Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

Molecular size as the main determinant of solute maximum flux across the skin

One of the most important determinants of dermatological and systemic penetration after topical application is the delivery or flux of solutes into or through the skin. The maximum dose of solute able to be delivered over a given period of time and area of application is defined by its maximum flux (J(max), mol per cm(2) per h) from a given vehicle. In this work, J(max) values from aqueous solution across human skin were acquired or estimated from experimental data and correlated with solute physicochemical properties. Whereas epidermal permeability coefficients (k(p)) are optimally correlated to solute octanol-water partition coefficient (K-ow) and molecular weight (MW) was found to be the dominant determinant of J(max) for this literature data set: log J(max)=-3.90-0.0190MW (n=87, r(2)=0.847, p

A golden age of human pigmentation genetics

The zebrafish golden mutation is characterized by the production of small and irregular-shaped melanin granules, resulting in a lightening of the pigmented lateral stripes of the animal. The recent positional cloning and localization of the golden gene, combined with genotype-phenotype correlations of alleles of its human orthologue (SLC24A5) in African-American and African-Caribbean populations, provide insights into the genetic and molecular basis of human skin colour. SLC24A5 promotes melanin deposition through maturation of the melanosome, highlighting the importance of ion-exchange in the function of this organelle.

Expression of p53 tumor suppressor protein in sun-exposed skin and associations with sunscreen use and time spent outdoors: A community-based study

The p53 gene is a tumor suppressor gene that is commonly mutated in skin cancer and sun-exposed skin, and this can be detected through immunohistochemical expression of the p53 protein. The authors hypothesized that time spent outdoors is associated with p53 protein expression in human skin and that sunscreen use counteracts the association. In 1996, they investigated this in a community-based cross-sectional study in Australia. Detailed information about skin type, time spent outdoors, and sunscreen use was collected from 139 residents of a subtropical township who also provided a skin biopsy from the back of the hand for measurement of p53 expression. Increasing time spent outdoors was positively associated with immuno reactivity in the whole epidermis and in the basal layer of the epidermis. After adjustment for confounders, p53 immunoreactivity was twice as high for people who used sunscreen 1 or 2 days per week as for those who used sunscreen daily (whole epidermis: ratio estimate = 2.0, 95% confidence interval: 1.1, 3.6; basal layer: ratio estimate = 1.7, 95% confidence interval: 0.9, 3.1). The authors conclude that p53 immunoreactivity in the skin is a marker of exposure to ultraviolet light in the past 6 months, but this may be mitigated by regular application of sunscreen.

Enhanced chlorhexidine skin penetration with eucalyptus oil

Background Chlorhexidine digluconate (CHG) is a widely used skin antiseptic, however it poorly penetrates the skin, limiting its efficacy against microorganisms residing beneath the surface layers of skin. The aim of the current study was to improve the delivery of chlorhexidine digluconate (CHG) when used as a skin antiseptic. Method Chlorhexidine was applied to the surface of donor skin and its penetration and retention under different conditions was evaluated. Skin penetration studies were performed on full-thickness donor human skin using a Franz diffusion cell system. Skin was exposed to 2% (w/v) CHG in various concentrations of eucalyptus oil (EO) and 70% (v/v) isopropyl alcohol (IPA). The concentration of CHG (µg/mg of skin) was determined to a skin depth of 1500 µm by high performance liquid chromatography (HPLC). Results The 2% (w/v) CHG penetration into the lower layers of skin was significantly enhanced in the presence of EO. Ten percent (v/v) EO in combination with 2% (w/v) CHG in 70% (v/v) IPA significantly increased the amount of CHG which penetrated into the skin within 2 min. Conclusion The delivery of CHG into the epidermis and dermis can be enhanced by combination with EO, which in turn may improve biocide.

Studies on enhanced surface disinfection and skin antisepsis using chlorhexidine and eucalyptus oil

Healthcare associated infections may arise from many sources, including patient?s own skin flora and the clinical environment, and inflict a significant burden within the health service. Adequate and effective skin antisepsis and surface disinfection are therefore essential factors in infection control. Current EPIC guidelines recommend 2 % chlorhexidine (CHG) in 70 % isopropyl alcohol (IPA) for skin antisepsis however poor penetration has been reported. Eucalyptus oil (EO) is a known permeation enhancer, producing synergistic antimicrobial activity when combined with CHG. In this current study, the antimicrobial efficacy of EO and its main constituent 1,8-cineole were assessed against a panel of clinically relevant microorganisms, alone and in combination with CHG. The superior antimicrobial efficacy of EO compared with 1,8-cineole, and synergistic effects with CHG against planktonic and biofilm cultures, confirmed its suitability for use in subsequent studies within this thesis. Impregnation of EO, CHG and IPA onto prototype hard surface disinfectant wipes demonstrated significantly improved efficacy compared with CHG/IPA wipes, with clear reductions in the time required to eliminate biofilms. Optimisation of the EO/CHG/IPA formulation resulted in the development of Euclean® wipes, with simulated-use and time kill studies confirming their ability to remove microbial surface contamination, prevent cross contamination and eliminate biofilms within 10 minutes. The employment of isothermal calorimetry provided additional information on the type and rate of antimicrobial activity possessed by Euclean® wipes. A clinical audit of the Euclean® wipes at Birmingham Children?s Hospital, Birmingham, U.K. revealed divided staff opinion, with the highest cited advantage and disadvantage concerning the odour. Finally, skin penetration and cell toxicity studies of EO/CHG biopatches and Euclean® solution developed during this study, revealed no permeation into human skin following biopatch application, and no significant toxicity. These current studies enhance the knowledge regarding EO and its potential applications.

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model

Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3's effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3's role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal keratinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Commiphora gileadensis sap extract induces cell cycle-dependent death in immortalized keratinocytes and human dermoid carcinoma

We would like to dedicate this article to the memory of Professor Yoram Milner, who has passed away before this study was achieved. We are immeasurably indebted to him, professionally and personally. The study was supported by grants from the ICA Foundation and the Ministry of Science, Technology and Space.

Inter-Species Microbial Sharing in Rural Madagascar: A Study of Environmental Influences on the Skin Microbiome

The skin is home to trillions of microbes, many of which are recently implicated in immune system regulation and various health conditions (33). The skin is continuously exposed to the outside environment, inviting microbial transfer between human skin and the people, animals, and surfaces with which an individual comes into contact. Thus, the aim of this study is to assess how different environmental exposures influence skin microbe communities, as this can strengthen our understanding of how microbial variation relates to health outcomes. This study investigated the skin microbial communities of humans and domesticated cattle living in rural Madagascar. The V3 region of the 16S rRNA gene was sequenced from samples of zebu (the domesticated cattle of Madagascar), zebu owners, and non-zebu owners. Overall, human armpits were the least diverse sample site, while ankles were the most diverse. The diversity of zebu samples was significantly different from armpits, irrespective of zebu ownership (one-way ANOVA and Tukey’s HSD, p<0.05). However, zebu owner samples (from the armpit, ankle forearm, and hand) were more similar to other zebu owner samples than they were to zebu, yet no more similar to other zebu owner samples than they were to non-zebu owner samples (unweighted UniFrac distances, p<0.05). These data suggest a lack of a microbial signature shared by zebu owners and zebu, though further taxonomic analysis is required to explain the role of additional environmental variables in dictating the microbial communities of various samples sites. Understanding the magnitude and directionality of microbial sharing has implications for a breadth of microbe-related health outcomes, with the potential to explain mosquito host preference and mitigate the threats of vector-borne diseases.

Ziziphus mauritiana leaf extract emulsion for skin rejuvenation

Purpose: To formulate stable water in oil (W/O) emulsion containing hydroalcoholic crude extract of Ziziphus mauritiana leaves for skin rejuvenation. Methods: Placebo (base) without any plant extract and formulation with 4 % Ziziphus mauritiana extract were prepared by mixing. Samples of the emulsions were subjected to varying storage conditions, i.e., 8, 25, 40 oC and 40 oC + 75 % relative humidity for a period of 4 weeks to predict their stability. During this period, stability parameters, including liquefaction, phase separation, color, electrical conductivity, centrifugation and pH were monitored at specified time intervals. Skin rejuvenation was evaluated using 13 healthy human volunteers over a period of 8 weeks. During this period, various skin parameters such as erythema, melanin level, moisture content, elasticity and sebum content of the skin were evaluated at specified intervals. Results: Both the active formulation and placebo were stable in terms of liquifaction, phase separation and color at all the storage conditions of temperature and humidity. Active formulation showed statistically significant (p < 0.05) improvement in skin melanin as well as in skin moisture and sebum levels, whereas these properties were reduced or even absent in the placebo formulation (p > 0.05). Both active and placebo formulations changed skin elasticity and erythema significantly (p < 0.05). Conclusion: It is evident from the findings that the leaf extract of Ziziphus mauritiana possesses antiaging properties as well as exert skin lightening, moisturizing and viscoelastic effects on human skin.

Reliability of infrared thermography in skin temperature evaluation of wheelchair users

To examine the reliability of infrared thermography (IRT) in wheelchair users (WCUs), as a noninvasive and risk-free technique to detect the natural thermal radiation emitted by human skin and to allow subsequent interpretations of temperature distributions.

Low-Level Laser Intensity Application in Masseter Muscle for Treatment Purposes

Objective: This study evaluated with histochemical analysis how the number of laser applications can affect the masseter muscle. Background: In dentistry today, the laser is used in patients with temporomandibular disorders (TMDs), mainly for radiating pain in the masticatory muscles, whose origins may be associated with malocclusion, although the laser effects are not well understood on the cellular level. Materials and Methods: Thirty mice (HRS/J lineage) were randomly distributed into groups according to the number of laser applications (three, six, and 10). For each group of laser applications (experimental, n = 5), it was considered the control group (n = 5), which was not irradiated. All animals inhaled halothane (2-bromo-2-chloro-1, 1, 1-trifluoroethane, minimum 99%, Sigma Aldrich, India) before each laser irradiation performed on the left masseter muscle region, on alternate days with 20 J/cm(2), 40mW, for 20 sec. The muscle samples were collected for histochemical analysis with succinate dehydrogenase (SDH) enzyme 72 h after the last application. Results: (a) A decrease in area of light fibers type (35.91% +/- 6.9%; 32.08% +/- 6.3%, and 27.88% +/- 6.3%), according to the increase of laser applications (p < 0.05); (b) significant increase (p < 0.05) in the area of intermediate fibers, with an increase of laser application (11.08% +/- 3.9%; 16.52% +/- 5.7%, and 15.96% +/- 3.9%), although the increase with 10 applications was small; (c) area increase of dark fibers in the group with three laser applications (0.16% +/- 0.3%) (p < 0.05), and in groups with six and 10 laser applications, respectively (9.68% +/- 6.0% and 9.60% +/- 4.0%). Conclusions: The SDH enzyme activity revealed that the number of laser applications increases the metabolic pattern of the muscle fibers. A minimal difference in metabolic activity between six and 10 applications of a laser suggests that further analyses should be done to confirm that six applications are enough to produce the same clinical effects, thereby contributing data to professionals from different fields in regard to the cost-benefit ratio of this therapy.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.