Normal Human Skin Cells Research Articles

Transcriptomic analysis of human dermal fibroblast cells reveals potential mechanisms underlying the protective effects of visible red light against damage from ultraviolet B light

BackgroundUltraviolet B (UVB) radiation is a major cause of skin photodamage, including the damage associated with photodermatoses, aging, and cancer. Although many studies have shown that red light has photoprotective effects on skin, the mechanisms underlying these effects are still poorly understood. ObjectiveThe aim of this study was to identify the photoprotective effects of visible red light against UVB-induced skin damage in normal human dermal fibroblast cells using a transcriptomic approach. MethodsNext-generation sequencing-based transcriptomic analyses were used to profile transcriptomic alterations and identify genes that are differentially expressed by visible red light and by UVB exposure. To understand the biological networks among identified genes, a literature-based biological pathway analysis was performed. Quantitative real-time polymerase chain reaction assays were used for mRNA-level validation of selected key genes. ResultsWe observed that visible red light contributes to skin cell protection against UVB by modulating gene expression that enhances the adaptive response to redox and inflammatory balancing and by upregulating genes involved in DNA excision repair processes. We also identified that several key genes in the red light-induced biological network were differentially regulated. ConclusionsVisible red light enhanced the UVB-protective effects in normal human skin cells via the transcriptomic modulation of genes involved in cell-protective processes. Our findings from this next-generation sequencing analysis may lead to a better understanding of the cytoprotective effects of visible red light and provide direction for further molecular or mechanistic studies.

Alignment-based design and synthesis of new antimicrobial Aurein-derived peptides with improved activity against Gram-negative bacteria and evaluation of their toxicity on human cells.

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram-positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment-based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium-upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8-64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128-256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8-500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram-negative bacteria by using machine-learning algorithm and other in silico methods.

Anti-cancer potential of the lipoidal and flavonoidal compounds from Pisum sativum and Vicia faba peels

For a long time, plant secondary metabolites have been strongly examined for their antitumor and cytotoxic impacts. These days, there is another pattern of making utilization of the waste products of plants because of their extravagance of numerous phytochemical components and adequacy on human wellbeing. This research work is handling the effect of diversity of lipoidal and phenolic compounds found in the peels of two common edible plants in the Middle East; Pisum sativum and Vicia faba L. for their assesment as anticancer agents. The GC/MS of the n-hexane extract of both plant peels led to identification of twenty compounds (82.99%) and seventeen compounds (85.97%) of the total lipoidal contents from P. sativum and V. faba, respectively. While the HPLC analysis of the ethyl acetate fraction of the two plant peels resulted in recognition of 17 flavonoids and 18 phenolics from P. sativum and 16 flavonoids and 17 phenolics from V. faba. Moreover, four flavonoidal compounds were isolated to our knowledge for the first time from the peels and tested separately against different human cancer cell lines and the mode of action of the most potent compound has been determined. P. sativum ethyl acetate fraction possessed the highest scavenging activity (31.2%) as well as the most cytotoxic effect on breast carcinoma cell line. Apigenin proved to be the most potent tested compound on (MCF-7) and has no cytotoxic effect on normal human skin cell lines.

P-10 - Oxidative stress induced by UV-B and sunlight mediated photoactivation of carbazole inhibits normal human skin cells physiology

Solar ultraviolet (UV) radiation causes skin cancer after prolongs exposure. Carbazole (CBZ) has a tricyclic structure and is produced from fossil fuels, cigarette smoke, eye kohl, black tattoo ink, coal and wood combustion. We have studied the photomodification of CBZ under UV-B (0.9mw/cm2) and sunlight. Phototoxicity of CBZ was done on human skin keratinocytes (HaCaT) through MTT, NRU and cell migration assays. CBZ generates reactive oxygen species (ROS) through type-I photodynamic reaction and measured through H2DCFDA and DHE fluorescence in cells. Photosensitized CBZ induced apoptosis which was confirmed through sub-G1 fraction, morphological changes, cytochrome c and caspase-12 release from mitochondria and ER, PS translocation, CPDs/6,4PPs/MN formation. Our RT-PCR and western blot results strongly supports our view point of apoptotic cell death through up-regulation of pro-apoptotic genes followed by antioxidant genes keap-1, nrf-2 and hmox-1. Therefore, much attention should be paid to concomitant exposure of carbazole and UV-R for its total environmental impact.

Cellular senescence regulated by SWI/SNF complex subunits through p53/p21 and p16/pRB pathway

SWI/SNF complex is an evolutionarily well-conserved chromatin-remodeling complex, which is implicated in the nucleosomes removing or sliding, impacting on the DNA repair, replication and genes expression regulation. The SWI/SNF complex consists up to 12 protein subunits. The catalytic subunits are BRG1 or BRM, which are exclusive ATPase subunits. BRG1 has been reported to play an important role in cellular senescence. However, The function of non-catalytic subunits involved in cellular senescence is rarely investigated. Therefore, we focused on the senescence regulation roles of SWI/SNF non-catalytic subunits in cellular senescent model induced by H2O2. H2O2 treatment was used to induce cellular senescence models in vitro. Screening the candidate subunits involved in this process by comparing the expression levels of SWI/SNF subunits with/without H2O2 treatment. Over-expression and knockdown the candidate subunits were utilized to investigate the functions and mechanism of the subunits involved in senescence regulation. The expressions of BAF57, BAF60a and SNF5 were changed significantly after H2O2 treatment. Overexpression of the three subunits separately induced cell growth arrest in both HaCaT and GLL19 cells, while knockdown of the subunits separately eased the senescence induced by H2O2 treatment. Results further showed that BAF57, BAF60a and SNF5 regulated cellular senescence via both p53/p21 and p16/pRB pathways, and the three subunits all had a directly interaction with p53. These results indicated that BAF57, BAF60a and SNF5 might act as novel pro-senescence factors in both normal and tumor human skin cells. Therefore, inhibiting expression of the three factors might delay the cellular senescence process.

Targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin squamous cell carcinoma cell growth in vitro and in vivo

Abnormal activation of PI3K-AKT-mTOR signaling is detected in human skin squamous cell carcinoma (SCC). LY3023414 is a novel, potent, and orally bio-available PI3K-AKT-mTOR inhibitor. Its activity against human skin SCC cells was tested. We demonstrated that LY3023414 was cytotoxic when added to established (A431 line) and primary (patient-derived) human skin SCC cells. LY3023414 induced G0/1-S arrest and inhibited proliferation of skin SCC cells. Moreover, LY3023414 induced activation of caspase-3/-9 and apoptosis in skin SCC cells. Intriguingly, LY3023414 was yet non-cytotoxic nor pro-apoptotic to normal human skin cells (melanocytes, keratinocytes and fibroblasts). At the molecular level, LY3023414 blocked PI3K-AKT-mTOR activation in skin SCC cells, as it dephosphorylated PI3K-AKT-mTOR substrates: P85, AKT and S6K1. In vivo studies showed that oral administration of LY3023414 at well-tolerated doses inhibited A431 xenograft tumor growth in severe combined immunodeficiency (SCID) mice. AKT-mTOR activation in LY3023414-treated tumors was also largely inhibited. Together, these results suggest that targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin SCC cell growth in vitro and in vivo, establishing the rationale for further clinical testing.

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma.

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.

449 In situ mapping of innate lymphoid cells in normal and inflamed human skin

449 In situ mapping of innate lymphoid cells in normal and inflamed human skin

The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases

Many human diseases arise from or have pathogenic contributions from a dysregulated immune response. One pathway with immunomodulatory ability is the tryptophan metabolism pathway, which promotes immune suppression through the enzyme indoleamine 2,3-dioxygenase (IDO) and subsequent production of kynurenine. However, in patients with chronic inflammatory skin disease, such as psoriasis and atopic dermatitis (AD), another tryptophan metabolism enzyme downstream of IDO, L-kynureninase (KYNU), is heavily upregulated. The role of KYNU has not been explored in patients with these skin diseases or in general human immunology. We sought to explore the expression and potential immunologic function of the tryptophan metabolism enzyme KYNU in inflammatory skin disease and its potential contribution to general human immunology. Psoriatic skin biopsy specimens, as well as normal human skin, blood, and primary cells, were used to investigate the immunologic role of KYNU and tryptophan metabolites. Here we show that KYNU(+) cells, predominantly of myeloid origin, infiltrate psoriatic lesional skin. KYNU expression positively correlates with disease severity and inflammation and is reduced on successful treatment of psoriasis or AD. Tryptophan metabolites downstream of KYNU upregulate several cytokines, chemokines, and cell adhesions. By mining data on several human diseases, we found that in patients with cancer, IDO is preferentially upregulated compared with KYNU, whereas in patients with inflammatory diseases, such as AD, KYNU is preferentially upregulated compared with IDO. Our results suggest that tryptophan metabolism might dichotomously modulate immune responses, with KYNU as a switch between immunosuppressive versus inflammatory outcomes. Although tryptophan metabolism is increased in many human diseases, how tryptophan metabolism is proceeding might qualitatively affect the immune response in patients with that disease.

Dermal fibroblast expression of stromal cell-derived factor-1 (SDF-1) promotes epidermal keratinocyte proliferation in normal and diseased skin.

Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0198-5) contains supplementary material, which is available to authorized users.

7-Hydroxydehydronuciferine induces human melanoma death via triggering autophagy and apoptosis.

Melanoma is the deadliest cancer. We identified 7-hydroxydehydronuciferine (7-HDNF) isolated from the leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a bio-active agent that antagonizes melanoma tumor growth in mice xenograft model in vivo. Cell proliferation assay demonstrated strong anticancer effects of 7-HDNF to exhibit a dose-dependent behaviour and displayed minor cytotoxicities on normal human skin cells, including epidermal keratinocytes and melanocytes, and dermal fibroblasts. With acridine orange (AO) staining and flow analysis, we found 7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained to confirm the cellular membrane asymmetry lost. One-dimensional flow cytometric analysis showed 7-HDNF increased the cellular arrest in cell cycle at the G2/M phase. Through Western blot examinations, protein expressions were discovered to verify autophagy and apoptosis response mechanisms sharing the associated pathways. Finally, 7-HDNF presented a high-quality antimigratory activity in wound-healing assay. Overall, 7-HDNF presented high-quality anticancer bio-functions and inhibited melanoma tumor growth in vivo and in vitro.

Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes

At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

Cytotoxicity and non-specific cellular uptake of bare and surface-modified upconversion nanoparticles in human skin cells

The cytotoxicity and non-specific cellular uptake of the most popular composition of upconversion nanoparticle (UCNP), NaYF4:Yb3+:Er3+, is reported using normal human skin cells, including dermal fibroblasts and immortalized human epidermal linear keratinocytes (HaCaT). A new hydrophilization reaction of as-synthesized UCNPs based on tetramethylammonium hydroxide (TMAH) enabled evaluation of the intrinsic cytotoxicity of bare UCNPs. The cytotoxicity effects of the UCNP surface-coating and polystyrene host were investigated over the concentration range 62.5–125 μg/mL with 24-h incubation, using a MTT test and optical microscopy. The fibroblast viability was not compromised by UCNPs, whereas the viability of keratinocytes varied from 52% ± 4% to 100% ± 10% than the control group, depending on the surface modification. Bare UCNPs reduced the keratinocyte viability to 76% ± 3%, while exhibiting profound non-specific cellular uptake. Hydrophilic poly(D,L-lactide)- and poly(maleic anhydride-alt-1-octadecene)-coated UCNPs were found to be least cytotoxic among the polymer-coated UCNPs, and were readily internalized by human skin cells. Polystyrene microbeads impregnated with UCNPs remained nontoxic. Surprisingly, no correlation was found between UCNP cytotoxicity and the internalization level in cells, although the latter ranged broadly from 0.03% to 59%, benchmarked against 100% uptake level of TMAH-UCNPs.

Study of common cytotoxicity of aminoglycoside and fluoroquinolone antibiotics in cell cultures

The aim of the study was to identify the overall cytotoxicity of aminoglycosides netilmicin and tobramycin, and fluoroquinolone ciprofloxacin. We used three types of the cells: constantly transformed cell line CHO-K1, normal human skin fibroblasts and cells of normal human conjunctiva Clone 1-5C-4. Antibiotics activity was detected by their influence on cell viability. Quantitative and qualitative methods of evaluation have used to determine viability of the cells (quantitative assessment: a method of cloning cells and colorimetric method for assessing cell proliferation; qualitative assessment: lifetime visual observation under an inverted microscope for morphological status of cells in culture with photofixing). The most toxic effect for all types of the cells was shown in tobramycin. The least degree of toxicity for all types of the cells was determined in netilmicin. Studied antibiotics exert a cytotoxic effect in vitro and differ in their cytotoxic potential.

Photoluminescence and photostability investigations of biocompatible semiconductor nanocrystals coated with glutathione using low laser power

Great efforts are currently devoted to fabricate high-quality quantum dots (QDs) in aqueous solutions for biomedical applications. Two biocompatible systems consisting of core (CdSe) and core/shell (CdSe/ZnS) QDs surface modified with glutathione (GSH), named CdSe-GSH and CdSe/ZnS-GSH, respectively, were built. Upon photoirradiation using low laser power, both systems in HEPES buffer (pH 7.2) showed significant photoluminescence (PL) enhancements. CdSe/ZnS-GSH showed much less blue shifts in excitonic absorption and emission peaks without photobleaching compared with CdSe-GSH QDs system. X-ray diffraction showed that there is no change in the crystalline phase structure of the CdSe/ZnS-GSH QDs system after 1 h irradiation. Cell viability assessment, in the dark, demonstrated that no cytotoxic effects were shown upon incubation CdSe/ZnS-GSH QDs with normal human skin cell line for periods up to 72 h and at concentrations up to 100 nM. The present study demonstrates that CdSe/ZnS-GSH QDs system exhibits a high photostability with a relatively high PL efficiency in aqueous medium following low intensity photoexcitation, without dark cytotoxicity, making this system attractive for several important biomedical applications.

The interrelation between a pro-inflammatory milieu and fluorescence diagnosis or photodynamic therapy of human skin cell lines

As carcinogenesis is often accompanied by inflammation, fluorescence diagnosis (FD) of tumours reportedly leads to false positive results. However, an influence of inflammation on the efficiency of photodynamic therapy (PDT) is not known. Therefore we investigated whether LPS-induced expression of the pro-inflammatory cytokine interleukin-6 (IL-6) influences hypericin and protoporphyrin IX (PpIX) accumulation, and their photocytotoxicity. As in vitro models served normal and malignant human skin cell lines. Additionally we analyzed whether low-dose photodynamic treatment as found during FD and sublethal PDT could modulate the expression of IL-6 and other cytokines as low-dose PDT has the potential for treatment of inflammatory skin diseases. Cells were pre-incubated with LPS to induce a pro-inflammatory milieu. Photosensitizer accumulation and toxicity were measured by a microplate reader, IL-6 via Sandwich ELISA and the other cytokines via a Multiplex Immunoassay. Neither hypericin uptake nor PpIX accumulation was influenced by the proinflammatory state. The false positive classification in FD of patients could rather be due to sensitizer-loaded immune cells. On the other hand, low dose PDT is able to significantly reduce the level of IL-6 in all cell lines. Under differing PDT-conditions, PDT can enhance IL-6 in malignant cells, which could elicit a specific anti-tumour immune response. Furthermore, hypericin alone is able to up-regulate the anti-inflammatory cytokines IL-4, -5 and -10, which could be useful to reduce excessive inflammation. LPS-induction has no additional influence in most cases. Some of these findings could contribute to abate inflammatory diseases on skin or promote wound healing.

Dicephalic ionic surfactants in fabrication of biocompatible nanoemulsions: Factors influencing droplet size and stability

The present work has been carried out to explore the potential of two novel ionic dicephalic-type surfactants, i.e., positively charged N,N-bis[3,3′-(trimethylammonio)propyl]-dodecanamide dimethylsulphate, C12(TAPAMS)2 and anionic disodium N-dodecyliminodiacetate, C12(COONa)2 for spontaneous formation of biocompatible and stable oil-in-water (o/w) nanoemulsions. Initially, we examined ternary phase diagrams of surfactant–oil–water (SOW) systems containing different ratios of C12(TAPAMS)2 or C12(COONa)2 as the surfactant entity (S), oleic acid, isopropyl palmitate or isopropyl myristate as the oil phase (O) and the water phase (W) by the titration method. Then, all obtained nanoemulsions were characterized by visual and microscopic observations, as well as dynamic light scattering (DLS) measurements of particle sizes and size distributions. Results of cryo-TEM and DLS showed that the studied droplets making up the nanoemulsions have a nearly monodisperse size distribution (DH<100nm, PdI<0.2). Backscattered profiles obtained by the turbidimetric technique proved high kinetic stability of the obtained systems whereas Doppler electrophoresis revealed their large positive or negative ζ-potential. In order to test the nanoemulsions biocompatibility under the surfactants concentration, cytotoxicity of the optimized formulations was evaluated in vitro upon two normal human skin cell lines, i.e., cutaneous keratinocytes (HaCaT) and gingival fibroblasts (HGF). The analyzed parameters – oil composition, surfactant type, and surfactant-to-oil ratio – were all found to influence the droplet size, charge and stability of the systems produced. Our results prove that the studied nanoemulsions, stabilized by dicephalic ionic surfactants, may provide new biocompatible colloidal systems, designated mainly for cosmetics, personal and household care products.

Brown and Goldstein: The Cholesterol Chronicles

When his parents took three-year-old John Despota to his primary care physician in Chicago in 1964, orange-tinted fat-laden bumps lined the skin on the back of his lower legs. Eruptions caused by excess cholesterol, the bumps spread across Despota’s body, mirroring a total blood cholesterol level that hovered above six-times the normal range. By age 12, Despota experienced shortness of breath upon slight exertions, frequent fatigue, and chest pain: cardinal signs of a cardiac illness called angina, caused by a short supply of oxygen-rich blood to the heart muscles. “Compared with normal kids, I would get tired too soon playing baseball and hockey,” recalls Despota. Fig. 1. LDL in FH: A comparative look. Courtesy of Brown and Goldstein. Fig. 2. Partners for life: Brown and Goldstein circa 1975. Courtesy of Brown and Goldstein. Fig. 3. Electron micrographs of normal human skin cells ( A ) showing labeled LDL particles and the LDL receptor clustered in cell membrane pockets called coated pits. In Despota’s skin cells ( B ), however, most of the LDL receptor was found to be remote from the coated pits and randomly scattered across the membrane. Reprinted by permission of Macmillian Publishers, Ltd: Nature (3), copyright 1977. Under cardiologist Neil Stone’s care at a lipid clinic at the now-renamed Lurie Children’s Hospital of Chicago, Despota received a diagnosis of familial hypercholesterolemia (FH), a metabolic disorder marked by high blood levels of cholesterol, artery-clogging plaques, and premature heart attacks. “When John first came to the clinic, his local cardiologist had given him a year to live,” says Stone. At 51, Despota leads a quiet life of early retirement in Tarpon Springs, a coastal town near Florida’s Tampa Bay, his ailment held in abeyance by an array of drugs and medical procedures. Described in the early 1960s as a disease that manifests in one of two genetic forms, FH …

Comparative histology and immunohistochemistry of porcine versus human skin

Porcine skin is increasingly being employed as a model of human skin in various research fields, including pharmacology, toxicology and immunology, with particular interest in percutaneous permeation and organ transplantation. Porcine skin shows several anatomical and physiological similarities, but also some differences, with human skin, but few in depth comparative studies are so far available. To study the immunohistochemical properties of normal porcine skin in comparison with human skin. We performed a histological and immunohistochemical study on frozen and formalin-fixed, paraffin-embedded skin biopsies from domestic swine and normal human skin, using a panel of 93 monoclonal or polyclonal antibodies recognizing various human and porcine skin cell types or structures. We found that several antibodies used to detect normal human skin cells showed equivalent immunoreactivity on normal porcine skin. However, some antibodies commonly used to detect human skin antigens remained unreactive on porcine skin. Our findings highlight the main immunohistochemical properties of porcine skin in comparison with those of human skin and provide a morphological and immunohistochemical basis useful to researchers using porcine skin.

Hydrogen water intake via tube-feeding for patients with pressure ulcer and its reconstructive effects on normal human skin cells in vitro

BackgroundPressure ulcer (PU) is common in immobile elderly patients, and there are some research works to investigate a preventive and curative method, but not to find sufficient effectiveness. The aim of this study is to clarify the clinical effectiveness on wound healing in patients with PU by hydrogen-dissolved water (HW) intake via tube-feeding (TF). Furthermore, normal human dermal fibroblasts OUMS-36 and normal human epidermis-derived cell line HaCaT keratinocytes were examined in vitro to explore the mechanisms relating to whether hydrogen plays a role in wound-healing at the cellular level.MethodsTwenty-two severely hospitalized elderly Japanese patients with PU were recruited in the present study, and their ages ranged from 71.0 to 101.0 (86.7 ± 8.2) years old, 12 male and 10 female patients, all suffering from eating disorder and bedridden syndrome as the secondary results of various underlying diseases. All patients received routine care treatments for PU in combination with HW intake via TF for 600 mL per day, in place of partial moisture replenishment. On the other hand, HW was prepared with a hydrogen-bubbling apparatus which produces HW with 0.8-1.3 ppm of dissolved hydrogen concentration (DH) and −602 mV to −583 mV of oxidation-reduction potential (ORP), in contrast to reversed osmotic ultra-pure water (RW), as the reference, with DH of < 0.018 ppm and ORP of +184 mV for use in the in vitro experimental research. In in vitro experiments, OUMS-36 fibroblasts and HaCaT keratinocytes were respectively cultured in medium prepared with HW and/or RW. Immunostain was used for detecting type-I collagen reconstruction in OUMS-36 cells. And intracellular reactive oxygen species (ROS) were quantified by NBT assay, and cell viability of HaCaT cells was examined by WST-1 assay, respectively.ResultsTwenty-two patients were retrospectively divided into an effective group (EG, n = 12) and a less effective group (LG, n = 10) according to the outcomes of endpoint evaluation and the healing criteria. PU hospitalized days in EG were significantly shorter than in LG (113.3 days vs. 155.4 days, p < 0.05), and the shortening rate was approximately 28.1%. Either in EG or in LG, the reducing changes (EG: 91.4%; LG: 48.6%) of wound size represented statistically significant difference versus before HW intake (p < 0.05, p < 0.001). The in vitro data demonstrate that intracellular ROS as quantified by NBT assay was diminished by HW, but not by RW, in ultraviolet-A (UVA)-irradiated HaCaT cells. Nuclear condensation and fragmentation had occurred for UVA-irradiated HaCaT cells in RW, but scarcely occurred in HW as demonstrated by Hoechst 33342 staining. Besides, under UVA-irradiation, either the mitochondrial reducing ability of HaCaT cells or the type-I collagen construction in OUMS-36 cells deteriorated in RW-prepared culture medium, but was retained in HW-prepared culture medium as shown by WST-1 assay or immunostain, respectively.ConclusionsHW intake via TF was demonstrated, for severely hospitalized elderly patients with PU, to execute wound size reduction and early recovery, which potently ensue from either type-I collagen construction in dermal fibroblasts or the promoted mitochondrial reducing ability and ROS repression in epidermal keratinocytes as shown by immunostain or NBT and WST-1 assays, respectively.