Messenger RNA electroporation of human monocytes,followed by rapid in vitro differentiation,leads to highly stimulatory antigen-loaded mature dendritic cells

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.     

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.     

Biogenic Silver for Disinfection of Water Contaminated with Viruses

The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag⁰. The antiviral action of biogenic Ag⁰ was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter⁻¹ biogenic Ag⁰ caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg⁰) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter⁻¹ ionic Ag⁺ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag⁰ were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag⁰ was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag⁰ m⁻² on the filter (135 mg biogenic Ag⁰ kg⁻¹ filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag⁰ and its promising opportunities for continuous water disinfection.At least 1 billion people do not have access to safe drinking water, according to the WHO (41). Contamination of drinking water and the subsequent outbreak of waterborne diseases are the leading cause of death in many developing nations. Moreover, the spectrum and incidence of some infectious diseases are increasing worldwide (40). Among them, the transmission of waterborne human noroviruses is considered to be the major cause of acute nonbacterial gastroenteritis (22). Numerous outbreaks of norovirus-associated gastroenteritis have been linked with ingestion of contaminated drinking water, in developed countries also (6; M. Kukkula, L. Maunula, E. Silvennoinen, and C. H. von Bonsdorff, presented at the International Workshop on Human Caliciviruses, Atlanta, GA, 29 to 31 March 1999). Therefore, the development of innovative drinking water quality control strategies is of the utmost importance in this decade.Recent interest has arisen in the use of nanotechnology for water disinfection (20). In particular the formation of by-products by conventional disinfection techniques (e.g., chlorination), has encouraged researchers to explore the antimicrobial activity of several nanomaterials, such as silver (18, 31). Silver-containing nanoparticles have previously been demonstrated to be effective against bacteria and viral particles (10, 28, 34). Several mechanisms of the antiviral activity have been ascribed to (chemically produced) zerovalent silver nanoparticles (nAg⁰) but still remain not fully understood. On the one hand, nAg⁰ can release Ag⁺ ions, which interact with thiol groups in proteins and interfere with DNA replication (11, 21, 24). On the other hand, the adhesion of nAg⁰ as such is responsible for the inactivation of HIV-1 virions (10).Previous studies showed that chemically produced nAg⁰ were unstable in solution and would easily aggregate with average particle sizes of <40 nm or at high concentrations (23). As a consequence, the specific surface of the nanomaterial decreases. Moreover, there is a need for environmentally friendly approaches to production of nanoparticles. To cope with these demands, biological processes have been developed using microorganisms. Microbial approaches to obtain nanoscale Ag⁰ have been demonstrated for the bacterium Pseudomonas stutzeri AG259 (17) and for fungi, e.g., Verticillium sp. (26), Phoma sp. (5), Fusarium sp. (2, 16), and Aspergillus sp. (12, 29, 39). However, these enzymatic reduction processes are slow and yield low concentrations of silver. Moreover, if the nanoparticles are produced intracellularly, specific treatments (e.g., heat treatment at 600°C for 6 h) are necessary to make the nanoparticles accessible for antibacterial or antiviral applications (39).Recently, lactic acid bacteria have been used as reducing agents for the fast, nonenzymatic, and extracellular production of nanoscale-sized Ag⁰ particles (33). The bacterial cell wall hereby serves as a microscale carrier matrix for the nanoparticles. The unique association of the nanoparticles with the (dead) bacterial carrier matrix, called biogenic Ag⁰, prevents them from aggregating and makes the association promising for disinfection technologies. In the case of virus inactivation, smaller nanoparticles are known to be more efficient due to a more effective binding to the glycoproteins of the viral envelope (10, 28). For biogenic Ag⁰ production using lactic acid bacteria, it was demonstrated that different particle sizes could be obtained, depending on the species used (33). Production by Lactobacillus fermentum resulted in the smallest average diameter and a narrow size distribution, potentially favorable for antimicrobial applications (33).The objective of the present study was to examine the inactivation of a bacteriophage (UZ1), isolated from hospital sewage, by biogenic Ag⁰. This DNA phage, a T7-like coliphage of the genus Podovirida (order Caudovirales) (38), is infective for Enterobacter aerogenes BE1, a species belonging to the normal digestive microbiota (30). The virucidal action of biogenic Ag⁰ was evaluated in drinking water and compared with the use of ionic Ag⁺ and chemically produced nAg⁰. To test the antiviral activity of biogenic Ag⁰ against noroviruses as well, the murine norovirus 1 (MNV-1) was used as a surrogate organism for human noroviruses (43). Finally, continuous disinfection by the biogenic nanoparticles was evaluated in a flowthrough system with a coated cartridge filter. To our knowledge, this is the first report to demonstrate the antiviral effect of extracellular biogenic Ag⁰.     

Human faecal microbiota display variable patterns of glycerol metabolism

Significant amounts of glycerol reach the colon microbiota daily through the diet and/or by in situ microbial production or release from desquamated epithelial cells. Some gut microorganisms may anaerobically reduce glycerol to 1,3-propanediol (1,3-PDO), with 3-hydroxypropanal as an intermediate. Accumulation of the latter intermediate may result in the formation of reuterin, which is known for its biological activity (e.g. antimicrobial properties). To date, glycerol metabolism in mixed cultures from the human colon has received little attention. Using in vitro batch incubations of faeces from 10 human individuals, we demonstrated that glycerol addition (140 mM) significantly affects the metabolism and composition of the microbial community. About a third of the samples exhibited rapid glycerol conversion, yielding proportionally higher levels of acetate and 1,3-PDO. In contrast, a slower glycerol metabolism resulted in higher levels of propionate. Furthermore, rapid glycerol metabolism correlated with significant shifts in the Lactobacillus-Enterococcus community, which were not observed in slower glycerol-metabolizing samples. As the conversion of glycerol to 1,3-PDO is a highly reducing process, we infer that the glycerol metabolism may act as an effective hydrogen sink. Given the importance of hydrogen-consuming processes in the gut, this work suggests that glycerol may have potential as a tool for modulating fermentation kinetics and profiles in the gastrointestinal tract.     

The Candida albicans GAP Gene Family Encodes Permeases Involved in General and Specific Amino Acid Uptake and Sensing

The Saccharomyces cerevisiae general amino acid permease Gap1 (ScGap1) not only mediates the uptake of most amino acids but also functions as a receptor for the activation of protein kinase A (PKA). Fungal pathogens can colonize different niches in the host, each containing various levels of different amino acids and sugars. The Candida albicans genome contains six genes homologous to the S. cerevisiae GAP1. The expression of these six genes in S. cerevisiae showed that the products of all six C. albicans genes differ in their transport capacities. C. albicans Gap2 (CaGap2) is the true orthologue of ScGap1 as it transports all tested amino acids. The other CaGap proteins have narrower substrate specificities though CaGap1 and CaGap6 transport several structurally unrelated amino acids. CaGap1, CaGap2, and CaGap6 also function as sensors. Upon detecting some amino acids, e.g., methionine, they are involved in a rapid activation of trehalase, a downstream target of PKA. Our data show that CaGAP genes can be functionally expressed in S. cerevisiae and that CaGap permeases communicate to the intracellular signal transduction pathway similarly to ScGap1.     

Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis

Aim

We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage.

Methods

This study included 10 patients on standard (3x4h/week) high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V₁), total distribution volume (V_tot) and intercompartment clearance (K₂₁). This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies.

Results

Dialyzer clearance K, V₁ and V_tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K₂₁was 2.7–5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3x4h dialysis at 300mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated.

Conclusion

When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.