Damage to the leech or mammalian CNS increases nitric oxide (NO) production and causes accumulation of phagocytic microglial cells at the injury site. The aim of this study was to determine whether NO plays a role in microglial migration and accumulation at lesions in which NO is generated by a rapidly appearing endothelial nitric oxide synthase (eNOS) in leeches. Immunohistochemistry and cytochemistry demonstrated active eNOS before and throughout the period of microglial accumulation at the lesion. Decreasing NO synthesis by application of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (1 mM) significantly reduced microglial accumulation, whereas its inactive enantiomer N(w)-nitro-D-arginine methyl ester (1 mM) resulted in microglial accumulation similar to that in crushed controls. Increasing NO with the donor spermine NONOate (SPNO) (1 mM) also inhibited accumulation, but not in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5, 5-teramethylimidazoline-oxyl-3-oxide (50 microM). The effect of SPNO was reversed by washout. SPNO application reduced average microglial migratory speeds and even reversibly arrested cell movement, as measured in living nerve cords. These results suggest that NO produced at a lesion may be a stop signal for microglia to accumulate there and that it can act on microglia early in their migration. Thus, NO may assume a larger role in nerve repair and recovery from injury by modulating accumulation of microglia, which appear to be important for axonal regeneration.

It is known that nitric oxide (NO) is produced by injured tissues of the mammalian central nervous system (CNS) within days of injury. The aim of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understanding the role of NO in nerve repair. We report that within minutes after crushing the nerve cord of the leech, the region of damage stained histochemically for NADPH diaphorase, which is indicative of nitric oxide synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detected a band with a relative molecular mass of 140 000, which is approximately the size of vertebrate eNOS. Cells expressing eNOS immunoreactivity as a result of injury were identi¢ed after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crushing. Immunoreactive cells included connective glia and some microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocked by calmodulin antagonist W7 (25 mM), but not by similar concentrations of the less potent calmodulin antagonist W12. Thus in the leech CNS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons.

An early step in repair of the leech CNS is the appearance of endothelial nitric oxide synthase (eNOS) immunoreactivity and NOS activity, but coincident generation of NO at the lesion after injury has not been shown. This is important because NO can regulate microglial cell motility and axon growth. Indirect measurement of NO with the standard citrulline assay demonstrated that NO was generated within 30 min after nerve cord injury. A polarographic NO-selective self-referencing microelectrode that measures NO flux noninvasively was developed to obtain higher spatial and temporal resolution. With this probe, it was possible to demonstrate that immediately after the leech CNS was injured, NO left the lesion with a mean peak efflux of 803 Ϯ 99 fmol NO cm Ϫ2 sec Ϫ1 . NO efflux exponentially declined to a constant value, as described through the equation f(t) ϭ y o ϩ ae Ϫt/ , with ϭ 117 Ϯ 30 sec. The constant y o ϭ 15.8 Ϯ 4.5 fmol cm Ϫ2 represents a sustained efflux of NO. Approximately 200 pmol NO cm Ϫ2 is produced at the lesion (n ϭ 8). Thus, injury activates eNOS already present in the CNS and precedes the accumulation of microglia at the lesion, consistent with the hypothesis that NO acts to stop the migrating microglia at the lesion site.

Some cytokines can reengineer anti-tumor immunity to modify the tumor micro-environment. Interleukin-27 (IL-27) can partially reduce tumor growth in several animal models, including prostate cancer. We hypothesized that addition of IL-18, which can induce the proliferation of several immune effector cells through inducing IFNγ could synergize with IL-27 to enhance tumor growth control. We describe our findings on the effects of IL-27 gene delivery on prostate cancer cells and how sequential therapy with IL-18 enhanced the efficacy of IL-27. The combination of IL-27 followed by IL-18 (27→18) successfully reduced cancer cell viability, with significant effects in cell culture and in an immunocompetent mouse model. We also examined a novel chimeric cytokine, comprising an IL-27 targeted at the C-terminus with a short peptide, LSLITRL (27pepL). This novel cytokine targets a receptor upregulated in tumor cells (IL-6Rα) via the pepL ligand. Interestingly, when we compared the 27→18 combination with the single 27pepL therapy, we observed a similar efficacy for both. This efficacy was further enhanced when 27pepL was sequenced with IL-18 (27pepL→18). The observed reduction in tumor growth and significantly enriched canonical pathways and upstream regulators, as well as specific immune effector signatures (as determined by bioinformatics analyses in the tumor microenvironment) supported the therapeutic design, whereby IL-27 or 27pepL can be more effective when delivered with IL-18. This cytokine sequencing approach allows flexible incorporation of both gene delivery and recombinant cytokines as tools to augment IL-27’s bioactivity and reengineer efficacy against prostate tumors and may prove applicable in other therapeutic settings.

Gene delivery as a therapeutic tool continues to advance toward impacting human health, with several gene therapy products receiving FDA approval over the past 5 years. Despite this important progress, the safety and efficacy of gene therapy methodology requires further improvement to ensure that nucleic acid therapeutics reach the desired targets while minimizing adverse effects. Synthetic polymers offer several enticing features as nucleic acid delivery vectors due to their versatile functionalities and architectures and the ability of synthetic chemists to rapidly build large libraries of polymeric candidates equipped for DNA/RNA complexation and transport. Current synthetic designs are pursuing challenging objectives that seek to improve transfection efficiency and, at the same time, mitigate cytotoxicity. This Perspective will describe recent work in polymer-based gene complexation and delivery vectors in which cationic polyelectrolytes are modified synthetically by introduction of additional components�including hydrophobic, hydrophilic, and fluorinated units�as well as embedding of degradable linkages within the macromolecular structure. As will be seen, recent advances employing these emerging design strategies are promising with respect to their excellent biocompatibility and transfection capability, suggesting continued promise of synthetic polymer gene delivery vectors going forward.