Isolation and characterization of neurotoxic astrocytes derived from adult triple transgenic Alzheimer's disease mice.

Alzheimer's disease has been considered mostly as a neuronal pathology, although increasing evidence suggests that glial cells might play a key role in the disease onset and progression. In this sense, astrocytes, with their central role in neuronal metabolism and function, are of great interest for increasing our understanding of the disease. Thus, exploring the morphological and functional changes suffered by astrocytes along the course of this disorder has great therapeutic and diagnostic potential. In this work we isolated and cultivated astrocytes from symptomatic 9-10-months-old adult 3xTg-AD mice, with the aim of characterizing their phenotype and exploring their pathogenic potential. These "old" astrocytes occurring in the 3xTg-AD mouse model of Alzheimer's Disease presented high proliferation rate and differential expression of astrocytic markers compared with controls. They were neurotoxic to primary neuronal cultures both, in neuronal-astrocyte co-cultures and when their conditioned media (ACM) was added into neuronal cultures. ACM caused neuronal GSK3ß activation, changes in cytochrome c pattern, and increased caspase 3 activity, suggesting intrinsic apoptotic pathway activation. Exposure of neurons to ACM caused different subcellular responses. ACM application to the somato-dendritic domain in compartmentalised microfluidic chambers caused degeneration both locally in soma/dendrites and distally in axons. However, exposure of axons to ACM did not affect somato-dendritic nor axonal integrity. We propose that this newly described old 3xTg-AD neurotoxic astrocytic population can contribute towards the mechanistic understanding of the disease and shed light on new therapeutical opportunities.

Comparative In Vitro Study of 11C-Methionine and 11C-Deuterodeprenyl Uptake in Three Human Glioma Cell Lines.

AIM: To compare the uptake of 11C-deuterodeprenyl (11C-DED) and 11C-methionine (11C-MET) in three human glioma cell lines and study the relationship with glial fibrillary acid protein (GFAP) and monoamine oxidase B (MAO B) expression. 11C-DED is used in positron emission tomography imaging as a marker of astrocytosis in various central nervous system pathologies. It binds irreversibly to MAO B, a glial dimeric enzyme with increased activity in some neurological pathologies. MATERIALS AND METHODS: Binding and internalization studies of 11C-MET and 11C-DED were performed in astrocytoma grade III, glioblastoma grade IV, and radio-resistant glioblastoma grade IV cells. Immunofluorescence was used. RESULTS: 11C-MET specific activity bound to membrane was 9.0%-11.1% and that internalized was 88.9%-91.0%. 11C-DED specific activity bound to membrane was 34.8%-58.0% and that internalized was 38.7%-65.2%. Immunocytochemistry revealed GFAP and MAO B expression. CONCLUSIONS: The expression of MAO B measured by 11C-DED uptake or immunocytochemistry was not significantly different in grade III or IV cells. The GFAP signal was higher for grade IV compared to grade III. 11C-MET uptake was high in all the tumor cells. 11C-DED is a dopamine analogue and the transport across cell membranes is expected to be mediated by DAT receptors present in astrocytes. Reactive astrocytes surround tumor lesions; so the authors suggest that the 11C-DED uptake might be caused by the reactive astrocytosis and not by MAO B expression in tumor cells.

68Ga-NOTA-UBI-29-41 as a PET Tracer for Detection of Bacterial Infection.

UNLABELLED: The cationic peptide (68)Ga-NOTA-UBI-29-41 was synthesized and characterized. Biodistribution and PET/CT examinations were performed for evaluation of its biologic behavior. Differentiation of infection from sterile inflammation was investigated using microbiology methods at the sites of bacterial infections. METHODS: Labeling of UBI-29-41 conjugated with NOTA with (68)Ga was optimized at 20°C-100°C and pH 3.5-5.5. Radiochemical purity, stability up to 260 min, and binding to serum proteins were determined. In vitro binding to Staphylococcus aureus was evaluated from 9.14 × 10(7) to 1.17 × 10(10) cfu/mL. Of 3 groups of Mus musculus Swiss male mice, the first was inoculated intramuscularly with 1.2 × 10(8) cfu of S. aureus to provoke infection, and the second, with 1.2 × 10(8) cfu of heat shock-treated S. aureus to generate sterile inflammation. The third mouse was not treated and served as a control. After 24 h, (68)Ga-NOTA-UBI-29-41 was administrated intravenously, and biodistribution was performed at 30, 60, and 120 min. PET/CT dynamic studies (120 min) were acquired. Sinograms were reconstructed using 3D maximum-likelihood expectation maximization and analyzed with software. Infected or inflamed muscles were dissected, homogenized, and cultured in tryptic soy agar medium. Recovered S. aureus was calculated as cfu/g. RESULTS: (68)Ga-NOTA-UBI-29-41 showed high renal excretion (83.2% ± 7.3%) of injected dose and rapid blood clearance. More than 95% was bound in vitro to 5 × 10(9) cfu/mL. A significantly higher (P< 0.05) accumulation of (68)Ga-NOTA-UBI-29-41 was observed at sites of S. aureus inoculation in infected mice (ratio of target to nontarget, 5.0 at 60 min and 4.1 at 120 min) compared with animals with inflammation (ratio of target to nontarget, 1.6 at 60 min and 1.2 at 120 min). CONCLUSION: The difference in uptake of (68)Ga-NOTA-UBI-29-41 in the infected muscles compared with the inflamed muscles was clearly observed in the PET/CT images and positively correlated with the degree of infection.

ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel.

Previous work from our laboratory and others has shown that, in some epithelia, the epithelial sodium channel (ENaC) increases its expression during wound healing. In these cases, inhibition of the channel determines a decrease in the healing rate, a result suggesting a role for ENaC in the overall healing process. To understand further this role of ENaC in epithelia, we explored the participation of ENaC in wound healing in four cultured epithelial cell lines selected on the basis of their different embryonic origins, function and modality of healing, i.e., by lamellipodial cell crawling or by actin cable formation. Three of the cell lines (bovine corneal endothelial cells, rabbit corneal epithelial cells and Madin-Darby canine kidney cells) exhibited an increase in ENaC expression and consequent membrane potential depolarization and an increase in cytosolic sodium and calcium, whereas one line (bovine aortal endothelial cells, BAEC) did not exhibit any of these changes. In all of the cell lines, however, ENaC inhibition determined a similar decrease in the rate of wound healing. In BAEC monolayers, the increase in ENaC activity produced plasma membrane depolarization, increased cytosolic sodium and calcium, and augmented the velocity of healing. These novel findings contribute to the idea that ENaC plays a critical role in wound healing in various epithelia, independently of the modality of healing and of any increase in the expression of the channel.