Downregulation of inflammatory markers by conjugated linoleic acid isomers in human cultured astrocytes.

OBJECTIVES: Conjugated linoleic acid (CLA) isomers have been shown to possess anti-inflammatory activity in the central nervous system. In this study, we aimed to evaluate whether modulation of the fatty acid profile by the CLA isomers c9,t11 or t10,c12CLA was associated with changes in the expression of pro-inflammatory molecules in human astrocytes. METHODS: Cultured astrocytes were treated for 6 days with 100 µM fatty acids (c9,t11CLA or t10,c12CLA or oleic acid). Following the treatment, the fatty acid profile of the cell and pro-inflammatory molecule expression were assessed. RESULTS: Only the t10,c12CLA isomer induced a significant decrease in arachidonic acid and increased the ratio of docosahexaenoic acid/eicosapentaenoic acid, which constitutes indirect evidence of peroxisome proliferator-activated receptor alpha activation. Inhibition of tumour necrosis factor-α, interleukin-1ß, and RANTES expression was observed in astrocytes treated with c9,t11CLA and t10,c12CLA. DISCUSSION: Current data demonstrate that CLA isomers, particularly t10,c12, may affect neuroinflammation by reducing the pro-inflammatory molecules in cultured astrocytes, suggesting a potential nutritional role of CLA isomers in modulating the astrocyte inflammatory response.

Thyrospheres from B-CPAP Cell Line with BRAF and TERT Promoter Mutations have Different Functional and Molecular Features than Parental Cells.

Human thyroid cancer derived cell lines are widely used to study the mechanisms involved in thyroid carcinogenesis. However, there is limited availability of non-cross-contaminated cancer cell lines derived from papillary thyroid carcinoma (PTC), and the B-CPAP cell line is one of the few such lines. B-CPAP cells have been genetically and cytogenetically well-characterized, but details of their stemness features remain uncertain. Considering that this cell line is extensively used for in vitro studies on thyroid tumorigenesis, we broaden its functional and molecular profiles as well as the tumorigenic capacity. We used functional assays (sphere-forming capacity and efficiency), assessed self-renewal and propagation efficiency and tested in vivo tumorigenicity in Hsd:Athymic Nude-Foxn1nu mice. Expression of markers of stemness, differentiation, and epithelial-mesenchymal transition were estimated at RNA and protein levels in adherent parental cells and sphere-forming cells. Functional aspects and stemness features were compared with normal thyrocytes. Protein expression of xenograft tumors was evaluated by immunohistochemistry. B-CPAP sphere-forming cells were able to form thyrospheres theoretically indefinitely in an appropriate serum-free medium, reverting to the adherent parental cell phenotype when cultured in differentiation medium. Different expression of ALDH1-A1 and CD44 stemness markers and TTF-1 and CK19 differentiation markers allowed discrimination between isolated sphere-forming cells and adherent parental cells, indicating that sphere-forming cells retained stem-like features. In keeping with these observations, tumorigenicity assays confirmed that, relative to parental adherent cells, thyrospheres had enhanced capacity to initiate xenograft tumors. Thyrospheres from normal cell line retained very low functional capacity, as well as different stemness markers expression compared to tumor thyrospheres. Our findings may constitute a useful background to develop an in vitro model for assessing the origin and progression of papillary thyroid carcinoma bearing BRAFV600E and TERT promoter mutations.

Gamma aminobutyric acid (GABA) modulators for amyotrophic lateral sclerosis/motor neuron disease.

BACKGROUND: Imbalance of gamma aminobutyric acid (GABA) and related modulators has been implicated as an important factor in the pathogenesis of amyotrophic lateral sclerosis (ALS), which is also known as motor neuron disease (MND). In this context, the role and mechanism of action of gabapentin and baclofen have been extensively investigated, although with conflicting results. This is the first systematic review to assess clinical trials of GABA modulators for the treatment of ALS. OBJECTIVES: To examine the efficacy of gabapentin, baclofen, or other GABA modulators in delaying the progression of ALS, and to evaluate adverse effects of these interventions SEARCH METHODS: On 16 August 2016, we searched the Cochrane Neuromuscular Specialised Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, CINAHL Plus, AMED, and LILACS. In addition, we checked the bibliographies of the trials found in order to identify any other trials, and contacted trial authors to identify relevant unpublished results or additional clinical trials. On 30 August 2016, we searched two clinical trials registries. SELECTION CRITERIA: Types of studies: double-blind randomized controlled trials (RCTs) or quasi-RCTsTypes of participants: adults with a diagnosis of probable or definite ALSTypes of interventions: gabapentin, baclofen, or other GABA modulators compared with placebo, no treatment, or each otherPrimary outcome: survival at one year from study enrollmentSecondary outcomes: individual rate of decline of maximum voluntary isometric contraction (MVIC), expressed as arm megascore; rate of decline of per cent predicted forced vital capacity (FVC); rate of decline of ALS Functional Rating Scale (ALSFRS); health-related quality of life; survival evaluated by pooling hazards; and adverse events DATA COLLECTION AND ANALYSIS: At least two review authors independently checked titles and abstracts identified by the searches. The review authors obtained and independently analyzed original individual participant data from each included study; additional review authors and the Cochrane Neuromuscular Managing Editor checked the outcome data. Two authors independently assessed the risk of bias in included studies. Data collection and analysis: At least two review authors independently checked titles and abstracts identified by the searches. The review authors obtained and independently analyzed original individual participant data from each included study; additional review authors and the Cochrane Neuromuscular Managing Editor checked the outcome data. Two authors independently assessed the risk of bias in included studies. MAIN RESULTS: We identified two double-blind RCTs of gabapentin treatment in ALS for inclusion in this review. We found no eligible RCTs of baclofen or other GABA modulators. The selected studies were phase II and phase III trials, which lasted six and nine months, respectively. They were highly comparable because both were comparisons of oral gabapentin and placebo, performed by the same investigators. The trials enrolled 355 participants with ALS: 80 in the gabapentin group and 72 in the placebo group in the first (phase II) trial and 101 in the gabapentin group and 102 in the placebo group in the second (phase III) trial. Neither trial was long enough to report survival at one year, which was our primary outcome. We found little or no difference in estimated one-year survival between the treated group and the placebo group (78% versus 77%, P = 0.63 by log-rank test; high-quality evidence). We also found little or no difference in the rate of decline of MVIC expressed as arm megascore, or rate of FVC decline (high-quality evidence). One trial investigated monthly decline in the ALSFRS and quality of life measured using the 12-Item Short Form Survey (SF-12) and found little or no difference between groups (moderate-quality evidence). The trials reported similar adverse events. Complaints that were clearly elevated in those taking gabapentin, based on analyses of the combined data, were light-headedness, drowsiness, and limb swelling (high-quality evidence). Fatigue and falls occurred more frequently with gabapentin than with placebo in one trial, but when we combined the data for fatigue from both trials, there was no clear difference between the groups. We assessed the overall risk of bias in the included trials as low. AUTHORS' CONCLUSIONS: According to high-quality evidence, gabapentin is not effective in treating ALS. It does not extend survival, slow the rate of decline of muscle strength, respiratory function and, based on moderate-quality evidence, probably does not improve quality of life or slow monthly decline in the ALSFRS. Other GABA modulators have not been studied in randomized trials.

S100B modulates growth factors and costimulatory molecules expression in cultured human astrocytes.

S100B is a Ca(2+)-binding protein expressed in the nervous system. Increased levels of S100B in the extracellular space have been detected in several neurological disorders. We investigated the response of human astrocytes to micromolar chronic concentrations of this protein measuring the expression of some costimulatory molecules, such as CD137, CD137-L, CD40, CD40-L, the chemokine RANTES and two growth factors FGF-2 and TGF-ß2. Our findings suggest that high levels of S100B in the brain parenchyma may modulate the activation status of astrocytes decreasing their neuroprotective role and modifying their interaction with microglia and other inflammatory cells. This effect may contribute to evolution of some neurological disorders.

Human astrocytes can be induced to differentiate into cells with neuronal phenotype.

Several recent studies have proposed that astrocytes may contribute to neurogenesis, not only as a source of trophic substances regulating it, but also as stem cells themselves. In order to better understand these mechanisms, primary astrocyte cultures were established from human fetal brain. After 3-4 weeks in culture, astrocytes (about 95% GFAP+; neurofilament, NF-; neuro-specific enolase, NSE-) were treated with a cocktail of protein kinase activators and FGF-1. After 5 h of treatment, most cells showed morphological changes that increased progressively up to 24-48 h, exhibiting a round cell body with long processes. Immunocytochemistry showed that treatment-induced NF and NSE expression in about 40% of cells. Nestin expression increased after treatment, whereas GFAP immunostaining was not significantly modified. Western blot and RT-PCR confirmed the results. No neuronal electrophysiological properties were observed after treatment, suggesting an incomplete maturation under these experimental conditions. Understanding the regenerative capability and neurogenic potential of astrocytes might be useful in devising therapeutic approaches for a variety of neurological disorders.

Differentiation of human bone marrow stem cells into cells with a neural phenotype: diverse effects of two specific treatments.

BACKGROUND: It has recently been demonstrated that the fate of adult cells is not restricted to their tissues of origin. In particular, it has been shown that bone marrow stem cells can give rise to cells of different tissues, including neural cells, hepatocytes and myocytes, expanding their differentiation potential. RESULTS: In order to identify factors able to lead differentiation of stem cells towards cells of neural lineage, we isolated stromal cells from human adult bone marrow (BMSC). Cells were treated with: (1) TPA, forskolin, IBMX, FGF-1 or (2) retinoic acid and 2-mercaptoethanol (BME). Treatment (1) induced differentiation into neuron-like cells within 24 hours, while a longer treatment was required when using retinoic acid and BME. Morphological modifications were more dramatic after treatment (1) compared with treatment (2). In BMSC both treatments induced the expression of neural markers such as NF, GFAP, TUJ-1 and neuron-specific enolase. Moreover, the transcription factor Hes1 increased after both treatments. CONCLUSION: Our study may contribute towards the identification of mechanisms involved in the differentiation of stem cells towards cells of neural lineage.

Differentiation of human adult CD34+ stem cells into cells with a neural phenotype: role of astrocytes.

It has recently been reported that adult hematopoietic stem cells can differentiate into neural cells, opening new frontiers in therapy for neurodegenerative diseases. In this study, adult human hematopoietic stem cells (HSCs) were isolated via magnetic bead sorting, using a specific CD34 antibody and cultured with human astrocyte culture conditioned medium (ACM). In order to evaluate their differentiation into neurons and/or astrocytes, ACM-treated cultures were probed for the expression of several neural markers. We observed morphological modifications and, after 20 days of treatment, cell morphology displayed extending processes. Immunocytochemistry, Western blotting and RT-PCR showed the expression of neuronal markers such as neurofilaments, neuron specific enolase (NSE) and NeuN in ACM-treated HSCs cultured in poly-L-lysine-coated dishes. On the contrary, when the same ACM-treated cells were grown on a plastic substrate, they expressed high levels of glial fibrillary acidic protein (GFAP), with only weak expression of neuronal markers. Nestin, a neural progenitor cell marker, was present in treated cells, regardless of the substrate. These results demonstrate that astrocytes can generate a suitable microenvironment for inducing HSCs to differentiate into neural cells. Therefore, adult bone marrow may represent a readily accessible source of cells for treating neurodegenerative diseases.

S100b counteracts effects of the neurotoxicant trimethyltin on astrocytes and microglia.

Central nervous system degenerative diseases are often characterized by an early, strong reaction of astrocytes and microglia. Both these cell types can play a double role, protecting neurons against degeneration through the synthesis and secretion of trophic factors or inducing degeneration through the secretion of toxic molecules. Therefore, we studied the effects of S100B and trimethyltin (TMT) on human astrocytes and microglia with two glial models, primary cultures of human fetal astrocytes and a microglia cell line. After treatment with 10(-5) M TMT, astrocytes showed morphological alterations associated with an increase in glial fibrillary acidic protein (GFAP) expression and changes in GFAP filament organization. Administration of S100B before TMT treatment prevented TMT-induced changes in morphology and GFAP expression. A decrease in inducible nitric oxide synthase expression was observed in astrocytes treated with TMT, whereas the same treatment induced iNOS expression in microglia. In both cases, S100B prevented TMT-induced changes. Tumor necrosis factor-alpha mRNA expression in astrocytes was not modified by TMT treatment, whereas it was increased in microglia cells. S100B pretreatment blocked the TMT-induced increase in TNF-alpha expression in microglia. To trace the mechanisms involved in S100B activity, the effect of BAY 11-7082, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and of PD98059, an inhibitor of MEK-ERK1/2, were investigated. Results showed that the protective effects of S100B against TMT toxicity in astrocytes depend on NF-kappaB, but not on ERK1/2 activation. These results might help in understanding the role played by glial cells in brain injury after exposure to chemical neurotoxicants and support the view that S100B may protect brain cells in case of injury. (c) 2005 Wiley-Liss, Inc.