Myxoma Virus Expressing a Fusion Protein of Interleukin-15 (IL15) and IL15 Receptor Alpha Has Enhanced Antitumor Activity

Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1^-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8⁺ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.     

Temperature dependence of ESR spectra of spin-labelled met-hemoglobin.

The maleimide spin-label, firmly fixed to the protein, was used to study conformation changes of various met-hemoglobin derivatives. The temperature dependence of the rotational correlation time shows a distinct conformation change in aquomet-hemoglobin at about 25 degrees C. The other met-hemoglobin derivatives studied (fluoro-, cyano-, aquomet-complexed with inositol hexaphosphate) and carbonmonoxy-hemoglobin exhibit no conformation changes in the temperature range from 0-50 degrees C.     

Influence of various structural domains of fibrinogen and fibrin on the potentiation of plasminogen activation by recombinant tissue plasminogen activator

Fibrinogen, fibrin, and related fragments have varying stimulatory effects on the initial rate of the activation of human plasminogen ([Glu¹]Pg) by recombinant tissue plasminogen activator (rt-PA). A detailed analysis of this enhancement was undertaken using various purified and complexed forms of the known domains of fibrin(ogen) with a view to gaining additional knowledge regarding the substructures of fibrinogen and fibrin that are important for their stimulatory capacities. Both arvin-mediated fibrin, as well as fibrinogen fragments generated as a result of its cleavage with CNBr, stimulate the activation in a biphasic manner, most likely as a result of changes in the promoter molecule accompanying the denaturation processes that are normally employed to either solubilize or generate these particular promoters. Using purified fibrinogen and fibrin fragments, it was found that fragment E, which binds to [Glu¹]Pg, does not enhance the activation reaction, while fragment D₁ has a potentiating effect. This suggests that the binding of [Glu¹]Pg to fibrin(ogen) alone is not, in itself, sufficient for stimulation of activation to occur, but that the rt-PA-fibrin(ogen) interaction is fundamental to this same process. All purified and mixtures of fragments containing the fragment D domain (e.g., D₂E, X-oligomer, fragment X) stimulate the reaction to a greater degree than fibrinogen and fragment D₁. It is concluded that the fibrinogen D domain is asine qua non for the enhancement reaction, while structures containing the E domain had a symbiotic effect on enhancement.On study leave from the National Institute for Biological Standards and Control, South Mimms, HERTS EN6 3QG, England.     

Sprouted and Freeze‐Dried Wheat and Oat Seeds – Phytochemical Profile and in Vitro Biological Activities

This research was carried out to study phytochemical profile, in vitro antioxidant capacity, reducing power, anti‐hyperglycemic, anti‐inflammatory activities and simulated gastrointestinal digestion of 7‐day old cereal sprouts: spelt wheat ‘Nirvana’ (WSSpe), wheat ‘Simonida’ (WSSim), oat ‘Golozrni’ (OSG) and oat ‘Jadar’ (OSJ). OSG expressed significantly higher (P ≤ 0.05) total phenols (TPC) and flavonoids content (TFC), antioxidant capacities (DPPH and ABTS assays) and reducing power (EC₅₀^DPPH = 2.12 mg/ml; EC₅₀^ABTS = 0.87 mg/ml; EC_0.5^RP = 12.24 mg/ml) as well as anti‐hyperglycemic activity (EC₅₀^AHgA = 0.96 mg/ml). WSSpe had the highest content of chlorophyll (131.23 mg/100 g) and carotenoids (22.84 mg/100 g). WSSim possessed the most potent anti‐inflammatory activity (2.71 mg/ml), though not significantly different from OSG (2.77 mg/ml). The in vitro simulation of gastro‐intestinal digestion showed higher release of phenolic compounds in intestinal than in gastric fluid.     

Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.     

Apparent survival rates of a long-lived partial migrant: the Yellow-legged Gull Larus michahellis

Capsule: In Yellow-legged Gull Larus michahellis populations from the eastern Adriatic Sea, there are considerable differences in the ratio of migratory birds and apparent survival among the colonies. Survival was dependent on age, colony and area of dispersal.

Aims: To identify the ratio of migratory birds within populations of Yellow-legged Gulls, and to compare apparent survival of gulls during migration in central and northern Europe and around the Adriatic Sea.

Methods: We analysed 15 years of resighting data of Yellow-legged Gulls using a MARK multi-state model to calculate survival rates. The effects of age, natal colony and area of dispersal were examined.

Results: Almost 60% of Adriatic Yellow-legged Gulls were migratory but the ratio varied among colonies (10.3–78.3%). Survival was dependent on age, colony and area of dispersal, with average values per group ranging between 0.599 (se 0.093) and 0.684 (se 0.084).

Conclusion: The ratio of migratory and dispersive Yellow-legged Gulls from different Adriatic colonies might be affected by both inheritance and food availability.     

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression “signals” intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.     

From Static to Interactive: Transforming Data Visualization to Improve Transparency

Data presentation for scientific publications in small sample size studies has not changed substantially in decades. It relies on static figures and tables that may not provide sufficient information for critical evaluation, particularly of the results from small sample size studies. Interactive graphics have the potential to transform scientific publications from static reports of experiments into interactive datasets. We designed an interactive line graph that demonstrates how dynamic alternatives to static graphics for small sample size studies allow for additional exploration of empirical datasets. This simple, free, web-based tool (http://statistika.mfub.bg.ac.rs/interactive-graph/) demonstrates the overall concept and may promote widespread use of interactive graphics.