Differential Roles of Hyperglycemia and Hypoinsulinemia in Diabetes Induced Retinal Cell Death: Evidence for Retinal Insulin Resistance

Diabetes pathology derives from the combination of hyperglycemia and hypoinsulinemia or insulin resistance leading to diabetic complications including diabetic neuropathy, nephropathy and retinopathy. Diabetic retinopathy is characterized by numerous retinal defects affecting the vasculature and the neuro-retina, but the relative contributions of the loss of retinal insulin signaling and hyperglycemia have never been directly compared. In this study we tested the hypothesis that increased retinal insulin signaling and glycemic normalization would exert differential effects on retinal cell survival and retinal physiology during diabetes. We have demonstrated in this study that both subconjunctival insulin administration and systemic glycemic reduction using the sodium-glucose linked transporter inhibitor phloridzin affected the regulation of retinal cell survival in diabetic rats. Both treatments partially restored the retinal insulin signaling without increasing plasma insulin levels. Retinal transcriptomic and histological analysis also clearly demonstrated that local administration of insulin and systemic glycemia normalization use different pathways to counteract the effects of diabetes on the retina. While local insulin primarily affected inflammation-associated pathways, systemic glycemic control affected pathways involved in the regulation of cell signaling and metabolism. These results suggest that hyperglycemia induces resistance to growth factor action in the retina and clearly demonstrate that both restoration of glycemic control and retinal insulin signaling can act through different pathways to both normalize diabetes-induced retinal abnormality and prevent vision loss

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

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

Green Tea Polyphenol Epigallocatechin Gallate Reduces Endothelin-1 Expression and Secretion in Vascular Endothelial Cells: Roles for AMP-Activated Protein Kinase, Akt, and FOXO1

Epigallocatechin gallate (EGCG), a green tea polyphenol, promotes vasodilation by phosphatidylinositol 3-kinase-dependent activation of Akt and endothelial nitric oxide synthase to stimulate production of nitric oxide. Reduction in endothelin-1 (ET-1) synthesis may also increase bioavailability of nitric oxide. We hypothesized that the phosphatidylinositol 3-kinase-dependent transcription factor FOXO1 may mediate effects of EGCG to regulate expression of ET-1 in endothelial cells. EGCG treatment (10 μm, 8 h) of human aortic endothelial cells reduced expression of ET-1 mRNA, protein, and ET-1 secretion. We identified a putative FOXO binding domain in the human ET-1 promoter 51 bp upstream from the transcription start site. Trans-activation of a human ET-1 (hET-1) promoter luciferase reporter was enhanced by coexpression of a constitutively nuclear FOXO1 mutant, whereas expression of a mutant FOXO1 with disrupted DNA binding domain did not trans-activate the hET-1 promoter. Disrupting the hET-1 putative FOXO binding domain by site-directed mutagenesis ablated promoter activity in response to overexpression of wild-type FOXO1. EGCG stimulated time-dependent phosphorylation of Akt (S473), FOXO1 (at Akt phosphorylation site T24), and AMP-activated protein kinase α (AMPKα) (T172). EGCG-induced nuclear exclusion of FOXO1, FOXO1 binding to the hET-1 promoter, and reduction of ET-1 expression was partially inhibited by the AMPK inhibitor Compound C. Basal ET-1 protein expression was enhanced by short interfering RNA knock-down of Akt and reduced by short interfering RNA knock-down of FOXO1 or adenovirus-mediated expression of dominant-negative Foxo1. We conclude that EGCG decreases ET-1 expression and secretion from endothelial cells, in part, via Akt- and AMPK-stimulated FOXO1 regulation of the ET-1 promoter. These findings may be relevant to beneficial cardiovascular actions of green tea

"In dubious Battle on the Plains of Heav'n": The Politics of Possession in Jerusalem's Holy Sepulchre

Debates about the "divisibility" or "sharing" of religious sites continue to engage historians, political scientists and anthropologists. This paper assesses the issue of agency in two of the more salient critiques of religious coexistence before approaching the Holy Sepulchre, or Anastasis, as a site for investigating the way various constituencies, most significantly visiting pilgrims and resident monks, have dealt with issues of "sharing". It contends that inter-communal antagonisms there originate with elite struggles over the possessions of places - struggles which tend to engage political actors far from the site of the conflicts rather than local communities - and concludes that, rather than accept an "identitarian politics" which assumes a profound "civilisational" attachment of cultures to religious identities, we must carefully assess the "politics of possession" which variously play out in sites of inter-communal engagement