A phase II, randomized, double-blind, placebo-controlled, dose-ranging study to evaluate the efficacy and safety of VT-1161 oral tablets in the treatment of patients with distal and lateral subungual onychomycosis of the toenail.

BACKGROUND: Onychomycosis is a fungal disease that affects the fingernails and toenails and is predominantly caused by dermatophytes. VT-1161 is a novel inhibitor of fungal CYP51 through the inhibition of lanosterol demethylase, and has demonstrated potent activity against Trichophyton rubrum and Trichophyton mentagrophytes. OBJECTIVES: To evaluate the safety and efficacy of four dosing regimens of orally administered VT-1161 compared with placebo in patients with moderate-to-severe distal and lateral subungual onychomycosis of the toenail. METHODS: This was a phase II, randomized, double-blind, placebo-controlled, multicentre study (ClinicalTrials.gov identifier NCT02267356). Patients aged 18-70 years (n = 259) who had 25-75% mycotic involvement were randomized to five treatment groups. They received 300 mg VT-1161 as a 2-week daily dose, followed by a once-weekly dose for either 10 or 22 weeks, or 600 mg VT-1161 as a 2-week daily dose, followed by a once-weekly dose for either 10 or 22 weeks. All treatments were followed by a nontreatment period of 36 weeks. A matching placebo arm was included. RESULTS: In the intent-to-treat population, at week 48 the complete cure rates were 0% in the placebo group and ranged from 32% to 42% in the VT-1161 treatment groups (P < 0·001 vs. placebo). VT-1161 was well tolerated, with no evidence of an adverse effect on liver function or QT intervals. CONCLUSIONS: VT-1161 treatment led to high nail clearance rates and a favourable safety profile. VT-1161 exhibits characteristics that appear promising for the treatment of this chronic and difficult-to-treat condition and warrants further evaluation in larger studies.

Safety and tolerability of luliconazole solution 10-percent in patients with moderate to severe distal subungual onychomycosis.

The study objective was to evaluate the safety, tolerability, systemic exposure, and pharmacokinetics (PK) of 10% luliconazole solution (luliconazole) when topically applied once daily to all 10 toenails and periungual areas in patients with moderate to severe distal subungual onychomycosis. In this single-center, open-label study, 24 patients applied 20 mg/ml of luliconazole (twice the clinical dose) for 29 days with a 7-day follow-up. Complete PK profiles were determined on days 1, 8, 15, and 29. Safety/tolerability assessments included application site reactions, adverse events, vital signs, clinical laboratory findings, and electrocardiograms. Mean luliconazole plasma concentrations remained around the lower limit of quantitation (0.05 ng/ml) and were comparable on days 8, 15, and 29 (range, 0.063 to 0.090 ng/ml), suggesting steady state occurred by day 8. Every patient had undetectable plasma luliconazole levels for at least 11% of the time points, and 12 of the 24 patients had undetectable levels for at least 70% of the time points. The maximum plasma concentration of luliconazole (C(max)) observed in any patient was 0.314 ng/ml and the maximum area under the concentration-time curve from 0 to 24 h (AUC0-24) was 4.34 ng · h/ml. Five patients (21%) had measureable luliconazole levels in the plasma 7 days after the last dose. The median concentration of luliconazole in the nail at this time point was 34.65 mg/g (from 42 of 48 collected toenail samples). There was one mild incidence of skin erythema on day 5 that resolved on day 8, there were no reports of drug-induced systemic side effects, and there was no evidence of QT prolongation. Luliconazole, when applied once daily to all 10 fungus-infected toenails for 29 days, is generally safe and well tolerated and results in significant accumulation of drug in the nail. Systemic exposure is very low, with no evidence of drug accumulation.

Terbinafine in the treatment of dermatophyte toenail onychomycosis: a meta-analysis of efficacy for continuous and intermittent regimens.

OBJECTIVE: To compare mycological and complete cures of terbinafine continuous and intermittent regimens in the treatment of toenail onychomycosis. METHODS: The PubMed database was searched using the terms "terbinafine", "onychomycosis", "continuous" and "pulse(d)" or "intermittent". The inclusion criteria were head-to-head comparison of terbinafine pulse and continuous regimens for dermatophyte toenail infections. Risk ratios were calculated for intention-to-treat and evaluable patient analyses, when possible. Pooled estimates for total and subgroup analyses were calculated using a random effect model, Mantel-Haenszel method and their probabilities were calculated with z-statistics. RESULTS: Nine studies from eight publications were included. Two continuous regimens and four intermittent regimens were investigated. A pooled risk ratio of 0.87 was obtained for intention-to-treat (95% CI: 0.79-0.96, P = 0.004, n = 6) and evaluable patient (95% CI: 0.80-0.96, P = 0.003, n = 8) analyses of mycological cure, favouring continuous terbinafine. For complete cure, pooled risk ratios of 0.97 (95% CI: 0.77-1.23, P = 0.82, n = 7) for intention-to-treat and 0.93 (95% CI: 0.76-1.13, P = 0.44, n = 9) for evaluable patient analyses showed equality of the two regimens. The pulse regimen that demonstrated consistently comparable results to the continuous terbinafine regimen was two pulses of terbinafine 250 mg/day for 4 weeks on/4 weeks off. CONCLUSIONS: Meta-analysis of published studies of toenail onychomycosis showed that a continuous terbinafine regimen is generally significantly superior to a pulsed terbinafine regimen for mycological cure. In contrast, some pulse terbinafine regimens were as effective as continuous terbinafine regimens for complete cure.

A randomized, placebo- and active-controlled, parallel-group, multicentre, investigator-blinded study of four treatment regimens of posaconazole in adults with toenail onychomycosis.

BACKGROUND: Onychomycosis accounts for up to 50% of all onychopathies. OBJECTIVES: To evaluate the efficacy of four posaconazole regimens compared with placebo in the treatment of toenail onychomycosis, to assess the safety and tolerability of posaconazole, and to estimate the relative efficacy of posaconazole against terbinafine. METHODS: A phase 2B, randomized, placebo- and active-controlled, parallel-group, multicentre, investigator-blinded (double blind for placebo) study (ClinicalTrials.gov identifier: NCT00491764). Onychomycosis patients aged 18-75years (n=218) were randomized equally to one of six treatment regimens: posaconazole (oral suspension) 100, 200 or 400mg once daily (24weeks); posaconazole 400 mg once daily (12weeks); terbinafine (tablets) 250mg once daily (12weeks); or placebo (24weeks). The primary efficacy variable was complete cure (negative mycology and 0% nail involvement) at week 48. RESULTS: All posaconazole treatment arms had a significantly (P≤0·012) greater proportion of patients with complete cure at week 48 compared with placebo. The proportions of patients with complete cure were numerically higher for posaconazole 200mg/24weeks (54·1%) and 400mg/24weeks (45·5%), but lower for 400mg/12weeks (20%) compared with terbinafine (37%; differences were not statistically significant). Posaconazole was well tolerated. Seven patients receiving posaconazole withdrew because of asymptomatic liver enzyme increases, as mandated by protocol discontinuation criteria. CONCLUSIONS: The efficacy and favourable safety profile of posaconazole suggest a potential new treatment for onychomycosis. The availability of low-cost generic terbinafine may limit posaconazole use to second-line treatment of infections refractory to, or patients intolerant of, terbinafine, or nondermatophyte mould infections.

Effects of environmentally realistic levels of ozone on stratum corneum function.

The impact of troposphere ozone (O(3)), the major oxidant in photochemical smog, on the overall wellbeing of skin is of considerable interest. To date, limited information is available on the impact of O(3) on human skin. Using a specially designed O(3) exposure chamber, we provide the first evidence that exposure of human skin to O(3) (0.8 ppm, 2-h time-weighted average) significantly reduced vitamin E by 70% and concomitantly increased lipid hydroperoxides by 2.3 fold in the superficial stratum corneum (SC). Although the dose of O(3) used here reduced the resident microflora population by 50% and created a state of oxidative stress within the SC, it did not affect several key enzymes involved in SC homeostasis including the redox-sensitive transglutaminase and the SC tryptic (KLK5) and chymotryptic (KLK7) proteases. Importantly, no signs of skin dryness or erythema were observed. We hypothesize that the limited effects of low doses of O(3) on SC function is attributable to several factors including: (i) protection provided by the anti-oxidant defence system; (ii) inability of O(3) to penetrate the SC; and (iii) limited water available to catalyse the Criegee reaction. Although chronic exposure to O(3) may produce a different outcome than that reported here, our data suggest that exposure to environmentally relevant doses of O(3), at best, induces a moderate state of oxidative stress, without producing a visible clinical response. In our opinion, exposure of skin to UV radiation is a much more significant threat than exposure to ground-level O(3).

Treatment of chronic rhinosinusitis with high-dose oral terbinafine: a double blind, placebo-controlled study.

OBJECTIVES: To evaluate antifungal terbinafine in patients with chronic rhinosinusitis. STUDY DESIGN: Randomized, double-blind, placebo-controlled multicenter pilot study. METHODS: Fifty-three adults with chronic rhinosinusitis received terbinafine 625 mg/day (n = 25) or placebo (n = 28) once daily for 6 weeks. Sinus secretions were collected at screening for mycology. Computed tomography was graded for extent of opacification at baseline and at week 6 using a modification of the Lund-Mackay scoring system. Patients recorded rhinosinusitis symptoms on a visual analogue scale and completed the Rhinosinusitis Disability Index. RESULTS: Positive fungal cultures were found in 41 of 53 patients (17 terbinafine, 24 placebo). (Two subjects from the Terbinafine group and one subject from the control group had no week 6 data). The mean opacification scores pre- and posttreatment for the entire study group improved from 24.2 to 22.5 in placebo (n = 26) and from 26.3 to 24.2 in terbinafine group (n = 23). The least squares means for percent change from baseline (SE) were -6.0 (8.7) for placebo compared with -7.2 (8.1) for terbinafine; 95% confidence interval for treatment difference (-18.9, 21.1); P = .91. Results were similar when only patients with positive fungal cultures were evaluated in the efficacy analysis. Investigator therapeutic evaluations and sinus symptom scores were not significantly different between the two groups at baseline or at treatment completion. CONCLUSION: Treatment with terbinafine failed to improve the symptoms or radiographic appearance of chronic rhinosinusitis even when nasal irrigation samples were positive for fungus on culture. One consideration is that the fungi isolated were not a major pathologic factor in this cohort. It is also possible that, even at high dose, terbinafine may not have maintained therapeutic levels in the nasal secretions.

Vitamin E delivery to human skin by a rinse-off product: penetration of alpha-tocopherol versus wash-out effects of skin surface lipids.

alpha-Tocopherol, the major biologically active form of vitamin E, represents a frequently added lipophilic compound of skin care products. Despite its emerging use in rinse-off formulations, little is known on its efficacy with respect to its deposition or its antioxidant potential in human skin. The objective of this study was to investigate whether the single use of an alpha-tocopherol-enriched rinse-off product provides effective deposition of alpha-tocopherol on human stratum corneum. To test this, forearm skin of 13 volunteers was washed either with an alpha-tocopherol-enriched rinse-off product (test product, TP) or with an alpha-tocopherol-free vehicle control (control product, CP) (contralateral arm) using a standardized wash protocol. Thereafter, skin surface lipids were extracted with pure ethanol after the wash procedure as well as after 24 h. Additionally, one group of volunteers was subjected to irradiation of their forearms with low-dose UVA (8 J/cm(2)) prior to lipid extraction. Skin lipid extracts were analyzed by high performance liquid chromatography using electrochemical detection for vitamin E and UV detection for squalene (SQ) and squalene monohydroperoxide. The results of this in vivo study demonstrated that (1) while CP treatment lowers, TP treatment strongly increases alpha-tocopherol levels of skin barrier lipids; (2) increased vitamin E deposition levels were maintained for a period of at least 24 h, and (3) TP treatment significantly inhibited photooxidation of SQ. In conclusion, the use of alpha-tocopherol-enriched rinse-off products may help to maintain the integrity of the skin barrier by providing protection against photooxidative stress at the level of skin surface lipids.

The use of ozone as an oxidizing agent to evaluate antioxidant activities of natural substrates.

Ozone, the main component of photochemical smog and air pollution, can damage the skin by oxidizing stratum corneum enzymes, lipids and structural proteins. We have developed a rapid screening assay to determine free radical scavenging capacity of various active ingredients that are frequently used in personal care products. Several known antioxidants including vitamin C, vitamin E analog Trolox, walnut seed extract, lipoic acid and ergothioneine inner salt were assayed for their ability to neutralize ozone-induced oxidation of beta-phycoerythrin, a fluorescent reporter protein derived from algae. The free radical scavenging capacities of these antioxidants were quantified and compared. The results demonstrate that this assay is a valuable primary screening tool for identifying antioxidant activity of natural or synthetic substrates that can be used in personal care products to protect the uppermost layer of our skin from oxidizing damage induced by O3.

Maintenance of human skin in organ culture: role for insulin-like growth factor-1 receptor and epidermal growth factor receptor.

Recent studies have shown that adult skin incubated in low-Ca2+ (0.15 mM) medium rapidly degenerates but that normal architecture is maintained when the tissue is incubated in high-Ca2+ medium (1.4 mM Ca2+). To investigate whether the skin cell-produced growth factors insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF) play a role in these events, 2-mm skin punch biopsies were obtained and maintained for 8 to 10 days in a basal medium containing 0.15 mM Ca2+ with and without growth factors, or containing 1.4 mM Ca2+ with and without antibodies to the same growth factors. In parallel experiments, cultured human keratinocytes were incubated for 2 days in the same basal medium in the presence or absence of the same growth factors and antibodies. Consistent with previous reports, organ cultures incubated in the low-Ca2+ (0.15 mM) medium rapidly degenerated. Neither IGF-1 nor EGF prevented the complete degeneration of epidermis and dermis in these organ cultures. Interestingly, the addition of an anti-IGF-1 receptor (IGF-1R) antibody to the organ cultures maintained in high-Ca2+ medium induced changes reminiscent of those seen when the organ cultures were maintained in low-Ca2+ medium, i.e. tissue degeneration. In contrast, antibodies to EGF receptor, used for comparison, only produced focal areas of epidermal necrosis. In vitro, IGF-1 is a known mitogen for keratinocytes. In cultured human keratinocytes, anti-IGF-1R antibody partially inhibited the IGF-1-mediated stimulation of human keratinocyte proliferation without affecting normal spontaneous growth. Additionally, IGF-1R immunolocalized to basal keratinocytes in vivo, exhibited specific binding to IGF-1 in vitro. This indicated a critical role for IGF-1R in both organ cultures ex vivo and cultured cells in vitro. Messenger RNA encoding both IGF-1 and IGF-1R were readily detected by RT-PCR in organ cultures incubated in both low- and high-Ca2+ medium. There were no detectable differences in IGF-1 mRNA in organ cultures growing in the low- or high-Ca2+ medium, but lower levels of IGF-1R mRNA were observed in the organ cultures maintained in low-Ca2+ medium than in those in high Ca2+ medium. These findings are consistent with homeostatic changes in the tissue grown under different calcium concentrations. IGF-1 mRNA was detected in several skin cell populations in vitro, even though it was undetectable in cultured keratinocytes. Taken together these findings indicate that (1) the IGF-1/ IGF-1R loop is critically involved in maintenance of human skin organ cultures ex vivo, and (2) IGF-1, locally produced by skin cells other than keratinocytes, interacts with its receptor, predominantly expressed in basal keratinocytes, to maintain tissue homeostasis.

The effects of prolonged use of surfactants on the skin of normal and photo-exposed hairless mice.

Laboratory tests to assess the irritant potential of materials, such as skin cleansers, which are normally used over a long period by humans, fail to mimic actual use. Most washing tests last a few days or at most a few weeks. Skin sites and techniques are often not standardized. The more standardized patch test involves occlusion and results in exaggerated reactions, since even water and blank patches produce visible and pathophysiologic changes. All of these tests rely on visual assessment despite strong evidence that similarly appearing skin can be very different histologically. The primary objective of this study was to use a well-defined animal model to evaluate the cumulative effects of repeated skin exposure to low levels of surfactants of varying skin irritation potential. A secondary aim was to examine whether or not surfactant-induced skin changes were exacerbated by suberythemal UV radiation. Test materials were applied topically, 2x daily to the dorsal areas of normal and low-dose solar simulator exposed mice for 15 weeks. Our results show that, with conditions mimicking typical normal use, these surfactants and skin cleansers produce little or very mild histological changes in the skin. UV irradiation alone produced the greatest change in all histological parameters examined, with no synergistic or additive effects with the topical treatments.

Topical all-trans retinoic acid (RA) induces an early, coordinated increase in RA-inducible skin-specific gene/psoriasin and cellular RA-binding protein II mRNA levels which precedes skin erythema.

Separation of specific and nonspecific "irritant" effects of topical all-trans retinoic acid (RA) is a key to understanding the mechanism of retinoid action in skin. Cellular RA-binding protein (CRABP) II has been found to be a marker of RA activity in human skin. We have also previously identified a skin-specific gene (RIS-1/psoriasin) which is rapidly induced in human skin treated with RA. Here we compared the kinetics and time-course of RIS-1 and CRABP II gene activation by RA, sodium lauryl sulfate (SLS), a classical irritant, and their vehicle (VH), using a quantitative reverse transcription polymerase chain reaction. RIS-1 and CRABP II were both expressed at very low levels in untreated normal human skin, and in RA-treated skin the kinetics and time course of RIS-1 and CRABP II mRNA induction were similar. Relative to VH-treated skin, RA induced RIS-1 mRNA levels within 6 h, which further increased to 6.4-fold by 24 h (n = 4). Similarly, CRABP II mRNA levels increased from 2.6-fold at 6 h to 7.8-fold after 24 h. At 48 h the relative mRNA levels for both genes decreased towards the steady-state levels. Relative to SLS-treated skin, RIS-1 mRNA increased by 3.2-fold after 6 h and by 5.1-fold after 12 h (n = 3). Also, a 2.6-fold higher CRABP II mRNA observed after 6 h increased to 6-fold after 12 h. After 24 and 48 h RA treatment the relative mRNA levels for both genes decreased towards the steady-state levels. RA-induced skin erythema was not obvious until 24 to 48 h. We conclude, therefore, that induction of RIS-1 and CRABP II mRNA levels by topical RA in human skin are early, coordinated molecular events which precede the clinical cutaneous erythematous response to RA.

Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation.

We investigated the clinical, histologic, and molecular responses of normal human skin to all-trans-retinol (ROL) application, compared to those induced by topical all-trans-retinoic acid (RA), and measured ROL-derived metabolites. Up to 1.6% ROL, 0.025% RA in vehicle (70% ethanol/30% propylene glycol), or vehicle alone were applied in a double-blind fashion to normal buttock skin and occluded for 4 d. ROL produced from none to only trace erythema, which was clinically and statistically insignificant, whereas RA induced a significant 3.7-fold increase in erythema score compared to vehicle (n = 10, p < 0.01). However, ROL induced significant epidermal thickening (1.5-fold at 1.6% ROL, p < 0.01), similar to RA (1.6-fold at 0.025% RA, p < 0.01), relative to the vehicle. ROL, compared with vehicle, also increased mRNA levels of cellular retinoic acid binding protein (CRABP-II) and cellular retinol binding protein (CRBP) genes as determined by Northern analysis (5-6-fold and 6-7-fold, respectively) and riboprobe in situ hybridization. CRABP-II and CRBP protein levels were also higher following ROL than vehicle treatment, as measured by ligand binding (3.2-fold, p < 0.001; n = 7) and Western analysis (3.6-fold, p < 0.003; n = 6), respectively. Epidermal retinyl ester (RE) content, measured after removal of stratum corneum, rose 240-fold (p < 0.005, n = 5) by 24 h of ROL occlusion. RA content, however, was undetectable or detectable only at trace amounts in all samples obtained at 0, 6, 24, and 96 h after ROL occlusion. Detectability of RA was not correlated with ROL treatment (compared to untreated normal skin, p = 0.86) or baseline skin ROL levels (average r = -0.1, p > 0.3). These data demonstrate that ROL application 1) produces trace erythema not significantly different from vehicle, whereas RA causes erythema; 2) induces epidermal thickening and enhances expression of CRABP-II and CRBP mRNAs and proteins as does RA; 3) causes marked accumulation of retinyl ester; and 4) does not significantly increase RA levels. Taken together, the data are compatible with the idea that ROL may be a prohormone of RA, because it produces changes in skin similar to those produced by RA but without measurable RA or irritation.

Clinical improvement following dermabrasion of photoaged skin correlates with synthesis of collagen I.

BACKGROUND AND DESIGN: The ability of superficial dermabrasion to improve clinical features of photoaged skin is well known, but the specific biological mechanisms involved are poorly understood. The so-called repair zone, as visualized by routine histologic examination, has been attributed to new collagen formation within the papillary dermis and may be responsible for clinical improvement following dermabrasion. We investigated molecular and histologic events occurring in dermabraded skin and correlated them with clinical improvement. Ten photoaged patients (mean age, 59 years) underwent facial dermabrasion to the level of the papillary dermis. Clinical severity of photoaging was graded in a blinded manner at baseline and 12 weeks after dermabrasion. Biopsy specimens obtained at baseline and 3 and 12 weeks after dermabrasion were analyzed histologically and by in situ hybridization for fibroblast procollagen I mRNA, immunohistologically and by Western blotting with a monoclonal antibody specific for the aminoterminal cleavage site of procollagen I. RESULTS: Masson's trichrome staining demonstrated an increase in collagen from baseline (as an upper dermal band in the dermabrasion "repair zone") at 3 and 12 weeks' postdermabrasion. Immunohistologic examination demonstrated papillary dermal fibroblast staining for procollagen I at baseline that increased by threefold at 3 weeks' postdermabrasion and by 1.5-fold at 12 weeks' postdermabrasion. Western blotting demonstrated an average-fold increase in pN collagen I of 4.2 +/- 1.5 at 3 weeks and of 2.7 +/- 0.7 at 12 weeks. By in situ hybridization, baseline levels of procollagen I mRNA in papillary dermal fibroblasts increased sixfold at weeks 3 and 12 postdermabrasion. Increase in procollagen I mRNA correlated with clinical improvement, ie, reduction in wrinkling. CONCLUSION: Superficial dermabrasion clinically improves photoaged skin, and this improvement correlates strongly with increased collagen I gene expression.

Storage phosphor imaging technique improves the accuracy of RNA quantitation using 32P-labeled cDNA probes.

The use of the storage phosphor imaging technique to quantitate radioactivity on blots generated by hybridization with 32P-cDNA probes was evaluated and compared with screen-enhanced x-ray film autoradiography. Quantitation of RNA dot blots hybridized with a 28S ribosomal RNA-specific cDNA probe showed that storage phosphor imaging was more sensitive than screen-enhanced x-ray film autoradiography in identifying low amounts of total RNA (1-10 micrograms). Evaluation of Northern blots containing 30 micrograms of total RNA from human skin biopsies hybridized with a cDNA probe for the human acidic ribosomal phosphoprotein, PO, showed that both techniques detected random biological variability of this housekeeping gene in a similar manner. The two techniques exhibited a strong linear correlation in their ability to quantitate mRNA levels of a retinoic acid-inducible gene (RIS-1). This correlation was stronger at levels corresponding to 1-fold to 30-fold increases of signal and decreased beyond this range because of the insensitivity of the x-ray film. In conclusion, storage phosphor imaging is more accurate than screen-enhanced x-ray film autoradiography in identifying different RNA amounts (higher sensitivity) and in detecting increasing RNA signals at high levels of radioactivity (higher dynamic range).

A retinoic acid-inducible skin-specific gene (RIS-1/psoriasin): molecular cloning and analysis of gene expression in human skin in vivo and cultured skin cells in vitro.

A retinoic acid (RA) inducible skin-specific gene transcript (RIS-1) was isolated by differential hybridization screening of a RA-treated human skin cDNA library. The library was constructed from pooled RNA derived from normal adult human skin treated with all trans-RA for 4 h (n = 6) and 12 h (n = 6) in vivo. RIS-1 cDNA corresponded to a 0.6 kb transcript that was barely detectable in normal adult human skin but was significantly induced by 8 h in RA-treated compared to vehicle-treated skin (range 1.1-3.6 fold). Prolonged RA treatment for up to 24 h further increased relative RIS-1 mRNA levels by 1.3-5.5 fold. HPLC analysis of the RA content of 0.1% RA-treated skin in vivo revealed significant levels at 6 h (18.8-120.6 ng RA/g wet weight tissue; approximately 240 nM), immediately preceding the time point at which the increased RIS-1 mRNA level was first seen. This concentration of RA also induced the mRNA levels for cellular RA binding protein II (1.6-19 fold), a marker of RA activity in human skin. RIS-1 mRNA was detected by Northern and dot blotting only in normal skin but not in any other normal human tissues examined, indicating a tissue-specific pattern of gene expression. RIS-1 transcripts were detected at very low levels in untreated cultured human epidermal keratinocytes, while no expression was seen in dermal fibroblasts and melanocytes, the other major cell types in skin. Southern analysis of human and mouse DNA indicated the existence of evolutionarily conserved sequences for RIS-1 between these two species. The polypeptide sequence derived from the partial RIS-1 cDNA was found to be identical to the calcium binding domain found in 'psoriasin', a gene whose expression appears to be increased in the skin of psoriasis patients.

Differential expression of protein kinase C isoenzymes in normal and psoriatic adult human skin: reduced expression of protein kinase C-beta II in psoriasis.

Psoriatic lesions contain elevated levels of 1,2-diacylglycerol, the physiologic activator of protein kinase C (PKC), suggesting that PKC activation may be aberrant in psoriasis. We therefore have investigated the expression and properties of PKC isozymes in normal and psoriatic skin and in human skin cells. Chromatographic and immunoblot analyses revealed the presence of the calcium-dependent PKC isozymes PKC-alpha and -beta, but not -gamma, in normal human epidermis. PKC-beta was more prominent, constituting two thirds of the total calcium-dependent PKC activity. In psoriatic lesions, expression of both PKC-alpha and -beta was decreased, with preferential reduction (80%) of PKC-beta. Northern analysis and semi-quantitative polymerase chain reaction (PCR) indicated no change in the mRNA levels of PKC-alpha and -beta between normal and psoriatic epidermis. In normal epidermis, PKC-alpha was expressed mainly in the lower epidermis, whereas PKC-beta was localized to the upper cell layers, with very intense staining of CD1a+ Langerhans cells. In psoriasis, PKC-alpha staining was present in the lower epidermis, whereas PKC-beta staining was essentially absent, with the exception of some positive inflammatory cells. In addition to PKC-alpha and beta, immunoblot and Northern/PCR analysis revealed expression of four calcium-independent PKC isozymes, delta, epsilon, zeta, and eta, in both normal and psoriatic skin. There were no significant differences in mRNA levels among any of these PKC isozymes, between normal and psoriatic skin. Soluble PKC-zeta protein was modestly increased (twofold) in psoriatic, compared to normal, skin, whereas the levels of PKC-delta, epsilon, and eta were unchanged. Analysis of PKC isozyme expression in the three major cell types of human epidermis revealed that Langerhans cells and keratinocytes were the major sources of PKC-beta and PKC-zeta, respectively. These data demonstrate the diversity of PKC isozyme expression in human skin, and suggest that alterations of PKC-beta and -zeta may participate in the aberrant regulation of growth and differentiation observed in psoriasis.

Retinoic acid metabolites exhibit biological activity in human keratinocytes, mouse melanoma cells and hairless mouse skin in vivo.

Topical all-trans retinoic acid (RA) modulates growth and differentiation of skin and is used in the treatment of various dermatological disorders. RA is metabolized to 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, which are believed to be markedly less active than RA. 3,4-didehydroretinoic acid (ddRA) is a metabolite of 3,4-didehydroretinol which is present in skin. ddRA is biologically active and acts as a morphogen. We have determined the relative biological activity of ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA as assessed by three retinoid responsive systems relevant to skin. RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA (10-100 nM) reduced epidermal transglutaminase activity in human keratinocytes to similar extents, and inhibited alpha-melanocyte-stimulating hormone-isobutylmethylxanthine-inducible tyrosinase activity in Cloudman S-91 mouse melanoma cells by 67, 39, 48, 51 and 19%, respectively, at 100 nM. Daily topical application of the retinoids to hairless mouse skin for 4 days resulted in dose-dependent changes in epidermal thickness and global histological score. The relative potencies of RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, as calculated by parallel line assay, were 1.0, 0.60, 0.34, 0.29 and 0.18, respectively, for epidermal hyperplasia and 1.0, 0.78, 0.23, 0.14 and 0.08, respectively, for global histological score. Interestingly, the compounds exhibited a similar rank order of potency with respect to induction of cellular retinoic acid binding protein-II mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Human dermal fibroblast interleukin-1 receptor antagonist (IL-1ra) and interleukin-1 beta (IL-1 beta) mRNA and protein are co-stimulated by phorbol ester: implication for a homeostatic mechanism.

Although the major functions of fibroblasts are to produce extracellular matrix and to maintain a structural framework for organ systems, recent studies have demonstrated that fibroblasts are active participants in inflammatory processes by synthesizing various inflammatory mediators. In this report, we provide evidence that fibroblasts may contribute to the regulation of inflammation by the synthesis of both the intracellular form and the secretory form of interleukin-1 receptor antagonists in conjunction with interleukin-1 beta production. Indirect immunofluorescence microscopy localized interleukin-1 receptor antagonist and interleukin-1 beta proteins primarily in the fibroblast cytoplasm. Polymerase chain reaction amplification of reverse-transcribed mRNA with primers specific for the intracellular form of interleukin-1 receptor antagonist detected cDNA fragments present in both unstimulated and phorbol ester-stimulated fibroblasts, identical in molecular size to that in unstimulated keratinocytes. Amplification with primers specific for the secretory form of interleukin-1 receptor antagonist, however, detected cDNA fragments in phorbol ester-stimulated fibroblasts and phytohemagglutinin-stimulated peripheral mononuclear cells, but not in unstimulated fibroblasts or keratinocytes. The amplified fibroblast cDNA sequences for both intracellular and secretory interleukin-1 receptor antagonists were confirmed by digestion with three restriction endonucleases. By ethidium bromide visualization of amplified cDNA derived from serially diluted total cellular RNA and by Southern blot hybridization analysis of amplified cDNA, we have demonstrated that fibroblast interleukin-1 receptor antagonist mRNA and interleukin-1 beta mRNA were co-stimulated by phorbol ester. Similarly, ELISA demonstrated that fibroblast cytoplasmic interleukin-1 receptor antagonist protein and interleukin-1 beta protein were co-stimulated by phorbol ester. Our data suggests that the intracellular form of interleukin-1 receptor antagonist may be important in maintaining physiologic homeostasis in fibroblasts during interleukin-1 beta induction and release.

Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin.

A cDNA corresponding to the membrane receptor for growth hormone (GH) was amplified by polymerase chain reaction (PCR) directly from human skin. The cDNA was cloned and found to have complete sequence homology to the extracellular domain of human liver GH receptor (GH-R). Northern analysis, using the cloned GH-R as probe, revealed relatively higher levels of GH-R transcripts in cultured human dermal fibroblasts compared to cultured keratinocytes or keratome biopsies. Semi-quantitative PCR analysis indicated that the level of GH-R mRNA in cultured melanocytes was similar to that in fibroblasts. The receptor protein encoded by GH-R mRNA in fibroblasts was shown by affinity cross-linking to have an apparent M(r) of 115-120 kDa, similar to that of 3T3-F442A fibroblasts used as a control. mRNA transcripts for the major mediator of GH actions, insulin-like growth factor 1 (IGF-1), were detected by PCR in fibroblasts, melanocytes, and keratome biopsies, but not in keratinocytes. In contrast, IGF-1 receptor mRNA were abundant in cultured keratinocytes and skin biopsies, as determined by Northern analysis. IGF-1 but not GH (5-50 ng/ml) promoted clonal proliferation of cultured keratinocytes. In contrast, GH (10 ng/ml) after 5 d markedly increased fibroblast cell numbers (70%, p less than 0.009) over 0.2% serum control. These data indicate that human skin cells possess the molecular elements necessary to respond to GH and raise the possibility that GH may influence skin growth in vivo.