The influence of fractionation on cell survival and premature differentiation after carbon ion irradiation

Carbon ion radiotherapy/Fractionated irradiation/R-BE/Premature terminal differentiation. To investigate the influence of fractionation on cell survival and radiation induced premature differentiation as markers for early and late effects after X-rays and carbon irradiation. Normal human fibroblasts NHDF, AG1522B and WI-38 were irradiated With 250 kV X-rays, or 266 MeV/u, 195 MeV/u and I I MeV/u carbon ions. Cytotoxicity was measured by a clonogenic survival assay or by determination of the differentiation pattern. Experiments with high-energy carbon ions show that fractionation induced repair effects are similar to photon irradiation. The RBE10 values for clonogenic survival are 1.3 and 1.6 for irradiation in one or two fractions for NHDF cells and around 1.2 for AG1522B cells regardless of the fractionation scheme. The RBE for a doubling of post mitotic fibroblasts (PMF) in the population is I for both single and two fractionated irradiation of NHDF cells. Using I I MeV/u carbon ions, no repair effect can be seen in WI-38 cells. The RBE10 for clonogenic survival is 3.2 for single irradiation and 4.9 for two fractionated irradiations. The RBE for a doubling of PMF is 3.1 and 5.0 for single and two fractionated irradiations, respectively. For both cell lines the effects of high-energy carbon ions representing the irradiation of the skin and the normal tissue in the entrance channel are similar to the effects of X-rays. The fractionation effects are maintained. For the lower energy, which is representative for the irradiation of the tumor region. RBE is enhanced for clonogenic survival as well as for premature terminal differentiation. Fractionation effects are not detectable. Consequently, the therapeutic ratio is significantly enhanced by fractionated irradiation with carbon ions.

Los diferentes tipos de láser y sus aplicaciones en podología

Los diferentes tipos de láseres, sobre todo el láser de diodo, irrumpen en la terapéutica podológica para proporcionar una alternativa más de tratamiento en muchas patologías que son el día a día de las consultas. El buen manejo y el conocimiento de sus características son requisitos imprescindibles para no tener efectos secundarios indeseados y poder llevar a cabo tratamientos poco dolorosos, minimizando el tiempo total, y muchas veces proporcionando una solución a diversas patologías.

Transient receptor potential melastatin 8 (TRPM8) channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

Design, optimisation and characterisation of polymeric microneedle arrays prepared by a novel laser-based micromoulding technique.

PURPOSE:
Design and evaluation of a novel laser-based method for micromoulding of microneedle arrays from polymeric materials under ambient conditions. The aim of this study was to optimise polymeric composition and assess the performance of microneedle devices that possess different geometries.
METHODS:
A range of microneedle geometries was engineered into silicone micromoulds, and their physicochemical features were subsequently characterised.
RESULTS:
Microneedles micromoulded from 20% w/w aqueous blends of the mucoadhesive copolymer Gantrez® AN-139 were surprisingly found to possess superior physical strength than those produced from commonly used pharma polymers. Gantrez® AN-139 microneedles, 600 µm and 900 µm in height, penetrated neonatal porcine skin with low application forces (>0.03 N per microneedle). When theophylline was loaded into 600 µm microneedles, 83% of the incorporated drug was delivered across neonatal porcine skin over 24 h. Optical coherence tomography (OCT) showed that drug-free 600 µm Gantrez® AN-139 microneedles punctured the stratum corneum barrier of human skin in vivo and extended approximately 460 µm into the skin. However, the entirety of the microneedle lengths was not inserted.
CONCLUSION:
In this study, we have shown that a novel laser engineering method can be used in micromoulding of polymeric microneedle arrays. We are currently carrying out an extensive OCT-informed study investigating the influence of microneedle array geometry on skin penetration depth, with a view to enhanced transdermal drug delivery from optimised laser-engineered Gantrez® AN-139 microneedles.

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9 +/- 0.5 h and a slow component with a half-life of 16 +/- 9 h. For a particles, a fast component with a half-life of 0.7 +/- 0.4 h and a slow component with a half-life of 12 5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37 +/- 0.12) relative to low-LET radiation (0.22 +/- 0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites. (c) 2005 by Radiation Research Society.

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of ‘Pathogenic’, ‘Commensal’ and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two ‘putative virulence’ genes (eMLST) that provides improved high resolution
typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA₁ clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA₂ strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA₁ and IA₂ strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.

Laser-engineered dissolving microneedles for active transdermal delivery of nadroparin calcium

There is an urgent need to replace the injection currently used for low molecular weight heparin (LMWH) multidose therapy with a non- or minimally invasive delivery approach. In this study, laser-engineered dissolving microneedle (DMN) arrays fabricated from aqueous blends of 15% w/w poly(methylvinylether-co-maleic anhydride) were used for the first time in active transdermal delivery of the LMWH nadroparin calcium (NC). Importantly, an array loading of 630 IU of NC was achieved without compromising the array mechanical strength or drug bioactivity. Application of NC-DMNs to dermatomed human skin (DHS) using the single-step 'poke and release' approach allowed permeation of approximately 10.6% of the total NC load over a 48-h study period. The cumulative amount of NC that permeated DHS at 24 h and 48 h attained 12.28 ± 4.23 IU/cm and 164.84 ± 8.47 IU/cm , respectively. Skin permeation of NC could be modulated by controlling the DMN array variables, such as MN length and array density as well as application force to meet various clinical requirements including adjustment for body mass and renal function. NC-loaded DMN offers great potential as a relatively low-cost functional delivery system for enhanced transdermal delivery of LMWH and other macromolecules. © 2012 Elsevier B.V. All rights reserved.

S-(2-Succinyl)cysteine:a novel chemical modification of tissue proteins by a Krebs cycle intermediate

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

Glycation, oxidation, and lipoxidation in the development of the complications of diabetes: a carbonyl stress hypothesis

Modifications of extant plasma proteins, structural proteins,and other macromolecules are enhanced in diabetes because of increased glycation (secondary to increased glucose concentrations) and perhaps because of increased oxidative stress, Increased glycation is present from the time of onset of diabetes, but the relation between diabetes and oxidative stress is less clear: increased oxidative stress may occur later in the course of disease, as vascular damage becomes established, or it may be a feature of uncomplicated diabetes, The combined effects of protein modification by glycation and oxidation may contribute to the development of accelerated atherosclerosis in diabetes and to the development of microvascular complications, Thus, even if not increased by diabetes, variations in oxidative stress may modulate the consequences of hyperglycemia in individual diabetic patients, In this review, the close interaction between glycation and oxidative processes is discussed, and the theme is developed that the most significant modifications of proteins are the result of interactions with reactive carbonyl groups, While glucose itself contains a carbonyl group that is involved in the initial glycation reaction, the most important and reactive carbonyls are formed by free radical-oxidation reactions damaging either carbohydrates (including glucose itself) or lipids, The resulting carbonyl-containing intermediate products then modify proteins, yielding "glycoxidation" and "lipoxidation" products, respectively, This common pathway for glucose and lipid-mediated stress, which may contribute to diabetic complications, is the basis for the carbonyl stress hypothesis for the development of diabetic complications.

Microneedles for intradermal and transdermal drug delivery

The formidable barrier properties of the uppermost layer of the skin, the stratum corneum, impose significant limitations for successful systemic delivery of broad range of therapeutic molecules particularly macromolecules and genetic material. Microneedle (MN) has been proposed as a strategy to breach the stratum corneum barrier function in order to facilitate effective transport of molecules across the skin. This strategy involves use of micron sized needles fabricated of different materials and geometries to create transient aqueous conduits across the skin. MN, alone or with other enhancing strategies, has been demonstrated to dramatically enhance the skin permeability of numerous therapeutic molecules including biopharmaceuticals either in vitro, ex vivo or in vivo experiments. This suggested the promising use of MN technology for various possible clinical applications such as insulin delivery, transcutaneous immunisations and cutaneous gene delivery. MN has been proved as minimally invasive and painless in human subjects. This review article focuses on recent and future developments for MN technology including the latest type of MN design, challenges and strategies in MNs development as well as potential safety aspects based on comprehensive literature review pertaining to MN studies to date. (C) 2013 Elsevier B.V. All rights reserved.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

Towards an understanding of bacterial metabolites prodigiosin and violacein and their potential for use in commercial sunscreens

Synopsis
Objectives

To exploit the microbial ecology of bacterial metabolite production and, specifically, to: (i) evaluate the potential use of the pigments prodigiosin and violacein as additives to commercial sunscreens for protection of human skin, and (ii) determine antioxidant and antimicrobial activities (against pathogenic bacteria) for these two pigments.

Methods
Prodigiosin and violacein were used to supplement extracts of Aloe vera leaf and Cucumis sativus (cucumber) fruit which are known to have photoprotective activity, as well as some commercial sunscreen preparations. For each, sunscreen protection factors (SPFs) were determined spectrophotometrically. Assays for antimicrobial activity were carried out using 96-well plates to quantify growth inhibition of Staphylococcus aureus and Escherichia coli.
Results
For the plant extracts, SPFs were increased by an order of magnitude (i.e. up to ~3.5) and those for the commercial sunscreens increased by 10–22% (for 4% w/w violacein) and 20–65% (for 4% w/w prodigiosin). The antioxidant activities of prodigiosin and violacein were approximately 30% and 20% those of ascorbic acid (a well-characterized, potent antioxidant). Violacein inhibited S. aureus (IC_{506.99 ± 0.146 μM) but not E. coli, whereas prodigiosin was effective against both of these bacteria (IC50 values were 0.68 ± 0.06 μM and 0.53 ± 0.03 μM, respectively).}

Conclusion
The bacterial pigments prodigiosin and violacein exhibited antioxidant and antimicrobial activities and were able to increase the SPF of commercial sunscreens as well as the extracts of the two plant species tested. These pigments have potential as ingredients for a new product range of and, indeed, represent a new paradigm for sunscreens that utilize substances of biological origin. We discussed the biotechnological potential of these bacterial metabolites for use in commercial sunscreens, and the need for studies of mammalian cells to determine safety.

A proposed model membrane and test method for microneedle insertion studies

A commercial polymeric film (Parafilm M (R), a blend of a hydrocarbon wax and a polyolefin) was evaluated as a model membrane for microneedle (MN) insertion studies. Polymeric MN arrays were inserted into Parafilm M (R) (PF) and also into excised neonatal porcine skin. Parafilm M (R) was folded before the insertions to closely approximate thickness of the excised skin. Insertion depths were evaluated using optical coherence tomography (OCT) using either a force applied by a Texture Analyser or by a group of human volunteers. The obtained insertion depths were, in general, slightly lower, especially for higher forces, for PF than for skin. However, this difference was not a large, being less than the 10% of the needle length. Therefore, all these data indicate that this model membrane could be a good alternative to biological tissue for MN insertion studies. As an alternative method to OCT, light microscopy was used to evaluate the insertion depths of MN in the model membrane. This provided a rapid, simple method to compare different MN formulations. The use of Parafilm M (R), in conjunction with a standardised force/time profile applied by a Texture Analyser, could provide the basis for a rapid MN quality control test suitable for in-process use. It could also be used as a comparative test of insertion efficiency between candidate MN formulations. 

Variations in the Processing of DNA Double-Strand Breaks Along 60-MeV Therapeutic Proton Beams

PURPOSE: To investigate the variations in induction and repair of DNA damage along the proton path, after a previous report on the increasing biological effectiveness along clinically modulated 60-MeV proton beams.

METHODS AND MATERIALS: Human skin fibroblast (AG01522) cells were irradiated along a monoenergetic and a modulated spread-out Bragg peak (SOBP) proton beam used for treating ocular melanoma at the Douglas Cyclotron, Clatterbridge Centre for Oncology, Wirral, Liverpool, United Kingdom. The DNA damage response was studied using the 53BP1 foci formation assay. The linear energy transfer (LET) dependence was studied by irradiating the cells at depths corresponding to entrance, proximal, middle, and distal positions of SOBP and the entrance and peak position for the pristine beam.

RESULTS: A significant amount of persistent foci was observed at the distal end of the SOBP, suggesting complex residual DNA double-strand break damage induction corresponding to the highest LET values achievable by modulated proton beams. Unlike the directly irradiated, medium-sharing bystander cells did not show any significant increase in residual foci.

CONCLUSIONS: The DNA damage response along the proton beam path was similar to the response of X rays, confirming the low-LET quality of the proton exposure. However, at the distal end of SOBP our data indicate an increased complexity of DNA lesions and slower repair kinetics. A lack of significant induction of 53BP1 foci in the bystander cells suggests a minor role of cell signaling for DNA damage under these conditions.

An optimized reverse-phase high performance liquid chromatographic method for evaluating percutaneous absorption of glucosamine hydrochloride

A relatively simple, selective, precise and accurate high performance liquid chromatography (HPLC) method based on a reaction of phenylisothiocyanate (PITC) with glucosamine (GL) in alkaline media was developed and validated to determine glucosamine hydrochloride permeating through human skin in vitro. It is usually problematic to develop an accurate assay for chemicals traversing skin because the excellent barrier properties of the tissue ensure that only low amounts of the material pass through the membrane and skin components may leach out of the tissue to interfere with the analysis. In addition, in the case of glucosamine hydrochloride, chemical instability adds further complexity to assay development. The assay, utilising the PITC-GL reaction was refined by optimizing the reaction temperature, reaction time and PITC concentration. The reaction produces a phenylthiocarbamyl-glucosamine (PTC-GL) adduct which was separated on a reverse-phase (RP) column packed with 5 microm ODS (C18) Hypersil particles using a diode array detector (DAD) at 245 nm. The mobile phase was methanol-water-glacial acetic acid (10:89.96:0.04 v/v/v, pH 3.5) delivered to the column at 1 ml min-1 and the column temperature was maintained at 30 degrees C. Galactosamine hydrochloride (Gal-HCl) was used as an internal standard. Using a saturated aqueous solution of glucosamine hydrochloride, in vitro permeation studies were performed at 32+/-1 degrees C over 48 h using human epidermal membranes prepared by a heat separation method and mounted in Franz-type diffusion cells with a diffusional area 2.15+/-0.1 cm2. The optimum derivatisation reaction conditions for reaction temperature, reaction time and PITC concentration were found to be 80 degrees C, 30 min and 1% v/v, respectively. PTC-Gal and GL adducts eluted at 8.9 and 9.7 min, respectively. The detector response was found to be linear in the concentration range 0-1000 microg ml-1. The assay was robust with intra- and inter-day precisions (described as a percentage of relative standard deviation, %R.S.D.) <12. Intra- and inter-day accuracy (as a percentage of the relative error, %RE) was <or=-5.60 and <or=-8.00, respectively. Using this assay, it was found that GL-HCl permeates through human skin with a flux 1.497+/-0.42 microg cm-2 h-1, a permeability coefficient of 5.66+/-1.6x10(-6) cm h-1 and with a lag time of 10.9+/-4.6 h.