Single-cycle viral gene expression, rather than progressive replication and oncolysis, is required for VSV therapy of B16 melanoma

A fully intact immune system would be expected to hinder the efficacy of oncolytic virotherapy by inhibiting viral replication. Simultaneously, however, it may also enhance antitumor therapy through initiation of proinflammatory, antiviral cytokine responses at the tumor site. The aim of this study was to investigate the role of a fully intact immune system on the antitumor efficacy of an oncolytic virus. In this respect, injection of oncolytic vesicular stomatitis virus (VSV) into subcutaneous B16ova melanomas in C57Bl/6 mice leads to tumor regression, but it is not associated with viral replicative burst in the tumor. In contrast, intratumoral delivery of VSV induces an acute proinflammatory reaction, which quickly resolves concomitantly with virus clearance. Consistent with the hypothesis that therapy may not be dependent on the ability of VSV to undergo progressive rounds of replication, a single-cycle VSV is equally effective as a fully replication-competent VSV, whereas inactivated viruses do not generate therapy. Even though therapy is dependent on host CD8+ and natural killer cells, these effects are not associated with interferon-γ-dependent responses against either the virus or tumor. There is, however, a strong correlation between viral gene expression, induction of proinflammatory reaction in the tumor and in vivo therapy. Overall, our results suggest that acute innate antiviral immune response, which rapidly clears VSV from B16ova tumors, is associated with the therapy observed in this model. Therefore, the antiviral immune response to an oncolytic virus mediates an intricate balance between safety, restriction of oncolysis and, potentially, significant immune-mediated antitumor therapy

Combination therapy with reovirus and anti-PD-1 blockade controls tumor growth through innate and adaptive immune responses.

Oncolytic reovirus can be delivered both systemically and intratumorally, in both pre-clinical models and in early phase clinical trials. Reovirus has direct oncolytic activity against a variety of tumor types and anti-tumor activity is directly associated with immune activation by virus replication in tumors. Immune mechanisms of therapy include both innate immune activation against virally infected tumor cells, and the generation of adaptive anti-tumor immune responses as a result of in vivo priming against tumor-associated antigens. We tested the combination of local oncolytic reovirus therapy with systemic immune checkpoint inhibition. We show that treatment of subcutaneous B16 melanomas with a combination of intravenous (i.v.) anti-PD-1 antibody and intratumoral (i.t.) reovirus significantly enhanced survival of mice compared to i.t. reovirus (p<0.01) or anti-PD-1 therapy alone. In vitro immune analysis demonstrated that checkpoint inhibition improved the ability of NK cells to kill reovirus-infected tumor cells, reduced Treg activity, and increased the adaptive CD8(+) T cell dependent anti-tumor T cell response. PD-1 blockade also enhanced the anti-viral immune response but through effector mechanisms which overlapped with, but also differed from those affecting the antitumor response. Therefore, combination with checkpoint inhibition represents a readily translatable next step in the clinical development of reovirus

The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual TDNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity

Experimental nitrogen addition alters structure and function of a boreal poor fen: Implications for critical loads

Bogs and fens cover 6 and 21%, respectively, of the 140,329 km2 Oil Sands Administrative Area in northern Alberta. Regional background atmospheric N deposition is low (b2 kg N ha−1 yr−1 ), but oil sands development has led to increasing N deposition (as high as 17 kg N ha−1 yr−1 ). To examine responses to N deposition, over five years, we experimentally applied N (as NH4NO3) to a poor fen near Mariana Lake, Alberta, unaffected by oil sands activities, at rates of 0, 5, 10, 15, 20, and 25 kg N ha−1 yr−1 , plus controls (no water or N addition). At Mariana Lake Poor Fen (MLPF), increasing N addition: 1) progressively inhibited N2-fixation; 2) had no effect on net primary production (NPP) of Sphagnum fuscum or S. angustifolium, while stimulating S. magellanicum NPP; 3) led to decreased abundance of S. fuscum and increased abundance of S. angustifolium, S. magellanicum, Andromeda polifolia, Vaccinium oxycoccos, and of vascular plants in general; 4) led to an increase in stem N concentrations in S. angustifolium and S. magellanicum, and an increase in leaf N concentrations in Chamaedaphne calyculata, Andromeda polifolia, and Vaccinium oxycoccos; 5) stimulated root biomass and production;6) stimulated decomposition of cellulose, but not of Sphagnum or vascular plant litter; and 7) had no or minimal effects on net N mineralization in surface peat, NH4 +-N, NO3 −-N or DON concentrations in surface porewater, or peat microbial composition. Increasing N addition led to a switch from new N inputs being taken up primarily by Sphagnum to being taken up primarily by shrubs. MLPF responses to increasing N addition did not exhibit threshold triggers, but rather began as soon as N additions increased. Considering all responses to N addition, we recommend a critical load for poor fens in Alberta of 3 kg N ha−1 yr−1

Direct Gene Transfer for the Understanding and Treatment of Human Disease

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75762/1/j.1749-6632.1994.tb21709.x.pd

Prodrug converting enzyme gene delivery by L. monocytogenes

<p>Abstract</p> <p>Background</p> <p><it>Listeria monocytogenes </it>is a highly versatile bacterial carrier system for introducing protein, DNA and RNA into mammalian cells. The delivery of tumor antigens with the help of this carrier into tumor-bearing animals has been successfully carried out previously and it was recently reported that <it>L. monocytogenes </it>is able to colonize and replicate within solid tumors after local or even systemic injection.</p> <p>Methods</p> <p>Here we report on the delivery of two prodrug converting enzymes, purine-deoxynucleoside phosphorylase (PNP) and a fusion protein consisting of yeast cytosine deaminase and uracil phosphoribosyl transferase (FCU1) into cancer cells in culture by <it>L. monocytogenes</it>. Transfer of the prodrug converting enzymes was achieved by bacterium mediated transfer of eukaryotic expression plasmids or by secretion of the proteins directly into the host cell cytosol by the infecting bacteria.</p> <p>Results</p> <p>The results indicate that conversion of appropriate prodrugs to toxic drugs in the cancer cells occured after both procedures although <it>L. monocytogenes</it>-mediated bactofection proved to be more efficient than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging the constitutively P_CMV-promoter with the melanoma specific P_4xTETP-promoter resulted in melanoma cell-specific expression of the prodrug converting enzymes but reduced the efficiencies.</p> <p>Conclusion</p> <p>These experiments open the way for bacterium mediated tumor specific activation of prodrugs in live animals with tumors.</p