UVA-induced metabolic changes in non-malignant skin cells and the potential role of pyruvate as antioxidant.

The exposure to UVA (320-400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts-normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.

Investigation of the HelioVital filter foil revealed protective effects against UVA1 irradiation-induced DNA damage and against UVA1-induced expression of matrixmetalloproteinases (MMP) MMP1, MMP2, MMP3 and MMP15.

The damaging effects of solar ultraviolet (UV) radiation exposure to human skin are well known and can reach from accelerated skin aging (photoaging) to skin cancer. Much of the damaging effects of solar UVA (320-400 nm) radiation is associated with the induction of reactive oxygen species (ROS), which are capable to cause oxidative damage to DNA like the oxidized guanosine 8-hydroxy-2' -deoxyguanosine (8-OHdG). Therefore, new UV protective strategies, have to be tested for their efficiency to shield against UV induced damage. We investigated the protective effects of HelioVital sun protection filter foil against UVA1 irradiation in skin cells. It could be shown, that HelioVital sun protection filter foil has protective effects against UVA1 irradiation induced changes in matrix metalloproteinase (MMP) expression. Furthermore a UVA1-dependant regulation of MMP15 in human fibroblasts could be shown for the first time in this context. In addition, this study demonstrated the protective effect of the HelioVital filter film against UVA1-induced ROS production and DNA damage. These results could pave the way for clinical studies with HelioVital filter foil shielding against the damaging effects of phototherapy and other forms of irradiation therapy, thereby increasing the safety and treatment opportunities of these forms of therapy.

Investigation of toxicity and mutagenicity of cold atmospheric argon plasma.

Cold atmospheric argon plasma is recognized as a new contact free approach for the decrease of bacterial load on chronic wounds in patients. So far very limited data are available on its toxicity and mutagenicity on eukaryotic cells. Thus, the toxic/mutagenic potential of cold atmospheric argon plasma using the MicroPlaSter ß® , which has been used efficiently in humans treating chronic and acute wounds, was investigated using the XTT assay in keratinocytes and fibroblasts and the HGPRT (hypoxanthine guanine phosphoribosyl transferase) assay with V79 Chinese hamster cells. The tested clinical parameter of a 2 min cold atmospheric argon plasma treatment revealed no relevant toxicity on keratinocytes (viability: 76% ± 0.17%) and on fibroblasts (viability: 81.8 ± 0.10) after 72 hr as compared to the untreated controls. No mutagenicity was detected in the HGPRT assay with V79 cells even after repetitive CAP treatments of 2-10 min every 24 hr for up to 5 days. In contrast, UV-C irradiation of V79 cells, used as a positive control in the HGPRT test, led to DNA damage and mutagenic effects. Our findings indicate that cold atmospheric plasma using the MicroPlaSter ß® shows negligible effects on keratinocytes and fibroblasts but no mutagenic potential in the HGPRT assay, indicating a new contact free safe technology. Environ. Mol. Mutagen. 58:172-177, 2017. © 2017 Wiley Periodicals, Inc.

Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model.

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l(-1) sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0-53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.

Investigation of the mutagenic potential of cold atmospheric plasma at bactericidal dosages.

In the past few years, cold atmospheric plasma (CAP) has evolved into a new tool in the fight against nosocomial infections and antibiotic-resistant microorganisms. The products generated by the plasma-electrons, ions, reactive species and UV light-represent a 'lethal cocktail' for different kinds of pathogen, which opens up possible applications in hygiene and medicine. Nevertheless, to ensure the safe usage of CAP on skin (e.g., to treat wounds or skin diseases) several pre-clinical in vitro studies have to be performed before implementing clinical trials on humans. In the study presented here, inactivation experiments with Escherichia coli were carried out to identify the necessary plasma dosage for a 5 log reduction: with a small hand-held battery-operated CAP device, these disinfection properties were achieved after application during 30s. This and higher plasma dosages were then used to analyze the mutagenicity induced in V79 Chinese hamster cells-to furthermore define a 'safe application window'-with the HPRT (hypoxanthine-guanine phosphoribosyl transferase) mutation assay. The results show that a CAP treatment of up to 240 s and repeated treatments of 30s every 12h did not induce mutagenicity at the Hprt locus beyond naturally occurring spontaneous mutations.

The decrease of pigment concentration in red tattooed skin years after tattooing.

BACKGROUND: Tattooing entails the injection of high amounts of colourants into skin. Excepting black inks, red azo pigments are the most frequent colourant used. Part of the pigment is transported away via lymphatic system. Another part can be decomposed in skin, which might be responsible for many known adverse skin reactions. OBJECTIVE: The aim of this study was to estimate the extent of decomposition and transportation by measuring the decrease of pigment concentration in human skin under in vivo conditions. METHODS: Red pigments were extracted from nine tattooed skin specimen and attempted quantification by using HPLC technology. To optimize quantification, we synthesized five common red azo pigments with purity at 98% and used them as HPLC reference substances. RESULTS: In five of the nine skin specimens, we were able to identify and subsequently to quantify the red tattoo pigments such as Pigment Red 22 or Pigment Red 112. The mean pigment concentration in skin was 0.077 ± 0.046 mg/cm². As the pigment concentration in skin ranges from 0.60 to 9.42 mg/cm² (mean: 2.53) directly after tattooing, we estimate a decrease of 87 to 99% of pigment concentration in skin after tattooing. CONCLUSION: Millions of people have many and large tattoos, whereas a single tattoo frequently covers a skin area of more than 300 cm². Thus, the major part of more than 760 mg of azo pigments either decomposes in skin or migrates in the body. That may pose a health risk on tattooed individuals, in particular may cause severe skin reactions.

Photodynamic inactivation of microorganisms as an innovative approach to kill mucocutaneous and skin microorganisms.

In 2002, the first documented case of a vancomycin-resistant S. aureus strain (MIC>or=32 microg/mL) was reported. Nowadays approximately 20% of S. aureus isolates in Europe are reported as methicillin-resistant. Besides bacteria infections, the emergence of fungal infections has increased considerably due factors such as immunosuppressive medications, broad-spectrum antibiotics, neutropenia and HIV infections. These tremendous effects underline the importance and the urgency to develop new alternative treatment approaches that are effective against infections caused by multi-resistant pathogens. Photodynamic inactivation of microorganisms (PDIM) is considered as a new approach, which utilizes a photoactive dye, oxygen and visible light to generate reactive oxygen species, which damage irreversible the pathogens during illumination. Cutaneous diseases caused by methicillin-resistant S. aureus or by fungal species are ideally suited to the treatment by PDIM for eradicating localized infections and for modulating wound healing due to the ability to deliver photosensitizer and light with topical application. The challenge of PDIM is to find a therapeutic window in vivo where multi-resistant microorganisms can be killed efficiently, thereby not harming the surrounding tissue or disturbing the residual bacteria-flora of the tissue. Different chemical classes of photosensitizers have demonstrate their potential to photoinactive Gram(+), Gram(-) and fungal cells. This review will focus on general photobiological and photochemical aspects of microbial inactivation by the photodynamic effect as well as to summarize the current knowledge about the possible application modalities of PDIM on localized infectious diseases in dermatology.

XF drugs: A new family of antibacterials.

The emerging increase of antibiotic resistance constitutes an important risk to human health. Two million patients acquire nosocomial infections in U.S. hospitals each year. Of these infections, 60% involve resistant bacteria. In the last decade, only a few new antibiotics with new mechanisms of action were approved by the FDA, but additional costs for preventing the spread of bacteria, side effects and resistance may limit their long-term usefulness. Therefore, the number of therapeutic options is limited and necessitates exploration of novel antibacterial agents/approaches to treat hospital- and community-acquired infections. The challenge in antibacterial research is to find appropriate structurally novel antibacterial agents inhibiting bacterial targets. The XF drug series, having a dicationic porphyrin structure, which is distinct from all other known antibiotic classes, are rapidly active against a broad range of bacteria. Another new strategy is called photodynamic inactivation of bacteria (PDIB), which utilizes visible light in combination with photosensitizing molecules to efficiently kill bacteria via reactive oxygen species. The XF drugs act additionally as photosensitizers to inactivate bacteria upon light activation. This review summarizes the efficacy of the XF series and describes it as a new class of antibacterial agents or as new photo-sensitizers.

Combination of 10% EDTA, Photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro.

AIMS: The goal of this study was to investigate the phototoxicity of Photosan in combination with EDTA and a hand-held photopolymerizer used in dentistry for light-curing resins against leading key pathogens in caries, endodontic treatment failures, and periodontitis respectively. METHODS AND RESULTS: Cellular uptake of Photosan was detected by fluorescence spectroscopy for Streptococcus mutans and Enterococcus faecalis but not for Aggregatibacter actinomycetemcomitans. Addition of 10% EDTA enabled the uptake of Photosan by A. actinomycetemcomitans. Killing of S. mutans and E. faecalis mediated by Photosan and blue light was concentration and light dose dependent, achieving a >or=99.9% (>or=3 log(10) reduction) efficacy of bacteria killing. In the presence of 10% EDTA, Photosan induced a reduction of >or=4 log(10) in the viability of A. actinomycetemcomitans at a concentration of 50 microg ml(-1), upon activation at a dose of 9.65 J cm(-2) for 60 s. EDTA alone, light alone, and Photosan alone were not able to kill bacteria. CONCLUSIONS: Ten per cent EDTA and Photosan cause a potent phototoxicity against oral bacteria upon illumination with a photopolymerizer. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing antibiotic resistance and insufficient drug concentrations within the sulcus fluid are responsible for lacking antimicrobial efficacy. This study provides useful information that combination of Photosan, EDTA, and a photopolymerizer may be a potentially powerful tool for the efficient destroying of key oral bacteria.

Variable pulsed light is less painful than light-emitting diodes for topical photodynamic therapy of actinic keratosis: a prospective randomized controlled trial.

BACKGROUND: Photodynamic therapy (PDT) of actinic keratosis (AK) using methylaminolaevulinate (MAL) is an effective and safe treatment option, but the procedure is painful. OBJECTIVES: To evaluate the efficacy and pain associated with variable pulsed light (VPL), a prospective, randomized, controlled split-face study was performed. METHODS: Topical MAL-PDT was conducted in 25 patients with AK (n = 238) who were suitable for two-sided comparison. After incubation with MAL, irradiation was performed with a light-emitting diode (LED) (50 mW cm(-2); 37 J cm(-2)) vs. VPL (80 J cm(-2), double pulsed at 40 J cm(-2), pulse train of 15 impulses each with a duration of 5 ms, 610-950 nm filtered hand piece) followed by re-evaluation up to 3 months. RESULTS: The pain during and after therapy was significantly lower with VPL irradiation [t (d.f. = 24) = 4.42, P < 0.001]. The overall mean +/- SD infiltration and keratosis score at 3 months after treatment was 0.86 +/- 0.71 (LED system) vs. 1.05 +/- 0.74 (VPL device) (no statistically significant difference; P = 0.292). Patient satisfaction following both treatment modalities did not significantly differ at the 3-month follow up (P = 0.425). CONCLUSIONS: VPL used for MAL-PDT is an efficient alternative for the treatment of AK that results in complete remission and cosmesis equivalent to LED irradiation but causes significantly less pain.

Determination of the antibacterial efficacy of a new porphyrin-based photosensitizer against MRSA ex vivo.

Following extensive in vitro screening of new photosensitizers the purpose of the present study was to examine penetration as well as antibacterial efficacy of a lead photosensitizer against MRSA using an ex vivo porcine skin model. Two different applications were performed: (i) preincubation of bacteria in solution with a porphyrin-based photosensitizer XF73 and subsequent application on the ex vivo porcine skin; (ii) application of pure bacteria on the explants followed by an incubation with XF73 in a water-ethanol formulation for up to 60 min under occlusion. The localisation of XF73 was restricted to the stratum corneum. Different concentrations (0-10 microM) of XF73 and different incubation times (5-60 min) were used to determine phototoxicity against methicillin-resistant and methicillin-sensitive S. aureus, which was applied on the explants. Preincubation of S. aureus with 0.1 microM XF73 in solution prior to the application of these XF73-incubated bacteria on the skin demonstrates a higher efficacy (>3 log10) after irradiation. Antibacterial photodynamic inactivation resulted in a approximately 1 log10 (0.1 microM)-3.64+/-0.035 (10 microM) log10 growth reduction independently of the antibiotic resistance pattern of used S. aureus strains. Irradiation of applied bacteria without photosensitizer incubation did not show any marked decrease (<1 log10) of bacteria cell number, indicating a significant phototoxicity of the XF73. Histological evaluations of untreated and treated skin areas upon irradiation within 24 h showed no significant degree of necrosis or apoptosis determined by TUNEL-assay indicating that the porcine skin is still vital. This study demonstrates that this XF73 porphyrin-based photosensitizer had concentration-dependent differences in killing efficacy of MRSA in comparison to skin cells using an ex vivo porcine skin model. The results described here imply that topical delivery of XF73 may be considered as a possible treatment in patients with superficial infections of the skin.

In vitro and in vivo comparison of two different light sources for topical photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an effective and safe treatment option for the treatment of actinic keratosis (AK). Incoherent lamps are often used, matching the absorption maxima of ALA. OBJECTIVES: A comparative trial was performed to evaluate the efficacy of recently developed light-emitting diodes (LEDs). METHODS: Human epidermal keratinocytes were incubated for 24 h with ALA (100, 200, 300, 400 or 500 micromol L(-1)) and irradiated consecutively using either an incoherent halogen lamp (lambda(em) = 580-750 nm; 24 J cm(-2); 40 mW cm(-2)) or an LED system (lambda(em) = 633 +/- 3 nm; 3, 6, 12 or 24 J cm(-2); 40 mW cm(-2)). Topical ALA-PDT was performed on 40 patients with AK (n = 584) in a symmetrical distribution suitable for two-sided comparison. After incubation with ALA (20% in cream base) irradiation was performed with the incoherent lamp (100 J cm(-2); 160 mW cm(-2)) on one side and the LED system (40 J cm(-2); 80 mW cm(-2)) on the opposite side followed by re-evaluation up to 6 months. RESULTS: No significant differences between the LED system (3, 6, 12 or 24 J cm(-2)) and the incoherent light source (24 J cm(-2)) regarding cytotoxicity was found in vitro. The complete remission rate yielded in the in vivo investigation was also not significantly different at 6 weeks (P = 0.95), 3 months (P = 0.75) and 6 months (P = 0.61) following therapy. Six weeks following therapy complete remission rates of 84.3% (LED system) and 82.8% (incoherent lamp) were achieved. There was also no significant difference between both light sources regarding pain during light treatment (P = 0.67), patient satisfaction (P = 1.0) or cosmesis (P = 1.0) following therapy. CONCLUSIONS: These results show the efficacy of an LED system for ALA-PDT both in vitro and in vivo. ALA-PDT with the LED system showed a noninferiority regarding the clinical outcome in the treatment of AK compared with the incoherent lamp.

[Antibacterial photodynamic therapy. A new treatment for superficial bacterial infections?]. / Antibakterielle photodynamische Therapie. Ein neues Verfahren zur Behandlung von oberflächlichen bakteriellen Infektionen?

The basis of "antibacterial photodynamic therapy" involves the killing of bacteria by reactive oxygen species in the presence of a photosensitizer and light. Possible dermatologic indications include inactivation of bacteria in skin and wound infections and reduction in density of nosocomial multi-resistant infections. The chief advantage of antibacterial photodynamic therapy is that regardless of the resistance pattern of a bacteria, inactivation can be achieved, analogous to the use of antiseptics. The aim of the present review is to describe the physicochemical and biological mechanisms of antibacterial photodynamic therapy as well as possible clinical indications in dermatology.

Photodynamic effects of novel XF porphyrin derivatives on prokaryotic and eukaryotic cells.

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to the killing of gram-positive antibiotic-resistant bacteria of the skin uses light in combination with a photosensitizer to induce a phototoxic reaction. Different concentrations (0 to 100 microM) of porphyrin-based photosensitizers (CTP1, XF70, and XF73) and different incubation times (5 min, 1 h, and 4 h) were used to determine phototoxicity against two methicillin-resistant Staphylococcus aureus strains, one methicillin-sensitive S. aureus strain, one methicillin-resistant Staphylococcus epidermidis strain, one Escherichia coli strain, and human keratinocytes and fibroblasts. Incubation with 0.005 microM XF70 or XF73, followed by illumination, yielded a 3-log10 (> or = 99.9%) decrease in the viable cell numbers of all staphylococcal strains, indicating that the XF drugs have high degrees of potency against gram-positive bacteria and also that the activities of these novel drugs are independent of the antibiotic resistance pattern of the staphylococci examined. CTP1 was less potent against the staphylococci under the same conditions. At 0.005 microM, XF70 and XF73 demonstrated no toxicity toward fibroblasts or keratinocytes. No inactivation of E. coli was detected at this concentration. XF73 was confirmed to act via a reactive oxygen species from the results of studies with sodium azide (a quencher of singlet oxygen), which reduced the killing of both eukaryotic and prokaryotic cells. When a quencher of superoxide anion and the hydroxyl radical was used, cell killing was not inhibited. These results demonstrate that the porphyrin-based photosensitizers had concentration-dependent differences in their efficacies of killing of methicillin-resistant staphylococcal strains via reactive oxygen species without harming eukaryotic cells at the same concentrations.