BackgroundThe vasculature of the brain is composed of endothelial cells, pericytes and astrocytic processes. The endothelial cells are the critical interface between the blood and the CNS parenchyma and are a critical component of the blood-brain barrier (BBB). These cells are innately programmed to respond to a myriad of inflammatory cytokines or other danger signals. IL-1β and TNFα are well recognised pro-inflammatory mediators, and here, we provide compelling evidence that they regulate the function and immune response profile of human cerebral microvascular endothelial cells (hCMVECs) differentially.MethodsWe used xCELLigence biosensor technology, which revealed global differences in the endothelial response between IL-1β and TNFα. xCELLigence is a label-free impedance-based biosensor, which is ideal for acute or long-term comparison of drug effects on cell behaviour. In addition, flow cytometry and multiplex cytokine arrays were used to show differences in the inflammatory responses from the endothelial cells.ResultsExtensive cytokine-secretion profiling and cell-surface immune phenotyping confirmed that the immune response of the hCMVEC to IL-1β was different to that of TNFα. Interestingly, of the 38 cytokines, chemokines and growth factors measured by cytometric bead array, the endothelial cells secreted only 13. Of importance was the observation that the majority of these cytokines were differentially regulated by either IL-1β or TNFα. Cell-surface expression of ICAM-1 and VCAM-1 were also differentially regulated by IL-1β or TNFα, where TNFα induced a substantially higher level of expression of both key leukocyte-adhesion molecules. A range of other cell-surface cellular and junctional adhesion molecules were basally expressed by the hCMVEC but were unaffected by IL-1β or TNFα.ConclusionsTo our knowledge, this is the most comprehensive analysis of the immunological profile of brain endothelial cells and the first direct evidence that human brain endothelial cells are differentially regulated by these two key pro-inflammatory mediators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0346-0) contains supplementary material, which is available to authorized users.

Neuromedin U (NMU) has been associated with the regulation of food-intake and energy balance in rats. The objective of this study was to identify the sites of gene expression for NMU and the NMU receptor-2 (NMU2R) in the mouse and rat hypothalamus and ascertain the effects of nutritional status on the expression of these genes. In situ hybridization studies revealed that NMU is expressed in several regions of the mouse hypothalamus associated with the regulation of energy balance. Analysis of NMU expression in the obese ob/ob mouse revealed that NMU mRNA levels were elevated in the dorsomedial hypothalamic (DMH) nucleus of obese ob/ob mice compared to lean litter-mates. In addition, NMU mRNA levels were elevated in the DMH of mice fasted for 24 h relative to ad libitum fed controls. The pattern of expression of NMU and NMU2R were more widespread in the hypothalamus of mice than rats. These data provide the first detailed anatomical analysis of the NMU and NMU2R expression in the mouse and advance our knowledge of expression in the rat. The data from the obese rodent models supports the hypothesis that NMU is involved in the regulation of nutritional status.

The xCELLigence technology is a real-time cellular biosensor, which measures the net adhesion of cells to high-density gold electrode arrays printed on custom-designed E-plates. The strength of cellular adhesion is influenced by a myriad of factors that include cell type, cell viability, growth, migration, spreading and proliferation. We therefore hypothesised that xCELLigence biosensor technology would provide a valuable platform for the measurement of drug responses in a multitude of different experimental, clinical or pharmacological contexts. In this manuscript, we demonstrate how xCELLigence technology has been invaluable in the identification of (1) not only if cells respond to a particular drug, but (2) the window of drug responsiveness. The latter aspect is often left to educated guess work in classical end-point assays, whereas biosensor technology reveals the temporal profile of the response in real time, which enables both acute responses and longer term responses to be profiled within the same assay. In our experience, the xCELLigence biosensor technology is suitable for highly targeted drug assessment and also low to medium throughput drug screening, which produces high content temporal data in real time.

BackgroundTransforming growth factor beta 1 (TGFβ1) is strongly induced following brain injury and polarises microglia to an anti-inflammatory phenotype. Augmentation of TGFβ1 responses may therefore be beneficial in preventing inflammation in neurological disorders including stroke and neurodegenerative diseases. However, several other cell types display immunogenic potential and identifying the effect of TGFβ1 on these cells is required to more fully understand its effects on brain inflammation. Pericytes are multifunctional cells which ensheath the brain vasculature and have garnered recent attention with respect to their immunomodulatory potential. Here, we sought to investigate the inflammatory phenotype adopted by TGFβ1-stimulated human brain pericytes.MethodsMicroarray analysis was performed to examine transcriptome-wide changes in TGFβ1-stimulated pericytes, and results were validated by qRT-PCR and cytometric bead arrays. Flow cytometry, immunocytochemistry and LDH/Alamar Blue® viability assays were utilised to examine phagocytic capacity of human brain pericytes, transcription factor modulation and pericyte health.ResultsTGFβ1 treatment of primary human brain pericytes induced the expression of several inflammatory-related genes (NOX4, COX2, IL6 and MMP2) and attenuated others (IL8, CX3CL1, MCP1 and VCAM1). A synergistic induction of IL-6 was seen with IL-1β/TGFβ1 treatment whilst TGFβ1 attenuated the IL-1β-induced expression of CX3CL1, MCP-1 and sVCAM-1. TGFβ1 was found to signal through SMAD2/3 transcription factors but did not modify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) translocation. Furthermore, TGFβ1 attenuated the phagocytic ability of pericytes, possibly through downregulation of the scavenger receptors CD36, CD47 and CD68. Whilst TGFβ did decrease pericyte number, this was due to a reduction in proliferation, not apoptotic death or compromised cell viability.ConclusionsTGFβ1 attenuated pericyte expression of key chemokines and adhesion molecules involved in CNS leukocyte trafficking and the modulation of microglial function, as well as reduced the phagocytic ability of pericytes. However, TGFβ1 also enhanced the expression of classical pro-inflammatory cytokines and enzymes which can disrupt BBB functioning, suggesting that pericytes adopt a phenotype which is neither solely pro- nor anti-inflammatory. Whilst the effects of pericyte modulation by TGFβ1 in vivo are difficult to infer, the reduction in pericyte proliferation together with the elevated IL-6, MMP-2 and NOX4 and reduced phagocytosis suggests a detrimental action of TGFβ1 on neurovasculature.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0503-0) contains supplementary material, which is available to authorized users.

Tanycytes in the ependymal layer of the third ventricle act both as a barrier and a communication gateway between the cerebrospinal fluid, brain and portal blood supply to the pituitary gland. However, the range, importance and mechanisms involved in the function of tanycytes remain to be explored. In this study, we have utilized a photoperiodic animal to examine the expression of three unrelated gene sequences in relation to photoperiod-induced changes in seasonal physiology and behaviour. We demonstrate that cellular retinoic acid-binding protein 1 (CRBP1), a retinoic acid transport protein, GPR50, an orphan G-protein-coupled receptor and nestin, an intermediate filament protein, are down-regulated in short-day photoperiods. The distribution of the three sequences is very similar, with expression located in cells with tanycyte morphology in the region of the ependymal layer where tanycytes are located. Furthermore, CRBP1 expression in the ependymal layer is shown to be independent of a circadian clock and altered testosterone levels associated with testicular regression in short photoperiod. Pinealectomy of Siberian hamsters demonstrates CRBP1 expression is likely to be dependent on melatonin output from the pineal gland. This provides evidence that tanycytes are seasonally responsive cells and are likely to be an important part of the mechanism to facilitate seasonal physiology and behaviour in the Siberian hamster.

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function.

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Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights-on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night-time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.