Marina Frušić-Zlotkin

Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just ‘witnesses of disease'?

Abstract:  Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ‐specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyones satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re‐examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti‐desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert ‘witnesses’ in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome ‘food for thought’ as well as very specific suggestions for important future research directions – within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting – and clinically very relevant! – immunopathogenesis of PV in all its complexity.

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

Abstract:  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.

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.

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.

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.

High resolution SEM imaging of gold nanoparticles in cells and tissues.

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.

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.

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–1β, 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.

Enhanced cutaneous bioavailability of dehydroepiandrosterone mediated by nano-encapsulation.

Polymeric nanocarriers, especially nanospheres (NSs) and nanocapsules (NCs), can promote the penetration of their cargo through the skin barrier, towards improved cutaneous bioavailability. Dehydroepiandrosterone (DHEA), an endogenous hormone exhibiting poor aqueous solubility, was shown to be effective in modulating skin-aging processes following topical application. In this study, we designed adequate DHEA preparations, in an attempt to enable local delivery of the active ingredient to the viable skin layers. In addition, the potential efficiency of DHEA NCs on dermal collagen synthesis was evaluated. Cryo-TEM observations and thermal analysis indicated that DHEA was successfully incorporated within a stable NC-based delivery system. Moreover, higher [(3)H]-DHEA levels were recorded in the viable skin layers following different incubation periods of NCs on excised pig skin specimens as compared to DHEA oil solution (free molecule). Furthermore, significantly higher (4-fold) skin flux values were observed for the DHEA NCs as compared to the values elicited by the oil control solution. Finally, collagen synthesis in human skin organ culture, assessed by the incorporation of [(3)H]-proline, was up to 42% higher for DHEA NCs 48h post-topical application than for the untreated specimens. Overall, these results suggest that poly lactic-co-glycolic acid (PLGA)-based NCs have promising potential to be used topically for various skin disorders.

Retinyl palmitate–loaded poly(lactide-co-glycolide) nanoparticles for the topical treatment of skin diseases

Nanoparticles were prepared with poly(lactide-co-glycolide), Pluronic F127, and phospholipids and loaded with retinyl palmitate. Morphology and physicochemical properties of these nanoparticles were determined by atomic force microscopy, light scattering, and zeta potential. The elasticity and deformability of the nanoparticles were correlated to Tg values measured by differential scanning calorimetry. The in vitro cytotoxicity and genotoxicity of the nanosystems were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell membrane asymmetry, and Ames tests with BALB/3T3 mouse embryo fibroblasts and HaCaT human keratinocytes cell lines. The reactive oxygen species levels and cytokine production in response to the exposure of cells to these nanoparticles were investigated, as well as the penetration in human skin culture.