Culture and Characterization of Microglia from the Adult Murine Retina

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay. Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of cells per cm2 in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-α and express CD86, CD40, and MHC-II. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.

Dense Multiperson Tracking with Robust Hierarchical Linear Assignment

We introduce a novel dual-stage algorithm for online multi-target tracking in realistic conditions. In the first stage, the problem of data association between tracklets and detections, given partial occlusion, is addressed using a novel occlusion robust appearance similarity method. This is used to robustly link tracklets with detections without requiring explicit knowledge of the occluded regions. In the second stage, tracklets are linked using a novel method of constraining the linking process that removes the need for ad-hoc tracklet linking rules. In this method, links between tracklets are permitted based on their agreement with optical flow evidence. Tests of this new tracking system have been performed using several public datasets.

Dissolving Microneedle Delivery of Nanoparticle Encapsulated Antigen Elicits Efficient Cross-Priming and Th1 Immune Responses by Murine Langerhans Cells

Dendritic cells (DCs) of the skin play an important role in skin-mediated immunity capable of promoting potent immune responses. We availed of polymeric dissolving microneedle (MN) arrays laden with nano-encapsulated antigen to specifically target skin DC networks. This modality of immunization represents an economic, efficient and potent means of antigen delivery directly to skin DCs, which are inefficiently targeted by more conventional immunization routes. Following MN immunization, Langerhans cells (LCs) constituted the major skin DC subset capable of cross-priming antigen-specific CD8(+) T cells ex-vivo. While all DC subsets were equally efficient in priming CD4(+) T cells, LCs were largely responsible for orchestrating the differentiation of CD4(+) IFN-γ and IL-17 producing effectors. Importantly, depletion of LCs prior to immunization had a profound effect on CD8(+) CTL responses in vivo, and vaccinated animals displayed reduced protective anti-tumour and viral immunity. Interestingly, this cross-priming bias was lost following MN immunization with soluble antigen, suggesting that processing and cross-presentation of nano-particulate antigen is favoured by LCs. Therefore, these studies highlight the importance of LCs in skin immunization strategies and that targeting of nano-particulate immunogens through dissolvable polymeric MNs potentially provides a promising technological platform for improved vaccination strategies.Journal of Investigative Dermatology accepted article preview online, 22 September 2014. doi:10.1038/jid.2014.415.

Dense spectrum of resonances and spin-1/2 particle capture in a near-black-hole metric

We show that a spin-1/2 particle in the gravitational field of a massive body of radius R which slightly exceeds the Schwarzschild radius rs, possesses a dense spectrum of narrow resonances. Their lifetimes and density tend to infinity in the limit R → rs. We determine the cross section of the particle capture into these resonances and show that it is equal to the spin-1/2 absorption cross section for a Schwarzschild black hole. Thus black-hole properties may emerge in a non-singular static metric prior to the formation of a black hole.

Bright Subcycle Extreme Ultraviolet Bursts from a Single Dense Relativistic Electron Sheet

Double-foil targets separated by a low density plasma and irradiated by a petawatt-class laser are shown to be a copious source of coherent broadband radiation. Simulations show that a dense sheet of relativistic electrons is formed during the interaction of the laser with the tenuous plasma between the two foils. The coherent motion of the electron sheet as it transits the second foil results in strong broadband emission in the extreme ultraviolet, consistent with our experimental observations.

Warm Dense Aluminum Plasma generated by the Free-Electron-Laser FLASH

We report on experiments aimed at the generation and characterization of solid density plasmas at the free-electron laser FLASH in Hamburg. Aluminum samples were irradiated with XUV pulses at 13.5 nm wavelength (92 eV photon energy). The pulses with duration of a few tens of femtoseconds and pulse energy up to 100 mu J are focused to intensities ranging from 10(13) to 10(17) W/cm(2). We investigate the absorption and temporal evolution of the sample under irradiation by use of XUV spectroscopy. We discuss the origin of saturable absorption, radiative decay, bremsstrahlung and ionic line emission. Our experimental results are in good agreement with hydrodynamic simulations.

X-ray Polarization Measurements of Dense Plasmas Heated by Fast Electrons

The detailed knowledge of fast electron energy transport following interaction with high-intensity, ultra-short laser pulses is a key area for secondary source generation for ELI. We demonstrate polarization spectroscopy at laser intensities up to 10(21) Wcm(-2). This is significant as it suggests that in situ emission spectroscopy may be used as an effective probe of fast electron velocity distributions in regimes relevant to electron transport in solid targets. Ly-alpha doublet emission of nickel (Z = 28) and sulphur (Z = 16) is observed to measure the degree of polarization from the Ly-alpha(1) emission. Ly-alpha(2) emission is unpolarized, and as such acts as a calibration source between spectrometers. The measured ratio of the X-ray sigma- and pi-polarization allows the possibility to infer the velocity distribution function of the fast electron beam.

The complex ion structure of warm dense carbon measured by spectrally resolved x-ray scattering

We present measurements of the complex ion structure of warm dense carbon close to the melting line at pressures around 100 GPa. High-pressure samples were created by laser-driven shock compression of graphite and probed by intense laser-generated x-ray sources with photon energies of 4.75 keV and 4.95 keV. High-efficiency crystal spectrometers allow for spectrally resolving the scattered radiation. Comparing the ratio of elastically and inelastically scattered radiation, we find evidence for a complex bonded liquid that is predicted by ab-initio quantum simulations showing the influence of chemical bonds under these conditions. Using graphite samples of different initial densities we demonstrate the capability of spectrally resolved x-ray scattering to monitor the carbon solid-liquid transition at relatively constant pressure of 150 GPa. Showing first single-pulse scattering spectra from cold graphite of unprecedented quality recorded at the Linac Coherent Light Source, we demonstrate the outstanding possibilities for future high-precision measurements at 4th Generation Light Sources.

SN2012ca: a stripped envelope core-collapse SN interacting with dense circumstellar medium

We report optical and near-infrared observations of SN2012ca with the Public ESO Spectroscopy Survey of Transient Objects (PESSTO), spread over one year since discovery. The supernova (SN) bears many similarities to SN1997cy and to other events classified as Type IIn but which have been suggested to have a thermonuclear origin with narrow hydrogen lines produced when the ejecta impact a hydrogen-rich circumstellar medium (CSM). Our analysis, especially in the nebular phase, reveals the presence of oxygen, magnesium and carbon features. This suggests a core-collapse explanation for SN2012ca, in contrast to the thermonuclear interpretation proposed for some members of this group. We suggest that the data can be explained with a hydrogen- and helium-deficient SN ejecta (Type I) interacting with a hydrogen-rich CSM, but that the explosion was more likely a Type Ic core-collapse explosion than a Type Ia thermonuclear one. This suggests that two channels (both thermonuclear and stripped envelope core-collapse) may be responsible for these SN 1997cy-like events.

Intravitreal AAV2.COMP-Ang1 prevents neurovascular degeneration in a murine model of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the United States. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable to non-diabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally-delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Laboratory measurements of resistivity in warm dense plasmas relevant to the microphysics of brown dwarfs

Since the observation of the first brown dwarf in 1995, numerous studies have led to a better understanding of the structures of these objects. Here we present a method for studying material resistivity in warm dense plasmas in the laboratory, which we relate to the microphysics of brown dwarfs through viscosity and electron collisions. Here we use X-ray polarimetry to determine the resistivity of a sulphur-doped plastic target heated to Brown Dwarf conditions by an ultra-intense laser. The resistivity is determined by matching the plasma physics model to the atomic physics calculations of the measured large, positive, polarization. The inferred resistivity is larger than predicted using standard resistivity models, suggesting that these commonly used models will not adequately describe the resistivity of warm dense plasma related to the viscosity of brown dwarfs.

Optically controlled dense current structures driven by relativistic plasma aperture-induced diffraction

The collective response of charged particles to intense fields is intrinsic to plasma accelerators and radiation sources, relativistic optics and many astrophysical phenomena. Here we show that a relativistic plasma aperture is generated in thin foils by intense laser light, resulting in the fundamental optical process of diffraction. The plasma electrons collectively respond to the resulting laser near-field diffraction pattern, producing a beam of energetic electrons with a spatial structure that can be controlled by variation of the laser pulse parameters. It is shown that static electron-beam and induced-magnetic-field structures can be made to rotate at fixed or variable angular frequencies depending on the degree of ellipticity in the laser polarization. The concept is demonstrated numerically and verified experimentally, and is an important step towards optical control of charged particle dynamics in laser-driven dense plasma sources.

Ectopic bone formation in rapidly fabricated acellular injectable dense collagen-Bioglass hybrid scaffolds via gel aspiration-ejection

Gel aspiration-ejection (GAE) has recently been introduced as an effective technique for the rapid production of injectable dense collagen (IDC) gel scaffolds with tunable collagen fibrillar densities (CFDs) and microstructures. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass® (IDC-BG) hybrid gel scaffolds for potential bone tissue engineering applications. The efficacy of GAE in generating mineralizable IDC-BG gels (from an initial 75-25 collagen-BG ratio) produced through needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD) was investigated. Second harmonic generation (SHG) imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number. In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite was detected as early as day 1 in simulated body fluid, which progressively increased up to day 14. In vivo mineralization of, and host response to, acellular IDC and IDC-BG gel scaffolds were further investigated following subcutaneous injection in adult rats. Mineralization, neovascularization and cell infiltration into the scaffolds was enhanced by the addition of BG and at day 21 post injection, there was evidence of remodelling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of explanted scaffolds indicated collagen fibril remodelling through cell infiltration and mineralization over time. In sum, the results suggest that IDC-BG hybrid gels have osteoinductive properties and potentially offer a novel therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions