Selenite reduction by a denitrifying culture: batch- and packed-bed reactor studies

Selenite reduction by a bacterial consortium enriched from an oil refinery waste sludge was studied under denitrifying conditions using acetate as the electron donor. Fed-batch studies with nitrate as the primary electron acceptor showed that accumulation of nitrite led to a decrease in the extent of selenite reduction. Also, when nitrite was added as the primary electron acceptor, rapid selenite reduction was observed only after nitrite was significantly depleted from the medium. These results indicate that selenite reduction was inhibited at high nitrite concentrations. In addition to batch experiments, continuous-flow selenite reduction experiments were performed in packed-bed columns using immobilized enrichment cultures. These experiments were carried out in three phases: in phase I, a continuous nitrate feed with different inlet selenite concentration was applied; in phase II, nitrate was fed in a pulsed fashion; and in phase III, nitrate was fed in a continuous mode but at much lower concentrations than the other two phases. During the phase I experiments, little selenite was removed from the influent. However, when the column was operated in the pulse feed strategy (phase II) or in the continuous mode with low nitrate levels (phase III), significant quantities of selenium were removed from solution and retained in the immobilization matrix in the column. Thus, immobilized denitrifying cultures can be effective in removing selenium from waste streams, but nitrate-limited operating conditions might be required.     

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

Device Thrombogenicity Emulator (DTE) − Design optimization methodology for cardiovascular devices: A study in two bileaflet MHV designs

Patients who receive prosthetic heart valve (PHV) implants require mandatory anticoagulation medication after implantation due to the thrombogenic potential of the valve. Optimization of PHV designs may facilitate reduction of flow-induced thrombogenicity and reduce or eliminate the need for post-implant anticoagulants. We present a methodology entitled Device Thrombogenicty Emulator (DTE) for optimizing the thrombo-resistance performance of PHV by combining numerical and experimental approaches. Two bileaflet mechanical heart valves (MHV) designs, St. Jude Medical (SJM) and ATS, were investigated by studying the effect of distinct flow phases on platelet activation. Transient turbulent and direct numerical simulations (DNS) were conducted, and stress loading histories experienced by the platelets were calculated along flow trajectories. The numerical simulations indicated distinct design dependent differences between the two valves. The stress loading waveforms extracted from the numerical simulations were programmed into a hemodynamic shearing device (HSD), emulating the flow conditions past the valves in distinct ‘hot-spot’ flow regions that are implicated in MHV thrombogenicity. The resultant platelet activity was measured with a modified prothrombinase assay, and was found to be significantly higher in the SJM valve, mostly during the regurgitation phase. The experimental results were in excellent agreement with the calculated platelet activation potential. This establishes the utility of the DTE methodology for serving as a test bed for evaluating design modifications for achieving better thrombogenic performance for such devices.     

Characterization of the biological activities of uridine diphosphate in human dendritic cells: Influence on chemotaxis and CXCL8 release

Uridine nucleotides are endogenous nucleotides which are released into the extracellular space from mechanical stressed endothelial and epithelial cells as well as lipopolysaccharide (lps)-stimulated monocytes. Here, we studied the biological activity of the selective purinoreceptor P2Y6 (P2YR6) agonist Uridine 5'diphosphate (UDP) as well as the P2YR2- and P2YR4-activating uridine 5'triphosphate (UTP) on human dendritic cells (DC). These cells in their immature state have the ability to migrate from blood to peripheral target sites where they sense dangerous signals and capture potential antigens. Moreover, mature DC induce innate immune responses and migrate from peripheral tissues to secondary lymphoid organs in order to activate naive T cells and initiate adaptive immunity. Here, we were able to show that uridine nucleotides stimulated Ca(2+) transients, actin polymerization, and chemotaxis in immature DC. Experiments with pertussis toxin, the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPgammaS) and receptor antagonists, as well as desensitization studies suggested that these uridine nucleotides activities were mediated by different G(i) protein-coupled receptors. During lps-induced maturation, DC lost their ability to respond towards uridine nucleotides with these activities. Instead, UDP, but not UTP, stimulated the release of the CXC-chemokine 8 (CXCL8) from mature DC in a reactive blue sensitive manner. Moreover, our study indicates that UDP stimulates different signaling pathways in immature and mature DC in order to favor the accumulation of immature DC and to augment the capacity to secrete CXCL8 in mature DC.     

The serotoninergic receptors of human dendritic cells: identification and coupling to cytokine release

The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is stored at peripheral sites in mast cells and released from this peripheral source upon IgE cross-linking. In this study, we investigated the expression of serotoninergic receptors (5-HTR), the signaling pathway, and biological activity of 5-HT on human dendritic cells (DC), showing that immature and mature DC expressed mRNA for different serotoninergic receptors. Thereby, the mRNA of 5-HTR(1B), 5-HTR(1E), 5-HTR(2A), 5-HTR(2B), one splicing variant of the 5-HTR(3), 5-HTR(4), and 5-HTR(7) receptors were detected. Immature DC preferentially expressed mRNA for the heptahelical 5-HTR(1B), 5-HTR(1E), and 5-HTR(2B) receptors, while mature DC mostly expressed 5-HTR(4) and 5-HTR(7). The mRNA expression level of the ligand-gated cation channel 5-HTR(3) and the heptahelical 5-HTR(2A) did not significantly change during maturation. Isotype-selective receptor agonists allowed us to show that 5-HT stimulated 5-HTR(3)-dependent Ca(2+) influx in immature and mature DC. Moreover, we revealed that 5-HTR(1) and 5-HTR(2) receptor stimulation induced intracellular Ca(2+) mobilization via G(i/o) proteins in immature, but not mature, DC. Activation of 5-HTR(4) and 5-HTR(7) induced cAMP elevation in mature DC. Functional studies indicated that activation of 5-HTR(4) and 5-HTR(7) enhanced the release of the cytokines IL-1beta and IL-8, while reducing the secretion of IL-12 and TNF-alpha in mature DC. In summary, our study shows that 5-HT stimulated, in a maturation-dependent manner, different signaling pathways in DC. These data point to a role for 5-HT in regulating the immune response at peripheral sites.     

LED Fluorescence Microscopy for the Diagnosis of Pulmonary Tuberculosis: A Multi-Country Cross-Sectional Evaluation

Background

The diagnosis of tuberculosis (TB) in resource-limited settings relies on Ziehl-Neelsen (ZN) smear microscopy. LED fluorescence microscopy (LED-FM) has many potential advantages over ZN smear microscopy, but requires evaluation in the field. The aim of this study was to assess the sensitivity/specificity of LED-FM for the diagnosis of pulmonary TB and whether its performance varies with the timing of specimen collection.

Methods and Findings

Adults with cough ≥2 wk were enrolled consecutively in Ethiopia, Nepal, Nigeria, and Yemen. Sputum specimens were examined by ZN smear microscopy and LED-FM and compared with culture as the reference standard. Specimens were collected using a spot-morning-spot (SMS) or spot-spot-morning (SSM) scheme to explore whether the collection of the first two smears at the health care facility (i.e., “on the spot”) the first day of consultation followed by a morning sample the next day (SSM) would identify similar numbers of smear-positive patients as smears collected via the SMS scheme (i.e., one on-the-spot-smear the first day, followed by a morning specimen collected at home and a second on-the-spot sample the second day). In total, 529 (21.6%) culture-positive and 1,826 (74.6%) culture-negative patients were enrolled, of which 1,156 (49%) submitted SSM specimens and 1,199 (51%) submitted SMS specimens. Single LED-FM smears had higher sensitivity but lower specificity than single ZN smears. Using two LED-FM or two ZN smears per patient was 72.8% (385/529, 95% CI 68.8%–76.5%) and 65.8% (348/529, 95% CI 61.6%–69.8%) sensitive (p<0.001) and 90.9% (1,660/1,826, 95% CI 89.5%–92.2%) and 98% (1,790/1,826, 95% CI 97.3%–98.6%) specific (p<0.001). Using three LED-FM or three ZN smears per patient was 77% (408/529, 95% CI 73.3%–80.6%) and 70.5% (373/529, 95% CI 66.4%–74.4%, p<0.001) sensitive and 88.1% (95% CI 86.5%–89.6%) and 96.5% (95% CI 96.8%–98.2%, p<0.001) specific. The sensitivity/specificity of ZN smear microscopy and LED-FM did not vary between SMS and SSM.

Conclusions

LED-FM had higher sensitivity but, in this study, lower specificity than ZN smear microscopy for diagnosis of pulmonary TB. Performance was independent of the scheme used for collecting specimens. The introduction of LED-FM needs to be accompanied by appropriate training, quality management, and monitoring of performance in the field.

Trial Registration

Current Controlled Trials ISRCTN53339491 Please see later in the article for the Editors'' Summary     

Inosine stimulates chemotaxis, Ca2+-transients and actin polymerization in immature human dendritic cells via a pertussis toxin-sensitive mechanism independent of adenosine receptors

Inosine is an endogenous purine nucleoside, which is formed by adenosine deaminidase during adenosine breakdown and is released into the extracellular space from the sympathetic nervous system or injured cells. Here, we studied the biological activity of inosine on human dendritic cells (DC), which are specialized antigen presenting cells characterized by their ability to migrate from the blood to peripheral tissues, and then to secondary lymphoid organs where they initiate adaptive immune responses. In immature DC, inosine concentration-dependently stimulated Ca(2+)-transients, actin polymerization, and chemotaxis. Experiments with adenosine receptor antagonists and pertussis toxin (PTX) as well as desensitization studies suggested that the activity of inosine was mediated by a G protein-coupled receptor pathway independent of adenosine receptors. DC, induced to mature by lipopolysaccharide, lost their ability to respond towards inosine with these activities. Moreover, inosine did neither influence membrane expression of CD54, CD80, CD83, CD86, HLA-DR, and MHC class I molecules nor modulated secretion of interleukin (IL)-12, IL-10, and tumor necrosis factor alpha in immature and lipopolysaccharide-matured DC. In aggregate, our study indicates that inosine may be involved in the trafficking control system of immature DC, and mediates its chemotactic activity by a PTX-sensitive mechanism independent of adenosine receptors.