Digital ELISA for the quantification of attomolar concentrations of Alzheimer's disease biomarker protein Tau in biological samples.

The close correlation between Tau pathology and Alzheimer's disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ±â€¯7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ±â€¯29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau values. The presented digital ELISA technology has great capacity in unlocking the potential of Tau as biomarker for early AD diagnosis.

Pretargeted PET Imaging Using a Bioorthogonal 18F-Labeled trans-Cyclooctene in an Ovarian Carcinoma Model.

In cancer research, pretargeted positron emission tomography (PET) imaging has emerged as an effective two-step approach that combines the excellent target affinity and selectivity of antibodies with the advantages of using short-lived radionuclides such as fluorine-18. One possible approach is based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between tetrazines and trans-cyclooctene (TCO) derivatives. Here, we report the first successful use of an 18F-labeled small TCO compound, [18F]1 recently developed in our laboratory, to perform pretargeted immuno-PET imaging. The study was performed in an ovarian carcinoma mouse model, using a trastuzumab-tetrazine conjugate.

Visualization of delayed release of compounds from pH-sensitive capsules in vitro and in vivo in a hamster model.

Delayed controlled release is an innovative strategy to locally administer therapeutic compounds (e.g. chemotherapeutics, antibodies etc.). This would improve efficiency and reduce side effects compared with systemic administration. To enable the evaluation of the efficacy of controlled release strategies both in vitro and in vivo, we investigated the release of contrast agents ((19)F-FDG and BaSO4) to the intestinal tract from capsules coated with pH-sensitive polymers (EUDRAGIT L-100) by using two complementary techniques, i.e. (19)F magnetic resonance imaging (MRI) and computed tomography (CT). Using in vitro (19)F-MRI, we were able to non-destructively and dynamically establish a time window of 2 h during which the capsules are resistant to low pH. With (19)F-MRI, we could establish the exact time point when the capsules became water permeable, before physical degradation of the capsule. This was complemented by CT imaging, which provided longitudinal information on physical degradation of the capsule at low pH that was only seen after 230 min. After oral administration to hamsters, (19)F-MRI visualized the early event whereby the capsule becomes water permeable after 2 h. Additionally, using CT, the integrity and location (stomach and small intestines) of the capsule after administration could be monitored. In conclusion, we propose combined (19)F-MRI and CT to non-invasively visualize the different temporal and spatial events regarding the release of compounds, both in an in vitro setting and in the gastrointestinal tract of small animal models. This multimodal imaging approach will enable the in vitro and in vivo evaluation of further technical improvements to controlled release strategies.

Development of a universal anti-adalimumab antibody standard for interlaboratory harmonization.

BACKGROUND: Therapeutic drug monitoring of adalimumab (ADM) has been introduced recently. When no detectable ADM serum concentrations can be found, the formation of antidrug antibodies (ADA) should be investigated. A variety of assays to measure the occurrence of ADA have been developed. Results are expressed as arbitrary units or as a titration value. The aim was to develop a monoclonal antibody (MA) that could serve as a universal calibrator to quantify the amount of ADA in ADM-treated patients. METHODS: Hybridoma technology was used to generate a MA toward ADM. The functionality of the MA was tested in a bridging enzyme linked immunosorbent assay (ELISA) setup and in a cell-based assay. Sera from 25 anti-tumor necrosis factor naive patients with inflammatory bowel disease were used to determine the cutoff values. Sera from 9 ADM-treated patients with inflammatory bowel disease, with undetectable serum concentrations of ADM were used to quantify the ADA response. RESULTS: In this study, MA-ADM6A10, an IgG1 that can be used as a calibrator in both an ELISA to quantify the amount of binding antibodies and in a cell-based assay to quantify the amount of neutralizing antibodies, was generated. Combining the results of both assays showed that the sera with high concentrations of anti-ADM binding antibodies also had the highest neutralizing capacity. CONCLUSIONS: The availability of a universal calibrator could facilitate the interlaboratory harmonization of antibody titers in patients who develop anti-adalimumab antibodies.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

Down-regulation of the epidermal growth factor receptor by altering N-glycosylation: emerging role of ß1,4-galactosyltransferases.

BACKGROUND/AIM: Blocking N-glycosylation of the epidermal growth factor receptor (EGFR) by tunicamycin inhibits its cellular accumulation. Due to the toxic potential of this drug, finding less drastic routes to reduce EGFR expression is desirable. MATERIALS AND METHODS: Four glycosylation mutants of Chinese hamster ovary (CHO) cells with defects in N-glycan processing and branch-end maturation were tested for EGFR gene expression, production, functionality and routing after transfection with a vector encoding for human EGFR. RESULTS: Lack of conversion of paucimannosidic to hybrid/complex-type N-glycans and drastic reductions in sialylation/galactosylation did not lead to major effects. In contrast, EGFR expression in a mutant with reduced presence of ß1,4-galactosyltransferases-I-VI was markedly reduced. Misrouting or defects in transfection/transcription were excluded. CONCLUSION: ß1,4-Galactosyl-transferases warrant for further attention as effector(s) in order to attenuate EGFR-dependent signaling.

Activation of the BRCA1/Chk1/p53/p21(Cip1/Waf1) pathway by nitric oxide and cell cycle arrest in human neuroblastoma NB69 cells.

Nitric oxide (NO) works as a bi-modal effector of cell proliferation, inducing either the increase or decrease of cell growth when cells are exposed, respectively, to low or high NO concentrations. To get further insight into the action of NO, we tested the effect of short- and long-lived NO donors on the control of the cell cycle in human neuroblastoma NB69 cells. We demonstrated that long-time exposure of cells to NO not only decreased the expression and/or the phosphorylation of elements involved in the control of the G(1)/S transition, such as the transcriptional repressor pRb and cyclin D1, but also down-regulated systems controlling the S and G(2)/M phases, such as the phosphorylation of Cdk1(cdc2) and the expression of cyclins A and B1. Increasing concentrations of NO also induced a biphasic effect on the expression of cyclins D1, A and B1, while this effect was less pronounced for cyclin E expression, but the levels of mRNAs of those cyclins changed in a distinct and complex manner. NO also changed the phosphorylation pattern of cyclin E and decreased the levels of phospho-cyclins D1 and B1. Moreover, NO decreased the expression of the Cdk inhibitors p16(Ink4a) and p19(Ink4d), without affecting p27(Kip1). In contrast, NO induced a biphasic effect on p21(Cip1/Waf1) expression. The BRCA1/Chk1/p53 pathway mediated the upregulation of p21(Cip1/Waf1). We also demonstrated that the NO-mediated up-regulation of p21(Cip1/Waf1) was inversely correlated with the activation status of the p38MAPK pathway.

Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells.

It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including α2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation.

Protective role of the inhibitor of apoptosis protein, survivin, in toxin-induced acute renal failure.

Acute renal failure (ARF) is a major worldwide cause of morbidity and mortality, lacking specific targeted, effective therapies. Renal tubular cell apoptosis has been recognized to play a critical role in the pathogenesis of ARF, yet few studies have evaluated whether intervention in apoptotic pathways can ameliorate the deterioration in renal function associated with ARF. Using transgenic mice with diminished levels of the inhibitor of apoptosis protein, survivin, we show that survivin is required to protect the kidney from apoptosis, to suppress renal expression of p53, and to ameliorate renal dysfunction in two models of ARF. Gene delivery of survivin to wild-type mice and mice with 50% levels of survivin, prior to or at the time of induction of ARF, interferes with the deterioration of renal function and preserves the integrity of the kidneys and the renal tubular cells by inhibiting activation of apoptotic pathways in the kidneys and suppressing expression of p53. These results encourage further evaluation of survivin, its active structural domains, and other inhibitors of apoptosis proteins, for preventing and/or treating acute renal failure.

Thrombomodulin-protein C-EPCR system: integrated to regulate coagulation and inflammation.

Late in the 18th century, William Hewson recognized that the formation of a clot is characteristic of many febrile, inflammatory diseases (Owen C. A History of Blood Coagulation. Rochester, Minnesota: Mayo Foundation; 2001). Since that time, there has been steady progress in our understanding of coagulation and inflammation, but it is only in the past few decades that the molecular mechanisms linking these 2 biologic systems have started to be delineated. Most of these can be traced to the vasculature, where the systems most intimately interact. Thrombomodulin (TM), a cell surface-expressed glycoprotein, predominantly synthesized by vascular endothelial cells, is a critical cofactor for thrombin-mediated activation of protein C (PC), an event further amplified by the endothelial cell protein C receptor (EPCR). Activated PC (APC), in turn, is best known for its natural anticoagulant properties. Recent evidence has revealed that TM, APC, and EPCR have activities that impact not only on coagulation but also on inflammation, fibrinolysis, and cell proliferation. This review highlights recent insights into the diverse functions of this complex multimolecular system and how its components are integrated to maintain homeostasis under hypercoagulable and/or proinflammatory stress conditions. Overall, the described advances underscore the usefulness of elucidating the relevant molecular pathways that link both systems for the development of novel therapeutic and diagnostic targets for a wide range of inflammatory diseases.

Novel functions of thrombomodulin in inflammation.

The objective of this study was to review the mechanisms by which thrombomodulin (TM) may modulate inflammation. The data were taken from published research performed by other laboratories and our own experimental results. TM is a transmembrane glycoprotein receptor and cofactor for thrombin in the protein C anticoagulant system. Recent studies have revealed that TM has activities, both dependent and independent of either protein C or thrombin, that affect biological systems beyond the coagulation pathway. This review highlights recent insights, provided by in vitro and in vivo analyses, into how the unique structural domains of TM effectively modify coagulation, fibrinolysis, and inflammation in health and disease. A paradigm is presented to describe how these apparently distinct functions are integrated to maintain homeostasis under stress conditions. Finally, we explore the potential diagnostic and therapeutic utility of dissecting out the structure-function correlates of TM. We conclude that TM plays a central role in regulating not only hemostasis but also inflammation, thus providing a close link between these processes. Elucidation of the molecular mechanisms by which TM functions will likely provide novel targets for therapeutic intervention.

The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways.

Thrombomodulin (TM) is a vascular endothelial cell (EC) receptor that is a cofactor for thrombin-mediated activation of the anticoagulant protein C. The extracellular NH(2)-terminal domain of TM has homology to C-type lectins that are involved in immune regulation. Using transgenic mice that lack this structure (TM(LeD/LeD)), we show that the lectin-like domain of TM interferes with polymorphonuclear leukocyte (PMN) adhesion to ECs by intercellular adhesion molecule 1-dependent and -independent pathways through the suppression of extracellular signal-regulated kinase (ERK)(1/2) activation. TM(LeD/LeD) mice have reduced survival after endotoxin exposure, accumulate more PMNs in their lungs, and develop larger infarcts after myocardial ischemia/reperfusion. The recombinant lectin-like domain of TM suppresses PMN adhesion to ECs, diminishes cytokine-induced increase in nuclear factor kappaB and activation of ERK(1/2), and rescues ECs from serum starvation, findings that may explain why plasma levels of soluble TM are inversely correlated with cardiovascular disease. These data suggest that TM has antiinflammatory properties in addition to its role in coagulation and fibrinolysis.