Dimethyloxalylglycine stabilizes HIF-1α in cultured human endothelial cells and increases random-pattern skin flap survival in vivo

The goal of this study was to evaluate in vitro and in vivo the effects of up-regulation of the proangiogenic hypoxia inducible factor (HIF)-1α induced by dimethyloxalylglycine on endothelial cell cultures and on skin flap survival.

Plakins, a Versatile Family of Cytolinkers: Roles in Skin Integrity and in Human Diseases

The plakin family consists of giant proteins involved in the cross-linking and organization of the cytoskeleton and adhesion complexes. They further modulate several fundamental biological processes, such as cell adhesion, migration, and polarization or signaling pathways. Inherited and acquired defects of plakins in humans and in animal models potentially lead to dramatic manifestations in the skin, striated muscles, and/or nervous system. These observations unequivocally demonstrate the key role of plakins in the maintenance of tissue integrity. Here we review the characteristics of the mammalian plakin members BPAG1 (bullous pemphigoid antigen 1), desmoplakin, plectin, envoplakin, epiplakin, MACF1 (microtubule-actin cross-linking factor 1), and periplakin, highlighting their role in skin homeostasis and diseases.

Human TH9 cells are skin-tropic and have autocrine and paracrine proinflammatory capacity.

T helper type 9 (TH9) cells can mediate tumor immunity and participate in autoimmune and allergic inflammation in mice, but little is known about the TH9 cells that develop in vivo in humans. We isolated T cells from human blood and tissues and found that most memory TH9 cells were skin-tropic or skin-resident. Human TH9 cells coexpressed tumor necrosis factor-α and granzyme B and lacked coproduction of TH1/TH2/TH17 cytokines, and many were specific for Candida albicans. Interleukin-9 (IL-9) production was transient and preceded the up-regulation of other inflammatory cytokines. Blocking studies demonstrated that IL-9 was required for maximal production of interferon-γ, IL-9, IL-13, and IL-17 by skin-tropic T cells. IL-9-producing T cells were increased in the skin lesions of psoriasis, suggesting that these cells may contribute to human inflammatory skin disease. Our results indicate that human TH9 cells are a discrete T cell subset, many are tropic for the skin, and although they may function normally to protect against extracellular pathogens, aberrant activation of these cells may contribute to inflammatory diseases of the skin.

First-in-Human Proof-of-Concept Study: Intralesional Administration of BQ788, an Endothelin Receptor B Antagonist, to Melanoma Skin Metastases

BACKGROUND This first-in-human proof-of-concept study aimed to check whether safety and preclinical results obtained by intratumoral administration of BQ788, an endothelin receptor B (EDNRB) antagonist, can be repeated in human melanoma patients. METHODS Three patients received a single intralesional BQ788 application of 3 mg. After 3-7 days, the lesions were measured and removed for analysis. The administered dose was increased to a cumulative dosage of 8 mg in patient 4 (4 × 2.0 mg, days 0-3; lesion removed on day 4) and to 10 mg in patient 5 (3 × 3.3 mg, days 0, 3, and 10; lesion removed after 14 days). Control lesions were simultaneously treated with phosphate-buffered saline (PBS). All samples were processed and analyzed without knowledge of the clinical findings. RESULTS No statistical evaluation was possible because of the number of patients (n = 5) and the variability in the mode of administration. No adverse events were observed, regardless of administered dose. All observations were in accordance with results obtained in preclinical studies. Accordingly, no difference in degree of tumor necrosis was detected between BQ788- and PBS-treated samples. In addition, both EDNRB and Ki67 showed decreased expression in patients 2 and 5 and, to a lesser extent, in patient 1. Similarly, decreased expression of EDNRB mRNA in patients 2 and 5 and of BCL2A1 and/or PARP3 in patients 2, 3, and 5 was found. Importantly, semiquantitatively scored immunohistochemistry for CD31 and CD3 revealed more blood vessels and lymphocytes, respectively, in BQ788-treated tumors of patients 2 and 4. Also, in all patients, we observed inverse correlation in expression levels between EDNRB and HIF1A. Finally, in patient 5 (the only patient treated for longer than 1 week), we observed inhibition in lesion growth, as shown by size measurement. CONCLUSION The intralesional applications of BQ788 were well tolerated and showed signs of directly and indirectly reducing the viability of melanoma cells.

Reliability and reproductibility of skin temperature of overweight subjects by an infrared thermograpy software designed for human beings

The technical improvement and new applications of Infrared Thermography (IRT) with healthy subjects should be accompanied by results about the reproducibility of IRT measurements in different popula-tion groups. In addition, there is a remarkable necessity of a larger supply on software to analyze IRT images of human beings. Therefore, the objectives of this study were: firstly, to investigate the reproducibility of skin temperature (Tsk) on overweight and obese subjects using IRT in different Regions of Interest (ROI), moments and side-to-side differences (?T); and secondly, to check the reliability of a new software called Termotracker®, specialized on the analysis of IRT images of human beings. Methods: 22 overweight and obese males (11) and females (11) (age: 41,51±7,76 years; height: 1,65±0,09 m; weight: 82,41±11,81 Kg; BMI: 30,17±2,58 kg/m²) were assessed in two consecutive thermograms (5 seconds in-between) by the same observer, using an infrared camera (FLIR T335, Sweden) to get 4 IRT images from the whole body. 11 ROI were selected using Termotracker® to analyze its reproducibility and reliability through Intra-class Correlation Coefficient (ICC) and Coefficient of Variation (CV) values. Results: The reproducibility of the side-to-side differences (?T) between two consecutive thermograms was very high in all ROIs (Mean ICC = 0,989), and excellent between two computers (Mean ICC = 0,998). The re-liability of the software was very high in all the ROIs (Mean ICC = 0,999). Intraexaminer reliability analysing the same subjects in two consecutive thermograms was also very high (Mean ICC = 0,997). CV values of the different ROIs were around 2%. Conclusions: Skin temperature on overweight subjects had an excellent reproducibility for consecutive ther-mograms. The reproducibility of thermal asymmetries (?T) was also good but it had the influence of several factors that should be further investigated. Termotracker® reached excellent reliability results and it is a relia-ble and objective software to analyse IRT images of humans beings.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.

Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk

The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer

The development and characterization of an enhanced composite skin substitute based on collagen and poly(e-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 ± 16.1 µm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4 mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed. © 2013 Jess Rollason et al.