Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA).

Early diagnosis of acute myocardial infarction (AMI) is of outmost importance to reduce the mortality rate, and cardiac troponins are considered the gold standard biomarkers of myocardial necrosis. In this scenario, the characterization of two troponin T (TnT)-binding aptamers as viable alternative to antibodies employed on clinical immunoassays is here reported for the first time. Their recognition ability was first investigated through surface plasmon resonance (SPR). Subsequently, an enzyme-linked oligonucleotide assay (ELONA) was developed on common 96-well polystyrene plates, both by direct and sandwich detection strategies for comparison. In both cases, the assay exhibits a detection ability of TnT in the range of low nanomolar but a great advantage on serum interference was obtained by using both aptamers in a sandwich format, with excellent reproducibility and recovery values. Despite the sensitivity needing to be enhanced to the low picomolar range, these results are encouraging for the development of new, low-cost, and rapid antibody-free colorimetric assays for AMI studies based on aptamer-Troponin T recognition.

Colorimetric determination of p-nitrophenol by using ELISA microwells modified with an adhesive polydopamine nanofilm containing catalytically active gold nanoparticles.

A microplate method is described for the quantification of p-nitrophenol (p-NPh) in urine samples where it can be found after exposure to certain insecticides such as methyl parathion or paraoxon. The assay is based on the use of a polydopamine (PDA) film doped with gold nanoparticles (AuNPs). The latter exerts a catalytic effect on the reduction of nitrophenols by NaBH4. PDA has adhesive properties and can be used to fix the AuNPs on several solid substrates, here ELISA polystyrene microwells. The optical and catalytic properties of different populations of AuNPs spontaneously grown on PDA films were investigated, mainly in terms of the relationship between AuNPs@PDA nanocomposite preparation and its catalytic activity and stability. The reduction of o-, m-, and p-nitrophenols by NaBH4 in aqueous solution was exploited as model study. The approach demonstrates that useful kinetic information on the catalytic effect can be obtained on 96-wells simultaneously by a conventional ELISA reader at a fixed wavelength of 415 nm. The method was successfully applied to the quantification of p-NPh in (spiked) urine samples and gave high reproducibility (RSD = 3.5%) and a 6.30 µM (836 µg/L) detection limit. Graphical abstract Schematic presentation of 96-wells preparation for optical quantification on ELISA reader of p-nitrophenol (p-NPh) catalytic reduction to p-aminophenol (p-APh), as model study, by NaBH4 and different population gold nanoparticles (AuNPs) grown on polydopamine (PDA) films attached onto polystyrene (PS) wells.

Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells.

The ability of a rigid α-Tn mimetic (compound 1) to activate murine invariant natural killer T (iNKT) and human natural killer (NK) cells, two subsets of lymphocytes involved in cancer immunesurveillance, was investigated. For this purpose, the mimetic 1 was properly conjugated to a stearic acid containing glycerol-based phospholipid (compound 5) to be presented, in the context of the conserved non polymorphic major histocompatibility complex class I-like molecules (CD1d), to iNKT cells. On the contrary, the mimetic 1 was conjugated to a multivalent peptide-based scaffold (compound 6) to induce NK cell activation.

Colorimetric determination of total protein content in serum based on the polydopamine/protein adsorption competition on microplates.

We report a facile, low-cost and safe analytical method for estimation of total protein content, even in complex matrix like human serum, by exploiting the competition between proteins and polydopamine (PDA) for surface binding. The surface coating has been examined by using a microplate reader taking the advantage of the PDA absorbance in the visible region, obtaining new insights into the modelling of polydopamine deposition and polymer/protein adsorption competition. This is helpful for rational development of imprinted biosensors, and potentially offering a with broad applications ranging from diagnostic tools in medicine to food analysis. The isothermal adsorption of polydopamine on polystyrene surface of multi-well plate displays a Langmuir-shaped curve. It allows the determination of the parameters of polymer film formation useful for any analytical assay depending on the surface coating. Among these, the molecular imprinting and the optical and acoustic evanescent sensing.

A replication-incompetent CD154/40L recombinant vaccinia virus induces direct and macrophage-mediated antitumor effects in vitro and in vivo.

CD40 triggering may result in antitumor effects of potentially high clinical relevance. To gain insights important for patient selection and to identify adequate targeting techniques, we investigated CD40 expression in human cancer tissues and generated a replication-incompetent recombinant vaccinia virus expressing CD40 ligand (rVV40L). Its effects were explored in vitro and in vivo upon direct CD40 targeting on malignant cells or macrophage activation. CD40 expression was analyzed by immunohistochemistry in tumor and stromal cells in a multi-tumor array including 836 specimens from 27 different tumor types. Established tumor cell lines were used to explore the capacity of rVV40L to induce malignant cell apoptosis and modulate functional profiles of polarized macrophages. CD40 expression was detectable in significantly higher numbers of stromal as compared to malignant cells in lung and breast cancers. CD40 ligation following rVV40L infection induced apoptosis in CD40(+) cancer cells, but only in the presence of intact specific signal transduction chain. Importantly, rVV40L infection promoted the induction of TNF-α-dependent antitumor activity of M1-like macrophages directed against CD40(-) targets. CD40-activated M1-like macrophages also displayed enhanced ability to CXCL10-dependently recruit CD8+ T cells and to efficiently present cancer cell intracellular antigens through cross-priming. Moreover, rVV-driven CD40L expression partially "re-educated" M2-like macrophages, as suggested by detectable CXCL10 and IL-12 production. Most importantly, we observed that intra-tumoral injection of rVV40L-infected human macrophages inhibits progression of human CD40(-) tumors in vivo. First evidences of anticancer activity of rVV40L strongly encourage further evaluations.

"In vitro" studies on galectin-3 in human natural killer cells.

Galectin-3 (Gal-3) is a ß-galactoside binding protein able to modulate both innate and adaptive immune responses. First identified in macrophages, Gal-3 has been studied widely in many mammalian immune cells, but scarcely in natural killer (NK) cells. The aim of this study was to analyze Gal-3 in human NK cells, isolated from peripheral blood mononuclear cells. Both PCR and RT-PCR analysis showed that resting human NK cells express Gal-3 mRNA, which can be modulated upon cytokine stimulation (100 U/ml IL-2 + 20 ng/ml IL-15) for different period of time (1-24 h). Western blot, cytofluorimetry, and confocal microscopy analysis clearly demonstrated that the Gal-3 gene can translate into the corresponding protein. From our results, resting NK cells, isolated from different healthy donors, can express high or low basal levels of Gal-3. In NK cells, Gal-3 was always intracellularly detected at both cytoplasm and nucleus levels, while never at the membrane surface, and its localization resulted independent from the cellular activation status. In addition, the intracellular Gal-3 can co-localize with perforin in exocytic vesicles. Cell treatment with a thiodigalactoside-based Gal-3 inhibitor (1-30 µM) slightly increased the number of degranulating NK cells, while it significantly increased the percentage of cells releasing high amounts of cytotoxic granules (+ 36 ±â€¯3% vs. inhibitor-untreated cells at 30 µM Gal-3). In conclusion, our results demonstrate that human resting NK cells express Gal-3 at both gene and protein levels and that the Gal-3 expression can be modulated upon cytokine stimulation. In the same cells, Gal-3 always localizes intracellularly and functionally correlates with the degree of NK cell degranulation.