Roads can fragment animal populations by reducing gene flow, which can lead to drift and the loss of genetic diversity. One of the principle signatures of reduced gene flow is increased genetic differentiation in isolated populations, and evidence that roads contribute to such differentiation has been reported for several species. We used microsatellites to examine whether six roads led to increased genetic differentiation in red-backed salamanders (Plethodon cinereus). These six roads included one divided interstate highway, one undivided four-lane highway, and four secondary roads. We found that the genetic distance between plots that were bisected by the interstate highway was significantly greater than the genetic distance between equidistant plots on the same side of the highway. However, for the five smaller roads, plots across the road were no more genetically distinct than were plots on the same side of the road. Bayesian clustering methods also supported both of these findings. The optimal clustering of plots for the interstate highway consisted of two clusters that corresponded to the two sides of highway. For the other five sites, the optimal grouping consisted of a single cluster containing all of the plots. Our findings suggest that gene flow across very large roads is rare and that bisected red-backed salamander populations are likely to diverge from one another. For smaller roads, our results imply that the indirect effects of roads on genetic population structure are probably less of a pressing concern for terrestrial salamanders than are the direct effects of mortality and habitat alteration.

To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting, We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 106 Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 μg of p53 intravenously) and gemcitabine (20 mg/kg Intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wt p53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight, Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wt p53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.

The inducible costimulator (ICOS) plays a key role in CD4+ Th17 cell development, but its role in CD8+ Tc17 cell development and self/tumor immunity remains unknown. We found that ICOS co-stimulation was important for the functional maintenance but not differentiation of Tc17 cells in vitro. Blocking the ICOS pathway using an antagonist antibody or by using mice genetically deficient in the ICOS ligand (ICOSL) reduced the antitumor activity of adoptively transferred Tc17 cells. Conversely, activating Tc17 cells with an ICOS agonist in vitro enhanced their capacity to eradicate melanoma and induce autoimmune vitiligo when infused into mice. However, ICOS stimulation did not augment the antitumor activity of IL-2 expanded T cells. Additional investigation revealed that ICOS stimulation not only increased IL-2Rα, CXCR3 and IL-23R expression on Tc17 cells, but also dampened their expression of suppressive molecule CD39. Although Tc17 cells activated with an ICOS agonist co-secreted heightened IL-17A, IL-9 and IFN-γ, their therapeutic effectiveness was critically dependent on IFN-γ production. Depletion of IL-17A and IL-9 had little impact of antitumor Tc17 cells activated with an ICOS agonist. Collectively, our work reveals that the ICOS pathway potentiates the antitumor activity of adoptively transferred Tc17 cells. This work has major implications for the design of vaccine, antibody and cell-based therapies for autoimmunity, infectious disease and cancer.