Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests. Nanoparticles were more toxic than micro-sized particles, and their effects were stronger when supplied in growth medium than in topical application. Still topically applied CuO nanoparticles induced inflammatory cytokine secretion and necrosis, especially in epidermis deprived of its protective cornea. Since nanoparticle penetration was not seen, we propose that they may adhere to skin surface, react with the local acidic environment, and generate soluble ions that make their way to inner sites. This work illustrates the abilities of skin organ culture to evaluate the biological effects of topically-applied materials on skin in vitro.

Penetration and biological effects of topically applied cyclosporin A nanoparticles in a human skin organ culture inflammatory model.

Systemic antipsoriatic therapies have potentially life-threatening, long-term side effects. The efficacy of topical drugs is poor, but may be improved by the use of delivery systems based on drug nanoparticles. To produce nanoparticles (NP) composed of cyclosporin A, a classical antipsoriatic drug, and to investigate their penetration and biological effects in human skin affected by psoriatic symptoms, poly-ε-caprolactone (PCL) and cyclosporin A (CsA) NP were prepared by the solvent evaporation method. Skin penetration was followed using fluorescently labeled NP in human skin organ cultures (hSOC). Psoriatic symptoms were mimicked in hSOC by the treatment with epidermal growth factor (EGF) and bacterial lipopolysaccharide (LPS). Cell viability in hSOC was evaluated by the resazurin test, and cytokine secretion into the growth medium was measured by immunodetection. We showed that topically applied NP diffused throughout the epidermis within two hours and through the dermis within the following day. They significantly reduced the secretion of inflammatory cytokines IL-1ß, IL-6, IL-8, IL-20 and IL-23. At active doses, no cytotoxicity was detected. This type of NP display relevant properties for the use as topical anti-inflammatory agents and may help to resorb psoriatic lesions.

Photo-damage protective effect of two facial products, containing a unique complex of Dead Sea minerals and Himalayan actives.

BACKGROUND: Skin appearance is badly affected when exposed to solar UV rays, which encourage physiological and structural cutaneous alterations that eventually lead to skin photo-damage. AIMS: To test the capability of two facial preparations, extreme day cream (EXD) and extreme night treatment (EXN), containing a unique complex of Dead Sea water and three Himalayan extracts, to antagonize biological effects induced by photo-damage. METHODS: Pieces of organ cultures of human skin were used as a model to assess the biological effects of UVB irradiation and the protective effect of topical application of two Extreme preparations. Skin pieces were analyzed for mitochondrial activity by MTT assay, for apoptosis by caspase 3 assay, and for cytokine secretion by solid phase ELISA. Human subjects were tested to evaluate the effect of Extreme preparations on skin wrinkle depth using PRIMOS and skin hydration by a corneometer. RESULTS: UVB irradiation induced cell apoptosis in the epidermis of skin organ cultures and increased their pro-inflammatory cytokine, tumor necrosis α (TNFα) secretion. Topical applications of both preparations significantly attenuated all these effects. Furthermore, in human subjects, a reduction in wrinkle depth and an elevation in the intense skin moisture were observed. CONCLUSIONS: The observations clearly show that EXD and EXN preparations have protective anti-apoptotic and anti-inflammatory properties that can attenuate biological effects of skin photo-damage. Topical application of the preparations improves skin appearance by reducing its wrinkles depth and increasing its moisturizing impact.

Non-invasive skin biomarkers quantification of psoriasis and atopic dermatitis: cytokines, antioxidants and psoriatic skin auto-fluorescence.

BACKGROUND: Psoriasis and atopic dermatitis (AD) are challenging to treat due to the absence of suitable monitoring procedure and their recurrences. Alteration of skin hydrophilic biomarkers (SHB) and structural elements occur in both disorders and may possess a distinct profile for each clinical condition. OBJECTIVE: To quantify skin cytokines and antioxidants non-invasively in psoriatic and in AD patients and to evaluate skin auto-fluorescence in psoriatic patients. METHODS: A skin wash sampling technique was utilized to detect the expression of SHB on psoriatic and AD patients and healthy controls. Inflammatory cytokine (TNFα, IL-1α and IL-6) levels, total antioxidant scavenging capacity and uric acid content were estimated. Additionally, measurement of the fluorescent emission spectra of tryptophan moieties, collagen cross-links and elastin cross-links were performed on psoriatic patients and healthy controls. RESULTS: Our findings demonstrate significant alterations of the SHB levels among psoriasis, AD and healthy skin. Differences were also observed between lesional and non-lesional areas in patients with psoriasis and AD. Ultra-structural changes were found in psoriatic patients both in lesional and non-lesional areas. CONCLUSION: Employing non-invasive measurements of skin wash sampling and skin auto-fluorescence might serve as complementary analysis for improved diagnosis and treatment of psoriasis and AD. Furthermore, they may serve as an additional monitoring tool for various diseases, in which skin dysfunction is involved.

Isothiocyanates inhibit psoriasis-related proinflammatory factors in human skin.

OBJECTIVE: 4-Methylthiobutylisothiocyanate (MTBI), the main rocket (Eruca sativa) seed isothiocyanate (ITC), and its oxidized form, sulforaphane (SFN), were assessed for their potential effects on psoriasis-related factors. METHODS: MTBI and SFN were evaluated for their effect on mRNA expression and cytokine secretion in vitro in human monocytes and macrophage-like cells and ex vivo in topically treated inflamed human skin. In addition, they were assayed in vivo for morphological changes in topically treated psoriasiform human skin in severe-combined immunodeficient (SCID) mice. RESULTS: MTBI and SFN contributed to the prevention of inflammation development and reduced ongoing inflammation by downregulating lipopolysaccharide (LPS)-induced mRNA expression of the psoriasis-related cytokines, interleukin (IL)-12/23p40 (25-58 %), tumor necrosis factor (TNF)-α (15-37 %) and IL-6 (25-71 %), in human macrophage-like cells. In monocytes, they tended to act additively on cytokine mRNA and reduced IL-12/23p40 (51 %) secretion. In an ex-vivo inflamed human skin organ culture, MTBI (1 µg/ml) reduced the secretion of IL-1 (39 %) and IL-6 (32 %). Moreover, 2/8 and 3/8 of the MTBI- and SFN-treated psoriasiform SCID mice, respectively, recovered partially or entirely from the psoriasiform process. CONCLUSIONS: Results from these models indicate the potential of rocket seed ITCs as biological agents in the therapy of psoriasis and inflammation-related skin diseases.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

BACKGROUND: Ultraviolet (UV) irradiation is a major cause of skin damage, of long-term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV-induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non-invasive procedures. OBJECTIVES: This work was aimed to reproduce the effects of UVB on whole-skin explants ex vivo and to study underlying biochemical mechanisms, especially in correlation with skin autofluorescence. METHODS: Human skin organ cultures were irradiated with UVB and subjected to enzyme assays, Western blots, solid-phase ELISA, HPLC and fluorescence measurements. RESULTS: UVB irradiation was found to enhance ROS production, to deplete the pool of low-molecular-weight antioxidants and to decrease the overall antioxidant capacity in the epidermis, in a manner dependent on xanthine-oxidase activity. Epidermal cell proliferation and mitochondrial activity were transiently stimulated. IκB-α was degraded, and the secretion of inflammatory cytokines was drastically increased. Inducible nitric oxide synthase activity was increased in non-irradiated controls, probably due to the mechanical stress of skin excision, and this phenomenon was suppressed by UVB. Autofluorescence measurements revealed alterations of dermal protein crosslinks following UVB irradiation. CONCLUSIONS: Skin organ culture proved to be an integrated model appropriate for in vitro analysis of UVB biologic effects and their correlations, and for the study of non-invasive diagnostic methods in cellular and molecular terms.

Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis.

Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a 'tombstone' appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin.

BACKGROUND: Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known. OBJECTIVES: To analyse the ability of Dermud, a leave-on skin preparation containing DS mud and other ingredients like DS water, zinc oxide, aloe-vera extract, pro-vitamin B5 and vitamin E, to antagonize biological effects induced by UVB irradiation in skin when topically applied in organ cultures. METHODS: We have used human skin organ cultures as a model to assess the biological effects of UVB irradiation and of Dermud cream topical application. Skin pieces were analysed for mitochondrial activity by MTT assay, for apoptosis by caspase 3 assay, for cytokine secretion by solid phase ELISA, for overall antioxidant capacity by ferric reducing antioxidant power and Oxygen radical absorbance capacity assays (epidermis) or by cyclic voltammetry (external medium), and for uric acid (UA) content by HPLC. RESULTS: We report that UVB irradiation decreases cell viability, total antioxidant capacity and UA contents in the epidermis of skin organ cultures, while increasing the levels of apoptosis in cells and their cytokine secretion. Topical application of Dermud decreased all these effects significantly. CONCLUSIONS: Our results clearly show that Dermud has protective, anti-oxidant and anti-inflammatory properties that can antagonize biological effects of UVB irradiation in skin. It may therefore be able to reduce skin photodamage and photoaging, and more generally to reduce oxidative stress and inflammation in skin pathologies.

Aged keratinocyte phenotyping: morphology, biochemical markers and effects of Dead Sea minerals.

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging. We showed that the expression of 16 biochemical markers was altered in aged cultured cells and in tissues, including caspases 1 and 3 and beta-galactosidase activities, immunoreactivities of p16, Ki67, 20S proteasome and effectors of the Fas-dependent apoptotic pathway. Aged cells diversity, and individual variability of aging markers, call for a multifunctional assessment of the aging phenomenon, and of its modulation by drugs. As a test case, we have measured the effects of Dead Sea minerals on keratinocyte cultures and human skin, and found that they stimulate proliferation and mitochondrial activity, decrease the expression of some aging markers, and limit apoptotic damage after UVB irradiation.

Method for chronological recording of antigen appearance in human head-hair shafts and its use for monitoring glycation products in diabetes.

We describe immunochemical assays of non-enzymatic glycation products in human head-hair protein extracts and hair cross sections using Western blots and a novel "dot-block" methodology. In the latter, groups of approximately 15 hair fibers, clipped at about 1 mm proximal to the scalp-skin were aligned, wound around, and attached to 3 mm diameter araldite screw rods. Up to 40 such rods were next embedded lengthwise in additional araldite polymer creating a solid block and the top surface of the block was sectioned off to the half-diameters of the screw rods thus exposing accurately transected hair cross sections at regular ( approximately 0.5 cm) intervals. Early- and advanced-glycation products (EGAs and AGEs, respectively) were determined in the exposed cross sections in-situ using specific antibodies and ECL densitometry as in conventional Western blots. Both Western blots and this technique demonstrated 3.1 fold EGAs increases in the proximal 2 cm of hair of diabetics as compared to non-diabetics. Dot-blocks, in addition, were less variable and demonstrated exponential EGAs decreases along fibers distally, with calculated intercepts (at the hair roots) of 4.9 fold increases in diabetics as opposed to non-diabetics and half-lives of 6.0, 5.9 and 9.0 months in hair of non-diabetics, gestational diabetics and diabetic patients, respectively. Correlations in amounts of BG vs. HbA1(c), BG vs. EGAs, and HbA1(c) vs. EGAs, using dot-block and clinical lab data were all significant (p<0.05). Acute onset T1D patients, defined as previously unsuspected patients diagnosed upon hospitalization due to diabetic complications, exhibited nearly identical EGAs levels in their proximal 0-9 cm hair as did T1D patients with long-established diabetes, thus supporting the notion of long and insidious T1D etiology. Removal of 1-2 microm layers from dot-block surfaces enabled their re-use for multiple assays. Applied anti-AGEs antibodies demonstrated slight decreases or no significant changes in CML and MGI along hair shafts of normal and diabetic subjects. Fluctuations in EGAs and AGEs along hair shafts, indicating alterations in glycemic control were also observed. We conclude that the dot-block method has a potential for early diagnosis and monitoring of diabetes, and more generally, as a long term "biological record" of various chronic medical conditions.

Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis--possible involvement of the EGF receptor.

Pemphigus is an autoimmune cutaneous disease characterized by circulating autoantibodies that cause blistering and erosions on skin and mucous membranes. Circulating autoantibodies bind to epidermal cell membrane and cause cell-cell detachment (acantholysis), leading to epidermal tissue damage and cell death. The principal target of pemphigus vulgaris autoantibodies (PV-IgG) is desmosomal cadherin desmoglein 3 (Dsg3), a constituent of desmosomes, mediating cell-cell adhesion. Several hypotheses for the mechanisms of acantholysis induction by PV-IgG exist, but the actual mechanism is not clear as yet. We have previously reported on apoptosis induction in PV-IgG-mediated epidermal tissue and cell damage as a possible mechanism of acantholysis and cell death (Wang et al. 2004, Apoptosis, 9:131-143). In this study we investigated the involvement of the EGFR and intracellular signal transduction pathways in the PV-IgG-induced apoptosis. We show here that PV-IgG induced activation/autophosphorylation of EGFR in cultured keratinocytes in vitro. The specific tyrosine kinase inhibitor AG1478 abrogated EGFR autophosphorylation, cell death, FasL appearance and acantholysis, all induced by PV-IgG, in parallel, confirming the involvement of EGFR in this Fas apoptotic cascade. Activation of EGFR was followed by phosphorylation of its downstream substrates, MAP kinase ERK and transcription factor c-Jun, and internalization of EGFR. Pharmacological inactivation of the EGFR and ERK kinase activities, by use of specific inhibitors AG1478 and PD98059 respectively, blocked PV-IgG-induced phosphorylation of EGFR, ERK and c-Jun and cellular apoptosis, measured by flow cytometry and caspase 3 activity. Prolonged activation of EGFR by PV-IgG led to dramatic internalization of this receptor, possibly reducing the ability of the cell to perform survival signals. This suggests that activation of EGFR, followed by its internalization, is pivotal for intracellular apoptotic signal transduction via ERK/c-Jun pathways, leading to acantholysis. Our experimental data indicate that the EGFR is instrumental in transducing apoptotic/acantholytic signals in keratinocytes cultures in response to PV-IgG treatment. The acantholytic effect caused by PV-IgG binding to cell surface receptors begins with and depends on cell surface receptor (EGFR) activation of intracellular signaling pathways (ERK pathway) and apoptosis induction (FasR pathway), which later lead to major cell-cell separation (acantholysis) and cell death.

The ubiquitin-proteasome system at the crossroads of stress-response and ageing pathways: a handle for skin care?

The regulation of gene expression at the transcriptional level has been considered for long as the main mechanism of cellular adaptive responses. Since the turn of the century, however, it is becoming clear that higher organisms developed a complex, sensitive and maybe equally important network of regulatory pathways, relying largely on protein interactions, post-translational modifications and proteolysis. Here we review the involvement of the ubiquitin-proteasome pathway of protein degradation at different levels of cellular life in relation with ageing, and with a special focus on skin. It comes out that the ubiquitin system plays a major role in signal transduction associated with stress and ageing, in skin in particular through the control of retinoid and NF-kappaB pathways. The understanding of specific proteolytic targeting by E3 ubiquitin-ligases paves the way for a new generation of active molecules that may control particular steps of normal and pathological ageing.

The interaction of pemphigus autoimmunoglobulins with epidermal cells: activation of the fas apoptotic pathway and the use of caspase activity for pathogenicity tests of pemphigus patients.

Pemphigus is a fatal autoimmune disease in which autoimmunoglobulins PV-IgG (binding to desmoglein 3) and PF-IgG (binding to desmoglein 1) in pemphigus vulgaris and pemphigus foliaceus, respectively, cause intraepidermal blisters, cell-cell separation (acantholysis), and cell death. The mechanism of acantholytic lesion formation has not yet been elucidated. Recently, we have reported that an apoptotic mechanism might be operative in PV-IgG-induced acantholysis: (1) in patients' lesional and some perilesional skin portions, the FasR pathway is activated as its components were enriched; (2) in cultured keratinocytes, PV-IgG upregulates effectors of the FasR pathway (including the mitochondrial loop), as found by immunodetermination (cytochemistry, Western blot of pathway effectors) and determination of caspases 1, 3, and 8 activity/activation; (3) in organ cultures of skin incubated with PV-IgG, activated caspase 8 was found also in perilesional cells and coaggregated with bound PV-IgG; (4) caspase 8 activation in DISCs precedes caspase 3 activation in keratinocytes in cultures upon incubation with PV-IgG. Because caspase activation was shown to accompany lesion formation in cell and organ cultures incubated with PV-IgG, we used caspase activity to monitor the pathogenicity of PV-IgG in relation to PV-IgG binding to epithelia. A rough correlation was found between sera titers, determined by IIF and by immunoblot binding to desmoglein 3, and activation of caspase 3.

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes.

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas-mediated apoptosis. Here, we describe the induction of the Fas-dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV-IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV-IgG-mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.

Enhancement of Fas-mediated apoptosis in ageing human keratinocytes.

Cellular senescence and apoptosis are two metabolically related and seemingly synergistic processes that are involved in tissue maintenance and homeostasis, anti-tumor protection, and age-related diseases. Despite this apparent co-operativity, senescence can inhibit apoptosis in certain conditions. Here, we describe senescence-apoptosis relationships in human epidermal cells by comparing apoptosis-related effector concentrations in keratinocyte cultures and epidermal skin cells at various stages of ageing. Using western blots, flow cytometry, enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence, we determined the amounts of apoptotic effectors in aged cells compared to young ones, in parallel with beta-galactosidase activity at neutral pH (senescence-associated beta-galactosidase, SA beta-gal), found to be a good indicator of cellular ageing. We observed increased levels of several Fas-mediated apoptosis effectors (Fas, Fas ligand, FADD, FLICE), both in cell cultures at advanced passages and in skin cells of aged donors (above 45 years). Furthermore, we found that while the pro-apoptotic p53 increased, the anti-apoptotic Bcl-2 declined. In spite of this, the extent of spontaneous apoptosis did not change in senescent keratinocyte cultures. The cells, however, became notably more susceptible to apoptosis when kept in exhausted growth medium, or upon Fas receptor activation by anti-Fas antibody binding. Our results are consistent with recent findings in senescent fibroblasts, showing that the death-signaling pathway is enhanced at senescence.

Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load.

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected. The observed velocities in situ (500pmoles/min/mg protein) were comparable to the best values of proteasome activity in crude cellular extracts. These features altogether allowed to identify the in situ activity as that of proteasome. To characterize proteasome complexes present in human keratinocytes, we analyzed cellular lysates by ultracentrifugation and gel filtration: most proteasome activity was associated with PA700-bound, presumably 26S, particles. PA28 activator was detected only when cells were treated by gamma interferon. Proteasome activities were determined using the in situ method in keratinocytes at different stages of replicative senescence. Only a slight decrease of proteasome activity per cell was seen at intermediate passages, followed by a slight increase in senescent cells. In the same time, the amount of total proteins increased notably with cellular ageing. Thus, proteasome activity decreased relatively to total proteins, but not relatively to cell numbers. Flow cytometry confirmed that the size of aged keratinocytes increased with the ageing marker beta-galactosidase.

Exogen, shedding phase of the hair growth cycle: characterization of a mouse model.

The hair growth cycle is generally recognized to comprise phases of growth (anagen), regression (catagen), and rest (telogen). Whereas, heretofore, the hair shedding function has been assumed to be part of the telogen phase, using a laboratory mouse model and newly developed techniques for quantitative collection and spectroscopic determination of shed hair, we found that shedding actually occurs as a distinct phase. Although some shedding occurs throughout the growth cycle, the largest peak is coupled to anagen. Using hair dye and rhodamine labeling we established that the shafts that shed arise during the previous hair cycle. We found that over the cycle the ratio of shed overfur to shed underfur hair shafts varies with the cycle phase and that the shed shaft base is unique morphologically, having a cylindrical shape with scalloped or "nibbled" edges. By electron microscopy the mooring cells of the exogen root show intercellular separation suggesting a proteolytic process in the final shedding step. This is the first report describing a distinct shedding, or exogen, phase of the hair cycle. This study supports the notion that this phase is uniquely controlled and that the final step in the shedding process involves a specific proteolytic step.