Fluoride Induces Neuroinflammation and Alters Wnt Signaling Pathway in BV2 Microglial Cells.

Fluoride is a common element in nature and our daily life, and excessive intake of this element can cause fluorosis and irreversible brain damage. The toxic effects of fluoride on the central nervous system may be attributed to the release of inflammatory cytokines and ROS. GSK3ß is a key protein that modulates NF-κB activity and inflammatory cytokine levels and plays an important role in the Wnt signaling pathway. In this study, we found that fluoride altered the inflammatory status and oxidative stress by inhibiting Wnt signaling pathway activity. This study thus provides a valid basis for the fluorine-induced neuroinflammation injury theory.

EGb-761 Attenuates the Anti-proliferative Activity of Fluoride via DDK1 in PC-12 Cells.

EGb-761 is commonly used as a treatment for ischemic brain injury, neurodegenerative diseases and some types of tumors (Christen and Maixent, in Cell Mol Biol 48(6):601-611, 2002). However, it is unclear whether EGb-761 affects the proliferation of cells exposed to fluoride. In this study, the proliferation and apoptosis of PC-12 cells exposed to fluoride were investigated and EGb-761 was used to protect PC-12 cells against the effects of fluoride. We found that the canonical Wnt signaling pathway was involved in the anti-proliferation of PC-12 cells exposed to fluoride. Furthermore, the results also showed that EGb-761 could attenuate the anti-proliferative activity of fluoride via DDK1 in PC-12 cells. This study may provide a new method for protecting against the inhibition of cell proliferation induced by fluoride.

Microneurosurgery and subsequent gamma knife radiosurgery for functioning pituitary macroadenomas or giant adenomas: One institution's experience.

OBJECTIVE: Functioning pituitary macroadenoma and giant adenoma have large growth volumes and endocrinological abnormalities, requiring proper medical intervention. In this retrospective study, microneurosurgery and subsequent gamma knife radiosurgery (GKRS) is assessed for efficacy and safety for the treatment of functioning pituitary macroadenoma and giant adenoma. METHODS: Between January 2007 and December 2011, 59 patients with functioning pituitary macroadenoma (n=38) or giant adenoma (n=21) received microneurosurgical resection, and after three months, GKRS with average maximum radiation dose ∼42Gy (range 30-66.7Gy). The median follow-up time was 54.3 months (range 36-85 months). RESULTS: The combined treatment controlled tumor growth in 81.4% (48/59) of patients, and improved the endocrinological status in 64.4% (38/59). Complications included hypopituitarism and visual deterioration (22 and 7 patients, respectively). Large tumor size at presentation was a risk factor for tumor recurrence, but not age, gender, invasion, radiosurgical dose, pituitary hormone status or follow-up period. Better outcomes were achieved by patients with macroadenoma than giant adenoma. CONCLUSIONS: Combined microneurosurgery and GKRS are safe and effective for functioning pituitary macroadenomas or giant adenomas. Tumor control and endocrinological improvement were satisfactory, with minimal complications.

The expression of Wnt-inhibitor DKK1 (Dickkopf 1) is determined by intercellular crosstalk and hypoxia in human malignant gliomas.

OBJECTIVE: Wnt signalling pathways regulate proliferation, motility and survival in a variety of human cell types. Dickkopf 1 (DKK1) gene codes for a secreted Wnt inhibitory factor. It functions as tumour suppressor gene in breast cancer and as a pro-apoptotic factor in glioma cells. In this study, we aimed to demonstrate whether the different expression of DKK1 in human glioma-derived cells is dependent on microenvironmental factors like hypoxia and regulated by the intercellular crosstalk with bone-marrow-derived mesenchymal stem cells (bmMSCs). METHODS: Glioma cell line U87-MG, three cell lines from human glioblastoma grade IV (glioma-derived mesenchymal stem cells) and three bmMSCs were selected for the experiment. The expression of DKK1 in cell lines under normoxic/hypoxic environment or co-culture condition was measured using real-time PCR and enzyme-linked immunoadsorbent assay. The effect of DKK1 on cell migration and proliferation was evaluated by in vitro wound healing assays and sulphorhodamine assays, respectively. RESULTS: Glioma-derived cells U87-MG displayed lower DKK1 expression compared with bmMSCs. Hypoxia led to an overexpression of DKK1 in bmMSCs and U87-MG when compared to normoxic environment, whereas co-culture of U87-MG with bmMSCs induced the expression of DKK1 in both cell lines. Exogenous recombinant DKK1 inhibited cell migration on all cell lines, but did not have a significant effect on cell proliferation of bmMSCs and glioma cell lines. CONCLUSION: In this study, we showed for the first time that the expression of DKK1 was hypoxia dependent in human malignant glioma cell lines. The induction of DKK1 by intracellular crosstalk or hypoxia stimuli sheds light on the intense adaption of glial tumour cells to environmental alterations.

Isolation and characterization of bone marrow-derived progenitor cells from malignant gliomas.

BACKGROUND: Malignant gliomas are highly-vascularised tumours. Neoangiogenesis is a crucial factor in the malignant behaviour of tumour and prognosis of patients. Several mechanisms are suspected to lead to neoangiogenesis, one of them is the recruitment of multipotent progenitor cells towards the tumour. Factors such as Vascular endothelial growth factor-A (VEGF-A) were described to recruit bone marrow-derived endothelial progenitor cells (EPCs) to the glioma stroma and vasculature. Little is known about isolating EPCs from normal or malignant tissues. MATERIALS AND METHODS: In this study, we addressed the topic of characterization of tumour-isolated EPCs and re-defined the clonal relationship between EPCs and hematopoietic stem cells (HSCs) in gliomas. We first checked public gene expression data of glioma for putative marker expression, pointing towards a prevalence of EPCs and HSCs in glioma. Immunohistochemical staining of glioma tissue confirmed the higher expression of these progenitor markers in glioma tissue. EPCs and HSCs were consequently isolated and characterized at the phenotypic and functional levels. We applied a new isolation method, for the first time, to specimen from patients with high grade glioma including seven grade IV glioblastoma, five-grade III astrocytoma, and three grade III oligoastrocytoma. RESULTS: In all samples, we were able to isolate the tumour-derived EPCs, which were positive for characteristic markers: CD31, CD34 and VEGFR2. The EPCs formed capillary networks in vitro and had the ability to take up acetylated low-density lipoprotein. Glioma-derived HSCs were positive for CD34 and CD45, but they were unable to form a capillary network in vitro. These findings on tumour-derived EPCs/HSCs were in concordance with the results, derived from peripheral blood of healthy volunteers. CONCLUSION: In our study, we established a new method for EPC/HSC isolation from human gliomas, defined the contribution of EPCs and HSCs to the tumour tissue, and highlighted the intense in vivo tumour host interaction.

Comparison of metabolite profiles in U87 glioma cells and mesenchymal stem cells.

Gas chromatography-mass spectrometry (GC-MS) profiles were generated from U87 glioma cells and human mesenchymal stem cells (hMSC). 37 metabolites representing glycolysis intermediates, TCA cycle metabolites, amino acids and lipids were selected for a detailed analysis. The concentrations of these metabolites were compared and Pearson correlation coefficients were used to calculate the relationship between pairs of metabolites. Metabolite profiles and correlation patterns differ significantly between the two cell lines. These profiles can be considered as a signature of the underlying biochemical system and provide snap-shots of the metabolism in mesenchymal stem cells and tumor cells.

CELL-SELEX: Novel perspectives of aptamer-based therapeutics.

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

Aptamer-based strategies for stem cell research.

Aptamers have been introduced to analytical applications, target validation, and drug discovery processes and, recently, applied directly as therapeutic agents. Aptamers can be generated by a method called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This is quite remarkable for such a young technology, which is only created in the early 1990s. This paper reviews recent new applications of aptamers in stem cell research and tissue engineering.

The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.

To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.

Combinations of TLR and NOD2 ligands stimulate rat microglial P2X4R expression.

As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-alpha production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.

A new technique for the isolation and surface immobilization of mesenchymal stem cells from whole bone marrow using high-specific DNA aptamers.

Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high-specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cell-sorting assay with streptavidin-coated magnetic microbeads. We also used 12-well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate-labeled aptamer to sort MSCs from bone marrow using high-speed fluorescence-activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29(+), CD44(+), CD45(-), CD90(+), SLA class I(+), SLA DQ(-), and SLA DR(-). Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC-derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine.

[Screening and characterization of DNA aptamers with hTNF-alpha binding and neutralizing activity].

Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.