Enhanced skin retention and permeation of a novel peptide via structural modification, chemical enhancement, and microneedles.

Hyperpigmentation is a common skin condition with serious psychosocial consequences. Decapeptide-12, a novel peptide, has been found to be safer than hydroquinone in reducing melanin content, with efficacy up to more than 50% upon 16 weeks of twice-daily treatment. However, the peptide suffers from limited transcutaneous penetration due to its hydrophilicity and high molecular weight. Therefore, decapeptide-12 was modified by adding a palmitate chain in an attempt to overcome this limitation. Molecular docking results showed that the two peptides exhibited similar biological activity towards tyrosinase. We also tested the effect of chemical penetration enhancers and microneedles to deliver the two peptides into and through skin, using an in vitro human skin permeation method. It was shown that the palm-peptide achieved the best skin retention owing to the increased lipophilicity. In addition, skin permeation of the palm-peptides was enhanced by the chemical skin penetration enhancers, namely, oleic acid and menthol. Skin permeation of the native peptide was enhanced by the microneedle patch but not the chemical skin penetration enhancers. Cutaneous absorption of the palm-peptides was estimated to have achieved its therapeutic concentration within skin. The combinatory approach of using molecular modification, chemical penetration enhancement, and microneedle patch proves to be useful to enhanceskin permeation of the peptides.

Heterologous expression of mutated HLA-G1 reduces alloreactivity of human dermal fibroblasts.

AIM: To engineer a stable HLA-G molecule and evaluate its immunomodulatory properties in transgenic human dermal fibroblasts (HDFs). MATERIALS & METHODS: A mutated HLA-G1 (mHLA-G1) molecule was generated by modifying the endoplasmic reticulum retrieval motif and 3'-untranslated region miRNA-binding sites of HLA-G1. Immunomodulatory properties of transgenic HDF-mHLA-G1 were evaluated in vitro. RESULTS: Stable mHLA-G1 expressing HDF cells were successfully generated and flow cytometry analysis revealed that mHLA-G1 efficiently localized to the cell surface. Natural killer cell-mediated cytolysis of HDF-mHLA-G1/green fluorescent protein (GFP) was reduced by 73% compared with HDF-GDP. HDF-mHLA-G1/GFP decreased phytohemagglutinin-activated peripheral blood mononuclear cell proliferation by 30% versus HDF-GFP. CONCLUSION: We are the first to successfully create a human fibroblast source with reduced alloreactivity using a novel mHLA-G1 construct. This approach may be extended to other cell types including human embryonic stem cells for use in allogeneic transplantation for cell-based regenerative medicine applications.

Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives.

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue, making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1α)-hESC lines using chEF1α promoter system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology, karyotype, and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers, OCT-3/4 and SSEA-4, in undifferentiated mHLA-G(EF1α)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors, expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1α)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives.

Inhibition of DNA methylation enhances HLA-G expression in human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are immunosuppressive multipotent cells under investigation for potential therapeutic applications in regenerative medicine and prevention of graft-versus-host disease. Human leukocyte antigen (HLA)-G contributes to the immunomodulatory properties of MSCs. HLA-G expression in MSCs is very low and diminishes during in vitro expansion. Epigenetic regulation activates HLA-G expression in some cancer cell lines but not in MSCs. In the present study, adipose- and bone marrow-derived MSCs were exposed to the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) and histone deacetylase inhibitor valproic acid (VPA) and HLA-G mRNA levels assessed using semi-quantitative reverse-transcription PCR. Exposure to 5-aza-dC resulted in HLA-G1 and -G3 upregulation in both early and late passage MSCs. VPA treatment did not induce HLA-G expression in both bone marrow and adipose derived MSCs. Our results provide the first evidence that HLA-G3 could be expressed in MSCs and that methylation-mediated repression is partly responsible for the observed low levels of HLA-G expression in MSCs. Our findings provide insight that treatment of MSCs with specific epigenetic regulatory modulators may improve their immunoregulatory capability for therapeutic applications.

Human embryonic stem cell derived-mesenchymal stem cells: an alternative mesenchymal stem cell source for regenerative medicine therapy.

AIM: To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS: hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics, colony-forming potential, as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS: hESC-MSC showed similar morphology, and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION: hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications.

Minireview: Peptide analogs and short sequence oligopeptides as modulators of skin pigmentation.

Short sequence amino acids or oligopeptides have recently garnered attention for use as treatments for a myriad of dermatologic disorders due to their ability to effect and modulate various biological processes in the epidermis and dermis, rendering them promising candidates as medical and cosmeceutical therapeutics. Major advantages include their relative ease of synthesis and multitude of modifications that can be applied to enhance potency, affinity, specificity, hydrophilicity or hydrophobicity and cytotoxicity. Given the photoprotective effects of eumelanin on skin, there has been substantial interest in developing agents, particularly α-MSH analogs, that can induce 'sunless tanning' helping reduce risk of melanoma and non-melanoma skin cancer. In this mini review, we present some of the recent and leading peptide modulators of melanogenesis with relevant clinical data and medical indications. Short sequence oligopeptides with tyrosinase inhibitory activity that can significantly reduce hyperpigmentation, as well α-MSH analogs that can enhance eumelanogenesis, are currently being clinically tested for treatment of erythropoietic protoporphyria, polymorphous light eruption, solar urticaria, actinic keratosis, and "sunless tanning". Success in developing such products can help reduce the incidence of skin cancer, one that surpasses that of all other human cancers combined.

Novel pentapeptide activators of mammalian and mushroom tyrosinase.

Melanoma incidence continues to rise due to intentional exposure to ultraviolet radiation (UVR) from sunlight and indoor tanning beds. Eumelanin exhibits photoprotective effects; thus, agents that induce its synthesis offer a means for sunless tanning without UVR damage. Herein, we report the development of two pentapeptides, P9 and P10, capable of enhancing melanin synthesis in B16 melanoma cells by activating mushroom and mouse tyrosinases without any effect on cell viability or proliferation. P9 and P10 significantly increased melanin content in a dose-dependent manner comparable to the positive controls, IBMX, scoparone, and α-MSH. However, unlike IBMX and scoparone, but similar to α-MSH, P9 and P10 were able to reverse 6BH4-dependent tyrosinase inhibition. We hypothesize that P9 and P10 allosterically activate tyrosinase and consequently enhance epidermal melanin synthesis. P9 and P10 may offer an alternative to tanning bed use and non-photoprotective tanning products. Moreover, sustained increase of melanin content in skin has the potential to reduce symptoms of photosensitivity disorders such as erythropoietic protoporphyria (EPP), solar urticaria (SU) and polymorphic light eruption (PLE), which lack fully effective treatments and result in significant morbidity.

Hydroquinone-induced depigmentation: case report and review of the literature.

Melasma is an acquired cutaneous disorder caused by an overproduction of melanin by the enzyme tyrosinase. Melasma remains a therapeutic challenge and no definitive standard therapy exists. Although hydroquinone (HQ) has emerged as the most common treatment, its popularity has recently waned because of concerns about its potential carcinogenicity and manufacturing challenges. The adverse effects of HQ range from the common irritant contact dermatitis to the less frequent exogenous ochronosis (EO). Previous reports suggest that the risk of leukoderma from HQ treatment is limited to individuals of African descent. Herein, we describe for the first time the development of depigmentation and paradoxical hyperpigmentation in 2 patients with Fitzpatrick skin type III/IV after brief treatment of their melasma with the HQ-containing Nu-Derm and Reverse systems.

Temporal HLA profiling and immunomodulatory effects of human adult bone marrow- and adipose-derived mesenchymal stem cells.

AIM: To investigate the temporal HLA expression profile and immunomodulatory function of mesenchymal stem cells (MSCs) during in vitro expansion. MATERIALS & METHODS: Adult bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (AMSCs) were cultured and HLA class I and II mRNA expression were investigated during serial expansion using semiquantitative reverse-transcription PCR. The immunomodulatory properties of MSCs were monitored using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS: Semiquantitative reverse-transcription PCR revealed that classical HLA class I molecules were highly expressed in MSCs and remained relatively stable during extended culture. Variable expression levels of HLA class II molecules were detected in both BMSCs and AMSCs across passages. AMSCs were more resistant to PBMC-mediated cytotoxicity and suppressed PBMC proliferation more than BMSCs, although the effect was diminished with increasing passage. CONCLUSION: These findings provide insight regarding the relationship between MSC passage number and MSC immunosuppressive properties and suggest that AMSCs hold advantages over BMSCs for immunomodulatory therapeutic purposes.

Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells.

HLA-G plays an important role in the induction of immune tolerance. Various attempts to produce good manufacturing practice levels of HLA-G as a therapeutic molecule have failed to date partly due to the complicated structure of full-length HLA-G1. Truncated HLA-G3 is simpler and easier to produce than HLA-G1 and contains the expected functional epitope in its only α1 monomorphic domain. In this study, we engineered the ER retrieval and retention signal on HLA-G3's cytoplasmic tail by replacing its RKKSSD motif with RAASSD. We observed that mutated HLA-G3 was highly expressed on the cell surface of transduced K562 cells but did not inhibit cytotoxicity of natural killer cells.

Sign and pseudo-sign of Leser-Trélat: case reports and a review of the literature.

BACKGROUND: Leser-Trélat is distinguished by a rare paraneoplastic sign that is characterized by the sudden eruption of multiple seborrheic keratoses (SKs), associated with underlying internal malignancies. Similar non-malignancy-associated SK eruptions are referred to as the "pseudo-sign of Leser-Trélat" (PLT). OBJECTIVE: Two cases of rapid SK eruptions, one the sign of Leser-Trélat (SLT) and one PLT, are presented, and the literature on SLT and PLT is reviewed. METHODS: A literature review of SLT/PLT was performed by searching the PubMed database for all related English published cases. RESULTS: We identified 109 cases of SLT and 12 cases of PLT, with a mean patient age of 61.8 years. SK eruptions were observed before (68.3%), after (22.1%), and at the time of (9.6%) malignancy diagnosis. The malignancy most frequently associated with SLT was gastric adenocarcinoma. The most common anatomical location of SK eruptions was the trunk (18.9%). Frequently reported associated signs and symptoms included pruritus (52%) and acanthosis nigricans (38.7%). The most common treatment included surgery (35.8%), chemotherapy (26.9%), and radiation therapy (26.9%). Treatment resulted in clinical improvement (45%), no change (30%), exacerbation (15%), or initial improvement followed by exacerbation of SKs. Patient outcomes included disease stability/ improvement (48.4%), recurrence (9.7%), exacerbation/metastasis/new malignancy (4.8%), and death (37.1%). LIMITATIONS: This was a retrospective study and excluded non-English published cases. CONCLUSION: This review updates the existing SLT literature and emphasizes the presence of PLT. Clinicians should be aware that SK eruptions may be early manifestations of an internal malignancy or other pathology. To our knowledge, this is the first review examining both SLT and PLT.

Minimally invasive bipolar fractional radiofrequency treatment upregulates anti-senescence pathways.

BACKGROUND: The sirtuin gene family has been implicated in various anti-senescence pathways. Its connection, if any, with the skin wound healing response has yet to be elucidated. OBJECTIVE: The goal of our study was to better understand the effects of FRF treatment on the sirtuin anti-senescence pathway in skin. METHODS: Human abdominal skin was treated with FRF, and then harvested at 0, 2, 14, and 28 days post-treatment to assess for temporal changes in gene expression levels. RESULTS: Decreased levels of SIRT1, 3, 5, and 7 were observed immediately post-FRF treatment. By Day 2, SIRT1, 6, and 7 expressions increased 50-100%. SIRT6 and 7 expression continued to increase through Day 28. Expression levels of apoptosis genes FoxO3 and p53 decreased, while Bax levels increased by Day 28. CONCLUSIONS: Our results raise the possibility that sirtuin activity may be used as an accurate corollary to clinical improvement in skin quality.

The expression and functional activity of membrane-bound human leukocyte antigen-G1 are influenced by the 3'-untranslated region.

Human Leukocyte Antigen (HLA)-G is an immunosuppressive molecule acting on both the innate and adaptive immune system. A 14 bp insertion/deletion polymorphism (rs66554220) in the 3'-untranslated region (3'UTR) of the HLA-G gene has been associated with a number of diseases, pregnancy complications, and graft rejection after organ transplantation. We have investigated the effect of HLA-G polymorphism in the 3'UTR on the processing and stability of the membrane-bound HLA-G1 (mHLA-G1) isoform, as well as its functional significance. Different HLA-G1 cDNA sequences were transduced into the human K562 cell line. Flow cytometry, immunohistochemistry, and ELISA were used to examine HLA-G1 protein expression. A quantitative RT-PCR assay was used to quantify transduced HLA-G1 DNA and mRNA transcript levels. Stability of mRNA and functional significance of HLA-G were investigated via Actinomycin D and NK cytotoxicity assays, respectively. Human leukocyte antigen-G mRNA from the 14 bp insertion K562-G1 cells showed a higher degree of stability than the other constructs, and increased mHLA-G1 expression relative to transductants lacking the 14 bp sequence. In line with this, transductants carrying the 14 bp insertion were the most efficient in inhibiting NK cytotoxicity but showed a lower soluble HLA-G1 per mHLA-G1 ratio than the HLA-G1 K562 cells lacking the 14 bp insertion. Our data suggest 3'UTR polymorphism may play an important role in HLA-G regulation with implications on a range of diseases.

Isolation and cultivation of human scalp interfollicular epidermal stem cells.

Skin regeneration is intricately controlled by epidermal stem cells. In human skin, the long-lived, slow-cycling, and highly proliferative stem cells are located in the basal layer of the interfollicular epidermis (IFE). The ability to isolate and culture human IFE stem cells (IFESCs) offers fascinating therapeutic potential for skin diseases as well as epithelial tissue engineering. Here we describe a straightforward strategy for generation of ß1 integrin(+)/CD24(-) IFESCs from scalp with defined, serum-free, feeder-free medium and collagen I-coated culture plates. The use of defined media throughout isolation and cultivation allows for detailed investigation of the molecular events involved in ESC self-renewal and differentiation as well as provides a safe source for clinical use.

Prospective multicenter clinical trial of a minimally invasive temperature-controlled bipolar fractional radiofrequency system for rhytid and laxity treatment.

BACKGROUND: A minimally invasive fractional bipolar radiofrequency (FRF) was developed. OBJECTIVE: To evaluate safety and efficacy of FRF in reducing face and neck rhytides and laxity. MATERIALS AND METHODS: This prospective, open-label, multicenter clinical trial enrolled 100 subjects with mild to severe facial and neck rhytides and laxity at seven centers in a per-protocol analysis. One single-pass FRF treatment was administered through five 32 g-needle electrode pairs at a preselected real-time fixed temperature of 62 to 78°C, energy duration for 3 to 5 seconds, and impedance restrictions of 200 to 3,000 Ohms, ensuring intradermal delivery. Five blinded dermatologists and plastic surgeons graded randomized standardized baseline and follow-up photographs of 53 and 42 subjects at 3- and 6-month follow-up intervals, respectively, using the Fitzpatrick wrinkle and Alexiades-Armenakas laxity scales. Subject assessments and adverse events were recorded in 100 subjects. RESULTS: Blinded evaluations revealed correct pre- and post-treatment identification in 100% of scored cases, mean improvement of 25.6% on the Fitzpatrick Wrinkle Scale and 24.1% on the Alexiades-Armenakas laxity scale at 6 months, and 100% response rate for rhytides and 95% for laxity. Subgroup analysis revealed maximal rhytid reduction in the mean target temperature of 66.7, energy duration of 4.2 seconds, and volume of denatured collagen of mm(3) denatured collagen group. Adverse events included transient erythema, edema, and ecchymoses, resolving within 1 to 5 days, and two incidents of temporary pinpoint depressions. More than 90% of subjects were satisfied or very satisfied. CONCLUSION: Real-time temperature-controlled FRF is a highly reproducible, safe, effective nonsurgical treatment of face and neck rhytides and laxity and provides important insights into neocollagenesis, neoelastogenesis, and clinical outcomes.

ß2-Microglobulin-free HLA-G activates natural killer cells by increasing cytotoxicity and proinflammatory cytokine production.

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule and plays a role in tissue specific immunoregulation. Many studies have addressed functional aspects of ß2-microglobulin (ß2m)-associated HLA-G1. ß2m-free HLA-G has been found in human placental cytotrophoblasts and pancreatic ß cells although its function remains unclear. In the present study, we investigated the function of ß2m-free HLA-G by transfecting HLA-G1 and -G3 into human ß2m deficient rat pancreatic ß cell carcinoma (BRIN-BD11) cells. RT-PCR and western blots studies confirmed high expression of HLA-G1 and -G3 in -G1 and -G3 transfectants, respectively. HLA-G1 and -G3 were detected mainly in intracellular compartments of BRIN-BD11 transductants by confocal fluorescent microscopy and flow cytometry. Functional analysis revealed that ß2m-free HLA-G promoted xenogeneic cytotoxic lysis of BRIN-BD11 cells by natural killer (NK) cells and increased production of IL-1ß, TNF-α, and IFN-γ. Stimulation of cytotoxic lysis was impaired by blocking the MAPK and DNA-PKcs pathways in NK cells. Importantly, treatment with 33mAb, a KLR2DL4 receptor agonist, induced NK-mediated cytotoxic lysis of BRIN-BD11 cells transfected with a mock vector. Our data suggest that ß2m-free HLA-G activates NK cells via engagement of KLR2DL4 receptors.

Microdermabrasion: molecular mechanisms unraveled, part 2.

Microdermabrasion (MDA) remains a common in-office procedure for many dermatologic practices. The procedure offers minimal downtime with a low incidence of side effects, making it a relatively desirable option for skin rejuvenation. Investigators have identified many of the molecular mechanisms behind this technology in an attempt to optimize clinical results. In particular, activation of the wound healing response plays a key role in the remodeling of post-MDA treated skin, although this response varies based on the type of MDA employed. In addition, advances in MDA technology offer new and promising ways to enhance transcutaneous penetration of active ingredients to improve clinical outcomes. Our review addresses innovative applications of MDA in the last 10 years of research.

Microdermabrasion: molecular mechanisms unraveled, part 1.

Microdermabrasion (MDA) remains a common in-office procedure for many dermatologic practices.The procedure offers minimal downtime with a low incidence of side effects, making it a relatively desirable option for skin rejuvenation. Investigators have identified many of the molecular mechanisms behind this technology in an attempt to optimize clinical results. In particular, activation of the wound healing response plays a key role in the remodeling of post-MDA treated skin, although this response varies based on the type of MDA employed.While many studies discuss the clinical applications of MDA and their relation to histologic changes found after treatment, few address the basic science behind the technology.Our review covers progress made in the last 10 years of research, with an emphasis on the molecular mechanisms.