B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells.

Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.

Source localization of averaged and single EEG spikes using the electric dipole model.

A scheme for localizing the epileptic focus is proposed. The scheme is on the basis of a model with an electric dipole inside a four-layer inhomogeneous spherical model of head and utilizes a nonlinear programming algorithm applying the gradient projection method. Various initial estimates are used to prove the stability of the implemented dipole model. Fourteen single spike data and the averaged spike data are used to localize the epileptic focus. The results of the usage of the averaged spike data show that the dipole position is compatible with visual inspection of experienced clinical physicians. The results of the usage of the single spike show that 11 of the 14 single spikes have dipole locations near the result of the averaged spike but the estimated dipole moments differ markedly from one another. The localization results of the other three single spikes show that the dipole position is strongly affected by the background EEG. This kind of interference generally causes the eccentricity of the dipole to deviate from its anatomically meaningful value. According to our results, the electric dipole model is concluded to be valuable for the clinical application of localizing epileptic foci.

Evaluation of parametric methods in EEG signal analysis.

In this paper, a well designed database, considering statistical characteristics and including all types of Electroencephalogram (EEG) is built. 900 EEG segments, each with a short interval (1.024 sec) in this database are clustered into eight classes. Three tests of white noise for evaluating the efficiency of autoregressive (AR) and autoregressive-moving average (ARMA) models are proposed. The Akaike information criterion (AIC) is used for determining orders of AR and ARMA models. The AR model requires a higher model order (8.67 on the average) than the ARMA model (6.17 on the average). However, it is found that about 96% of the 900 segments can be efficiently represented by the AR model, and only about 78% of them can be efficiently represented by ARMA model. We therefore conclude that the AR model is preferred for estimating EEG signals.

Photodynamic therapy of idiopathic subfoveal choroidal neovascularization in Taiwanese patients: a 2-year follow-up.

AIMS: To study the efficacy of verteporfin photodynamic therapy (VPDT) retrospectively in the treatment of idiopathic subfoveal choroidal neovascularization (ICNV) in an Asian population in correlation with number of treatments and age at treatment. This is the first report to compare the efficacy between single and multiple treatments. METHODS: VPDT was administered according to protocol to 45 eyes in 45 patients aged 18-55 years diagnosed with active subfoveal ICNV between September 2003 and December 2005. In total 28 patients received a single VPDT treatment and the remaining 17 received multiple treatments. Collected measurements of visual acuity (VA) were plotted on a time-course model, and later dichotomized by age (18-45 vs 46-55 years). RESULTS: The 28 patients receiving a single VPDT treatment showed significant improvement in VA at 3-month follow-up. The 17 patients, who did not show improvement after the first treatment, received multiple VPDT treatments. Those patients showed an even clearer trend in VA improvement although significance was detected only at the 24th month. All patients showed a significant improvement in mean VA of 0.46 logMAR (P<0.01 compared to baseline) by the end of the 24-month observation period, although VPDT treatment for subfoveal ICNV appears to stabilize vision more rapidly in younger patients. CONCLUSIONS: ICNV patients who did not benefit from single VPDT treatments could receive multiple treatments, and showed a more significant improvement in visual acuity. These results are the first of their kind in ICNV treatment.

Aggregation and concentration-dependent sorting of exocrine regulated secretory proteins.

Sorting between the regulated and the constitutive secretory pathway in exocrine cells is thought to involve aggregation of regulated secretory proteins. This study demonstrates that, unlike endocrine secretory proteins, exocrine secretory proteins, including amylase, do not undergo homotypic aggregation under the conditions found in the sorting organelles. Also, unlike other exocrine proteins, amylase does not aggregate with chondroitin sulfate. Since amylase exhibits heterotypic aggregation, the role of protein concentration in amylase sorting was tested in AR42J cells. Secretion was stimulated with substance P and cholecystokinin from both untreated and dexamethasone-treated cells, with more efficient stimulation from dexamethasone-treated cells. These results indicate that amylase sorting is enhanced when its expression is stimulated by dexamethasone treatment.

A highly sensitive affinity-column-mediated immunometric assay, as exemplified by digoxin.

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.

Enzyme-amplified rate conductimetric immunoassay.

A new immunoassay technique based on measurement of conductance changes in solutions is described. The assay employs an immobilized monoclonal antibody to capture a protein analyte along with a second antibody to the same analyte, conjugated to an enzyme capable of producing ions which are measured conductimetrically. Urease was selected as the enzyme, because it produces, from urea, four ions for each catalytic event. The analyte studied was human chorionic gonadotropin in serum. Higher concentrations of analyte during incubation with immobilized antibody and antibody-urease conjugate led to increased binding of the latter. After removal of unbound conjugate, urea solution was added and the rate of conductance change measured in the bulk substrate solution. Experiments, performed in polystyrene microtiter wells using a specially designed electrode, demonstrated the ability to measure 30 picomolar concentrations of human chorionic gonadotropin with a 30-s rate measurement. Urease proved to be an excellent labeling enzyme, retaining its activity under the nonionic conditions necessary to maintain low background conductance. Good agreement was obtained between observed rates and those expected from conductimetric theory and known physical parameters. The potential utility of the conductimetric immunoassay lies in the fabrication of biosensor devices for simplification and cost reduction of immunochemical-based instrumentation. Further improvements to the technique are proposed to achieve lower detection limits.

An homogeneous fluorescence polymerase chain reaction assay to identify Salmonella.

We have developed a semiquantitative homogeneous fluorescence assay that combines polymerase chain reaction (PCR) amplification with direct fluorescence detection (HF-PCR). The assay eliminates the need to perform gel electrophoresis on test samples. Using a set of Salmonella-specific primers, this system was used to verify suspect colonies from culture plates as Salmonella. The fluorescence signal is generated by a nucleic acid dye, YO-PRO-1, that is included in the amplification reaction. This homogeneous PCR assay was used to test 84 Salmonella strains picked from selective culture plates. All data indicated positive results when compared with 17 non-Salmonella strains (in general, Citrobacter, Hafnia, Proteus, and Escherichia). The HF-PCR assay is a sensitive, simple, accurate, and reproducible method that correlates well with size-exclusion high-performance liquid chromatography and gel electrophoresis techniques as a means to monitor PCR-mediated DNA amplification. This assay can confirm suspect colonies within 2.5 h.

Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin.

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.

Application of novel chromium dioxide magnetic particles to immunoassay development.

We have used chromium dioxide magnetic particles as the solid support in developing a series of immunological tests. The high surface area (greater than 40 m2/g) available on the magnetic particles and their easy dispersion throughout a solution allow for rapid and complete capture of the target antigen. The magnetic responsiveness of the particles allows for rapid, high-efficiency washing to reduce nonspecific binding, which often limits the sensitivity of serological assays. These features form the basis of extremely rapid and flexible assays for several hormones and markers of cancer and infectious disease. Most of the assays involve monoclonal antibodies. Here we describe specific performance characteristics for thyroxin, follitropin, creatine kinase isoenzyme MB, and antibody to human immunodeficiency virus (HIV). All of the assays are performed in less than 90 min, many in 30 to 45 min. The technology is highly flexible and is suitable for a variety of formats, from manual to fully automated.