Effect of a psoriatic microenvironment in a threedimensional model of normal human skin

Among the cytokines involved in the pathogenesis and in the progression of the disease, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 are the most relevant. A three dimensional model of organotypic human skin cultures is a valuable approach for exposing the whole skin to TNF-alpha and IL-17 as specific proinflammatory stimuli, thus mimicking a psoriatic microenvironment. Normal human skin explants were obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent. Bioptic fragments were cultured overnight in a DMEM medium and further divided before adding either 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines. Samples were harvested 24 hours after cytokine incubation. Each patient was represented in all experimental groups. Epidermal proliferation together with the expression of terminal differentiation biomarkers (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) were investigated by indirect immunofluorescence. Vibrational spectroscopy analysis by a confocal micro-Raman system (785nm laser) has been carried out in three skin samples to evaluate differences of the spectrum versus normal skin. Both cytokines induced a strong inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). A non-continuous occludin expression in the granular layer was observed after the TNF-alpha and IL-17 exposure. Immunolabelings for E-cadherin in tight junctions, for K10 in the suprabasal layers, and for K14 in the basal layer were similar in all experimental groups. The preliminary Raman results highlighted some biomolecules modifications in TNF-alpha- and IL-17-treated skin samples related to ceramide and amide III (keratin proteins) peaks. These results suggest that in this experimental model we reproduced a psoriatic microenvironment in which TNF-alpha and IL-17 induce an early alteration of the homeostasis of the inner proliferative layer, the upper granular layer, and stratum corneum as shown by cell proliferation inhibition, occludin expression, and the biomolecules Raman bands

How Many People Are Able to Control a P300-Based Brain-Computer Interface (BCI)?

An EEG-based brain-computer system can be used to control external devices such as computers, wheelchairs or Virtual Environments. One of the most important applications is a spelling device to aid severely disabled individuals with communication, for example people disabled by amyotrophic lateral sclerosis (ALS). P300-based BCI systems are optimal for spelling characters with high speed and accuracy, as compared to other BCI paradigms such as motor imagery. In this study, 100 subjects tested a P300-based BCI system to spell a 5-character word with only 5 min of training. EEG data were acquired while the subject looked at a 36-character matrix to spell the word WATER. Two different versions of the P300 speller were used: (i) the row/column speller (RC) that flashes an entire column or row of characters and (ii) a single character speller (SC) that flashes each character individually. The subjects were free to decide which version to test. Nineteen subjects opted to test both versions. The BCI system classifier was trained on the data collected for the word WATER. During the real-time phase of the experiment, the subject spelled the word LUCAS, and was provided with the classifier selection accuracy after each of the five letters. Additionally, subjects filled out a questionnaire about age, sex, education, sleep duration, working duration, cigarette consumption, coffee consumption, and level of disturbance that the flashing characters produced. 72.8% (N = 81) of the subjects were able to spell with 100% accuracy in the RC paradigm and 55.3% (N = 38) of the subjects spelled with 100% accuracy in the SC paradigm. Less than 3% of the subjects did not spell any character correctly. People who slept less than 8 h performed significantly better than other subjects. Sex, education, working duration, and cigarette and coffee consumption were not statistically related to differences in accuracy. The disturbance of the flashing characters was rated with a median score of 1 on a scale from 1 to 5 (1, not disturbing; 5, highly disturbing). This study shows that high spelling accuracy can be achieved with the P300 BCI system using approximately 5 min of training data for a large number of non-disabled subjects, and that the RC paradigm is superior to the SC paradigm. 89% of the 81 RC subjects were able to spell with accuracy 80-100%. A similar study using a motor imagery BCI with 99 subjects showed that only 19% of the subjects were able to achieve accuracy of 80-100%. These large differences in accuracy suggest that with limited amounts of training data the P300-based BCI is superior to the motor imagery BCI. Overall, these results are very encouraging and a similar study should be conducted with subjects who have ALS to determine if their accuracy levels are similar

Cream Formulation Impact on Topical Administration of Engineered Colloidal Nanoparticles

In order to minimize the impact of systemic toxicity of drugs in the treatment of local acute and chronic inflammatory reactions, the achievement of reliable and efficient delivery of therapeutics in/through the skin is highly recommended. While the use of nanoparticles is now an established practice for drug intravenous targeted delivery, their transdermal penetration is still poorly understood and this important administration route remains almost unexplored. In the present study, we have synthesized magnetic (iron oxide) nanoparticles (MNP) coated with an amphiphilic polymer, developed a water-in-oil emulsion formulation for their topical administration and compared the skin penetration routes with the same nanoparticles deposited as a colloidal suspension. Transmission and scanning electron microscopies provided ultrastructural evidence that the amphiphilic nanoparticles (PMNP) cream formulation allowed the efficient penetration through all the skin layers with a controllable kinetics compared to suspension formulation. In addition to the preferential follicular pathway, also the intracellular and intercellular routes were involved. PMNP that crossed all skin layers were quantified by inductively coupled plasma mass spectrometry. The obtained data suggests that combining PMNP amphiphilic character with cream formulation improves the intradermal penetration of nanoparticles. While PMNP administration in living mice via aqueous suspension resulted in preferential nanoparticle capture by phagocytes and migration to draining lymph nodes, cream formulation favored uptake by all the analyzed dermis cell types, including hematopoietic and non-hematopoietic. Unlike aqueous suspension, cream formulation also favored the maintenance of nanoparticles in the dermal architecture avoiding their dispersion and migration to draining lymph nodes via afferent lymphatics

Control of size and aspect ratio in hydroquinone-based synthesis of gold nanorods

In this article, we describe how it is possible to tune the size and the aspect ratio of gold nanorods obtained using a highly efficient protocol based on the use of hydroquinone as a reducing agent by varying the amounts of CTAB and silver ions present in the ‘‘seed-growth’’ solution. Our approach not only allows us to prepare nanorods with a four times increased Au3? reduction yield, when compared with the commonly used protocol based on ascorbic acid, but also allows a remarkable reduction of 50–60 % of the amount of CTAB needed. In fact, according to our findings, the concentration of CTAB present in the seed-growth solution do not linearly influence the final aspect ratio of the obtained nanorods, and an optimal concentration range between 30 and 50 mM has been identified as the one that is able to generate particles with more elongated shapes. On the optimized protocol, the effect of the concentration of Ag? ions in the seed-growth solution and the stability of the obtained particles has also been investigated.JRC.I.4-Nanobioscience

Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer

Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases

Micropatterned Dry Electrodes for Brain-Computer Interface

Electroencephalography (EEG) recordings can be used to help paralyzed patients by replacing functionality of damaged neural connections through the aid of a brain\u2013computer interface. Conventional electrodes used for EEG require long skin preparation due to the impedance of the outermost layers of skin. On the contrary, dry spiked electrodes allow overcoming of impedance problems and can be suitable for long-term measurements. This paper describes microfabrication and packaging of micropatterned dry electrodes built with deep X-ray lithography, electroplating and soft lithography. Preliminary measurements performed with electrodes having different geometries are also described

Fe₃O₄ nanoparticles (MNP, a) synthesized in organic solvent and transferred to a water solution using PMA amphiphilic polymer (PMNP, b).

<p>MNP and PMNP were highly monodisperse in size as it is shown by TEM images (scale bars = 40 nm,). Part of the highly concentrated PMNP suspension (8 mg mL^–1) was incorporated in a w/o cream (0.8 wt % concentration) (c).</p

Cytofluorimetric analysis showing PMNP nanoparticles uptake by mouse skin and lymph node cells.

<p>PMNP suspension (a, upper panels). Skin CD45-positive and negative cells showing CFSE incorporation. Note that most of the skin cells uptake PMNP nanoparticles administered with the cream formulation. (a, lower panels) CFSE-positive cells in the lymph nodes of mice that received PMNP nanoparticles via cream formulation or via sc administration. Note that only with sc PMNP administration, nanoparticle-positive cells can be detected in the draining lymph nodes. (b) Lymph node macrophages and dendritic cells, identified as CD11b- and CD11c-positive cells respectively, showing CFSE incorporation. Note that only when PMNP are administered sc, CFSE positive macrophages and dendritic cells can be detected in the lymph nodes.</p

Histological microphotograph of normal human skin section.

<p>Haematoxylin and eosin staining (original magnification 40×) (a). <i>In vitro</i> diffusion studies of PMNP colloidal suspension or cream in human skin were carried out using Franz diffusion cells and diffused PMNP were quantified by ICP-OES analysis (b).</p