Tape-stripped Skin Research Articles

IL-10 is critical for Th2 responses in a murine model of allergic dermatitis.

We found that mechanical injury to mouse skin, which can be caused by tape stripping, results in rapid induction of IL-10 mRNA. IL-10-/- mice were used to examine the role of IL-10 in a mouse model of allergic dermatitis induced by epicutaneous (EC) sensitization with OVA on tape-stripped skin. Skin infiltration by eosinophils and expression of eotaxin, IL-4, and IL-5 mRNA in OVA-sensitized skin sites were severely diminished in IL-10-/- mice. Following in vitro stimulation with OVA, splenocytes from EC-sensitized IL-10-/- mice secreted significantly less IL-4, but significantly more IFN-gamma, than splenocytes from WT controls. A similar skewing in cytokine secretion profile was observed in the splenocytes of IL-10-/- mice immunized intraperitoneally with OVA. IL-10-/- APCs skewed the in vitro response of OVA T cell receptor (TCR) transgenic T cells towards Th1. Examination of the Th response of WT and IL-10-/- mice immunized with OVA-pulsed WT or IL-10-/- DCs revealed that both DCs and T cells participate in IL-10 skewing of the Th2 response in vivo. These results suggest that IL-10 plays an important role in the Th2 response to antigen and in the development of skin eosinophilia in a murine model of allergic dermatitis.

IL-10 is critical for Th2 responses in a murine model of allergic dermatitis

We found that mechanical injury to mouse skin, which can be caused by tape stripping, results in rapid induction of IL-10 mRNA. IL-10–/– mice were used to examine the role of IL-10 in a mouse model of allergic dermatitis induced by epicutaneous (EC) sensitization with OVA on tape-stripped skin. Skin infiltration by eosinophils and expression of eotaxin, IL-4, and IL-5 mRNA in OVA-sensitized skin sites were severely diminished in IL-10–/– mice. Following in vitro stimulation with OVA, splenocytes from EC-sensitized IL-10–/– mice secreted significantly less IL-4, but significantly more IFN-γ, than splenocytes from WT controls. A similar skewing in cytokine secretion profile was observed in the splenocytes of IL-10–/– mice immunized intraperitoneally with OVA. IL-10–/– APCs skewed the in vitro response of OVA T cell receptor (TCR) transgenic T cells towards Th1. Examination of the Th response of WT and IL-10–/– mice immunized with OVA-pulsed WT or IL-10–/– DCs revealed that both DCs and T cells participate in IL-10 skewing of the Th2 response in vivo. These results suggest that IL-10 plays an important role in the Th2 response to antigen and in the development of skin eosinophilia in a murine model of allergic dermatitis.

Characterization of transdermal solute transport induced by low-frequency ultrasound in the hairless rat skin

Sonophoretic drug transport with low-frequency (41–445 kHz) and low-intensity (60–240 mW/cm 2) ultrasound was characterized using hydrophilic calcein and deuterium oxide (D 2O) as a solvent vehicle in excised hairless rat skin. The excised skin was mounted in vertical diffusion chambers for measurement of skin resistance and sonophoretic transport of calcein and D 2O. The calcein content of the skin was also measured after ultrasound application. When the stratum corneum (sc) side was exposed to ultrasound at an intensity of 60 mW/cm 2 for 30 min, the calcein flux in the sc-to-dermis direction was increased by 22.3-, 6.3-, and 3.8-fold from a baseline of 0.0088±0.0100 nmol/(cm 2·h) at frequencies of 41, 158, and 445 kHz, respectively, without significant changes in skin resistance. The ultrasonically-enhanced fluxes returned to baseline following cessation of the ultrasound application. At 41 kHz, there was a further increase in the magnitude of enhancement and a significant decrease in skin resistance (by 50% of the baseline resistance) on increasing the intensity from 60 to 120 mW/cm 2, whereas no further enhancement was observed at 158 and 445 kHz up to 240 mW/cm 2. Comparison of the calcein content in the skin before, during, and after ultrasound application at 41 kHz, 120 mW/cm 2, was consistent with a transient ultrasonically-induced increase in calcein flux. In the sonophoretic transport experiments at 41 kHz, 120 mW/cm 2, calcein transport correlated well with D 2O transport. When 41-kHz ultrasound was applied to the sc side at 120 mW/cm 2, the calcein and D 2O fluxes in the sc-to-dermis direction were 13.7- and 5.2-fold higher than those in the dermis-to-sc direction. Similar directionality was also observed in tape-stripped skin, suggesting possible induction of convection in the direction of sound propagation. However, dermal application under the same ultrasound conditions induced neither an increase in calcein and D 2O transport nor a decrease in skin resistance. These results demonstrate that low frequency sonophoresis is a potentially useful technique for controlling transdermal drug transport. Convective solvent flow as well as structural alteration of the skin induced by ultrasound are likely to be responsible for the observed sonophoretic transport enhancement.

In vivo study of the physiological impact of stratum corneum sampling methods

Several methods have been developed to study in vivo the stratum corneum with minimal 'invasion'. Cyanoacrylate adhesives employed in skin surface biopsy (SSB) and tape stripping are commonly used techniques that can be applied to study the different layers of the epidermis or even to permeation studies. However, depending on the type of adhesive tape used and pressure applied, different information is gathered. The purpose of this study was to identify and compare the impact of both techniques in the normal skin physiology to further establish its potential usefulness and limitations. Alterations were focused on basic structural related properties, such as epidermal barrier function (assessed by transepidermal water loss evaporimetry) and flow related changes (by laser Doppler velocimetry and colourimetry). Results indicate a more pronounced impact of SSB than that of tape stripping, attributable to the removal of a thicker layer of cells. The intertechnique correlation coefficients were good between transepidermal water loss and colourimetry, but poorer between these techniques and laser doppler flowmetry, which probably reflects the difficulties associated with this technique.

Optimization of epicutaneous immunization for the induction of CTL

The immune system of the skin has recently been exploited for the development of non-invasive vaccine technologies. However, one of the limitations of current vaccine protocols is the inefficient priming of cytotoxic T lymphocytes (CTL). In this study, we report that the application of either an immunodominant class I MHC restricted ovalbumin peptide or whole ovalbumin protein, to tape-stripped skin together with the co-application of the bacterial enterotoxin cholera toxin (CT) induces antigen-specific CTL. Tape-stripping (TS) was found to enhance the magnitude of antibody responses to co-administered protein and to promote the generation of antigen-specific IgG 2a responses. As well, both cholera toxin and tape-stripping enhanced epidermal dendritic cell (DC) immigration into draining lymph nodes. The adjuvant effect of co-administered cholera toxin and tape-stripping in promoting CTL priming was not dependent on IL-12. Epicutaneous immunization has previously been shown to induce robust antibody responses to administered protein antigen. We now demonstrate the induction of robust and persistent CTL responses to epicutaneously administered protein antigen. Epicutaneous immunization is cheap, simple and effective. These findings suggest the potential use of the skin for the generation of protective immune responses to both viral and tumor challenge.

Reverse iontophoretic monitoring in premature neonates: feasibility and potential

Premature neonates represent a fragile patient population, often subjected to intensive clinical care and multiple drug therapy, which must be monitored carefully and continuously. The difficult and painful nature of repetitive blood sampling, particularly in this population, has provided considerable impetus for the development of noninvasive methods for monitoring blood analytes. Reverse iontophoresis, a relatively new technology already used for the transdermal monitoring of blood glucose levels in adults, may be particularly well-suited to exploit the unique properties of preterm neonatal skin. The underdevelopment of the premature infant’s epidermis, and more specifically the stratum corneum (SC), results in an increased permeability to molecular transport. In this study, we have investigated the feasibility of reverse iontophoretic monitoring of two model drugs, caffeine and theophylline, which are often administered to premature neonates. To this purpose, tape-stripped porcine skin in vitro, which has been previously demonstrated to be an excellent model for premature neonatal skin, was employed. Reverse iontophoresis across intact membranes enabled a quantifiable extraction of both drugs predominantly at the cathode compartment. The mechanism of extraction of these essentially neutral drugs (caffeine and theophylline being uncharged at pH 7.4) was electroosmosis. However, when the SC was removed by progressive tape-stripping, the amounts of drugs extracted by reverse iontophoresis were equivalent to those obtained by passive diffusion. In these circumstances, therefore, the benefit and usefulness of the applied electric field had been lost. In summary, the absence of an at least partially functional skin barrier obviates, in the case of neutral molecules, the control (and directional transport) offered by iontophoresis; in contrast, for ionized species, where the principal iontophoretic transport mechanism is electromigration, the approach should be valid.

Physicochemical interaction of local anesthetics with lipid model systems—correlation with in vitro permeation and in vivo efficacy

In dermal/transdermal drug administration stratum corneum (SC) is often the rate-limiting step. Furthermore, the intercellular lipid domain of SC is nowadays widely accepted as the major contributor to the skin barrier. The current work investigates whether the difference in the level of topical efficacy of local anesthetic compounds correlates with the type of interaction between the drug and the intercellular lipids of SC. Therefore, local anesthetics of varying topical efficacy were evaluated with respect to their effect on the morphology of various model lipid systems using small and wide angle X-ray diffraction (SWAXD) and differential scanning calorimetry (DSC). The model lipids used were glyceryl monooleate, sphingomyelin and lipids isolated from human SC. Furthermore, partitioning into isolated human SC as well as permeation through isolated human SC and human tape-stripped skin were investigated in vitro. The results indicate that local anesthetics may act as their own permeation enhancers by increasing the degree of hydrocarbon chain fluidity of the intercellular lipids. Eventually these interactions may induce non-lamellar reversed types of liquid crystalline structures locally in SC, which further facilitate the drug mobility. The large difference in topical efficacy of the investigated local anesthetics could not be explained simply by looking at their effect on the phase behavior of lipid model systems. Despite the similarities in physicochemical properties of these substances, the in vitro skin permeability differed markedly (AD>EMLA>lidocaine>prilocaine>sameridine). Thus, it was concluded that sufficient drug permeability over SC is essential to obtain local anesthesia by blocking the superficial nociceptors.

Epicutaneous natural rubber latex sensitization induces localized Th2-dominate dermatitis and strong IgE response

1081 Epicutaneous Natural Rubber Latex Sensitization Induces Localized Th2-Dominated Dermatitis and Strong IgE Response Harri Tapio Alenius*, Maili Lehto*, /man Amghaiab*, Marja-Leena Majuri*, Guoying Wang*, Henrik Wolff*, Kristiina Turjanmaa§, Antti Lauerma¥ Timo Reunala§, Timo Palosuo~ *Finnish Institute of Occupational Health, Helsinki, Finland §Tampere University Hospital, Tampere, Finland ¥Helsinki University Hospital for Skin and Allergic Diseases, Helsinki ~National Public Health Institute, Helsinki, Finland In addition to immediate type I allergy symptoms, natural rubber latex (NRL) allergy may manifest as protein contact dermatitis on the hands of health-care workers and other NRL glove users. We examined the role of repeated epicutaneous (EC) sensitization with NRL proteins in the development of dermatitis and antibody response in a murine model. EC application with NRL or glove extract (GE) to the shaved and tape-stripped mouse skin, but not with phosphate buffered saline (PBS) control, produced significant influx of mononuclear cells, T cells and eosinophils to the sensitized skin sites (P<0.001). The number of degranulated mast cells in NRLor GE-sensitized skin sites was significantly higher compared to PBS-treated sites (p<0.0l). IL-4 mRNA expression was markedly increased in NRLand GE-sensitized skin sites compared to PBS-treated sites (p<0.001). Moreover, significant increase in MIPl-alpha and M1PI-beta mRNA was found in NRLor GE-sensitized skin sites (p<0.0 I). EC application with NRL induced a striking increase in total and specific IgE levels but not in IgG2a levels. In contrast, intraperitoneal (IP) immunization with NRL combined with aluminum hydroxide induced a strong IgG2a response. Interestingly, EC-application with NRL elicited strong IgE response against Hey b 6.01 but not against Hev bl. In contrast, IP immunization with NRL elicited strong IgG2a production to Hev bl but not to Hev b 6.01. These results demonstrate that EC-sensitization with NRL proteins induces Th2-dominated dermal inflammation and strong IgE response in a murine model. EC-sensitization to NRL proteins eluting from the gloves may therefore play also in man an important role in the development of hand dermatitis and NRL-specific IgE antibodies.

In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.

Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

Histopathologic effects of cutaneous tape stripping in pigs.

The stratum corneum (SC) is the major barrier to topical absorption of medications. Skin tape stripping (TS) removes the stratum corneum, allowing more rapid absorption of drugs such as local anesthetics. Prior to evaluating TS in human clinical trials, this study was performed to evaluate its immediate and delayed histopathologic effects in swine. This was a prospective, interventional, longitudinal, experimental animal study using two isoflurane-anesthetized young swine. Cellophane tape was applied to the skin of clipped swine flanks and peeled away 10, 15, 20, 25, or 30 times. Each level of tape stripping was performed three times in each pig. Full-thickness biopsies were taken at 30 minutes and two weeks later for blinded histopathologic evaluation by a dermatopathologist using randomly ordered hematoxylin-and-eosin-stained tissue sections and conventional light microscopy. The absolute thickness of the cornified layer was measured and compared with normal (unstripped) cornified layer. Tape stripping up to 30 times produces thinning of the SC without detectable changes in the underlying epidermis and dermis at 30 minutes and 14 days post-stripping. The degree of thinning was proportional to the number of tape applications. Complete recovery of the cornified layer was noted at two weeks. There were no adverse effects, such as infection, scarring, or inflammatory cell infiltrates. Under the conditions studied, TS not only appears safe, but appears to produce no long-term sequelae.

Barrier recovery rate varies time-dependently in human skin.

The recovery in cutaneous barrier functions, assessed in terms of transepidermal water loss, 1 h after tape stripping of volar forearm skin in human volunteers, was investigated at different times over the 24 h day. The barrier recovery rate was significantly lower between 20:00 h and 23:00 h than that at other time points. The skin surface temperature and the basal transepidermal water loss reached their highest values at about 03:00 h (33.6 degrees C and 0.30 mg cm-2 h-1), while the cortisol level in the saliva was highest at 09:00 h (7.8 pmol mL-1). These results suggest significant time-dependent variation in cutaneous barrier repair independent of changes in skin temperature and cortisol level.

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin.

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioid-receptor interaction by NTX for 4 h resulted in an elevation of 36-66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met5]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37-46% at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptide receptor interactions.

Coexpression of keratins 13 and 16 in human keratinocytes indicates association between hyperproliferation-associated and retinoid-induced differentiation.

In human skin, epidermal differentiation occurs in two ways: normal differentiation, characterized by keratin 10 expression, and alternative differentiation. Alternative differentiation can be hyperproliferation-associated differentiation (expression of keratin 16) or the reinduction of an embryonic type of differentiation (expression of keratin 13). This embryonic type of differentiation is also seen following treatment with retinoids. In the present study, the hypothesis that hyperproliferation-associated and retinoid-induced differentiation are separate processes was investigated. Two areas of normal skin were treated for 24 h with 0. 1% all-trans-retinoic acid. Subsequently, one of the areas was tape-stripped and treatment was continued for 48 h. Multiparameter flow cytometry permitted simultaneous measurement of two coexpressed differentiation markers (retinoid-induced and normal differentiation or retinoid-induced and hyperproliferation-associated differentiation) and the proliferation characteristics (cells in S/G(2)M phase). Concerning normal and retinoid-induced differentiation, the all-trans-retinoic acid-induced expression of keratin 13 was only seen in tape-stripped retinoid-treated skin and exclusively together with that of keratin 10. The assessment of hyperproliferation-associated and retinoid-induced differentiation showed slight expression of keratin 13 without expression of keratin 16 in tape-stripped skin. Coexpression of keratins 16 and 13 was exclusively seen in tape-stripped retinoid-treated skin. The finding that keratin 13 expression following treatment with all-trans-retinoic acid occurred exclusively in hyperproliferative skin suggests that retinoid-induced and hyperproliferation-associated differentiation are coupled. Coexpression of keratins 13 and 16 provides direct experimental evidence for this association.

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis.

H-2K(b)-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181-188 (TRP-2) and connexin 37 residues 52-59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Ia(b+) cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

Positive atopy patch test reactions to Pityrosporumorbiculare in atopic dermatitis patients

Pityrosporum orbiculare, although a part of our normal cutaneous microflora, can cause skin infections and induce specific immunoglobulin (Ig) E antibodies in atopic dermatitis patients. P. orbiculare is therefore considered to be one of the trigger factors for atopic dermatitis. To investigate if P. orbiculare can induce an eczematous reaction in atopic dermatitis patients, seborrhoeic dermatitis patients and healthy controls. Fifteen atopic dermatitis patients, eight seborrhoeic dermatitis patients and eight healthy controls were patch tested with extract of P. orbiculare on non-lesional, tape-stripped skin of the back. NaCl was used as a negative control. The patch tests were evaluated after 24, 48 and 72 h. Skin biopsies were taken from P. orbiculare patch test sites at 24 h and 72 h, from NaCl patch test sites at 72 h, from non-lesional skin and, in the atopic dermatitis patients, also from lesional skin. The skin biopsies were investigated with immunohistochemical techniques. P. orbiculare-specific IgE in serum was analysed with RAST. Specific IgE to P. orbiculare was found in serum from 13/15 atopic dermatitis patients and in eight of them a positive patch test reaction to P. orbiculare was observed, with a maximal reaction at 48 h. Significantly higher serum levels of P. orbiculare-specific IgE were detected in patch test-positive compared with patch test-negative atopic dermatitis patients (P < 0. 01). The seborrhoeic dermatitis patients and healthy controls were RAST and patch test-negative for P. orbiculare. In the patch test-positive atopic dermatitis patients an infiltration of CD4+ T cells and eosinophils was observed at the P. orbiculare patch test sites together with an upregulation of ICAM-1 and HLA-DR expression. P. orbiculare can induce an eczematous reaction in sensitized atopic dermatitis patients and may be an important trigger factor in these patients. The P. orbiculare patch test can be of diagnostic value in this subgroup of atopic dermatitis patients.

Effect of barrier perturbation on cutaneous penetration of salicylic acid in hairless rats: in vivo pharmacokinetics using microdialysis and non-invasive quantification of barrier function.

The penetration of topically applied drugs is altered in diseased or barrier-damaged skin. We used microdialysis in the dermis to measure salicylic acid (SA) penetration in hairless rats following application to normal (unmodified) skin (n = 11) or skin with perturbed barrier function from (1) tape-stripping (n = 5), (2) sodium lauryl sulphate (SLS) 2% for 24 h (n = 3) or (3) delipidization by acetone (n = 4). Prior to the experiment, transepidermal water loss (TEWL) and erythema were measured. Two microdialysis probes were inserted into the dermis on the side of the trunk and 5% SA in ethanol was applied in a chamber overlying the probes. Microdialysis sampling was continued for 4 h, followed by measurements of probe depth by ultrasound scanning. SA was detectable in all samples and rapidly increasing up to 130 min. Microdialysates collected between 80 and 200 min showed mean SA concentrations of 3 microg/ml in unmodified and acetone-treated skin, whereas mean SA concentrations were 280 microg/ml in SLS-pretreated skin and 530 microg/ml in tape-stripped skin (P < 0.001). The penetration of SA correlated with barrier perturbation measured by TEWL (P < 0.001) and erythema (P < 0.001). A correlation between dermal probe depth and SA concentration was found in unmodified skin (P = 0.04). Microdialysis sampling in anatomical regions remote from the dosed site excluded the possibility that SA levels measured were due to systemic absorption. Microdialysis sampling of cutaneous penetration was highly reproducible. Impaired barrier function, caused by irritant dermatitis or tape stripping, resulted in an 80- to 170-fold increase in the drug level in the dermis. This dramatic increase in drug penetration could be relevant to humans, in particular to topical treatment of skin diseases and to occupational toxicology.

The Response of Distant Uninvolved Psoriatic Skin to Standardised Injury Is Not Different from That in Normal Skin

The epidermis of uninvolved psoriatic skin is characterised by a slight hyperproliferation and an increase in inflammatory parameters, whereas no differentiation abnormalities are seen. Data with respect to the response of distant uninvolved psoriatic skin to standardised injury are not uniform. In this study, a recently developed multiparameter flow cytometric assay was used to compare the response to tape stripping of uninvolved psoriatic and normal skin. With this method, a parameter for proliferation, differentiation and inflammation was measured simultaneously. Concerning these parameters, no statistically significant differences were found between uninvolved psoriatic skin and normal skin. The mechanism that underlies hyperproliferation in distant uninvolved psoriatic skin does not indicate an intrinsic abnormality in keratinocytes. Inflammatory signals might play a role in this process.

Estimation of intradermal disposition kinetics of drugs: I. Analysis by compartment model with contralateral tissues.

The objectives of dermal application of drugs are not only systemic therapeutic, but also local ones. We would expect its intradermal kinetics to be dependent on its therapeutic purpose. To develop more efficient drugs for local or systemic therapeutics, it will be important to estimate quantitatively the intradermal disposition of drugs applied topically. We tried, therefore, to develop the compartment model to describe the intradermal disposition kinetics after topical application of drugs. In vivo percutaneous absorption study for antipyrine, a model compound, was performed using rats with tape-stripped skin, using the assumption that the stratum corneum permeability to drugs would be improved enough not to be a rate-limiting process. To analyze the results obtained, a 4-compartment model, composed of donor cell, viable skin, muscle, and plasma compartments, was applied. Although the fitting lines obtained could describe the concentration-time profiles of antipyrine in each compartment very well, the concentration profiles in the contralateral tissues were extensively overestimated. Therefore, we developed a 6-compartment model which included the viable skin and muscle in the contralateral site, and analysed the concentration-time curve of each compartment. The fitting curves were in good agreement with the experimental data for all the compartments including the contralateral viable skin and muscle, and thus, this model was recognized to be adequate for the estimation of intradermal kinetics after topical application. Judging from the obtained values of clearance from viable skin to plasma and from viable skin to muscle, about 80% of antipyrine penetrated into viable skin, which suggested it was absorbed into circulating blood and 20% was transported to muscle under viable skin. Pharmacokinetic analysis using the 6-compartment model would be very useful for the estimation of local and systemic availability after topical application of drugs.

Percutaneous Absorption of Salicylic Acid, Theophylline, 2,4-Dimethylamine, Diethyl Hexyl Phthalic Acid, andp-Aminobenzoic Acid in the Isolated Perfused Porcine Skin Flap Compared to Manin Vivo

Human risk assessment for topical exposure requires percutaneous absorption data to link environmental contamination to potential systemic dose. Human absorption data are not readily available, so absorption models are used.In vitrodiffusion systems are easy to use but have proved to be somewhat unreliable and are not validated to man. This study compares percutaneous absorption in the isolated perfused porcine skin flap (IPPSF) system with that in manin vivo.The study design utilized the same compounds and the same dose concentration and vehicle in both systems. Methodology for each system was that which is routinely used ineach system. The skin surface was not protected during the absorption dosing period. Percutaneous absorption values were, for man and the IPPSF system, respectively: salicylic acid (6.5 ± 5.0%; 7.5 ± 2.6%), theophylline (16.9 ± 11.3%; 11.8 ± 3.8%), 2,4-dimethylamine (1.1 ± 0.3%; 3.8 ± 0.6%), diethyl hexyl phthalic acid (1.8 ± 0.5%; 3.9 ± 2.4%), andp-aminobenzoic acid (11.5 ± 6.3%; 5.9 ± 3.7%) (correlation coefficient was 0.78;p< 0.04).The skin surface wash recovery postapplication was similar for salicylic acid in man (53.4 ± 6.3%) and the IPPSF system (48.2 ± 4.9%). With the other compounds the majority of surface chemical was recovered in the surface wash and skin tape strip in the IPPSF system. With man, other than salicylic acid, only a few percent applied dose was recovered with surface washing and tape stripping. Since the wash procedure was effective with pig skin, we can assume that these chemicals in man were lost to adsorption to any clothing or bedding with the volunteers. The absorption in man was not less than that in the IPPSF. Assuming the dose was lost in man, it seems plausible that whatever compound was to penetrate human skin in solvent vehicle did so in the period of time before the chemical was removed.The IPPSF system appears to be a good model for predicting percutaneous absorption relative to man. This study design should be used to validate other systems to humansin vivo.

Epidermal cell kinetics by combining in situ hybridization and immunohistochemistry.

Double labelling can serve as a useful tool for providing information about cell kinetics in normal and hyperproliferative tissues in general, and skin in particular. We have developed a double-labelling method that combines immunohistochemistry using the monoclonal antibody MIB1 and non-isotopic in situ hybridization using either a digoxigenin-labelled RNA probe specific for histone 3 mRNA sequences or a Fluorescein-labelled oligonucleotide probe specific for histone 2b, 3, 4 mRNA sequences. Double labelling was performed on normal, tape-stripped normal skin and psoriatic skin. The three proliferation markers were also examined by single labelling. The ratio of cells in the S-phase (Ns) and the growth fraction (Ncy) was determined. In normal skin, psoriatic skin and tape-stripped normal skin after 24 h and after 48 h, we calculated that 15%, 16%, 3% and 12% of growth fraction consisted of cells in the S-phase respectively. The S-phase lasts approximately 10 h, so the cell cycle time in normal and psoriatic skin is approximately 62.5 h. At present, the MIB1/H3 digoxigenin or MIB1/H2b-H3-H4 Fluorescein double-labelling technique cannot be used routinely. Therefore, in order to understand the cell kinetic processes better, experiments are recommended to optimize these methods. From a practical point of view and for reasons of specificity and sensitivity, we prefer the Fluorescein-labelled oligonucleotide probe method.