The effect of short term feeding with organic and conventional diets on selected immune parameters in rat

There is currently no evidence for beneficial health impacts being associated with the consumption of organic rather than conventional foods. This preliminary study was therefore aimed at using haematological parameters, white blood cell (WBC) number and splenocyte proliferation as sensitive assays to evaluate influence of the organic, low input and conventional components in the diet on rats’ immune system function. The results of a short term feeding trial with two rat generations indicates a potential effect on immune system function, which has to be confirmed by longer-term exposure studies

Frequency domain interferometer simulation with higher-order spatial modes

FINESSE is a software simulation that allows to compute the optical properties of laser interferometers as they are used by the interferometric gravitational-wave detectors today. It provides a fast and versatile tool which has proven to be very useful during the design and the commissioning of gravitational-wave detectors. The basic algorithm of FINESSE numerically computes the light amplitudes inside an interferometer using Hermite-Gauss modes in the frequency domain. In addition, FINESSE provides a number of commands to easily generate and plot the most common signals like, for example, power enhancement, error or control signals, transfer functions and shot-noise-limited sensitivities. Among the various simulation tools available to the gravitational wave community today, FINESSE is the most advanced general optical simulation that uses the frequency domain. It has been designed to allow general analysis of user defined optical setups while being easy to install and easy to use.Comment: Added an example for the application of the simulation during the commisioning of the GEO 600 gravitational-wave detecto

Regulation of RPE phenotype by Annexin A8 and Wnt signalling

Purpose. Fenretinide (FR), a retinoic acid derivative, is capable of trans-differentiating retinal pigment epithelial (RPE) cells into a neuronal-like phenotype in culture. Microarray analysis pre- and post-FR treatment revealed down-regulation of Annexin (Anx) A8 and various proteins involved in Wnt signalling in trans-differentiated cells. AnxA8, a member of a superfamily of calcium-dependent phospholipid-binding proteins, is expressed in RPE cells and involved in membrane and cytoskeletal organisation and cell proliferation. The purpose of this study was to analyse the role of AnxA8 and its relationship with Wnt signalling in epithelial trans-differentiation. Methods. At 10% confluence, human RPE cells were treated with 3% charcoal dextran-treated foetal bovine serum (FBS) for 24 h. 3 µM FR or vehicle (0.1% dimethylsulfoxide) was added to the cells every day for 7 days. As a second approach, AnxA8 was suppressed in RPE cells using short interfering RNA (siRNA). Cells were then analysed for expression of AnxA8, neuronal markers (Calbindin, Calretinin) and Wnt signalling proteins (β-Catenin, Frizzled-1, Frizzled-4, Wnt2b, Wnt3a) using immunofluorescence staining, qPCR and western blot analysis. Results. FR and AnxA8 siRNA treatment both induced a decrease in AnxA8 expression and inhibited cell proliferation. FR also led to trans-differentiation of ARPE-19 cells into neuron-like cells and a concomitant up-regulation of neuronal markers. Additionally, expression of proteins involved in Wnt signalling was decreased. The effect of FR was partially reversible by activating Wnt signalling using recombinant Wnt3a or SB216763, a glycogen synthase kinase-3β inhibitor. Conclusions. These data imply an important role for AnxA8 in maintaining RPE phenotype. Down-regulation of AnxA8 appears to be sufficient for neuronal trans-differentiation of RPE cells and the expression of neuronal markers. Further, the interdependence of AnxA8 and Wnt proteins suggests that AnxA8 might be an important regulator in Wnt signalling

Demonstration of detuned dual recycling at the Garching 30m laser interferometer

Dual recycling is an advanced optical technique to enhance the signal-to-noise ratio of laser interferometric gravitational wave detectors in a limited bandwidth. To optimise the center of this band with respect to Fourier frequencies of expected gravitational wave signals detuned dual recycling has to be implemented. We demonstrated detuned dual recycling on a fully suspended 30m prototype interferometer. A control scheme that allows to tune the detector to different frequencies will be outlined. Good agreement between the experimental results and numerical simulations has been achieved.Comment: 9 page

Regulation of epithelial cell phenotype by annexin A8

Background The retinoic acid derivative Fenretinide (Fr) is capable of trans-differentiating retinal pigment epithelial (RPE) cells towards a neuron-like phenotype in culture. Microarray analysis of Fr-treated ARPE-19 cells revealed down-regulation of annexin (anx) A8 in trans-differentiated cells. AnxA8, a calcium-dependent phospholipid-binding protein, is expressed in RPE cells, where it may be involved in membrane and cytoskeletal organisation and cell proliferation. Objectives The purpose of this study was to analyse the role of anxA8 in maintaining the RPE cell phenotype. Methods RPE cells were seeded at 2,200 cells/cm2 and treated with 3% charcoal dextran-treated foetal bovine serum (FBS) for 24 h. 1µM Fr or vehicle (0.1% dimethylsulfoxide) was added every day for 7 days. As a second approach, anxA8 was suppressed in RPE cells using short interfering RNA (siRNA). Further, an anxA8-GFP construct was used to overexpress anxA8 and to restore the anxA8 loss derived from Fr or anxA8 suppression. Cells were analysed for anxA8 and the neuronal markers Calbindin and Calretinin using immunofluorescence staining and qPCR. Results Fr and anxA8 siRNA treatment both induced a decrease in anxA8 expression and inhibited cell proliferation. They also led to RPE trans-differentiation into neuron-like cells and a concomitant up-regulation of neuronal markers. Overexpression of anxA8 led to a recovery of the anxA8 loss-induced neuron-like cell phenotype. Conclusions These data reveal an important role for anxA8 in maintaining RPE phenotype. Down-regulation of anxA8 appears not only to be sufficient, but also to be necessary for neuronal trans-differentiation of RPE cells and the expression of neuronal markers. Sources of funding: BBSRC Conflicts of interest: none declare

Complement increases release of proinflammatory and proangiogenic mediators by retinal pigment epithelial cells

Objectives. A mutation in complement factor H (CFH) gene, leading to augmented complement activation, is correlated with development of age-related macular degeneration (AMD). Therefore, the influence of complement on retinal pigment epithelial (RPE) cells was examined concerning their production of proinflammatory and proangiogenic mediators relevant in AMD. Methods. ARPE-19 cells were cultured with human or fetal calf serum (FCS). Therefore, complement containing native serum as well as the heat-inactivated form with inoperable complement was used. Further, RPE cells were treated with zymosan, a complement activating yeast particle. Serum and zymosan in combination was also tested. Levels of interleukin (IL)-6, -8 and vascular endothelial growth factor (VEGF) in supernatants were examined by ELISA. Results. Untreated RPE cells produced IL-6, -8 and VEGF constitutively. FCS or human serum led to a concentration dependent release of all mediators. Thereby, FCS increased the cytokine production stronger than human serum, native serum stronger than heat-inactivated. Zymosan only intensified IL-6 and -8 secretion. Combined treatment with serum and zymosan resulted in an additive release of IL-8 and VEGF. In contrast, secretion of IL-6 was synergistic. Conclusion. The enhanced expression of IL-6, -8 and VEGF by RPE after exposure to complement might explain the correlation between augmented complement production and inflammatory processes accompanying AMD. IL-6 production was strongly increased due to activation of complement within the serum by zymosan. Thus, complement activation could stimulate inflammatory processes by activated RPE cells leading to AMD

The ratio of pro- and anti-angiogenic cytokines produced by retinal pigment epithelial cells is shifted to support angiogenesis by complement

Purpose The complement system of age-related macular degeneration (AMD) patients is marginally but chronically over-activated. Retinal pigment epithelial (RPE) cells and photoreceptor cells undergo cell death during the development of this potentially blinding eye disease. In this study the balance between the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic pigment epithelium-derived factor (PEDF) by RPE cells in response to complement serum was analysed. Methods Increasing concentrations of complement competent human serum were incubated with human RPE cells. Controls with the addition of zymosan to activate the complement cascade, zymosan alone, and heat-treated serum with inoperative complement were included. The secretion of VEGF and PEDF was measured by sandwich ELISA. Immunocytochemistry was performed for the in situ detection of VEGF and PEDF. The experiments were supplemented by RT-PCR expression analysis and Western Blot detection of both antagonists. Results Human complement competent serum stimulated the RPE cells to produce enhanced amounts of VEGF while unspecific stimuli showed no influence on the secretion of VEGF. The combination of complement competent serum and zymosan was revealed as the most effective treatment for an increased VEGF production. The PEDF-specific staining of RPE cells decreased with augmented concentrations of complement competent serum. PCR data showed an enhanced amount of VEGF-encoding transcripts and an unaltered or lower amount of PEDF-specific transcripts. Western Blots confirmed the shift in favour of VEGF when compared to PEDF after complement treatment of RPE cells. Conclusions Activated complement may shift the balance between VEGF and PEDF produced by RPE cells towards the blood vessel chemoattractant VEGF. This finding may reveal a mechanism how enhanced complement activation might contribute to a pro-angiogenic retinal environment supporting neovascularisation during the late stage of exsudative AMD

Regulation of epithelial cell phenotype by annexin A8 and Wnt signalling

The retinoic acid derivative Fenretinide (FR) is capable of trans-differentiating retinal pigment epithelial (RPE) cells towards a neuron-like phenotype in culture. Microarray analysis of FR-treated ARPE-19 cells revealed down-regulation of Annexin (Anx)A8 and specific Wnt signalling proteins in transdifferentiated cells. AnxA8, a calcium-dependent phospholipid-binding protein, is expressed in the RPE cell cytosol, where it may be involved in membrane and cytoskeletal organisation and cell proliferation. The aim here was to analyse the role of AnxA8 and its interaction with Wnt signalling in RPE cell transdifferentiation. Human RPE cells were seeded at a concentration of 2,200/ml and treated with 3% charcoal dextran-treated foetal bovine serum (FBS) for 24h. 3µM FR or vehicle (0.1% dimethylsulfoxide) was added every day for 7 days. As a second approach, AnxA8 was suppressed in RPE cells using short interfering RNA (siRNA). FR and AnxA8 siRNA treatment both induced a decrease in AnxA8 expression and inhibited cell proliferation. It further led to transdifferentiation of ARPE-19 cells into neuron-like cells and a concomitant up-regulation of the neuronal markers Calbindin and Calretinin analysed by qPCR and immunofluorescence. Additionally, expression of Wnt signalling proteins such as β-Catenin, Frizzled-1, Frizzled-4, Wnt2b and Wnt3a was decreased. The reduction in AnxA8 and cell morphology changes induced by FR, were not reversible by inhibiting Wnt signalling using Dickkopf-1 and DAPT. Wnt signalling activators such as recombinant Wnt3a or SB216763 (glycogen synthase kinase-3β inhibitor) were able to partially reverse the FR effect. These data imply an important role for AnxA8 in maintaining RPE phenotype. Down-regulation of AnxA8 appears to be sufficient for neuronal transdifferentiation of RPE cells and the expression of neuronal markers. Further, the interdependence of AnxA8 and Wnt proteins suggests that AnxA8 might be an important regulator of Wnt signalling

Retinal pigment epithelial cells respond to complement by an augmented production of vitronectin

Objectives: Genetic studies have demonstrated the role of activated complement on the alternative pathway during the development of age-related macular degeneration (AMD). The extracellular matrix component vitronectin can protect against activated complement. Drusen appear in the retina between the retinal pigment epithelial (RPE) cell layer and Bruch’s membrane. Drusen are hallmarks of early and late AMD and contain high amounts of vitronectin. Therefore this study addressed the influence of complement on the vitronectin production by RPE cells. Methods: ARPE-19 cells as model for RPE cells were cultivated with increasing amounts of human serum as complement source in its naïve and heat (and thereby complement) inactivated form. In another series of experiments zymosan as an activator of the alternative pathway of complement was tested alone and in combination with naïve human serum. Vitronectin was assayed in situ by immunohistochemistry, on protein level by western blot and by PCR after reverse transcription of total RNA. Results: A constitutive production of vitronectin by RPE cells was detected by all three tests. With naïve human serum increased vitronectin protein was found by immunohistochemistry and western blot while the number of mRNA transcripts was not significantly altered. The vitronectin production was further enhanced with the combination of zymosan and naïve human serum while heat inactivated serum showed lesser effect. Conclusion: Activated complement lead to an augmented vitronectin production by RPE cells on post-transcriptional level. Enhanced complement activation during AMD might also contribute in vivo to an enhanced production of vitronectin by RPE cells. On the one hand this can cause protection against activated complement but on the other hand the increased retinal vitronectin might contribute to thickening of Bruch’s membrane and may facilitate the development of drusen

RPE Tissue-specific Factor H Deletion Induces AMD-like Features

Age-related macular degeneration (AMD) is a central vision-threatening disease, and its development is significantly associated with the Y402H variant in the complement factor H (Cfh) gene. Cfh is a soluble glycoprotein, and a negative regulator of complement activation. It is secreted by retinal pigment epithelium (RPE), liver and immune cells among others, but to date it is unclear whether the origin of the Cfh is of importance in regulating the alternative complement pathway and eye homeostasis. Here, Cfhflx/flx mice were crossed with Best1-cre mice, with the Best1 promoter providing RPE-specific expression of the Cre recombinase, inducing an RPE-specific deletion of loxP flanked Cfh. Cfh(RPE)-/- mice on a mixed C57BL/6 and 129 background exhibited 95% Cre-positive RPE cells in immunostained cryosections. qPCR revealed a concomitant decrease of 95% in Cfh mRNA. Compared to Cfh total knockout mice, Cfh(RPE)-/- animals preserved an intact complement system, with normal levels of circulating Cfh, C3 and Cfb, as well as sub-RPE deposition of the C3-breakdown product iC3b, as also seen in AMD patients. C5b-9 deposition was elevated in cryosections of 6 month old Cfh(RPE)-/- mice, and western blotting analysis of RPE and choroid protein revealed higher Cfh levels compared to controls. F4/80 and CD206 immunostaining revealed sub-RPE accumulation of activated macrophages in 6 month old Cfh(RPE)-/- animals compared to controls. Increased autofluorescence was visualised in 12 month old Cfh(RPE)-/- mice using a micron III, and yellow, drusen-like deposits were evident in fundus imaging relative to wild type and Cre controls. RPE-selective Cfh loss thus manifests in AMD-like changes and may provide insight into the source of secreted Cfh as target for therapeutics