Clinical impact of anti-inflammatory microglia and macrophage phenotypes at glioblastoma margins

Glioblastoma is a devastating brain cancer for which effective treatments are required. Tumour-associated microglia and macrophages promote glioblastoma growth in an immune-suppressed microenvironment. Most recurrences occur at the invasive margin of the surrounding brain, yet the relationships between microglia/macrophage phenotypes, T cells and programmed death-ligand 1 (an immune checkpoint) across human glioblastoma regions are understudied. In this study, we performed a quantitative immunohistochemical analysis of 15 markers of microglia/macrophage phenotypes (including anti-inflammatory markers triggering receptor expressed on myeloid cells 2 and CD163, and the low-affinity-activating receptor CD32a), T cells, natural killer cells and programmed death-ligand 1, in 59 human IDH1-wild-type glioblastoma multi-regional samples (n = 177; 1 sample at tumour core, 2 samples at the margins: the infiltrating zone and leading edge). Assessment was made for the prognostic value of markers; the results were validated in an independent cohort. Microglia/macrophage motility and activation (Iba1, CD68), programmed death-ligand 1 and CD4+ T cells were reduced, and homeostatic microglia (P2RY12) were increased in the invasive margins compared with the tumour core. There were significant positive correlations between microglia/macrophage markers CD68 (phagocytic)/triggering receptor expressed on myeloid cells 2 (anti-inflammatory) and CD8+ T cells in the invasive margins but not in the tumour core (P < 0.01). Programmed death-ligand 1 expression was associated with microglia/macrophage markers (including anti-inflammatory) CD68, CD163, CD32a and triggering receptor expressed on myeloid cells 2, only in the leading edge of glioblastomas (P < 0.01). Similarly, there was a positive correlation between programmed death-ligand 1 expression and CD8+ T-cell infiltration in the leading edge (P < 0.001). There was no relationship between CD64 (a receptor for autoreactive T-cell responses) and CD8+/CD4+ T cells, or between the microglia/macrophage antigen presentation marker HLA-DR and microglial motility (Iba1) in the tumour margins. Natural killer cell infiltration (CD335+) correlated with CD8+ T cells and with CD68/CD163/triggering receptor expressed on myeloid cells 2 anti-inflammatory microglia/macrophages at the leading edge. In an independent large glioblastoma cohort with transcriptomic data, positive correlations between anti-inflammatory microglia/macrophage markers (triggering receptor expressed on myeloid cells 2, CD163 and CD32a) and CD4+/CD8+/programmed death-ligand 1 RNA expression were validated (P < 0.001). Finally, multivariate analysis showed that high triggering receptor expressed on myeloid cells 2, programmed death-ligand 1 and CD32a expression at the leading edge were significantly associated with poorer overall patient survival (hazard ratio = 2.05, 3.42 and 2.11, respectively), independent of clinical variables. In conclusion, anti-inflammatory microglia/macrophages, CD8+ T cells and programmed death-ligand 1 are correlated in the invasive margins of glioblastoma, consistent with immune-suppressive interactions. High triggering receptor expressed on myeloid cells 2, programmed death-ligand 1 and CD32a expression at the human glioblastoma leading edge are predictors of poorer overall survival. Given substantial interest in targeting microglia/macrophages, together with immune checkpoint inhibitors in cancer, these data have major clinical implications

A novel method to visualise the three-dimensional organisation of the human cerebral cortical vasculature

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aβ42 were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states

The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms

Use of Chitosan as a Bioactive Implant Coating for Bone-Implant Applications

Chitosan is the deacetylated derivative of the natural polysaccharide, chitin. Chitosan has been shown to be biocompatible, biodegradable, osteoconductive, and to accelerate wound healing. These characteristics are largely due to its structural and chemical homology to hyaluronic acid and other proteoglycans found in extracellular matrices. Because of these properties, chitosan has been investigated as a coating for implant materials to promote osseointegration, and as a potential vehicle to deliver therapeutic agents to the local implant-tissue interface. The coating of chitosan onto implant alloy surfaces has been achieved via chemical reactions and electrodeposition mechanisms as well as by other methods such as dip coating and layer-by-layer assembly. This work examines the different mechanisms and bond strengths of chitosan coatings for implant alloys, coating composition and physiochemical properties, degradation, delivery of therapeutic agents, such as growth factors and antibiotics, and in vitro and in vivo compatibilities. © 2011 Springer-Verlag Berlin Heidelberg