The reconstructed skin model as a new tool for investigating <Emphasis Type="Italic">in vitro</Emphasis> dermal fillers: increased fibroblast activity by hyaluronic acid

Background

Clinical studies on dermal fillers have essentially focused upon visible improvement of skin quality and any eventual side effects, whereas very little is known about their detailed biological effects.

Objectives

New skin equivalent models were created to investigate the biological impact of hyaluronic acid (HA) fillers on the dermal compartment in vitro.

Materials and methods

Two different reconstructed skin models were developed to incorporate HA within the collagen fibers. In the mixed model, HA was distributed throughout the whole collagen gel whereas the HA was concentrated in the center of collagen gel in the inclusion model.

Results

A comparison of the addition of fillers in two models of reconstructed skin has permitted a better understanding of the biological impact of HA fillers. Protein profiling of supernatants from both models suggested a regulation of MMP-1 secretion by fibroblasts as a function of HA volume, distribution in the dermis and degree of cross-linking. Immunostaining of the inclusion model revealed increased production of type I and III procollagens close to the cross-linked HA. Fibroblasts located in this area showed a fusiform morphology as well as an increase in α-smooth actin expression. The observed increase in collagen production may thus result in part from tension in fibroblasts surrounding the cross-linked HA.

Conclusion

The inclusion reconstructed skin model, as compared to the mixed model, presented here, appears to be a useful tool for investigating the properties of various fillers in vitro and closer to the in vivo situation; our results show that HA fillers promote in vitro remodeling of the dermis by fibroblasts.

     

Skin tumours and skin aging in 209 French elderly people: the PROOF study

Background

Few studies have evaluated the prevalence of skin tumours in the geriatric population and none have analysed different skin aging parameters for whole-body skin in this population.

Objectives

To evaluate the prevalence of skin tumours and global skin aging in a French cohort of elderly people.

Materials and methods

In total, 209 subjects, 105 women and 104 men (mean age: 77.5; range: 74-81 years), were enrolled from the PROOF (PROgnostic indicator OF cardiovascular and cerebrovascular events) cohort. SCINEXA (SCore for INtrinsic and EXtrinsic skin Aging) was used to assess the degree of skin aging and the prevalence of skin tumours. Some additional cutaneous parameters were also studied. Skin aging in women and men was compared.

Results

Mean global SCINEXA was 24.3 (SD: 4.7; range: 8.2-35.3). Solar elastosis and lax appearance were more severe in women (t test; p<0.0001), whereas pseudoscars (t test; p = 0.0312) and coarse wrinkles (t test; p = 0.0479) were more severe in men. Erythrosis coli (chi-square test; p <0.0001) was more frequent in men, whereas varicous veins (chi-square test; p = 0.0026) and eyelid xanthomas (chi-square test; p = 0.0282) were more frequent in women. Twelve patients presented with cutaneous carcinomas and two patients had early melanomas.

Conclusion

This research describes in detail the main indices of skin aging in an old population and the differences related to gender. Moreover, it highlights the utility of systematic screening of old patients by dermatologists in order to diagnose skin cancers early.

     

The use of biodegradable microneedle patches to increase penetration of topical steroid for prurigo nodularis

Background

The stratum corneum is an almost impermeable barrier. Recently, microneedles have been used to increase drug delivery passing the stratum corneum by incorporating the drug within the microneedle or by coating the surface of the microneedle with the drug.

Objective

This study was performed to investigate whether applying a biodegradable microneedle patch after topical steroid application increases penetration of the steroid in vitro, as well as treatment efficacy in patients with prurigo nodularis.

Materials & methods

In vitro penetration of topical steroids after biodegradable microneedle patch application was measured using a 3D skin model. To evaluate the treatment efficacy of the combination of biodegradable microneedle and topical steroids, a split-body clinical study was performed.

Results

Penetration of topical steroid in the in vitro skin model was significantly greater in the microneedle-applied skin. In a split-body clinical study with prurigo nodularis patients, the area and height of skin lesions decreased after four weeks of treatment on both sides, however, the microneedle patch side exhibited a significantly greater decrease in both area and height, compared to the control side. The pruritus visual analogue scale was also significantly lower on the microneedle side.

Conclusion

We suggest that simply applying a microneedle patch after topical steroid application could be a useful strategy for treating refractory skin diseases such as prurigo nodularis.

     

Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin

Please cite this paper as: Functional characterization of highly adherent CD34+ keratinocytes isolated from human skin. Experimental Dermatology 2010; 19: 685–688. Abstract: Compared to murine models, data on cells responsible for the homeostasis of human epidermis are scarce and often contradictory. Given the conflicting results and the availability of clinical grade protocols to purify CD34 cells from a given tissue, we pursued to phenotypically characterize human epidermal CD34+ population. After magnetic separation of whole skin CD34+ and CD34? cell fractions and selection for cells highly adherent to extracellular matrix, both CD34± fractions retained the ability to form a stratified epidermis in organotypic cultures and presented similar in vitro migratory phenotypes. However CD34? cells showed higher clonogenic potential and in vitro proliferative capacity. These results indicated that CD34? cell fraction contains stem/early progenitor cells, while CD34+ cells might be a transit‐amplifying precursor for hair follicle (HF) sheath cells. The ability to isolate living cells using differential cell adhesion and surface markers provides an opportunity to study cells from different morphological regions of the HF.     

Targeting gene expression to the epidermis of transgenic mice: potential applications to genetic skin disorders

The ability to specifically target gene expression to the epidermis of transgenic mice offers the exciting possibility of creating animal models of certain skin disorders that are inherited in man. It may be possible to produce mouse models of dominantly inherited keratinization disorders by targeting the expression of mutant genes encoding the major differentiation products of the epidermis, such as the differentiation specific keratins, filaggrin and cell envelope proteins. Mouse models for other skin disorders associated with abnormal regulation of growth, such as psoriasis, may be generated by targeting the overexpression of cytokines and growth factors, which are thought to play important roles in the pathogenesis of this disease. The development of currently unavailable animal models for certain inherited human skin diseases would not only contribute to our understanding of the pathogenesis of these diseases at the molecular level, but also provide interesting models for therapeutic intervention.     

Increased expression of enhancer of Zeste Homolog 2 (EZH2) differentiates squamous cell carcinoma from normal skin and actinic keratosis

Background

Enhancer of Zeste Homolog 2 (EZH2) is a polycomb group protein that has been shown to be involved in the progression of multiple human cancers including melanoma. The expression of EZH2 in normal skin and in pre-malignant and malignant cutaneous squamous cell carcinoma (SCC) has not been studied.

Objectives

We examined the expression of EZH2 in normal skin, actinic keratosis (AK), SCC in situ, well-differentiated (SCC-WD), moderately-differentiated (SCC-MD) and poorly-differentiated SCC (SCC-PD) to ascertain whether EZH2 expression differentiates these conditions.

Materials and Methods

Immunohistochemical staining for EZH2 was performed on formalin-fixed paraffin-embedded biopsies and a tissue microarray containing normal skin, AK, SCC in situ, and SCC of different grades.

Results

In comparison to the normal skin, EZH2 expression in actinic keratosiswas increased (p = 0.03). Similarly, EZH2 expression in all of the neoplastic conditions studied (SCC in situ, SCC-WD, SCC-MD and SCC-PD) was greatly increased in comparison to both the normal skin and actinic keratosis (p≤0.001).

Conclusion

EZH2 expression increases incrementally from normal skin to AK and further to SCC, suggesting a role for EZH2 in the progression and differentiation of SCC. EZH2 expression may be used as a diagnostic marker for differentiating SCC from AK or normal skin.

     

Different cutaneous innate immunity profiles in acne patients with and without atrophic scars

Background

Acne is a chronic inflammatory disease associated with scar development in many patients.

Objectives

To check whether early inflammatory events in the epidermis via keratinocytes influence the development of scars in acne patients.

Methods

We investigated several immunological markers involved in epidermal innate immunity in both clinically normal skin and inflammatory early papules in acne patients prone to scars or not.

Results

In normal skin of acne patients prone to scars vs not prone to scars, TLR-4, IL-2, IL-10, TIMP-2 and JUN were significantly overexpressed and the MMP-9 protein level was decreased. Similar results were obtained in early inflammatory papules (no more than three days), except for TLR-4.

Conclusion

These results suggest for the first time a link between the early events of inflammation with levels of activation of innate immunity in normal epidermis of acne patients and the development of scars. These markers could be a target for drugs in the field of scar prevention.

     

<Emphasis Type="Italic">Chlamydia muridarum</Emphasis> plasmid induces mouse oviduct pathology by promoting chlamydial survival and ascending infection and triggering host inflammation

Background

Infection with plasmid-free Chlamydia trachomatis and Chlamydia muridarum fails to induce severe pathology, however, the mechanisms underlying this phenotype are unclear.

Objectives

To elucidate the mechanisms of chlamydial plasmid-mediated pathology in mouse oviducts.

Materials & methods

BALB/c mice were intravaginally infected with either plasmid-competent or plasmid-free C. muridarum strains. To compare the survival and ascending infection of these strains, vaginal swabs and genital tract tissues were collected and cultured with HeLa cells to monitor the recovery of live organisms. In addition, Chlamydia strains were intrabursally inoculated into the oviducts of mice to assess pathogenicity. Cytokine levels in the vaginal swabs collected from both the plasmid-competent and plasmidfree C. muridarum-infected mice were detected using Bio-Plex Pro Mouse Cytokine, Chemokine, and Growth Factor Assays.

Results

The plasmid-competent C. muridarum strain induced hydrosalpinx formation in mouse oviducts following intravaginal inoculation, however, this was not the case for the plasmid-free C. muridarum strain. The lack of hydrosalpinges in response to the plasmid-free C. muridarum strain correlated with its significantly reduced ability to survive and disseminate to the upper genital tract. Furthermore, the plasmid-free C. muridarum failed to induce hydrosalpinx formation in mice, even when the strain was intrabursally injected into oviducts. A comparison of the cytokine levels in mouse vaginal secretions showed that the plasmid-free C. muridarum strain induced less IL-15, LIF, MIP-2, IL-1-, IL-1α, TNF-α, and RANTES.

Conclusion

C. muridarum plasmid contributes to oviduct pathology by promoting bacterial survival and ascending infection, and triggering host inflammatory responses.

     

Ectopic expression of syndecan-1 in basal epidermis affects keratinocyte proliferation and wound re-epithelialization

Epidermal proliferation and differentiation can be regulated by soluble morphogens and growth factors. Heparan sulfate proteoglycans (HSPGs) modulate the action of several of these effector molecules, such as members of the fibroblast growth factor (FGF) and Wnt families. Syndecan-1 is a cell-surface proteoglycan that is expressed in differentiating keratinocytes and transiently upregulated in all layers of the epidermis upon tissue injury. To address the role of syndecan-1 in the regulation of keratinocyte proliferation and differentiation, we generated transgenic mice that overexpress syndecan-1 under K14 keratin promoter in the basal layer of the epidermis. We observed epidermal hyperproliferation in newborn transgenic mice, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers and by expression of keratin 6 in the interfollicular epidermis. Compared to both wild-type and syndecan-1-null animals, the transgene expression interfered with skin wound healing in adult mice by decreasing cell proliferation in the re-epithelialized epidermis. Thus, syndecan-1 regulates keratinocyte proliferation differently during skin development and in healing wounds.     

Muir-Torre syndrome caused by exonic deletion of <Emphasis Type="Italic">MLH1</Emphasis> due to homologous recombination

Background

Muir-Torre syndrome (MTS) is characterized by sebaceous neoplasms with internal malignancies and regarded as a variant of hereditary nonpolyposis colorectal cancer (HNPCC). Pathogenic variations of MTS have been identified in the MSH2, MLH1, and MSH6 genes, with the majority of variations located in MSH2.

Objectives

To present an MTS patient who was the only individual with skin malignancies within a cancer-prone pedigree and to showthe usefulness ofRNA-based genetic analysis in the investigation of MTS.

Materials & methods

A 77-year-old man who had operated X-ray equipment at his workplace in his twenties was clinically diagnosed with MTS and investigated by RNA-based analysis, multiplex ligation-dependent probe amplification, and genomic DNA sequencing.

Results

The patient had suffered from sebaceous tumours, squamous cell carcinomas of the skin, and colon cancer. The patient’s family history was remarkable for visceral malignant diseases. Genetic analysis revealed homologous recombination between two Alu elements within intron 4 and 5 of the MLH1 gene. The rearrangement caused a 1,222-bp deletion, including the entire exon 5. Deletion of exon 5 has previously been reported only in two patients with HNPCC, and not in patients with MTS.

Conclusions

For the genetic analysis of MTS, the possibility of rare copy number variations of MLH1, as well as MSH2 variations, should be considered. RNA-based screening using puromycin is recommended in order to identify such variations. It remains unclear why only the proband among the pedigree had skin malignancies, however, the skin carcinogenesis might have been related to occupational radiation exposure.

     

In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.     

Alterations in skin and stratified epithelia by constitutively activated PPARalpha

Peroxisome proliferator-activated receptor (PPAR)alpha is a pleiotropic regulator in many cell types and has recently been implicated in skin homeostasis. To determine the role of PPARalpha in skin physiology, transgenic mice were generated using the tetracycline Tet-off regulatory system to target constitutively activated PPARalpha to the epidermis and other stratified epithelia by the bovine keratin K5 promoter. Expression of the transgene during early development resulted in postnatal lethality within 2 days after birth. A thin epidermis, few hair follicles, and abnormal development of the tongue were observed in neonatal transgenic mice. Early mortality was not observed when transgenic PPARalpha expression was diminished by administration of doxycycline (dox) to the mothers. The alterations noted in neonatal mice were not observed in adult mice upon re-expression of the PPARalpha transgene by withdrawing dox. Attenuated hyperplasia of interfollicular epidermis after topical application of the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was observed in adult mice expressing the PPARalpha transgene. In addition, expression of the PPARalpha transgene in mammary gland during pregnancy resulted in abnormal development of this organ and impaired lactation. Further investigations using primary keratinocytes revealed that expression of the transgene in keratinocytes resulted in increased differentiation and decreased proliferation, which may contribute to the observed phenotype in the transgenic mice. Thus, these results indicate that PPARalpha plays an important role in the development of stratified epithelia including skin, tongue, and mammary gland.     

Estimation of individual cumulative ultraviolet exposure using a geographically-adjusted,openly-accessible tool

Background

Estimates of an individual’s cumulative ultraviolet (UV) radiation exposure can be useful since ultraviolet radiation exposure increases skin cancer risk, but a comprehensive tool that is practical for use in the clinic does not currently exist.The objective of this study is to develop a geographically-adjusted tool to systematically estimate an individual’s self-reported cumulative UV radiation exposure, investigate the association of these estimates with skin cancer diagnosis, and assess test reliability.

Methods

A 12-item online questionnaire from validated survey items for UV exposure and skin cancer was administered to online volunteers across the United States and results cross-referenced with UV radiation indices. Cumulative UV exposure scores (CUES) were calculated and correlated with personal history of skin cancer in a case–control design. Reliability was assessed in a separate convenience sample.

Results

1,118 responses were included in the overall sample; the mean age of respondents was 46 (standard deviation 15, range 18 – 81) and 150 (13 %) reported a history of skin cancer. In bivariate analysis of 1:2 age-matched cases (n?=?149) and controls (n?=?298), skin cancer cases were associated with (1) greater CUES prior to first skin cancer diagnosis than controls without skin cancer history (242,074 vs. 205,379, p?=?0.003) and (2) less engagement in UV protective behaviors (p?<?0.01). In a multivariate analysis of age-matched data, individuals with CUES in the lowest quartile were less likely to develop skin cancer compared to those in the highest quartile. In reliability testing among 19 volunteers, the 2-week intra-class correlation coefficient for CUES was 0.94. We have provided the programming code for this tool as well as the tool itself via open access.

Conclusions

CUES is a useable and comprehensive tool to better estimate lifetime ultraviolet exposure, so that individuals with higher levels of exposure may be identified for counseling on photo-protective measures.

     

Overexpression of IL-4 alters the homeostasis in the skin

IL-4 has been implicated to play an important role in the pathogenesis of many inflammatory diseases including skin diseases such as atopic dermatitis. Because it is not clear which pathologic features of atopic dermatitis are dependent on IL-4, we assessed the consequences of IL-4 overexpression in the skin, using transgenic mice overexpressing IL-4 ubiquitously. Although transgenic mice display no clinical signs of skin inflammation, IL-4 induced a wide spectrum of pathologies including an increased number of mast cells and Langerhans cells in dermis and epidermis, respectively, focal deposition of collagen and a considerably reduced adipocyte layer in the dermis as well as an increased mitotic activity of keratinocytes, reflected in acanthosis and hyperkeratosis. The increase in Langerhans cell number may be explained in part by the substantially reduced Langerhans cell emigration from the epidermis in transgenic mice. The molecular mechanism behind this phenomenon remains to be clarified. Under in vitro culture conditions, Langerhans cells from transgenic mice undergo a maturation process similar to that of Langerhans cells from control mice, and their immunostimulatory capacity is also comparable. In contrast, transgenic Langerhans cells are superior to control Langerhans cells in their antigen-processing capacity. We conclude that the overexpression of IL-4 in the skin is, by itself, not sufficient for the induction of a full-blown atopic dermatitis phenotype, but several changes seen in the skin of transgenic mice mirror the cardinal pathologic manifestations of this disease.     

Down-regulation of microRNA-196a in the sera and involved skin of localized scleroderma patients

Background

Localized scleroderma (LSc) exhibits fibrosis of the skin and subcutaneous tissue. LSc shows an excessive deposition of type 1 collagen.

Objectives

To elucidate the mechanism of type 1 collagen overexpression in LSc, we investigated the epigenetics, focusing on microRNA (miRNA).

Materials & Methods

miRNA expression profile was determined by PCR array analysis. The expression of microRNA-196a (miR-196a) in the skin tissuewas examined by in situ hybridization or real-time PCR. The serum levels of miR-196a were measured by real-time PCR.

Results

PCR array analysis demonstrated that the miR-196a level was markedly decreased in LSc skin tissue in vivo. The transfection of specific inhibitor for miR-196a into normal cultured human dermal fibroblasts led to the up-regulation of type 1 collagen protein in vitro. Furthermore, the serum levels of miR-196a were significantly decreased in LSc patients.

Conclusion

Down-regulation of miR-196a and subsequent overexpression of type 1 collagen in dermal fibroblasts may play a key role in the pathogenesis of LSc. The serum levels of miR-196a may be useful as a diagnostic marker of LSc.

     

Epithelial skin cancers after kidney transplantation: a retrospective single-centre study of 376 recipients

Background

Post-transplant non-melanoma skin cancers (NMSC) are the most common malignancies in kidney transplant recipients.

Objectives

To analyse risk factors associated with the occurrence of basal cell carcinomas (BBC) and squamous cell carcinomas (SCC) in kidney transplant recipients.

Materials and methods

Statistical analysis was performed on 376 kidney transplant recipients screened for NMSC in 2002-2009 and followed until 2013.

Results

NMSC developed in 23.67% of individuals with an SCC/BCC ratio of 2.15:1 and an age-standardised incidence ratio (IR) of 2.71 cases (95% CI: 1.97-3.46) per 100 patients/year. Based on multivariable analysis, NMSC occurrence significantly correlated with higher age (p<0.001), fair skin type (p = 0.01), and particularly SCCs with male gender (p = 0.001). Patients with >10 actinic keratoses were at higher risk of developing NMSCs (IRR = 2.95; 95% CI: 1.97-4.42; p<0.001) and more prone to SCCs, compared to BCCs (p = 0.04). Also, more SCC carriers had high counts of warty lesions (p = 0.006). Calcineurin inhibitors were associated with higher NMSC incidence (IRR = 2.81; 95% CI: 1.1-7.01; p = 0.03), while no difference was seen with the mammalian target of rapamycin (mTOR) inhibitors.

Conclusion

Our results confirm an influence of the individual immunosuppressive regimen, in addition to the duration of immunosuppression, and suggest that older patients, males, fair skinned recipients or those affected with high counts of actinic keratoses (field cancerisation) are particularly prone to development of NMSC.

     

T-cell-mediated injury to keratinocytes: insights from animal models of the lichenoid tissue reaction

The lichenoid tissue reaction is a histopathologic pattern of skin inflammation noted in a diverse group of clinical diseases. Although few of these diseases are reflected in animal models, select murine models of skin inflammation recapitulate salient characteristics of the lichenoid tissue reaction pattern. Animal models of predominantly T-cell-mediated skin inflammation induced by the adoptive transfer of T-cell clones, by the generation of autoreactive T-cell receptor transgenic T cells, and by genetic manipulation of the epidermis are reviewed. Their relevance for a better understanding of the lichenoid tissue reaction is discussed.