Maturation of autophagosomes and endosomes: A key role for Rab7

AbstractMacroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases

Attenuated Semliki Forest virus for cancer treatment in dogs : safety assessment in two laboratory Beagles

Background: Dogs suffer from spontaneous tumors which may be amenable to therapies developed for human cancer patients, and dogs may serve as large-animal cancer models. A non-pathogenic Semliki Forest virus vector VA7-EGFP previously showed promise in targeting human tumor xenografts in mice, but the oncolytic capacity of the virus in canine cancer cells and the safety of the virus in higher mammals such as dogs, are not known. We therefore assessed the oncolytic potency of VA7-EGFP against canine cancer cells by infectivity and viability assays in two dog solid tumor cell lines. Furthermore we performed a 3-week safety study in two adult Beagles which received a single intravenous injection of similar to 2 x 10(5) plaque forming units of parental A7(74) strain. Results: VA7-EGFP was able to replicate in and kill both canine cancer cell lines tested. No adverse events were observed in either of the two virus-injected adult Beagles and no infective virus could be recovered from any of the biological samples collected over the course of the study. Neutralizing antibodies to Semliki Forest virus became detectable in the dogs at 5 days post infection and remained elevated until study termination. Conclusions: Based on these results, testing of the oncolytic potential of attenuated Semliki Forest virus in canine cancer patients appears feasible.Peer reviewe

The initiation knot is a signaling center required for molar tooth development

Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.Peer reviewe

The Regulation of NFE2L2 (NRF2) Signalling and Epithelial-to-Mesenchymal Transition in Age-Related Macular Degeneration Pathology

Age-related macular degeneration (AMD) is a mounting cause of loss of sight in the elderly in the developed countries, a trend enhanced by the continual ageing of the population. AMD is a multifactorial and only partly understood, malady. Unfortunately, there is no effective treatment for most AMD patients. It is known that oxidative stress (OS) damages the retinal pigment epithelium (RPE) and contributes to the progression of AMD. We review here the potential importance of two OS-related cellular systems in relation to AMD. First, the nuclear factor erythroid 2-related factor 2 (NFE2L2; NRF2)-mediated OS response signalling pathway is important in the prevention of oxidative damage and a failure of this system could be critical in the development of AMD. Second, epithelial-to-mesenchymal transition (EMT) represents a change in the cellular phenotype, which ultimately leads to the fibrosis encountered in RPE, a characteristic of AMD. Many of the pathways triggering EMT are promoted by OS. The possible interconnections between these two signalling routes are discussed here. From a broader perspective, the control of NFE2L2 and EMT as ways of preventing OS-derived cellular damage could be potentially valuable in the therapy of AMD

Uusitalo H. Altered calcium signalling in an experimental model of glaucoma

1, 5, 6 PURPOSE. To investigate calcium signaling in a rat experimental model of glaucoma. METHODS. A method for labeling ganglion cell layer (GCL) neurons with the calcium indicator Fura-2 in flat-mounted retinas of adult rats was established. Pharmacologically evoked responses in laser-induced glaucomatous and control retinas were imaged 2 weeks after the initial laser treatment. The optic nerves of the same eyes were evaluated for neurodegenerative changes. RESULTS. After laser treatment, intraocular pressure (IOP) was elevated 1.5-to 4.9-fold (24.70 Ϯ 15.57 mm Hg) compared with control eyes (8.71 Ϯ 1.53 mm Hg), and the area of neurodegenerative axons in optic nerve sections of lasertreated eyes was increased by 1.2-to 13.3-fold. The basal intracellular Ca 2ϩ level, as revealed by the Fura-2 ratio, was elevated in GCL neurons of laser-treated eyes compared with controls. This might suggest a mild degree of damage at the level of the soma in the GCL neurons of eyes with elevated IOP. Although glaucomatous GCL neurons remained functional as assessed pharmacologically, analysis of imaging data revealed that responses evoked by a brief application of ATP were slightly reduced rather than increased in the cells of lasertreated eyes compared with controls. No significant relationships were found between IOP/optic nerve damage and functional characteristics (basal intracellular Ca 2ϩ level or response to carbachol/elevated K ϩ /ATP) within cells of laser-treated eyes. CONCLUSIONS. Ca 2ϩ imaging is a useful tool to map altered physiological characteristics of individual GCL neurons in the glaucomatous eye. (Invest Ophthalmol Vis Sci. 2010;51: 6387-6393

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.Peer reviewe