Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation

Porcine ear skin is widely used to study skin permeation and absorption of ester compounds, whose permeation and absorption profiles may be directly influenced by in situ skin esterase activity. Importantly, esterase distribution and activity in porcine ear skin following common protocols of skin handling and storage have not been characterised. Thus, we have compared the distribution and hydrolytic activity of esterases in freshly excised, frozen, heated and explanted porcine ear skin. Using an esterase staining kit, esterase activity was found to be localised in the stratum corneum and viable epidermis. Under frozen storage and a common heating protocol of epidermal sheet separation, esterase staining in the skin visibly diminished. This was confirmed by a quantitative assay using HPLC to monitor the hydrolysis of aspirin, in freshly excised, frozen or heated porcine ear skin. Compared to vehicle-only control, the rate of aspirin hydrolysis was approximately three-fold higher in the presence of freshly excised skin, but no different in the presence of frozen or heated skin. Therefore, frozen and heat-separated porcine ear skin should not be used to study the permeation of ester-containing permeants, in particular co-drugs and pro-drugs, whose hydrolysis or degradation can be modulated by skin esterases.

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Developing sensorized arm skin for an octopus inspired robot

Soft skin artefacts made of knitted nylon reinforced silicon rubber were fabricated mimicking octopus skin. A combination of ecoflex 0030 and 0010 were used as matrix of the composite to obtain the right stiffness for the skin artefacts. Material properties were characterised using static uniaxial tension and scissors cutting tests. Two types of tactile sensors were developed to detect normal contact; one used quantum tunnelling composite materials and the second was fabricated from silicone rubber and a conductive textile. Sensitivities of the sensors were tested by applying different modes of loading and the soft sensors were incorporated into the skin prototype. Passive suckers were developed and tested against squid suckers. An integrated skin prototype with embedded deformable sensors and attached suckers developed for the arm of an octopus inspired robot is also presented.

Role of NleH, a Type III Secreted Effector from Attaching and Effacing Pathogens, in Colonization of the Bovine, Ovine, and Murine Gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157: H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappa B to the nucleus. In this study we investigated the role of NleH during EHEC O157: H7 infection of calves and lambs. We found that while EHEC Delta nleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157: H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappa B reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappa B response elements, we found that NleH causes an increase in NF-kappa B activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Developing skin analogues for a robotic octopus

In order to fabricate a biomimetic skin for an octopus inspired robot, a new process was developed based on mechanical properties measured from real octopus skin. Various knitted nylon textiles were tested and the one of 10-denier nylon was chosen as reinforcement. A combination of Ecoflex 0030 and 0010 silicone rubbers was used as matrix of the composite to obtain the right stiffness for the skin-analogue system. The open mould fabrication process developed allows air bubble to escape easily and the artificial skin produced was thin and waterproof. Material properties of the biomimetic skin were characterised using static tensile and instrumented scissors cutting tests. The Young’s moduli of the artificial skin are 0.08 MPa and 0.13 MPa in the longitudinal and transverse directions, which are much lower than those of the octopus skin. The strength and fracture toughness of the artificial skin, on the other hand are higher than those of real octopus skins. Conically-shaped skin prototypes to be used to cover the robotic arm unit were manufactured and tested. The biomimetic skin prototype was stiff enough to maintain it conical shape when filled with water. The driving force for elongation was reduced significantly compared with previous prototypes.

Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system

BACKGROUND: Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. METHODS: We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. KEY RESULTS: The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to approximately 50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. CONCLUSIONS & INFERENCES: G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.

A reprocessing for climate of sea surface temperature from the Along-Track Scanning Radiometers: initial validation, accounting for skin and diurnal variability

An initial validation of the Along Track Scanning Radiometer (ATSR) Reprocessing for Climate (ARC) retrievals of sea surface temperature (SST) is presented. ATSR-2 and Advanced ATSR (AATSR) SST estimates are compared to drifting buoy and moored buoy observations over the period 1995 to 2008. The primary ATSR estimates are of skin SST, whereas buoys measure SST below the surface. Adjustment is therefore made for the skin effect, for diurnal stratification and for differences in buoy–satellite observation time. With such adjustments, satellite-in situ differences are consistent between day and night within ~ 0.01 K. Satellite-in situ differences are correlated with differences in observation time, because of the diurnal warming and cooling of the ocean. The data are used to verify the average behaviour of physical and empirical models of the warming/cooling rates. Systematic differences between adjusted AATSR and in-situ SSTs against latitude, total column water vapour (TCWV), and wind speed are less than 0.1 K, for all except the most extreme cases (TCWV < 5 kg m–2, TCWV > 60 kg m–2). For all types of retrieval except the nadir-only two-channel (N2), regional biases are less than 0.1 K for 80% of the ocean. Global comparison against drifting buoys shows night time dual-view two-channel (D2) SSTs are warm by 0.06 ± 0.23 K and dual-view three-channel (D3) SSTs are warm by 0.06 ± 0.21 K (day-time D2: 0.07 ± 0.23 K). Nadir-only results are N2: 0.03 ± 0.33 K and N3: 0.03 ± 0.19 K showing the improved inter-algorithm consistency to ~ 0.02 K. This represents a marked improvement from the existing operational retrieval algorithms for which inter-algorithm inconsistency is > 0.5 K. Comparison against tropical moored buoys, which are more accurate than drifting buoys, gives lower error estimates (N3: 0.02 ± 0.13 K, D2: 0.03 ± 0.18 K). Comparable results are obtained for ATSR-2, except that the ATSR-2 SSTs are around 0.1 K warm compared to AATSR

Self-assembly of palmitoyl lipopeptides used in skin care products

The self-assembly of three cosmetically active peptide amphiphiles C16-GHK, C16-KT, and C16-KTTKS (C16 denotes a hexadecyl, palmitoyl chain) used in commercial skin care products is examined. A range of spectroscopic, microscopic, and X-ray scattering methods is used to probe the secondary structure, aggregate morphology, and the nanostructure. Peptide amphiphile (PA) C16-KTTKS forms flat tapes and extended fibrillar structures with high β-sheet content. In contrast, C16-KT and C16-GHK exhibit crystal-like aggregates with, in the case of the latter PA, lower β-sheet content. All three PA samples show spacings from bilayer structures in small-angle X-ray scattering profiles, and all three have similar critical aggregation concentrations, this being governed by the lipid chain length. However, only C16-KTTKS is stained by Congo red, a diagnostic dye used to detect amyloid formation, and this PA also shows a highly aligned cross-β X-ray diffraction pattern consistent with the high β-sheet content in the self-assembled aggregates. These findings may provide important insights relevant to the role of self-assembled aggregates on the reported collagen-stimulating properties of these PAs.

Acts of estrangement: reading the psoriatic-skin

I am continually surprised by the acts of estrangement in which my body engages. All bodies do this to some extent – lungs breathe, hearts beat and so on, but with psoriasis, which my article is concerned with, these processes are highly visible. As a condition of the skin, it is a pathology which is enacted both within and without, visible to the social world as well emerging from some little understood inflammatory source within the body. This article will undertake a phenomenological exploration of my own skin and how my experience of my body is affected by its lack of compliance with medicine, cosmetics etc. By some unknown causation it flakes, breaks, itches constantly drawing attention to bodily limits and the limits of medical knowledge. I want to think through the meanings we ascribe to such inflammatory conditions in Western society, how my skin materialises at the nexus of industrialisation, medicine, class and capital. This investigation will emerge at the limit point, the thin skin between the subjective and objective, observing and theorising my skin in ways which dissolve disciplinary boundaries, to comprehend the cultural, biological and environmental forces that work upon my body, estranging it from me.

Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method

Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Relationships Among Murine CD11chigh Dendritic Cell Subsets as Revealed by Baseline Gene Expression Patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.