the desquamation process in plantar stratum corneum has recently been partially characterized. The aim of the present study was to elucidate whether a similar proteinase is also present in non-palmo-plantar stratum corneum. Stratum corneum was obtained by tape stripping of volar forearm skin after the skin surface had been painted with colourless nail varnish. The adherent tissue was released from the tape strips by acetone treatment, then extracted with diethyl ether and dried. Extracts of this acetone-ether powder were analyzed with respect to proteolytic activity by means of electrophoresis under non-reducing conditions in polyacrylamide gels containing sodium dodecyl sulphate and casein. The extracts were found to contain one major chymotrypsin-like proteinase with an apparent molecular weight of around 25 kDa, and several minor proteinases with trypsin-like activity. The 25 kDa proteinase was active at pH 5.5-8, and could be inhibited by aprotinin, chymostatin and zinc ion, but not by leupeptin. No difference could be found between the 25 kDa enzyme in forearm stratum corneum and the recently described chymotrypsin-like enzyme in dissociated plantar stratum corneum cells as regards electrophoretic mobility, pH dependency, and inhibitor profile. The fact that the enzyme could degrade casein at pH 5.5 and that it appears to be present in stratum corneum in general suggests that it may play a role in the desquamation process under in vivo conditions. The tentative name "stratum corneum chymotryptic enzyme" is proposed for this newly discovered proteinase.

Cell Adhesion↗

Levels of fluconazole in serum, stratum corneum, epidermis-dermis (without stratum corneum) and eccrine sweat.

The distribution in the skin of orally active antifungals and other drugs claimed to be active in the treatment of skin diseases is of major importance, but to date has been studied only rarely. In the present study the distribution of fluconazole in stratum corneum, epidermis-dermis (without stratum corneum), eccrine sweat and serum was studied in human male volunteers after dosages of 50 mg daily for 12 days and 150 mg once weekly for 2 weeks. In the present study high levels of fluconazole were found especially in the stratum corneum. At a dose of 50 mg once daily the concentration of fluconazole after 12 days was 73.0 micrograms/g and 7 days after cessation of treatment the concentration was still 5.8 micrograms/g. At the 150 mg once a week dose the concentration 7 days after the second dose was still 7.1 micrograms/g in stratum corneum indicating that medication once a week may be effective in the treatment of dermatomycoses. High concentrations, all above the serum concentrations, were also seen in sweat and epidermis/dermis. These results indicate that fluconazole is delivered to the stratum corneum, where it is accumulated, through sweat and by direct diffusion through the dermis-epidermis. These pharmacokinetic results indicate that the drug should be very effective in the treatment of dermatomycoses. An extremely high concentration of the drug in the stratum corneum combined with concentrations from 2.93 to 4.62 micrograms/g in the rest of the epidermis and dermis is important in the treatment of these diseases.

Adult↗

Relationship between cholesterol sulfate and intercellular cohesion of the stratum corneum: demonstration using a push-pull meter and an improved high-performance thin-layer chromatographic separation system of all major stratum corneum lipids.

To investigate the role of cholesterol sulfate (CS) as an intercellular glue or cement in the stratum corneum, we compared the relationship between CS levels and magnitude of the intercellular cohesion of the stratum corneum between the palm and the upper arm. Using a push-pull meter, the palm displayed approximately seven times the magnitude of cohesion of the stratum corneum as the upper arm (n = 11). CS and other stratum corneum lipids were extracted from the palm and the upper arm (n = 22) by a cup method and determined by our improved high-performance thin-layer chromatography (HPTLC). Despite a great difference in the magnitude of cohesion (p less than 0.01), CS levels and ratios of CS to ceramides and CS to cholesterol in the stratum corneum showed no significant differences between the palm and the upper arm. Our results suggest that differences in CS cannot account for the differences in cohesion between palm and upper arm.

Adult↗

Unitary IPSPs evoked by interneurons at the stratum radiatum-stratum lacunosum-moleculare border in the CA1 area of the rat hippocampus in vitro.

1. Hippocampal non-principal neurons at the stratum radiatum-stratum lacunosum-moleculare border (R-LM interneurons) of the CA1 area may constitute several cell classes and have been implicated in the generation of GABAergic unitary IPSPs. Using biocytin-filled electrodes we recorded R-LM interneurons intracellularly in vitro and determined their postsynaptic effects in concomitantly recorded pyramidal cells. 2. Light microscopic analysis revealed four populations of R-LM interneurons with distinct axons: (1) basket cells (n = 4) with axons predominantly ramifying in the pyramidal cell layer; (2) Schaffer collateral/commissural pathway-associated interneurons (n = 10) stratifying in stratum radiatum and, to a lesser extent, stratum oriens; (3) perforant pathway-associated interneurons (n = 6) innervating the perforant path termination zone in stratum lacunosum-moleculare of the CA1 area as well as equivalent portions of the dentate gyrus and subiculum; and (4) neurogliaform interneurons (n = 2) characterized by their dense, compact axonal and dendritic arbour. 3. Random electron microscopic sampling of synaptic targets revealed a preponderance of pyramidal neurons as postsynaptic elements. Basket cells had a synaptic target preference for somata and proximal dendrites, whereas the remainder of R-LM interneurons innervated dendritic shafts and spines. The axon of dendrite-targeting cells formed up to six putative contacts with individual postsynatpic pyramidal cells. 4. Anatomically recovered R-LM interneurons (n = 22) had a mean resting membrane potential of -56.7 +/- 3.6 mV, a membrane time constant of 12.9 +/- 7.7 ms and an input resistance of 86.4 +/- 29.2 M omega. Depolarizing current pulses generally elicited overshooting action potentials (70.8 +/- 6.9 mV) which had a mean duration, when measured at half-amplitude, of 0.7 +/- 0.1 ms. In response to prolonged (> 200 ms) depolarizing current pulses all R-LM interneurons displayed (a varying degree of) spike frequency adaptation. 5. Basket cells, Schaffer-associated and neurogliaform interneurons elicited small-amplitude (< 2 mV), short-latency IPSPs in postsynaptic pyramids (n = 5, 13 and 1, respectively). Those interactions in which an effect was elicited with the repetitive activation of the presynaptic neuron (n = 13) showed a substantial degree of postsynaptic response summation. Unitary IPSPs had fast kinetics and, whenever tested (n = 5; 1 basket cell and 4 Schaffer-associated interneurons), were abolished by the GABAA receptor antagonist bicuculline. 6. Thus, R-LM interneurons comprise several distinct populations which evoke fast GABAA receptor mediated IPSPs. The domain-specific innervation of postsynaptic pyramidal cells suggests functionally diverse effects on the integration of afferent information in functionally non-equivalent compartments of pyramidal cells.

Action Potentials↗

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes.

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.

Animals↗

Levels of terbinafine in plasma, stratum corneum, dermis-epidermis (without stratum corneum), sebum, hair and nails during and after 250 mg terbinafine orally once per day for four weeks.

The distribution of terbinafine in stratum corneum dermis-epidermis (without stratum corneum), sebum, hair, nails and plasma was studied in human male volunteers during and after 250 mg orally once daily for 28 days. The highest concentration was seen in sebum, 56.07 micrograms/g, after 14 days of therapy. The concentration was still 1.0 microgram/g 44 days after stop of medication. In stratum corneum the highest concentration, 14.4 micrograms/g, was seen 1 day after the last day of therapy, and it was 2.1 micrograms/g 44 days after stop of medication. The concentrations in hair and nails were lower with a maximum of 2.36 and 0.39 micrograms/g respectively, 1 day after stop of therapy, and still 0.21 microgram/g in hair and 0.09 microgram/g in nails 55 days after the last day of medication. With the exception of nails, all other tissue levels were at all times above the plasma concentrations. For nails, tissue levels exceeded that of plasma as early as 1 day after stop of medication, and this difference continued to increase until the last day of tissue sampling, 55 days after the last tablet. These results indicate that terbinafine is delivered to the stratum corneum through sebum and to a minor extent by direct diffusion through dermis-epidermis. Probably short-term therapy with terbinafine may be effective in the treatment of several dermatomycoses, due to the strong binding of terbinafine to stratum corneum for a long time after stop of medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

The role of proteases in stratum corneum: involvement in stratum corneum desquamation.

The effects of protease inhibitors on cell dissociation were studied in vitro in order to examine the involvement of proteases in stratum corneum desquamation. Stratum corneum sheet (peeled from human backs after sunburn) was incubated in a detergent mixture containing 8 mM N,N-dimethyldodecylamine oxide, 2 mM sodium lauryl sulphate and 60 micrograms/ml kanamycin with or without protease inhibitors, and the number of released cells was counted after incubation for 48 h. Cell dissociation was inhibited strongly by antipain or aprotinin, but not at all by N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin, N-ethylmaleimide or pepstatin, which suggests that only serine proteases are associated with desquamation. Furthermore, leupeptin and chymostatin each reduced cell dissociation about half as effectively as aprotinin or antipain, while a mixture of leupeptin and chymostatin prevented stratum corneum dissociation as potently as antipain or aprotinin. In addition, the activity of chymotrypsin-like protease in scaly skin was higher than that in normal skin, as we have previously found for trypsin-like protease. These results suggest that both trypsin-like and chymotrypsin-like serine proteases are involved in stratum corneum desquamation.

Antipain↗

Isolation, barrier properties and lipid analysis of stratum compactum, a discrete region of the stratum corneum.

We have isolated the lowest region of human and pig stratum corneum as an integral layer which we have termed the stratum compactum. This preparation is resistant to disruption by enzymes, 6 M urea, Triton X-100 and solvents. Our evidence suggests that all cells of the stratum corneum may be equally permeable to aqueous soluble materials but that penetration of materials through the corneum depends on the state of cohesion between cells and of the organization of intercellular lipid species. As the cells move up towards the outside of the stratum corneum the cohesive forces are reduced due to desmosome degradation and lipid modifications with ultimate dyshesion and sloughing of individual cells.

Animals↗

Cytokeratins of the stratum medium and stratum internum of the equine hoof wall in acute laminitis.

The cytoskeleton of living keratinocytes consists mainly of cytokeratins that have polymerised into intermediate filaments. The aim of this study was to describe the expression of cytokeratins in the living epidermal cells of the weight-bearing parts of the equine hoof wall during acute spontaneous laminitis. A total of 9 hooves from 3 horses subjected to euthanasia within 48 h of the first clinical signs of laminitis were sectioned and examined. The cytokeratins in the stratum medium and stratum internum of the hoof wall were characterized by 1- and 2-dimensional gel electrophoresis, and the tissue distribution of the cytokeratins was studied by immunohistochemical staining. The biochemical results showed the same set of cytokeratins as was seen in 8 normal horses, reported on previously, used as controls. The immunohistochemical results indicated a difference between normal horses and horses with acute laminitis in the content of cytokeratins in the basal cells of the matrix of the stratum medium of the hoof wall and in the basal and suprabasal cells in the stratum internum at the mid level of the hoof wall. However, no conclusion could be drawn as to whether this change in the cytokeratin distribution in laminitis was primary or was caused by the initiation of the local tissue-repairing process.

Acute Disease↗

Terbinafine levels in serum, stratum corneum, dermis-epidermis (without stratum corneum), hair, sebum and eccrine sweat.

We determined terbinafine levels in serum, stratum corneum, dermis-epidermis (without stratum corneum), hair, sebum and eccrine sweat before, during and after 250 mg doses orally to volunteers once daily. Terbinafine is concentrated rapidly in stratum corneum (up to 9.1 micrograms/g of tissue) primarily by diffusion from the vascular system through the dermisepidermis. It also reaches high concentration in sebum (up to 45.1 micrograms/ml) after several days and continue to concentrate in sebum for up to two days after discontinuation of drug. Hair concentration reach levels of 2.6 micrograms/g of tissue indicating high drug levels in and around the hair follicle. It is not found in sweat. Plasma levels range between 0.1 and 1.0 micrograms/ml. There is a tenfold accumulation of drug in stratum corneum by day 2. Elimination of drug from tissue occurs with a half-life of 4 to 5 days and with the potential for drug levels above fungicidal concentrations for dermatophytes for more than 3 weeks. The tissue pharmacokinetic profile of terbinafine is similar to that of another lipophilic drug, itraconazole, but is very different from ketoconazole and griseofulvin. Higher levels of terbinafine are achieved than of either of the imidazoles and remain longer than griseofulvin.

Adolescent↗