No abstract

Microglia, the resident macrophages of the central nervous system play vital roles in brain homeostasis through clearance of pathogenic material. Microglia are also implicated in neurological disorders through uncontrolled activation and inflammatory responses. To date, the vast majority of microglial studies have been performed using rodent models. Human microglia differ from rodent counterparts in several aspects including their response to pharmacological substances and their inflammatory secretions. Such differences highlight the need for studies on primary adult human brain microglia and methods to isolate them are therefore required. Our procedure generates microglial cultures of >95% purity from both biopsy and autopsy human brain tissue using a very simple media-based culture procedure that takes advantage of the adherent properties of these cells. Microglia obtained in this manner can be utilised for research within a week. Isolated microglia demonstrate phagocytic ability and respond to inflammatory stimuli and their purity makes them suitable for numerous other forms of in vitro studies, including secretome and transcriptome analysis. Furthermore, this protocol allows for the simultaneous isolation of neural precursor cells during the microglial isolation procedure. As human brain tissue is such a precious and valuable resource the simultaneous isolation of multiple cell types is highly beneficial.

In this paper, we demonstrate the application of electrical cell-substrate impedance sensing (ECIS) technology for measuring differences in the formation of a strong and durable endothelial barrier model. In addition, we highlight the capacity of ECIS technology to model the parameters of the physical barrier associated with (I) the paracellular space (referred to as Rb) and (II) the basal adhesion of the endothelial cells (α, alpha). Physiologically, both parameters are very important for the correct formation of endothelial barriers. ECIS technology is the only commercially available technology that can measure and model these parameters independently of each other, which is important in the context of ascertaining whether a change in overall barrier resistance (R) occurs because of molecular changes in the paracellular junctional molecules or changes in the basal adhesion molecules. Finally, we show that the temporal changes observed in the paracellular Rb can be associated with changes in specific junctional proteins (CD144, ZO-1, and catenins), which have major roles in governing the overall strength of the junctional communication between neighbouring endothelial cells.

The first two authors contributed equally to this work It is commonly accepted from gene expression studies that the CB2 receptor is expressed by most cell types of the rodent and human immune system. However, the exact identity of cells expressing CB2 receptor protein in human blood or the abundance of receptors expressed by each immune subset is not well characterised. We conducted a detailed analysis of CB2 protein levels expressed by bloodderived immune cells from healthy human donors. Flow-cytometry was conducted using 4 commercially available anti-CB2 polyclonal antibodies in conjunction with a selection ofimmune cell specific markers. Across multiple healthy subjects we observed that NK cells, B-Iymphocytes and monocytes expressed a higher level of CB2 receptor than CD4+or CD8+T-Iymphocytes. Neutrophils also expressed a low level of CB2 receptor. NK cells had the greatest variation in CB2 expression levels, whereas for each of the other cell types CB2 levels were relatively similar between subjects. In contrast to other methods, the high sensitivity of flow-cytometry revealed that CB2 receptors are present on resting T-Iymphocytes at low abundance in some healthy subjects. These data provide the first detailed analysis of CB2 protein levels in blood leukocyte subsets from healthy donors and identifies the cell types which could be targeted with CB2-mimetic drugs in humans.

Cannabinoid receptor 2 (CB 2 ) is a promising therapeutic target for immunological modulation. There is, however, a deficit of knowledge regarding CB 2 signaling and function in human primary immunocompetent cells. We applied an experimental paradigm which closely models the in situ state of human primary leukocytes (PBMC; peripheral blood mononuclear cells) to characterize activation of a number of signaling pathways in response to a CB 2 -selective ligand (HU308). We observed a "lag" phase of unchanged cAMP concentration prior to development of classically expected Gα i -mediated inhibition of cAMP synthesis. Application of G protein inhibitors revealed that this apparent lag was a result of counteraction of Gα i effects by concurrent Gα s activation. Monitoring downstream signaling events showed that activation of p38 was mediated by Gα i , whereas ERK1/2 and Akt phosphorylation were mediated by Gα i -coupled βγ. Activation of CREB integrated multiple components; Gα s and βγ mediated ∼85% of the response, while ∼15% was attributed to Gα i . Responses to HU308 had an important functional outcomesecretion of interleukins 6 (IL-6) and 10 (IL-10). IL-2, IL-4, IL-12, IL-13, IL-17A, MIP-1α, and TNF-α were unaffected. IL-6/IL-10 induction had a similar G protein coupling profile to CREB activation. All response potencies were consistent with that expected for HU308 acting via CB 2 . Additionally, signaling and functional effects were completely blocked by a CB 2 -selective inverse agonist, giving additional evidence for CB 2 involvement. This work expands the current paradigm regarding cannabinoid immunomodulation and reinforces the potential utility of CB 2 ligands as immunomodulatory therapeutics.

Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30–60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.

Some of the differences between recent estimates of the remaining budget of carbon dioxide (CO 2) emissions consistent with limiting warming to 1.5°C arise from different estimates of the level of warming to date relative to pre-industrial conditions, but not all. Here we show that, for simple geometrical reasons, the combination of both the level and rate of human-induced warming provides a remarkably accurate prediction of remaining emission budgets to peak warming across a broad range of scenarios, if budgets are expressed in terms of CO 2-forcing-equivalent emissions. These in turn predict CO 2 emissions budgets if (but only if) the fractional contribution of non-CO 2 drivers to warming remains approximately unchanged, as it does in some ambitious mitigation scenarios, indicating a best-estimate remaining budget for 1.5°C of about 22 years' current emissions with a 'likely' (1-standard-error) range of 13-32 years. This provides a simple, transparent, and modelindependent metric of progress towards an ambitious temperature stabilisation goal that could be used to inform the Paris Agreement stocktake process. It is less applicable to less ambitious goals. Alternate definitions of current warming and scenarios for non-CO 2 drivers give lower 1.5°C budgets. Lower budgets based on the MAGICC simple modelling system widely used in Integrated Assessment Studies reflect its relatively high simulated current warming rates. Recent discussion 1,2,3,4 of the "carbon budget" to limit warming to below 1.5°C relative to preindustrial levels highlights the impact of different geophysical constraints on emissions consistent with ambitious mitigation goals, including the definition of "preindustrial", 5 current level of warming, 6 committed warming due to past emissions, 7 interpretation of "exceedance", 8 and contribution of non-CO 2 forcing. 9,10 Many methods compute remaining carbon budgets as a residual, calculating an emission budget relative to some reference period and then subtracting estimated emissions since that time, which does not make optimal use of proximity to the target temperature. Differences in the estimated current level of warming explain discrepancies between budgets based on full-complexity climate models, but not between these budgets and those based on the MAGICC simple modelling system 11,12 which, by expressing temperatures relative to a recent reference period, simulates a current level of warming close to that observed. This has led to the suggestion 13 that the entire carbon budget concept is too uncertain to be relevant to very ambitious mitigation scenarios, and attention should focus instead on the pathway to achieving net zero emissions. This seems paradoxical, because the remaining Threshold Exceedance Budget 14 (TEB, or emissions to the time a

The significance of this work is to demonstrate how patterning hNT astrocytes replicates spatio-temporal clustering of Ca signalling that is observed in vivo but not in dissociated in vitro cultures. We therefore highlight the importance of the structure of astrocytic networks in determining ensemble Ca behaviour.