In vivo preclinical low field MRI monitoring of tumor growth following a suicide gene therapy in an orthotopic mice model of human glioblastoma

Purpose

The aim of this study was to monitor in vivo with low field MRI growth of a murine orthotopic glioma model following a suicide gene therapy.

Methods

The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (MVA) vector encoding for a suicide gene (FCU1) that transforms a non toxic prodrug 5-fluorocytosine (5-FC) to its highly cytotoxic derivatives 5-fluorouracil (5-FU) and 5’-fluorouridine-5’monophosphate (5’-FUMP). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing orthotopic human glioblastoma (U87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n = 4), sham group treated with 5-FC only (n = 4), sham group with injection of MVA-FCU1 vector only (n = 4), therapy group administered with MVA-FCU1 vector and 5-FC (n = 4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images.

Results

Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p < 0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of MVA-FCU1 vector in combination with 2 weeks per os 5-FC administration was demonstrated.

Conclusion

Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of orthotopic glioblastoma.     

Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

Biochemical and functional characteristics of the plasma membrane of macrophages from BCG-infected mice.

A set of quantitative signs of activation, previously developed in studies of inflammatory peritoneal macrophages, has been applied to the study of immunologically stimulated peritoneal cells. Mice that are infected systemically with Bacillus Calmette Guérin (BCG) and then challenged locally with soluble mycobacterial antigens generate populations of cells that spread rapidly in culture, display an elevated pinocytic rate, ingest IgMC-coated sheep erythrocytes, and have diminished levels of 5'-nucleotidase activity. These effects depend on sensitization with live organisms, require secondary antigenic challenge, and develop on the same schedule as does effective cell-mediated immunity. The challenge is antigen specific and cannot be replaced by a nonspecific inflammatory stimulus. Thus, the characteristics of inflammatory macrophages that were previously defined are applicable to the study of immunologically mediated macrophage activation. Cells sharing this pattern of characteristics, whatever their mode of generation, are proposed to represent a distinct class of differentiated macrophages, as compared with the resident macrophage population.     

Free-air CO2 enrichment in a wheat-weed assembly – effects on water relations

In a three-year free-air CO₂ enrichment study (Mini-FACE), spring wheat associated with typical arable weeds were grown under present and elevated atmospheric carbon dioxide concentrations [CO₂] (ambient air+150 μmol mol^?1). Analyses of plant stable carbon isotope ratios and in vivo measurements of leaf gas exchange were used to describe the CO₂ effects on water relations. For most species examined elevated [CO₂] significantly increased the intrinsic water-use efficiency (A/g_s) as derived from carbon isotope analyses. In some of the species, seasonal averages of the ratio between leaf internal to atmospheric CO₂ (c_i/c_a) were found to be significantly reduced by elevated [CO₂]. Periodic leaf gas exchange measurements confirmed the increased water-use efficiency, but significant CO₂ effects became evident only over the entire season by carbon isotope analysis. In both types of analysis conducted, spring wheat was found to react significantly different from all other species examined. The relation between A/g_s and biomass production was significantly influenced by elevated [CO₂] in all three years of the study. At the end of the drier growing seasons 2003 and 2004, the soil water content tended to be increased in the CO₂ enriched plots indicating a water saving effect. These observations demonstrate the impact of elevated [CO₂] on plant water relations with a likely positive feedback leading to higher soil water availability. Due to the differences in the CO₂ responses of spring wheat compared to the weeds we suggest that rising [CO₂] may cause shifts in the species composition of crop-weed communities.     

Identification of a proacrosin precursor in the cell-free translation of boar testicular poly(A)(+)-mRNA.

A cell-free translation system was used to determine the molecular mass of the protein component of precursor(s) to boar proacrosin. Poly(A)(+)-mRNA was extracted from freshly excised boar testis into phenol/chloroform, precipitated in chilled (-20 degrees C) ethanol, then translated in a cell-free, reticulocyte lysate system with Tran 35S-label. Analysis of the resulting products by SDS-PAGE followed by autoradiography demonstrated multiple bands of translated proteins. Both Western blotting and immunoprecipitation with a specific polyclonal antibody to boar proacrosin yielded a single major band with a relative molecular weight of approximately 64,000. These results suggest that proacrosin (Mr = 53,000-55,000), which contains both protein and carbohydrate moieties, results from the cellular processing of a proacrosin precursor molecule.     

Biomass responses in a temperate European grassland through 17 years of elevated CO2

Future increase in atmospheric CO₂ concentrations will potentially enhance grassland biomass production and shift the functional group composition with consequences for ecosystem functioning. In the “GiFACE” experiment (Giessen Free Air Carbon dioxide Enrichment), fertilized grassland plots were fumigated with elevated CO₂ (eCO₂) year‐round during daylight hours since 1998, at a level of +20% relative to ambient concentrations (in 1998, aCO₂ was 364 ppm and eCO₂ 399 ppm; in 2014, aCO₂ was 397 ppm and eCO₂ 518 ppm). Harvests were conducted twice annually through 23 years including 17 years with eCO₂ (1998 to 2014). Biomass consisted of C3 grasses and forbs, with a small proportion of legumes. The total aboveground biomass (TAB) was significantly increased under eCO₂ (p = .045 and .025, at first and second harvest). The dominant plant functional group grasses responded positively at the start, but for forbs, the effect of eCO₂ started out as a negative response. The increase in TAB in response to eCO₂ was approximately 15% during the period from 2006 to 2014, suggesting that there was no attenuation of eCO₂ effects over time, tentatively a consequence of the fertilization management. Biomass and soil moisture responses were closely linked. The soil moisture surplus (c. 3%) in eCO₂ manifested in the latter years was associated with a positive biomass response of both functional groups. The direction of the biomass response of the functional group forbs changed over the experimental duration, intensified by extreme weather conditions, pointing to the need of long‐term field studies for obtaining reliable responses of perennial ecosystems to eCO₂ and as a basis for model development.     

Effects of free air carbon dioxide enrichment and drought stress on the feed value of maize silage fed to sheep at different thermal regimes

Information about the effects of rising atmospheric CO2 concentration and drought on the feed value of maize silage and interactions with the thermal environment during feeding is limited. A free air carbon dioxide enrichment facility was operated in a maize field to generate an elevated CO2 concentration of 550 ppm. Drought was induced by the exclusion of precipitation in one half of all experimental plots. Plants were harvested, chopped and ensiled. In a balance experiment on sheep, the nutrient digestibility was determined for three climatic treatments (temperate, temperature humidity index (THI) 57-63; mild heat, THI 68-71; severe heat, THI 75-80). The CO2 concentration and drought did not alter the crude nutrient content of silage dry matter (DM) or nutrient and organic matter (OM) digestibility. Drought increased the concentration of deoxynivalenol (DON, p < 0.001). The drought-associated increase of DON was reduced by CO2 enrichment (p = 0.003). The lowest digestibility of acid detergent fibre (p = 0.024) and neutral detergent fibre (p = 0.005) was observed during the coldest climate. OM digestibility increased during mild heat (p = 0.023). This study did not indicate considerable alterations of the feed value of maize silage due to increased atmospheric CO2 and drought. Enriched CO2 may decrease DON contaminations during drought. The thermal environment during the balance experiment did not interact with feeding maize silage grown under elevated CO2, but may affect cell wall and OM digestibility.