25 years of UV-induced immunosuppression mediated by T cells-from disregarded T suppressor cells to highly respected regulatory T cells

For more than 25 years it is known that UV radiation, in particular the UVB range suppresses the immune system. In contrast to conventional immunosuppression by immunosuppressive drugs, UV radiation does not compromise the immune system in a general but rather in an antigen-specific fashion via induction of immunotolerance. This effect is mostly mediated via regulatory T cells (Treg) induced by UV. Several subtypes of UV-induced Treg may exist, the best characterized are those which inhibit contact hypersensitivity. Induction of these Tregs by UV radiation is an active process which requires antigen presentation by UV-damaged but still alive Langerhans cells (LC) in the lymph nodes. UV-induced Treg have recently been characterized as expressing CD4 and CD25 and as releasing upon activation the immunosuppressive cytokine interleukin (IL)-10. Once activated in an antigen-specific manner, they suppress immune responses in a general fashion via the release of IL-10, a phenomenon called bystander suppression. The further phenotypic and functional characterization of these cells will not only contribute to a better understanding of the impact of UV radiation on the immune system but will also determine whether they can be applied in the future therapeutically with the final aim of achieving specific immunosuppression.     

Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor

This paper describes a microquantitative method for glucose determination in situ of living cells in real-time. In this novel technique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injection lab-on-valve system (microSI-LOV). Glucose sensing is performed through the use of a two-step, NAD-linked enzymatic process. The course of the assay is monitored in real-time, by absorbance of NADH at 340 nm. The microsequential assay based on plug/nozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM. The design of the (microSI-LOV) system allows the assay to be carried out using only 40 microL of the enzyme reagent and 3 microL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopore beads. Rapid cellular glucose consumption, in this technique, is facilitated by a high cell density, which allows a large number of cells (10(4)-10(5)) to be retained in a very small volume (3 microL). In turn, this cell density results in the rapid depletion of glucose from the cell medium over short time periods (< 2 min). In conjunction with the assay development, the plug/nozzle design and its ramifications on mixing in general are presented and discussed.     

High efficient delivery of siRNA into tumor cells by positively charged carbon dots

Abstract

Nowadays development of safe and simple siRNA delivery system is still a great challenge. Carbon dots have attracted considerable attention in bioimaging, drug delivery, nanosensors, and other fields during the past few years. In this study, we successfully developed carbon dots (TEPA-CDs) as siRNA nanocarriers based on glucose and tetraethylene pentamine. TEPA-CDs have a mean diameter of less than 10?nm and positive charged decoration. TEPA-CDs could condense siRNA into stable complexes without no obvious premature release. Cellular uptake analysis clearly showed that Cy3-labeled siRNA could be uptake by HeLa cells. GFP expression in HeLa-EGFP cells could be significantly inhibited by TEPA-CDs/siRNA complexes. Our study indicated that TEPA-CDs could be used as a siRNA nanocarrier to tumor cells.     

Implementation of an integrated glucose POCT system

In response to a change of the Belgian National Directives whereby hospital laboratories became responsible for all point-of-care testing (POCT) performed within hospital walls a standardized and automated POC glucose-testing system was implemented in our hospital. The system consists of 50 AccuCheck Inform instruments (Roche Diagnostics, Vilvoorde, Belgium), 50 docking stations, a DataCare Server, and connections to the medical laboratory information system (MOLIS, Sysmex, Barchon, Belgium) and to the hospital information system. Implementation involved many parties and extensive preparation and communication. Key issues were bar-coded patient and user identification, training, and responsibilities. One year after the hospital wide implementation of this system the quality of POC glucose testing has significantly increased, thereby improving patient safety. This study describes a stepwise change over involving the medical laboratory and with a focus on hands-on quality.Presented at the ninth conference on Quality in the Spotlight, 18–19 March 2004, Antwerp, Belgium.     

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.     

Ferric chloride/tetraethyl orthosilicate as an efficient system for synthesis of dihydropyrimidinones by Biginelli reaction

An efficient method for the Biginelli reaction of aldehydes, acetoacetate esters and urea employing tetraethyl orthosilicate in the presence of ferric chloride is described. These improved reaction conditions allow the preparation of a wide variety of substituted dihydropyrimidinones (including sterically encumbered ones) in high yields and purity under mild reaction conditions.     

REMUS100 AUV with an integrated microfluidic system for explosives detection

Quantitating explosive materials at trace concentrations in real-time on-site within the marine environment may prove critical to protecting civilians, waterways, and military personnel during this era of increased threat of widespread terroristic activity. Presented herein are results from recent field trials that demonstrate detection and quantitation of small nitroaromatic molecules using novel high-throughput microfluidic immunosensors (HTMI) to perform displacement-based immunoassays onboard a HYDROID REMUS100 autonomous underwater vehicle. Missions were conducted 2–3 m above the sea floor, and no HTMI failures were observed due to clogging from biomass infiltration. Additionally, no device leaks were observed during the trials. HTMIs maintained immunoassay functionality during 2 h deployments, while continuously sampling seawater absent without any pretreatment at a flow rate of 2 mL/min. This 20-fold increase in the nominal flow rate of the assay resulted in an order of magnitude reduction in both lag and assay times. Contaminated seawater that contained 20–175 ppb trinitrotoluene was analyzed.

Higher infiltration by Th17 cells compared with regulatory T cells is associated with severe acute T-cell-mediated graft rejection

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were 11.6 ± 12.2 cells/mm2 and 5.6 ± 8.0 cells/mm2, respectively. The average Treg/Th17 ratio was 5.6 ± 8.2. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury (P < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.     

Development and application of an integrated system for monitoring ethanol content of fuels

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-aminophenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05–1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05–1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.     

Development of an integrated chromatographic system for on-line digestion and characterization of phosphorylated proteins

The development of an integrated chromatographic system for complete phosphoprotein analysis is described. The digestion of phosphoproteins with trypsin- or pronase-based monolithic bioreactors is carried out on-line with selective enrichment on a TiO(2) trap and separation of the produced phosphopeptides by reversed-phase liquid chromatography-multiple mass spectrometry (RPLC/MS(n)). A detailed study on the selective extraction of peptides with different degrees of phosphorylation on TiO(2) cartridges is discussed. This analytical strategy has been optimized using beta-casein as a standard phosphoprotein, and then applied to the identification of phosphorylation sites in insulin-like grow factor-binding protein 1 (IGFBP-1) isolated from amniotic fluid.     

Improved photocatalytic efficiency of a WO3 system by an efficient visible-light induced hole transfer

Amorphous Cu(II) nanoclusters grafted WO(3) particles were coated on a smooth TiO(2) film, and site selective depositions of PbO(2) and metal Ag particles by photocatalytic processes were observed on TiO(2) and WO(3) due to transfer of holes to TiO(2), and accumulation of electrons in WO(3) respectively. As a result, the photocatalytic activity of TiO(2) modified Cu(II)-WO(3) increased ~3.5 fold higher than that of Cu(II)-WO(3).     

Platinum-free tungsten carbides as an efficient counter electrode for dye sensitized solar cells

Mesoporous tungsten carbides displayed an excellent solar conversion efficiency (7.01%) as a counter electrode for dye sensitized solar cells under 100 mW cm(-2), AM 1.5G illumination, which corresponded to ca. 85% of the efficiency of the conventional platinum electrode.     

Methodological approach for an integrated environmental monitoring system relative to heavy metals from an incineration plant

The use of an Integrated Environmental Monitoring System is an innovative and very important approach for the determination of environmental impacts due to a contamination source. In the present work, the methodological approach is described and applied to the case study of a MSW incineration plant. Heavy metals were chosen as Environmental Indicators. Gaseous emissions were measured and correlated to wet and dry depositions, soil and vegetation samples. Results show a good correlation between stack emissions and atmospheric depositions; less with soil and vegetation, but these results are important in order to design a standard procedure for an Integrated Monitoring System.     

Biological fluid screening and confirmation of bile acids by use of an integrated flow-injection-LC-evaporative light-scattering system

Summary An integrated flow-injection-liquid chromatographic system is presented for the screening of biological fluids for bile acids, and for subsequent confirmation. It is based on the use of a flow-injection configuration with an evaporative light-scattering detector (ELSD), for routine screening for the total concentration of the analytes, followed by the individual determination, by liquid chromatography, of only those samples for which the total concentration is close to or above the normal level in healthy individuals. Bile acids are extracted from human serum and urine withn-hexane under acid conditions. After phase separation the organic layer is evaporated to dryness under a stream of nitrogen and redissolved in weakly alkaline solution (pH 8). The aqueous phase is then passed through a miniaturized Amberlite XAD-7 column for preconcentration and clean up, and eluted with acetonitrile-methanol, 65∶35 (v/v). The effluent is introduced directly into the ELSD for determination of the total bile-acid content of the sample. For confirmatory analysis another aliquot of the sample is processed in the screening module and the effluent from the adsorbent column is then directed to the chromatographic column to obtain the bile acid profile of the sample. Good agreement was obtained in the analysis of urine and serum samples by both procedures.     

Nuclear factor of activated T cells negatively regulates expression of the tumor necrosis factor receptor-related 2 gene in T cells

Tumor necrosis factor receptor-related 2 (TR2, HVEM or TNFRSF-14) plays an important role in immune responses, however, the mechanisms regulating its expression are unclear. To understand the control of TR2 gene expression, we studied the upstream region of the gene. Gel supershift assays revealed inducible binding of nuclear factor of activated T cells (NFAT) to a putative NFAT site within the TR2 promoter. Furthermore, cotransfection of a dominant negative NFAT construct, or siRNA for NFAT, resulted in increased expression of a TR2 reporter gene. Our findings demonstrate that NFAT negatively regulates TR2 expression in activated T cells.