Inflammatory mechanisms in focal cerebral ischaemia

Stroke is a complex pathophysiological process and the role of inflammation in its initiation and resolution has been much debated. Inflammation is now emerging as a contributor in the development of ischaemic brain damage. The use of anti -inflammatory strategies to reduce damage and improve functional outcome of stroke patients may be valuable in the treatment for a condition that currently has no effective treatment. The exact contribution of the inflammatory response and the involvement of the various components of the immune system are still under investigationIn this thesis, focal cerebral ischaemia was induced in three animal models in an attempt to investigate the contribution of the inflammatory response. The rat monofilament model of middle cerebral artery (MCA) occlusion, considered by some to be a pro- inflammatory model, was set up for the first time in Edinburgh and validated as suitable model for further investigation. Permanent and transient models were established to allow the evaluation of possible reperfusion injury. Both monofilament models were compared with the Endothelin -1 model already established and routinely in use in the laboratory. Analysis of the volume of damage and distribution of the lesion revealed no differences between the three models. However, this observation did not in itself rule out the possibility of different pathophysiological mechanisms in the three models that ultimately resulted in apparently similar sized lesions.FK506, a potent neuroprotectant widely used experimentally, exhibited different neuroprotective efficacies. In all models, FK506 significantly reduced the overall volume of both damage and oedema. The majority of the neuroprotection was observed in the cortex although striatal protection was seen in the transient rat monofilament model. The neuroprotection observed in the transient monofilament model was approximately twice that seen in the permanent model and similar to that in the Endothelin -1 model suggesting distinct pathways that lead to cell death. Data for FK506 administration in the mouse monofilament model demonstrated neuroprotection for the first time in this species was included as an interesting comparison with the rat data.In keeping with the investigation of inflammation in cerebral ischaemia, it was proposed that FK506 neuroprotection was in part mediated through modulation of the inflammatory response. The response of the microglia, the resident immune cells of the central nervous system was examined following FK506 administration. Although the drug appeared to have a noticeable effect the activation state of the microglia, the response was not consistent and difficult to quantify by histological methods. Microglial cultures were established to investigate the effect of FK506 in a less complex system. Ramified microglial cultures were established but the analysis of microglia in vitro by morphology also proved difficult and another method of assessing activation of the cells was pursued. The microglia are known to secrete noxious stimuli when activated amongst which are the pro- inflammatory cytokines. IL-1P, IL -6 and TNFa gene expression was investigated to assess microglial activation. Lipopolysaccharide treated animals and treated microglial preparations were used initially to refine the use of multiplex polymerase chain reaction (MPCR) analysis of gene products. This was extended to tissue from both monofilament models. IL -1(3, IL -6 and TNFa were detected in the cortex and striatum when measured at 3 and 24 hrs post occlusion and showed an earlier cytokine response where reperfusion occurred. It is suggested that the early cytokine response is associated with the endogenous inflammatory cells. Western analysis experiments were performed to verify the presence of the corresponding cytokine proteins with little success. The recent availability of improved cytokine antibodies enabled the examination of cytokines (IL -1f3 and TNFa) in ischaemic by enzyme linked immunosorbant assay (ELISA). No TNFa response was detected despite the presence of mRNA. IL -lß was detected at 3 and 24 hrs post -insult with greater expression at 24 hrs. It may be speculated that this increased expression at the later time is related to the peripheral inflammatory cell infiltration. There was no difference in expression levels between models and FK506 had no affect on the cytokine expression.In summary, the re- introduction of blood to ischaemic tissue appears to alter the response of the individual cells although this does not change their ultimate fate. In instances where reperfusion is established, the tissue appears to be more amenable to neuroprotection by FK506. It is suggested that this is associated with the blockade of the endogenous inflammatory mechanisms that respond acutely to an ischaemic insult

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Toward a Mechanistic Understanding of Linguistic Diversity

Our species displays remarkable linguistic diversity. Although the uneven distribution of this diversity demands explanation, the drivers of these patterns have not been conclusively determined. We address this issue in two steps: First, we review previous empirical studies whose authors have suggested environmental, geographical, and sociocultural drivers of linguistic diversification. However, contradictory results and methodological variation make it difficult to draw general conclusions. Second, we outline a program for future research. We suggest that future analyses should account for interactions among causal factors, the lack of spatial and phylogenetic independence of the data, and transitory patterns. Recent analytical advances in biogeography and evolutionary biology, such as simulation modeling of diversity patterns, hold promise for testing four key mechanisms of language diversification proposed here: neutral change, population movement, contact, and selection. Future modeling approaches should also evaluate how the outcomes of these processes are influenced by demography, environmental heterogeneity, and time

Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization