Differential Modulation of Retinal Degeneration by Ccl2 and Cx3cr1 Chemokine Signalling

Microglia and macrophages are recruited to sites of retinal degeneration where local cytokines and chemokines determine protective or neurotoxic microglia responses. Defining the role of Ccl2-Ccr2 and Cx3cl1-Cx3cr1 signalling for retinal pathology is of particular interest because of its potential role in age-related macular degeneration (AMD). Ccl2, Ccr2, and Cx3cr1 signalling defects impair macrophage trafficking, but have, in several conflicting studies, been reported to show different degrees of age-related retinal degeneration. Ccl2/Cx3cr1 double knockout (CCDKO) mice show an early onset retinal degeneration and have been suggested as a model for AMD. In order to understand phenotypic discrepancies in different chemokine knockout lines and to study how defects in Ccl2 and/or Cx3cr1 signalling contribute to the described early onset retinal degeneration, we defined primary and secondary pathological events in CCDKO mice. To control for genetic background variability, we compared the original phenotype with that of single Ccl2, Cx3cr1 and Ccl2/Cx3cr1 double knockout mice obtained from backcrosses of CCDKO with C57Bl/6 mice. We found that the primary pathological event in CCDKO mice develops in the inferior outer nuclear layer independently of light around postnatal day P14. RPE and vascular lesions develop secondarily with increasing penetrance with age and are clinically similar to retinal telangiectasia not to choroidal neovascularisation. Furthermore, we provide evidence that a third autosomal recessive gene causes the degeneration in CCDKO mice and in all affected re-derived lines and subsequently demonstrated co-segregation of the naturally occurring RD8 mutation in the Crb1 gene. By comparing CCDKO mice with re-derived CCl2−/−/Crb1Rd8/RD8, Cx3cr1−/−/Crb1Rd8/RD8 and CCl2−/−/Cx3cr1−/−/Crb1Rd8/RD8 mice, we observed a differential modulation of the retinal phenotype by genetic background and both chemokine signalling pathways. These findings indicate that CCDKO mice are not a model of AMD, but a model for an inherited retinal degeneration that is differentially modulated by Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Localisation of Epithelial Cells Capable of Holoclone Formation <i>In Vitro</i> and Direct Interaction with Stromal Cells in the Native Human Limbal Crypt

<div><p>Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used <i>in vitro</i> clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.</p></div

Melanocytes interact with putative LESCs in their niche.

<p>Immunohistochemistry reveals much more MelanA positive cells within the LCs than within the non-crypt rich limbal areas (A, B). Serial-block face scanning electron micrographs showing the edge of one limbal crypt (C, D). After manual segmentation and 3D reconstruction (E, F, G) putative LESC (smallest epithelial cell with a high nucleus/cytoplasm ratio) is represented in green, melanocyte (dendritic cell containing electron dense granules) in red, limbal stromal cell in yellow. Blue volumes correspond to nuclei. Continuous line in G corresponds to block face represented in C. Dashed line in G corresponds to block face represented in D. Scale bars: 50 μm (A, B) and 7 μm (C, D).</p

Limbal crypt ultrastructure observed by SBFSEM.

<p>Limbal crypt tangentially imaged through 70 μm from the corneal to the conjunctival side of the limbus. A, B and C represent non-sequential micrographs of the same 3D dataset. Manual segmentation followed by 3D reconstruction highlights the close proximity between the limbal epithelium (yellow volume in D) and a limbal stromal cell (white asterisks in B, blue area in C and blue volume in D) within the limbal crypt (Lc) suggesting a putative cell-to-cell contact. Lc: limbal crypt; Bv: blood vessel; St: Stroma. Arrowheads: melanocytes. Scale bars: 50 μm.</p

Observation of putative LESCs by TEM within the LCs.

<p>Transmission electron micrographs highlighting the interface between the limbal stroma and the limbal basal epithelial layer within the non-crypt rich limbus (A, B) and the limbal crypts (C, D, E, F). White arrowheads: hemidesmosomes; Black arrows: limbal stromal cell extensions in the vicinity of putative LESCs. Epi.: epithelium; St.: stroma; Bm: Basement membrane. White asterisks: putative LESCs. Black arrowhead: Contact between putative LESC and limbal stromal cell. Black box in E represents area in F. Scale bars A, B, C, D and E: 2 μm, F: 1 μm.</p

Data from: Regulation of cell protrusions by small GTPases during fusion of the neural folds

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development

Stabilised integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM

The inferior retinal degeneration in <i>CCDKO</i> mice is not dependent on light.

<p>(A) Comparison of autofluorescent SLO fundus images of <i>CCDKO</i> mice at 8 weeks of age that were raised from birth either in complete darkness (24 h dark, luminescence <0.5 lx) or in normal 12 h/12 h light/dark cycle (light 12 h/12 h, luminescence = 33±28 lx). As a comparison an AF-SLO fundus image from an aged-matched <i>C57Bl/6</i> wildtype mouse raised under normal 12 h/12 h light/dark cycle is shown. (B) Quantitative assessment of the number of autofluorescent lesions/per fundus image for the three animal groups. The number of autofluorescent lesions in <i>CCDKO</i> (24 h dark, n = 9) and <i>CCDKO</i> (12 h/12 h light/dark, n = 9) was not significantly different, but was significantly higher compared to that in <i>C57Bl/6</i> mice (12 h/12 h light/dark, n = 14). *: One-way-ANOVA (p<0.0001), Tukey's posthoc test for multiple comparison (p<0.05). This suggests that light is not necessary for the manifestation of the early onset inferior retinal degeneration in <i>CCDKO</i> mice and that ambient light (33+−28 lx) does not augment the number of inferior retinal autofluorescent lesions in <i>CCDKO</i> mice within the first 8 weeks of their life.</p