The effect of adhesive dressing edges on cutaneous irritancy and skin barrier function.

OBJECTIVE: To assess the effect of repeated application and removal of adhesive edges from wound-care products on cutaneous irritancy and barrier function in normal volunteer subjects. METHOD: This was a study using a 'repeat-insult patch test'. Adhesive edges from six commonly used wound-care products were applied continuously to the same site (six applications over a 14-day period) in 30 normal volunteer subjects. The test sites were assessed clinically before product reapplication using established ranking scales for cutaneous erythema. The cumulative irritancy score (CIS) for each test site was determined by adding the erythema scores at days 3, 5, 8, 10, 12 and 15. At the study end the barrier function of each test site was assessed by measuring transepidermal water loss (TEWL). RESULTS: The CIS showed that the products fall into two distinct groups, with Mepilex, Tielle and Allevyn giving low scores and Biatain, Comfeel and DuoDERM higher scores. Statistical analysis indicated significant differences (p < 0.05) between Mepilex and Biatain, Mepilex and Comfeel, Mepilex and DuoDERM, Tielle and Biatain, Allevyn and Biatain. The mean TEWL values also indicated that the products fall into two distinct groups: Mepilex, Tielle and Allevyn with low mean values close to that of normal adjacent back skin and Biatain, Comfeel and DuoDERM with much higher mean values. Statistical analysis indicated that Mepilex, Tielle and Allevyn were not significantly different from normal skin (p < 0.05), whereas Biatain, Comfeel and DuoDERM were significantly higher than normal skin and the other products tested. CONCLUSION: The results show clear differences between products; the clinical scores and TEWL measurements indicate that the products fall into two distinct groups. This novel approach seems able to discriminate between adhesive borders and may be useful during product development and in selecting products for clinical trials.

Measurement of skin thickness: a comparison of two in vivo techniques with a conventional histometric method.

Two in vivo techniques which are rapid, inexpensive, and reproducible have been investigated. The first is a standardized radiological (xerographic) technique which we have shown is capable of detecting small degrees of dermal atrophy after the application of topical corticosteroid preparations for only one month. The second technique employs the Harpenden Skinfold Caliper used in an unconventional manner so as to exclude subcutaneous fat. We have shown that this too is capable of detecting dermal atrophy from the application of topical corticosteroids and that there is a strong correlation between the two techniques (r = 0.82, p less than 0.001). Histometric techniques, on the other hand, give inaccurate and erroneous results for dermal thickness.

Plantar hyperkeratosis: a study of callosities and normal plantar skin.

Although callosities of the plantar skin are common and often disabling, little is known of their pathology or the reasons for their persistence. In this study plantar epidermal structure and cell renewal were investigated in patients with callosities and normal, age-, sex- and site-matched control subjects. Tritiated thymidine autoradiographic labeling indices were increased in the calluses but the dansyl chloride fluorescence clearance time was prolonged, reflecting the increased thickness of the stratum corneum. The number of corneocytes that could be removed from the surface of callosities by a standardized stimulus was considerably increased compared to controls but after adhesive tape stripping no such increase was observed. The density of corneocytes as measured on Percoll gradients was decreased in corneocytes from callus compared to normal plantar skin, and their volume was increased. These observations suggest that there are differences in epidermal differentiation due to an increased rate of epidermal cell production in plantar skin affected by callosity.

Preparation and characterization of the nonionic detergent-soluble proteins of human stratum corneum.

Nonionic detergent-soluble (NIDS) proteins from human stratum corneum have been prepared by the combined action of detergent and mechanical stimulation of normal human skin. SDS-polyacrylamide gel electrophoresis studies revealed approximately 15 components falling into the molecular weight ranges of 10,000-15,000, 24,000-37,000, and 44,000-68,000. Immunization of rabbits with this material gave antisera which demonstrated 3 or 4 antigenic components using 2-dimensional immunoelectrophoresis. Studies with 125I-labeled NIDS protein indicated that the main precipitate on 2-dimensional immunoelectrophoresis was associated with SDS-polyacrylamide gel components of molecular weight 15,000 and 30,000. Indirect immunofluorescence studies on human skin sections revealed localization of NIDS protein antigens throughout the suprabasal epidermis but concentrated in the stratum granulosum and stratum corneum. Localization studies using strips of stratum corneum obtained by the skin surface biopsy technique revealed a pericellular type of distribution of the NIDS protein antigens.

Epidermal metabolism in heredopathia atactica polyneuritiformis (Refsum's disease).

The epidermal metabolic activity of a patient with a marked generalized ichthyosis associated with heredopathia atactica polyneuritiformis has been investigated. Both the in vivo labeling index and the in vitro rates of incorporation of radioactively labeled thymidine, proline, histidine and acetate were increased relative to normal indicating a high rate of epidermopoiesis. Thin-layer chromatographic analysis of 14C-acetate containing lipid extracts revealed qualitative changes compared with normal. In particular, altered proportions of radioactivities were incorporated into the triglyceride and phospholipid moieties. However, as abnormal patterns of lipogenesis are also seen in autosomal dominant ichthyosis, these changes are probably a reflection of disordered keratinization.

Heat shock proteins in cultured human keratinocytes and fibroblasts.

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.

Keratinocyte growth stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF).

Human granulocyte-macrophage colony stimulating factor (GM-CSF) is a 22 kD glycoprotein derived from activated T cells, endothelial cells, fibroblasts and macrophages. It stimulates the proliferation and differentiation of haematopoietic cells and certain nonhaematopoietic cells. This study investigated the effect of GM-CSF on cultured human keratinocytes and determined whether these cells express GM-CSF receptors. Measurement of keratinocyte growth by image analysis showed that GM-CSF mildly stimulated the growth of keratinocytes. With 4 ng/ml GM-CSF the growth of primary keratinocytes and subcultured keratinocytes was only stimulated up to 25% and 18% of the control cell level, respectively. Human keratinocytes were incubated with GM-CSF at 4 ng/ml for 4 days to induce receptor expression. These cells showed only a weak positive reaction on affinity labelling using digoxigenated GM-CSF. We conclude that keratinocyte growth may be stimulated by GM-CSF but that the presence of GM-CSF receptors on these cells is equivocal.

The fatty acid composition of skin and plasma lipids in Refsum's disease.

The fatty acid composition of the skin and plasma lipids is described in a patient severely affected by Refsum's disease whose plasma phytanic acid concentration was very high (3.1 mg/ml). In the epidermal lipids, especially in the phospholipid fraction, phytanic acid tended to replace linoleic acid and to some extent arachidonic acid. In some respects the changes in the skin in Refsum's disease resemble those of essential fatty acid deficiency.

The effect of calcium on the initiation and growth of human epidermal cells.

The effect of the concentration of Ca2+ in the medium on the plating efficiency of human epidermal cells has been investigated. Primary cultures plated onto lethally irradiated mouse embryo fibroblasts showed not only increased colony formation at lowered (0.2 mM) Ca2+ levels but also increased growth rate as indicated by greater colony size. On the other hand, secondary cultures showed little variation in plating efficiency and only small differences in colony size were apparent over the range 0.2-1.64 mM Ca2+. Epidermal growth factor had no effect on the Ca2+ dependence of primary or the Ca2+ independence of secondary cultures.

Non identity of non ionic detergent soluble (NIDS) protein and stratum corneum autoantigens.

The relationship between naturally occurring circulating stratum corneum autoantibodies and experimentally produced antibodies to a non ionic detergent soluble (NIDS) protein fraction from human stratum corneum has been investigated. Using indirect immunofluorescence and indirect haemagglutination techniques, anti NIDS protein serum was shown not to inhibit the autoantibody-antigen reaction. Furthermore no direct interaction could be demonstrated between sheep red blood cells coated with stratum corneum autoantigen and anti NIDS protein serum. The NIDS protein antigens therefore represent a separate and distinct class of stratum corneum antigens.

Effects of all-trans retinoic acid on the morphology of human epidermal cells in vitro.

The effects of all-trans retinoic acid on human epidermal cell cultures were studied using ultrastructural techniques. Differentiation and stratification were reduced in retinoic acid treated epidermal cells. Treated cells developed a rounded appearance and seemed to contain more granules and vacuoles than usual. Desmosomes were not found in treated cells.

In vitro reconstruction of human skin: The use of skin equivalents as potential indicators of cutaneous toxicity.

A three-dimensional reconstruction of living skin-the skin equivalent-has been modified to accept materials that can be applied topically to the skin. Using an irritant gel containing 10% benzoyl peroxide, changes in the skin equivalent model were investigated. Histologically, epidermal necrosis and vacuolar change were observed within 4-6 hr after topical application. Using skin equivalents prelabelled with [(3)H]arachidonic acid, studies involving direct measurement using HPLC and radioimmunoassay have indicated the release of potent lipoxygenase products, such as leukotriene B(4) and 15-hydroxyeicosa-tetraenoic acid. These preliminary results suggest that the skin equivalent may prove to be a useful in vitro model for the prediction of cutaneous toxicity of topically applied substances.

Induction of heat shock proteins as a measure of chemical cytotoxicity.

Exposure of human keratinocytes to non-lethal heat shock treatment (43 degrees C for 90 min) followed by a recovery period of 2 hr at 37 degrees C resulted in the rapid accumulation of two proteins with polypeptide molecular weights of 72 and 90 kDa. Exposure of human keratinocytes to sodium arsenite (10-200 mug/ml) for 90 min at 37 degrees C resulted in the synthesis of proteins with polypeptide molecular weights of 110, 90, 72, 46 and 28 kDa. The 72 kDa heat- or sodium arsenite-induced protein was identified by immunoprecipitation as the 72 kDa heat shock protein. In contrast, the human epithelial tumour cell line (A431) synthesized only the 72 and 28 kDa heat shock proteins in response to arsenite treatment with all other stress proteins being expressed constitutively. General protein synthesis was inhibited in cells exposed to elevated temperature or sodium arsenite. Using immunofluorescence a rapid and reversible accumulation of the 72 kDa heat shock protein was demonstrated within the nucleolus of heat stressed human fibroblasts and keratinocytes.

A stepwise procedure for evaluating irritant materials in normal volunteer subjects.

1. The cutaneous response to a known irritant has been assessed in human volunteer subjects using both clinical scoring and two non-invasive instrumental methods; erythema measurement using an erythema meter and capillary blood flow using a laser Doppler device. 2. Aqueous solutions (0.5% and 1%) of sodium hydroxide were applied to back skin for 3, 15 and 60 min with assessments immediately after removal and at 1, 24 and 48 hours. 3. Increased erythema was seen with increasing duration of exposure and an increase was also seen at 1 h, 24 h and 48 h after removal of the patch. The results obtained with the erythema meter paralleled the clinical erythema scores. However, the laser Doppler device showed the greatest changes immediately after removal of the patch with subsequent readings showing a gradual decrease. 4. Statistical analysis of the data has been carried out to determine the accuracy and precision of the assessment procedures and to determine the minimum test panel size for detecting irritant reactions. 5. Comparison between back and forearm skin indicated a greater sensitivity to sodium hydroxide on the back. 6. The results of this study define an ethical approach to testing irritant materials in human subjects and provide the basis for the development of a classification system for cutaneous irritants.

The link between the peel force of adhesive dressings and subjective discomfort in volunteer subjects.

OBJECTIVE: The study compared the level of discomfort experienced by healthy volunteers on the removal of a range of adhesive wounds. METHOD: This was an open, within subject comparative study of six adhesive dressings in 24 volunteers. The test site was the lower back. Allocation of test materials to the test sites was randomised. The peel force of removal was recorded after 24 hours of application using a device that removed the dressing at a constant speed and angle to the skin surface. The discomfort experienced at each removal was assessed by the subjects themselves using an electronic visual analogue scale. RESULTS: Overall, Mepilex Border was given a significantly lower discomfort score (p < or = 0.01) by the subjects than the other dressings. There were no clear differences between the five other products tested. Tielle and Allevyn Adhesive had significantly higher (p < or = 0.05) peel force than the other products. Mepilex Border caused less discomfort on removal than Duoderm Extra Thin, Biatain and Versiva, even though the peel force was similar. Tielle and Allevyn had higher peel force, but the levels of discomfort were not significantly higher for these products. CONCLUSION: It may be that the level of discomfort experienced by subjects on removal of an adhesive dressing is not entirely dependent on the peel force and that other aspects of the interaction of the skin surface and adhesive play a role.

Effects of adhesive dressings on the stratum corneum of the skin.

Two human models were developed to quantify the stratum corneum removed by different adhesive dressings and to measure the peel force of dressing removal and relate this to stratum corneum removal. The first was an open study designed to compare the effects of applying Mepiform Safetac, Tielle and Duoderm Extra Thin to the skin of 12 normal volunteers aged 19-53 years. Treatments were applied once (one 24-hour application) or three times (three x 24-hour applications) to forearm skin which had been prestained with methylene blue. After dressing removal the dye left on the skin was sampled using the skin surface biopsy method and measured spectrophotometrically. The results show that, after one and three applications, the Mepiform Safetac sites had a higher level of dye than those on which the other dressings had been applied (p < 0.05, after three applications). Based on the assumption that the more dye is left on the skin, the less damage is caused, this suggests that Mepiform Safetac is less damaging to the skin surface than the other products tested. In the second study the peel force needed to remove adhesive dressings from prestained skin was measured and related to the amount of stratum corneum removed. Mepilex Border Safetac, Duoderm Extra Thin, Allevyn Adhesive, Biatain Adhesive and Tielle Hydropolymer Dressing were compared in 20 normal volunteers aged 23-64 years. Three consecutive 24-hour applications of each product were made, with measurements of peel force at 24, 48 and 72 hours. The amount of dye remaining on the skin at 72 hours was assessed by the surface biopsy method. Statistically significant differences between products were observed in terms of both peak force and steady state force of removal. Differences in the level of damage to the superficial stratum corneum were also detected. However, low levels of peel force were not always associated with low damage and, therefore, other factors must contribute to stratum corneum removal in this model.

What are meters measuring?

There is increasing pressure on manufacturers of cosmetic products to provide data to support claims. Data are available from many sources including historical (published literature), laboratory data, cell culture experiments and human studies. Undoubtedly, human studies are the most reliable, and there are a wide range of tests available. Many meters have been developed for measuring different aspects of skin physiology but an understanding of these devices is essential, otherwise the data generated is of little value. There is some confusion as to what exactly some meters measure, an example of which is transepidermal water loss (TEWL) and water content (hydration) of the stratum corneum. Measurement of TEWL is used mainly to support claims that a product may, in the short or long term, improve or repair the barrier function of skin. It is not an indicator of hydration of the stratum corneum. One way to measure hydration is to look for the changes in electrical properties of the stratum corneum that the increased water content produces, i.e. measure capacitance or conductance. It is important that we do not loose sight of the fact that meters may measure something that is imperceptible to the consumer or has no meaning to them. Reliance only on devices that give numbers may lead to problems. An example of a study where three facial cosmetic products were subjected to perceptual tests and to a standard volunteer test for moisturization will be discussed. The relationship between any measured parameter, and what it means to the consumer, needs to be understood. A moisturization claim may be technically supported by a study using a device such as the Corneometer. However, a 20% increase in water content almost certainly does not represent a 20% better moisturization as far as the consumer is concerned. The way forward is to relate the two approaches to product testing during product development. Hopefully, this will allow the product development process to be more systematic.

The distribution of melanin in skin determined in vivo.

BACKGROUND: There continues to be a need for objective, noninvasive methods to measure melanin concentration in vivo in human skin, independent of the confounding chromophore, haemoglobin. Existing methods are limited by a lack of specificity and inability to resolve the spatial distribution of these chromophores. OBJECTIVES: To validate and calibrate the measurement of eumelanin in vivo using SIAscopic techniques, relating this with histologically and analytically determined eumelanin concentrations in nonsun-exposed skin from subjects of Fitzpatrick skin types I-VI. METHODS: Observations were made in five subjects from each of the Fitzpatrick skin types I-VI using chromophore mapping by contact and noncontact SIAscopy and other noninvasive spectrophotometric means. Measurements were performed on the inner aspect of both upper arms. Subsequently two 4 mm punch biopsies were taken from the inner upper arm, one per arm after injection of local anaesthesia. One biopsy was fixed in formalin and processed for histology; specifically, sections were stained for melanin using a silver staining technique and the amount of melanin was graded microscopically. The other biopsy was subjected to an analytical assay to yield precise quantitative measures of melanin. The correlation between the different methods of melanin measurement was determined. RESULTS: Clear, significant correlations were obtained between contact and noncontact SIAscope-derived eumelanin values and actual eumelanin tissue content (determined both histologically and analytically), across the full range of Fitzpatrick skin types. There was no correlation between SIAscope-derived eumelanin and haemoglobin values, indicating efficient separation of the two chromophores. CONCLUSIONS: New contact and noncontact chromophore SIAscopic mapping techniques provide robust, rapid noninvasive measures of the concentration and spatial distribution of eumelanin in vivo, independent of haemoglobin, which correspond to true tissue values for this chromophore.

The role of menthol in skin penetration from topical formulations of ibuprofen 5% in vivo.

In vivo plasma profiles from formulations containing 5% ibuprofen were compared after a single topical application in a randomised, double-blind, cross-over trial. Ibuleve gel (Dermal Laboratories, UK) contained only ibuprofen whilst Deep Relief gel (Mentholatum, UK) also contained 3% menthol. In contrast to results obtained when these products were compared under in vitro conditions, there was no statistically significant difference in vivo between delivery of ibuprofen. Estimated relative bioavailability fraction (Deep Relief gel/Ibuleve gel) from log-transformed AUC((0-24h)) was 0.99 (95% CI: 0.94-1.04), estimated C(max )ratio was 0.96 (95% CI: 0.91-1.00) and estimated t(max) ratio was 1.01 (95% CI: 0.81-1.20). Menthol produces local vasodilation, which reduces skin barrier function, and these data demonstrate that it is inappropriate to extrapolate from in vitro data where formulation components produce biologically-mediated enhancement of permeation which cannot be modelled ex vivo. In clinical use, these products deliver comparable amounts of ibuprofen, but only Deep Reliefgel provides the secondary immediate benefit of the direct analgesic action of menthol.

1 -Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing.

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.