Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

Background: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence.

Methods: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues.

pH dependence of water anomaly temperature investigated by Eu(III) cryptate luminescence.

Although water has been extensively studied, not all of its unique properties have been fully understood. There is still controversy about the temperature at which hydrogen bonds are broken or weakened, producing the anomalous temperature dependence of many water properties. Different temperatures between 23 and 48 °C have been reported, but no study has scrutinized the reasons for this discrepancy.

Neuregulin 1 Allosterically Enhances the Antitumor Effects of the Noncompeting Anti-HER3 Antibody 9F7-F11 by Increasing Its Binding to HER3.

Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.

Examination of HER3 targeting in cancer using monoclonal antibodies.

The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity.

Evaluation of the dimerization profiles of HER tyrosine kinases by time-resolved Förster resonance energy transfer (TR-FRET).

Activation of receptor tyrosine kinases (RTK), such as those belonging to the human epidermal growth factor receptor (HER) family, occurs only after receptor dimerization, which is a crucial step for cellular signal transduction and diversification. The HER family includes four members (EGFR/HER1, HER2, HER3, and HER4) that can homodimerize or heterodimerize. Here, we describe immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET) to profile EGFR-EGFR, HER2-HER2, and EGFR-HER2 dimers directly in tumor samples.

HER3 as biomarker and therapeutic target in pancreatic cancer: new insights in pertuzumab therapy in preclinical models.

The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression.

Luminescent lanthanide cryptates: from the bench to the bedside.

The design and application of luminescent lanthanide cryptates for sensing biological interactions is highlighted through the review of the work performed in our laboratory and with academic collaborations. The path from the initial applications probing biochemical interaction in vitro to "state-of-the-art" cellular assays toward clinical applications using homogeneous time-resolved fluorescence technology is described. An overview of the luminescent lanthanide macrocyclic compounds developed at Cisbio in the recent past is given with an emphasis on specific constraints required by specific applications.

Endogenous morphine-6-glucuronide (M6G) is present in the plasma of patients: validation of a specific anti-M6G antibody for clinical and basic research.

Endogenous morphine and its derivatives (morphine-6-glucuronide [M6G]; morphine-3-glucuronide [M3G]) are formed by mammalian cells from dopamine. Changes in the concentrations of endogenous morphine have been demonstrated in several pathologies (sepsis, Parkinson's disease, etc.), and they might be relevant as pathological markers.

Synthesis and optical properties of macrocyclic lanthanide(III) chelates as new reagents for luminescent biolabeling.

The convenient and efficient synthesis of two macrocyclic ligands (15- and 18-membered) based on a dipyrido-6,7,8,9-tetrahydrophenazine (dpqc) or 2,2':6',2''-terpyridine (tpy) heterocycle and a DTTA (diethylenetriaminetriacetic acid) skeleton is described. In these ligands the DTTA skeleton contains an additional extracyclic functionality (NH(2) group) suitable for covalent attachment to bioactive molecules. These octa- and nonadentate ligands form very stable and luminescent neutral lanthanide complexes in aqueous solutions at physiological pH.

Quantification of HER expression and dimerization in patients' tumor samples using time-resolved Förster resonance energy transfer.

Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers.

In vivo phage display to identify new human antibody fragments homing to atherosclerotic endothelial and subendothelial tissues [corrected].

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues.

[History of vaccine refusal].

The human fight against infectious diseases is ancient and ongoing. It started with variolation, probably in China in the 17th century and in the West at the beginning of the 18th century. Like most innovations it aroused a good deal of opposition.

Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies.

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines.

Time-resolved FRET between GPCR ligands reveals oligomers in native tissues.

G protein-coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells.

Antimicrobial resistance of Listeria monocytogenes strains isolated from humans in France.

Susceptibility to antibiotics of 4,816 clinical L. monocytogenes strains isolated since 1926 was studied, and the temporal evolution of susceptibility to antibiotics was analyzed through several decades. The mechanisms of resistance in each resistant strain were studied.

A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.

Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin.

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities.

Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands.

A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods.

Luminescent Eu(III) and Gd(III) trisbipyridine cryptates: experimental and theoretical study of the substituent effects.

Synthesis, absorption spectra and luminescebce properties of a series of lanthanide trisbipyridine cryptates Ln within R-Bpy x R-Bpy x R-Bpy, where Ln = Eu, Gd and R = H, COOH, COOCH3, CONH(CH2)2NH2 are described. Comparison of the unsubstituted parent compound with the substituted compounds shows that bipyridine substitution doesn't alter significantly the photophysical properties of the lanthanide cryptate. The absorption maximum is slightly red-shifted when three bipyridines are substituted, whereas substituting one bipyridines has a negligible effect on the absorption spectra.

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation.

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses.

Positively charged GdIII cryptates: slow, associative water exchange.

A combined variable-temperature and multiple field 17O NMR, EPR and NMRD study has been performed for the first time on gadolinium(III) complexes of cryptand ligands, L1 and L2, where L1 contains three 2,2'-bipyridine units ([bpy.bpy.bpy]) and L2 is the disubstituted methyl ester derivative of L1.

Anti-CD2 monoclonal antibody and tacrolimus in adult liver transplantation.

Background: Blockade of costimulation and adhesion signaling is an attractive approach to interfere with graft rejection

Methods: Between January 1997 and May 1999, forty adults having benign liver diseases were included in a prospective, randomized study comparing tacrolimus plus low-dose short-term steroids without (n=20, TAC group) or with a 10-day course of antihuman CD2 monoclonal antibody (n=20, BTI group).

Results: At day 7, histological rejection expressed by mean Banff scores (2.3+/-1.

Microwaves synthesis of solid supports for the synthesis of 3'-aminoalkyl oligodeoxynucleotides.

Phthalimido-alkyl alcohol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 13-aminoalkyl oligodeoxynucleotides allowing a two step deprotection necessary to avoid aminolink alkylation.

A separation-free assay for the detection of mutations: combination of homogeneous time-resolved fluorescence and minisequencing.

Single nucleotide primer extension reaction has been widely used in DNA testing, and several detection methods based on this core allelic discrimination have been developed. Most of the reported formats are based on a two step protocol involving first, a liquid phase extension reaction, then a physical separation process (chromatography, electrophoresis, capture on solid support, mass spectrometry). Here we describe a new strategy based on homogeneous time-resolved fluorescence (HTRF), which does not involve any separation process and which allows a simple "mix and measure" protocol.

Optimized synthesis of functionalized fluorescent oligodeoxynucleotides for protein labeling.

Phthalimido-alkanol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 5'-fluorescent 3'-aminoalkyl oligodeoxynucleotides allowing a two-step deprotection necessary to avoid aminolink alkylation. After conversion into an NHS derivative using dissuccinimidyl suberate and an optimized isolation, they were conjugated with a protein.