Isolation and Quantification of Blood Apoptotic Bodies, a Non-invasive Tool to Evaluate Apoptosis in Patients with Ischemic Stroke and Neurodegenerative Diseases.

BACKGROUND: Improper regulation of apoptosis has been postulated as one of the main factors that contributes to the etiology and/or progression of several prevalent diseases, including ischemic stroke and neurodegenerative pathologies. Consequently, in the last few years, there has been an ever-growing interest in the in vivo study of apoptosis. The clinical application of the tissue sampling and imaging approaches to analyze apoptosis in neurological diseases is, however, limited. Since apoptotic bodies are membrane vesicles that are released from fragmented apoptotic cells, it follows that the presence of these vesicles in the bloodstream is likely due to the apoptotic death of cells in tissues. We therefore propose to use circulating apoptotic bodies as biomarkers for measuring apoptotic death in patients with ischemic stroke and neurodegenerative diseases. RESULTS: Since there is no scientific literature establishing the most appropriate method for collecting and enumerating apoptotic bodies from human blood samples. Authors, here, describe a reproducible centrifugation-based method combined with flow cytometry analysis to isolate and quantify plasma apoptotic bodies of patients with ischemic stroke, multiple sclerosis, Parkinson's disease and also in healthy controls. Electron microscopy, dynamic light scattering and proteomic characterization in combination with flow cytometry studies revealed that our isolation method achieves notable recovery rates of highly-purified intact apoptotic bodies. CONCLUSIONS: This easy, minimally time consuming and effective procedure for isolating and quantifying plasma apoptotic bodies could help physicians to implement the use of such vesicles as a non-invasive tool to monitor apoptosis in patients with cerebrovascular and neurodegenerative diseases for prognostic purposes and for monitoring disease activity.

Involvement of N-methylpurine DNA glycosylase in resistance to temozolomide in patient-derived glioma cells.

Chemotherapy for high-grade astrocytic tumors is mainly based on the use of temozolomide (TMZ), whose efficacy is limited by resistance mechanisms. Despite many investigations pointing to O6-methylguanine-DNA-methyltransferase (MGMT) as being responsible for tumor chemo-resistance, its expression does not predict an accurate response in most gliomas, suggesting that MGMT is not the only determinant of response to treatment. In this sense, several reports indicate that N-methylpurine-DNA-glycosylase (MPG) may be involved in that resistance. With that in mind, we evaluated for the first time the degree of resistance to TMZ treatment in 18 patient-derived glioma cells and its association with MGMT and MPG mRNA levels. Viability cell assays showed that TMZ treatment hardly caused growth inhibition in the patient-derived cells, even in high concentrations, indicating that all primary cultures were chemo-resistant. mRNA expression analyses showed that the TMZ-resistant phenotype displayed by cells is associated with an elevated expression of MPG to a greater extent than it is with transcript levels of MGMT. Our findings suggest that not only is MGMT implicated in resistance to TMZ but MPG, the first enzyme in base excision repair processing, is also involved, supporting its potential role as a target in anti-resistance chemotherapy for astrocytoma and glioblastoma.

The use of nanoparticles for gene therapy in the nervous system.

Nanoparticles represent an alternative to viral vectors for genetic material transfer to the nervous system. However, to increase transfection efficiency in the central nervous system and to decrease toxicity, the design of nanoparticles needs to be improved to enhance blood-brain barrier crossing and endosomal escape. This paper reviews the strategies used to solve these difficulties and covers the use of various nanoparticles including natural inorganic particles, natural polymers, cationic lipids, polyethylenimine derivatives, dendrimers, and carbon-based nanoparticles. The effectiveness, both in vivo and in vitro, of each method to deliver genetic material to neural tissue is discussed.

Aliskiren prevents the toxic effects of peritoneal dialysis fluids during chronic dialysis in rats.

The benefits of long-term peritoneal dialysis (PD) in patients with end-stage renal failure are short-lived due to structural and functional changes in the peritoneal membrane. In this report, we provide evidence for the in vitro and in vivo participation of the renin-angiotensin-aldosterone system (RAAS) in the signaling pathway leading to peritoneal fibrosis during PD. Exposure to high-glucose PD fluids (PDFs) increases damage and fibrosis markers in both isolated rat peritoneal mesothelial cells and in the peritoneum of rats after chronic dialysis. In both cases, the addition of the RAAS inhibitor aliskiren markedly improved damage and fibrosis markers, and prevented functional modifications in the peritoneal transport, as measured by the peritoneal equilibrium test. These data suggest that inhibition of the RAAS may be a novel way to improve the efficacy of PD by preventing inflammation and fibrosis following peritoneal exposure to high-glucose PDFs.

Enhanced docetaxel-mediated cytotoxicity in human prostate cancer cells through knockdown of cofilin-1 by carbon nanohorn delivered siRNA.

We synthesized a non-viral delivery system (f-CNH3) for small interfering RNA (siRNA) by anchoring a fourth-generation polyamidoamine dendrimer (G4-PAMAM) to carbon nanohorns (CNHs). Using this new compound, we delivered a specific siRNA designed to knockdown cofilin-1, a key protein in the regulation of cellular cytoskeleton, to human prostate cancer (PCa) cells. The carbon nanohorn (CNH) derivative was able to bind siRNA and release it in the presence of an excess of the polyanion heparin. Moreover, this hybrid nanomaterial protected the siRNA from RNAse-mediated degradation. Synthetic siRNA delivered to PCa cells by f-CNH3 decreased the cofilin-1 mRNA and protein levels to about 20% of control values. Docetaxel, the drug of choice for the treatment of PCa, produced a concentration-dependent activation of caspase-3, an increase in cell death assessed by lactate dehydrogenase release to the culture medium, cell cycle arrest and inhibition of tumor cell proliferation. All of these toxic effects were potentiated when cofilin-1 was down regulated in these cells by a siRNA delivered by the nanoparticle. This suggests that knocking down certain proteins involved in cancer cell survival and/or proliferation may potentiate the cytotoxic actions of anticancer drugs and it might be a new therapeutic approach to treat tumors.

Atorvastatin reduces high glucose toxicity in rat peritoneal mesothelial cells.

OBJECTIVE: Continuous exposure of the peritoneal membrane to high glucose dialysis solutions can produce functional alterations in this membrane. We studied the toxic effects of high glucose (50 mmol/L and 83 mmol/L) and its reversal by atorvastatin (0.5 - 5 µmol/L) on cultures of rat peritoneal mesothelial cells (PMCs). METHODS: Rat PMCs were harvested from the peritonea of male Sprague-Dawley rats and grown in M199 medium supplemented with 10% fetal bovine serum. The effects of high glucose (50 mmol/L and 83 mmol/L) on levels of reactive oxygen species (ROS), on caspase 3 activity, and on phospho-p38 mitogen-activated protein kinase (MAPK) in the cultures were evaluated. RESULTS: Exposure to high glucose (for 4, 8, and 24 hours) increased intracellular levels of ROS and phospho-p38 MAPK (indices of cellular toxicity). Atorvastatin blocked these toxic effects of high glucose, being more effective against 50 mmol/L glucose (protective effects were observed above 0.5 µmol/L) than against 83 mmol/L (protective effects were observed above 2.5 µmol/L). Atorvastatin was also able to prevent glucose-induced increase in caspase 3 activity. CONCLUSIONS: The present study shows that high glucose may promote oxidative stress and may activate apoptotic pathways in rat PMCs. These toxic effects could be reversed by atorvastatin.