Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation

Rapid flow cytometric immunodetection of bacteria to monitor aquatic environments

International audienceno abstrac

Isolation and characterization of two cyclin cDNAs from Pisum sativum L.

In order to investigate the role of cell division in plant development, we isolated two plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins. Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Pisum sativum sequence which showed homology to the ‘cyclin box°s functional domain found within cyclin proteins. Using this sequence as probe we isolated two different cyclin cDNAs, Pissa;CycA3;1 and Pissa;CycB1;3 from a Pisum sativum L. root tip cDNA library. The deduced amino acid sequences of both cDNAs showed the highest sequence similarity with mitotic cyclins. Analyses of Pissa;CycA3;1 and Pissa;CycB1;3 expression in different tissues, by reverse transcription-polymerase chain reaction (RT-PCR) using primers corresponding to unique regions of their cDNAs, showed their differential expression in relation to cell cycle activity. Furthermore, RT-PCR was used to analyze synchronized root tip cells; results revealed that Pissa;CycA3;1 is preferentially expressed in mid-S (SM) and during late S-G2 (Sl- G2) transition, whereas Pissa;CycB1;3 mRNA is only detectable in Sl and G2 phases

Radioanalytical Techniques in Studies of Trace Metals in Renal Insufficiency and Dialytic Treatment

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DNA content, morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus

Intervertebral disc regeneration: Influence of growth factors on differentiation of human mesenchymal stem cells (hMSC)

Introduction: One common cause of disability in modern society is low back pain. The main reason for this pain is the degeneration of the intervertebral disc (IVD), particular of the nucleus pulposus (NP). For an early degeneration stage cell-based therapy would be a minimal invasive method of treatment. Therefore, adequate cells are needed. As the usage of NP cells is limited because of their insufficient amount or vitality, a promising alternative is the application of human mesenchymal stem cells (hMSCs). Objective: To investigate the potential of various growth factors to induce the differentiation of hMSCs into NP cells and thereby to obtain an alternative cell source for the treatment of IVD degeneration. Methods: hMSC-TERT were cultivated three-dimensionally in a hydrogel for 21 days to form NP cells. Cell survival and proliferation were determined using SybrGreen/propidium iodide double staining and the WST-test. To investigate the ability of several growth factors to differentiate hMSCs into NP cells, fluorescence immunostaining of NP-specific marker proteins (e.g. chondroadherin (CHAD) and the recently discovered cytokeratin 19 [1]) was performed. Results: Following the procedure described above, cells are able to maintain their viability and proliferation capacity throughout the cultivation time. By using a previously established immunofluorescence protocol, we could indicate the ability of three different growth factors to differentiate hMSCs into NP-like cells. Conclusion: The expression of several marker proteins in all differentiation experiments indicates the ability of IGF-1, FGF-2 and PDGF-BB to differentiate hMSCs into NP-like cells apart from the usually applied TGF-β3. Furthermore, our findings preclude the application of Cytokeratin 19 as a specific marker protein for NP cells [1]. Further experiments have to be done to find real specific NP marker proteins to indisputable verify the differentiation of hMSCs into NP cells. If so, application of the mentioned three growth factors would possibly be an option to obtain sufficient NP cells for minimal invasive IVD regeneration

Wastewater Disinfection Alternatives: Chlorine, Ozone, Peracetic Acid and UV Light

Disinfection tests were carried out at pilot scale to compare the disinfection efficiency of ozone, sodium hypochlorite (NaOCl), peracetic acid (PAA), and UV irradiation. Total coliforms, fecal coliforms, and Escherichia coli were monitored as reference microorganisms. Total heterotrophic bacteria (THB) were also enumerated by cytometry. At similar doses, NaOCl was more effective than PAA, and its action was less affected by contact time. The results obtained by ozonation were comparable for total coliforms, fecal coliforms, and E. coli. On the contrary, some differences among the three indicators were observed for NaOCl, PAA, and UV. Differences increased with increasing values of the disinfectant concentration times contact time (Ct) and were probably the result of different initial counts, as total coliforms include fecal coliforms, which include E. coli. The UV irradiation lead to complete E. coli removals, even at low doses (10 to 20 mJ/cm2). Total heterotrophic bacteria appeared to be too wide a group to be a good disinfection indicator; no correlation was found among THB inactivation, dose, and contact time