A sensitive and less cytotoxic assay for identification of proliferating T cells based on bioorthogonally-functionalized uridine analogue.

Quantitative detection of T cell proliferation is an important readout in immunology research, as it is one of the hallmarks of T cell activation. Fluorescence-based methods for T cell proliferation mostly rely on the usage of probes that non-specifically conjugate to free primary amine groups in cells. Each cell division then results in a two-fold dilution of the probes which is detectable with flow cytometry. However, questions have been raised about cytotoxicity of these dilution-based T cell proliferation probes and they potentially affect T cell activation. An alternative assay relies on the incorporation of the uridine analog BrdU in the DNA of dividing T cells that can be detected with an antibody, but this requires harsh fixation and denaturation conditions. Recently, a new assay for detection of cell proliferation has been developed, based on the incorporation of the bioorthogonally-functionalized uridine analog 5-ethynyl-2'-deoxyuridine (EdU). Goal of this study was to compare the sensitivity and cytotoxicity of the EdU assay with a widely-used dilution-based T cell proliferation probe, CellTrace Far Red. We found that, compared to the dilution-based probe, the EdU-based assay better preserves T cell viability, is more sensitive for detecting T cell proliferation, and allows for better discernable interferon gamma responses.

Staphylococcus aureus food-poisoning outbreak associated with the consumption of ice-cream.

In April 2013, a food poisoning outbreak caused by staphylococcal enterotoxins (SEs) in ice-cream occurred in Freiburg, Germany, among the 31 participants of a christening party. Of the 13 cases, seven were hospitalized or obtained ambulatory treatment. Different types of ice-cream, which was freshly produced at the hotel where the party took place, were found to contain SE and high amounts of coagulase positive staphylococci. Enterotoxigenic Staphylococcus aureus strains isolated from ice-cream and human cases were of the same spa-type (t127), harboured the sea gene and displayed identical phenotypic resistance-, Fourier transform infrared spectroscopy- (FT-IR) and microarray-profiles. Despite the strong microbiological and epidemiological evidence of ice-cream being the incriminated food vehicle of the outbreak, a common source of S. aureus from the ice-cream could not be deduced. As none of the employees carried the outbreak strain, either the equipment used for the production of the ice-cream or a contaminated ingredient is the most likely introduction source.

Transfusion-transmitted hepatitis E in Germany, 2013.

The reported IgG seroprevalence against hepatitis E virus (HEV) in German blood donations is 6.8%, and HEV RNA detected in 0.08%, but documented evidence for HEV transmission is lacking. We identified two donations from a single donor containing 120 IU HEV RNA/mL plasma and 490 IU/mL. An infectious dose of 7,056 IU HEV RNA was transmitted via apheresis platelets to an immunosuppressed patient who developed chronic HEV. Further, transmission was probable in an immunocompetent child.

Sink or source? - The effect of hydrology on phosphorus release in the cultivated riverine wetland Spreewald (Germany).

The cultivated riverine wetland region Spreewald faces detrimental changes in the hydrological conditions due to a significant discharge reduction. With its dense network of impounded waterways and a forced tendency of sedimentation of soluble reactive phosphorus adsorbed to large amounts of FeOH/FeOOH available from mining water and groundwater discharges the 320 km2 region is favoured to accumulate large amounts of total phosphorus (TR) and thus act as an effective phosphorus sink. The change of conditions strongly challenges this function hereafter. This is especially important because eutrophication of lakes downstream the Spreewald region is controlled by phosphorus. Phosphorus balances at a testfield situated in a polder area typical for the central Spreewald region point out that hydrological and consequently hydraulic conditions are the key factors for the phosphorus sink or source behaviour. This is true for the main processes determine P retention and release at the sediment-surface water transition zone as well as for the dominant phosphorus release and retention pathways: groundwater emissions and sedimentation. In the context of hydrological changes in the Spree river catchment results from point scale and river reach scale point out the need for an adapted water management in the Spreewald region to prevent risk of extended eutrophication tendencies downstream due to forced SRP emissions.

Statistical-mechanical predictions and Navier-Stokes dynamics of two-dimensional flows on a bounded domain.

In this paper the applicability of a statistical-mechanical theory to freely decaying two-dimensional (2D) turbulence on a bounded domain is investigated. We consider an ensemble of direct numerical simulations in a square box with stress-free boundaries, with a Reynolds number that is of the same order as in experiments on 2D decaying Navier-Stokes turbulence. The results of these simulations are compared with the corresponding statistical equilibria, calculated from different stages of the evolution. It is shown that the statistical equilibria calculated from early times of the Navier-Stokes evolution do not correspond to the dynamical quasistationary states. At best, the global topological structure is correctly predicted from a relatively late time in the Navier-Stokes evolution, when the quasistationary state has almost been reached. This failure of the (basically inviscid) statistical-mechanical theory is related to viscous dissipation and net leakage of vorticity in the Navier-Stokes dynamics at moderate values of the Reynolds number.

Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum deltaH.

Methanobacterium thermoautotrophicum deltaH was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F420 (factor F390-A) and other methanogenic cofactors. In addition, cells collected from the chemostat were measured for methyl coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F420-dependent and H2-dependent methylenetetrahydromethanopterin dehydrogenase (F420-MDH and H2-MDH, respectively), total (viologen-reducing) and coenzyme F420-reducing hydrogenase (FRH), factor F390 synthetase, and factor F390 hydrolase. The experiments were performed to investigate how the intracellular F390 concentrations changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F390 varied in a way that is consistently understood from the biochemical mechanisms underlying its synthesis and degradation. Moreover, a remarkable correlation was observed between expression levels of MCR I and II, F420-MDH, and H2-MDH and the cellular contents of the factor. These results suggest that factor F390 is a reporter compound for hydrogen limitation and may act as a response regulator of methanogenic metabolism.

Phagocytic uptake and cytotoxicity of solid lipid nanoparticles (SLN) sterically stabilized with poloxamine 908 and poloxamer 407.

Solid lipid nanoparticles (SLN) as alternative intravenous colloidal drug carriers were produced by high pressure homogenisation of melted lipids (glycerolbehenate, cetylpalmitate). Their surface was modified by using hydrophilic poloxamine 908 and poloxamer 407 blockcopolymers in order to reduce the phagocytic uptake by the reticuloendothelial system (RES) after i. v. injection. The phagocytosis reducing effect of the polymers was investigated in vitro in cultures of human granulocytes, uptake was quantified by chemiluminescence. Modification of the SLN with poloxamine 908 and poloxamer 407 reduced the phagocytic uptake to appr. 8-15% compared to the phagocytosis of hydrophobic polystyrene particles. The modified SLN proved more efficient in avoiding phagocytic uptake than polystyrene particles surface-modified with these blockcopolymers (48% and 38%, respectively). Viability determinations revealed the SLN to be 10 fold less cytotoxic than polylactide nanoparticles and 100 fold less than butylcyanoacrylate particles.

Aztreonam in the treatment of bacterial meningitis.

The monobactam aztreonam was used to treat 22 young patients with meningitis caused by gram-negative bacilli. Haemophilus influenzae was isolated from the CSF of 21 patients and Salmonella heidelberg from the CSF of 1. Dosages ranged from 100 to 200 mg/kg/day in 4 doses at 6-hour intervals. Minimal inhibitory concentrations were determined by the broth dilution method for all isolated strains, and values ranged from 0.05 to 2.0 micrograms/ml. Blood and CSF drug levels were determined by a microbiologic plate diffusion method, and mean values for CSF and blood were 1.4 and 14.9 micrograms/ml, respectively. The outcome was good in 21 patients; 1 patient died. Complications were mild; subdural effusion occurred in 6 cases and was managed clinically; asymptomatic hydrocephalus was seen in 4; seizure during the acute phase occurred in 6 cases; hypoacusis was noted in 2, and motor impairment was detected at the follow-up in 1 case. Aztreonam achieved good blood and CSF penetration and performed well in the treatment of 20 cases of H. influenzae meningitis and in the one case of S. heidelberg meningitis.