Anti-Inflammatory Therapies for Treatment of Inflammation-Related Preterm Brain Injury.

Despite the prevalence of preterm brain injury, there are no established neuroprotective strategies to prevent or alleviate mild-to-moderate inflammation-related brain injury. Perinatal infection and inflammation have been shown to trigger acute neuroinflammation, including proinflammatory cytokine release and gliosis, which are associated with acute and chronic disturbances in brain cell survival and maturation. These findings suggest the hypothesis that the inhibition of peripheral immune responses following infection or nonspecific inflammation may be a therapeutic strategy to reduce the associated brain injury and neurobehavioral deficits. This review provides an overview of the neonatal immunity, neuroinflammation, and mechanisms of inflammation-related brain injury in preterm infants and explores the safety and efficacy of anti-inflammatory agents as potentially neurotherapeutics.

Melanoma Mediated Disruption of Brain Endothelial Barrier Integrity Is Not Prevented by the Inhibition of Matrix Metalloproteinases and Proteases.

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium.

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

An anti-inflammatory role for C/EBPδ in human brain pericytes.

Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1ß (IL-1ß). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1ß-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1ß-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1ß, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.