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1.
FASEB J ; 35(5): e21540, 2021 05.
Article in English | MEDLINE | ID: mdl-33817838

ABSTRACT

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the HCN and HCC subdomains of the BoNT/A1 receptor-binding domain (HC ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to HC confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Botulinum Toxins, Type A/immunology , Epitopes/immunology , Gangliosides/metabolism , Membrane Glycoproteins/metabolism , Nerve Tissue Proteins/metabolism , Neuromuscular Agents/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/metabolism , Botulinum Toxins, Type A/metabolism , Mice , Neuromuscular Agents/metabolism , Protein Conformation
2.
Alzheimers Dement (N Y) ; 6(1): e12046, 2020.
Article in English | MEDLINE | ID: mdl-32642550

ABSTRACT

INTRODUCTION: An effective therapy has not yet been developed for Alzheimer's disease (AD), in part because pathological changes occur years before clinical symptoms manifest. We recently showed that decreased plasma DYRK1A identifies individuals with mild cognitive impairment (MCI) or AD, and that aged mice have higher DYRK1A levels. METHODS: We assessed DYRK1A in plasma in young/aged controls and in elderly cognitive complainers with low (L) and high (H) brain amyloid load. RESULTS: DYRK1A level increases with age in humans. However, plasma from elderly individuals reporting cognitive complaints showed that the H group had the same DYRK1A level as young adults, suggesting that the age-associated DYRK1A increase is blocked in this group. L and H groups had similar levels of clusterin. DISCUSSION: These results are reflective of early changes in the brain. These observations suggest that plasma DYRK1A and not clusterin could be used to classify elderly memory complainers for risk for amyloid beta pathology.

3.
Chem Commun (Camb) ; 56(52): 7183-7186, 2020 Jul 04.
Article in English | MEDLINE | ID: mdl-32463407

ABSTRACT

In this article, we report the synthesis and use of iminosydnone-based profluorophores as bioorthogonal cleavable linkers for imaging applications. These linkers react with cycloalkynes via subsequent [3+2] cycloaddition and retro Diels-Alder reactions, allowing simultaneous release of two dyes in biological media.


Subject(s)
Fluorescent Dyes/chemical synthesis , Sydnones/chemical synthesis , Animals , CHO Cells , Catalysis , Click Chemistry , Cricetulus , Cycloaddition Reaction , Cyclooctanes/chemistry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/metabolism , Optical Imaging , Palladium/chemistry , Pyrazoles/chemistry , Solvents/chemistry , Structure-Activity Relationship , Sydnones/metabolism
4.
Biosensors (Basel) ; 9(3)2019 Aug 31.
Article in English | MEDLINE | ID: mdl-31480476

ABSTRACT

Inexpensive simple medical devices allowing fast and reliable counting of whole cells are of interest for diagnosis and treatment monitoring. Magnetic-based labs on a chip are one of the possibilities currently studied to address this issue. Giant magnetoresistance (GMR) sensors offer both great sensitivity and device integrability with microfluidics and electronics. When used on a dynamic system, GMR-based biochips are able to detect magnetically labeled individual cells. In this article, a rigorous evaluation of the main characteristics of this magnetic medical device (specificity, sensitivity, time of use and variability) are presented and compared to those of both an ELISA test and a conventional flow cytometer, using an eukaryotic malignant cell line model in physiological conditions (NS1 murine cells in phosphate buffer saline). We describe a proof of specificity of a GMR sensor detection of magnetically labeled cells. The limit of detection of the actual system was shown to be similar to the ELISA one and 10 times higher than the cytometer one.


Subject(s)
Biosensing Techniques/instrumentation , Magnetics/methods
5.
Angew Chem Int Ed Engl ; 58(19): 6366-6370, 2019 05 06.
Article in English | MEDLINE | ID: mdl-30856679

ABSTRACT

A bioorthogonal approach is explored to release the content of nanoparticles on demand. Exploiting our recently described click-and-release technology, we developed a new generation of cleavable micelles able to disassemble through a sequential enzymatic and bioorthogonal activation process. Proof-of-concept experiments showed that this new approach could be successfully used to deliver the substances encapsulated into micelles in living cells as well as in mice by two complementary targeted strategies.


Subject(s)
Micelles , Pharmaceutical Preparations/metabolism , Alkynes/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Click Chemistry , Cyclooctanes/chemistry , Drug Liberation , Glucuronides/chemistry , Humans , Kinetics , Mice , Nanoparticles/chemistry , Neoplasms/drug therapy , Neoplasms/pathology , Pharmaceutical Preparations/chemistry , Tetrazoles/chemistry , Transplantation, Heterologous
6.
MAbs ; 8(7): 1371-1385, 2016 10.
Article in English | MEDLINE | ID: mdl-27390909

ABSTRACT

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Endothelin B Receptor Antagonists/pharmacology , Melanoma , Receptor, Endothelin B/immunology , Cell Line, Tumor , Humans
7.
J Immunol Methods ; 428: 37-41, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26657944

ABSTRACT

Antibodies (Abs) specific to cell-surface receptors are attractive tools for studying the physiological role of such receptors or for controlling their activity. We sought to obtain such antibodies against the type 1 receptor for melatonin (MT1). For this, we injected mice with CHO cells transfected with a plasmid encoding human MT1 (CHO-MT1-h), in the presence or absence of an adjuvant mixture containing Alum and CpG1018. As we previously observed that the immune response to a protein antigen is increased when it is coupled to a fusion protein, called ZZTat101, we also investigated if the association of ZZTat101 with CHO-MT1-h cells provides an immunogenic advantage. We measured similar levels of anti-CHO and anti-MT1-h Ab responses in animals injected with either CHO-MT1-h cells or ZZTat101/CHO-MT1-h cells, with or without adjuvant, indicating that neither the adjuvant mixture nor ZZTat101 increased the anti-cell immune response. Then, we investigated whether the antisera also recognized murine MT1 (MT1-m). Using cloned CHO cells transfected with a plasmid encoding MT1-m, we found that antisera raised against CHO-MT1-h cells also bound the mouse receptor. Altogether our studies indicate that immunizing approaches based on MT1-h-expressing CHO cells allow the production of polyclonal antibodies against MT1 receptors of different origins. This paves the way to preparation of MT1-specific monoclonal antibodies.


Subject(s)
Antibodies/immunology , Antibodies/isolation & purification , Immunization , Receptor, Melatonin, MT1/biosynthesis , Receptor, Melatonin, MT1/immunology , Animals , CHO Cells , Cricetulus , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL
8.
MAbs ; 5(1): 56-69, 2013.
Article in English | MEDLINE | ID: mdl-23221682

ABSTRACT

Endothelin B receptor (ETBR) is a G protein-coupled receptor able to bind equally to the three identified human endothelin peptides. It is expressed primarily on vascular endothelial cells and involved in various physiological processes including vascular tone homeostasis, enteric nervous system development, melanogenesis and angiogenesis. Furthermore, overactivation or overexpression of ETBR have been associated with the development of various diseases such as cardiovascular disorders and cancers. Therefore, ETBR appears to be relevant target for the therapy or diagnosis of highly prevalent human diseases. In this study, we report the in vitro characterization of rendomab-B1, a monoclonal antibody (mAb) obtained by genetic immunization, which selectively recognizes the native form of human ETBR (hETBR). Rendomab-B1 is the first-reported mAb that behaves as a potent antagonist of hETBR. It recognizes an original extracellular conformational epitope on the receptor, distinct from the endothelin-1 (ET-1) binding site. Rendomab-B1 not only blocks ET-1-induced calcium signaling pathway and triggers rapid receptor internalization on recombinant hETBR-expressing cells, but also exerts pharmacological activities on human vascular endothelial cells, reducing both cell viability and ET-1-induced hETBR synthesis. In addition, binding experiments using rendomab-B1 on different melanoma cell lines reveal the structural and functional heterogeneity of hETBR expressed at the surface of these cancer cells, strongly suggesting the existence of tumor-specific receptors. Collectively, our results underscore the value of rendomab-B1 for research, therapeutic and diagnostic applications dealing with hETBR.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Endothelin B Receptor Antagonists , Receptor, Endothelin B/immunology , Animals , CHO Cells/metabolism , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cricetinae , DNA/administration & dosage , Female , HEK293 Cells/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunization , Melanoma/metabolism , Mice , Mice, Inbred C57BL , Receptor, Endothelin B/genetics , Receptor, Endothelin B/metabolism , Receptors, G-Protein-Coupled/metabolism
9.
DNA Cell Biol ; 30(9): 727-37, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21688998

ABSTRACT

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.


Subject(s)
Antibody Formation/immunology , DNA/immunology , Electroporation/methods , Immunization/methods , Protein Conformation , Receptor, Endothelin B/immunology , Animals , CHO Cells , Cricetinae , Cricetulus , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay/methods , Epitope Mapping , Flow Cytometry/methods , Humans , Mice , Microscopy, Confocal , Plasmids/genetics , Polymerase Chain Reaction , Receptor, Endothelin B/genetics
10.
DNA Cell Biol ; 29(3): 121-31, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20025536

ABSTRACT

Prion diseases (e.g., Creutzfeldt-Jakob disease in humans) are always fatal neurodegenerative disorders characterized by conversion of the ubiquitous cellular prion protein (PrP(c)) into a pathological conformer. Immunological strategies are considered as promising prophylactic or therapeutic approaches but, unfortunately, vaccination attempts until now have been very disappointing in wild-type animals because of immune tolerance to self PrP(c). Encouraging results have come from recent experiments carried out through genetic immunization (i.e., injection in mice of cDNA coding for murine prion protein [PrP]) or heterologous protein immunization (i.e., injection in mice of PrP from another species), albeit the levels of autoantibodies in wild-type animals remained generally low. Here we investigated whether combining the potential benefits of these two last approaches, namely using genetic immunization with the cDNA coding for a heterologous PrP, could more efficiently break immune tolerance. Wild-type mice were thus vaccinated with cDNA coding for human PrP(c), fused or unfused to a stimulatory T-cell epitope, using or not using electrotransfer of DNA. After three DNA injections, mice receiving electrotransferred DNA developed a strong immune response, oriented toward the humoral Th2 type, characterized not only by high IgG1 and IgG2a antibody titers against the heterologous human PrP(c), but also, as expected, by significant amounts of autoantibodies recognizing the native conformation of murine PrP(c) expressed on cell membranes as revealed by flow cytometry and immunofluorescence. These results hence open the way for investigation of the possible protective effects of anti-PrP(c) autoantibodies in infected mouse models. More generally, our results suggest that this original immunization strategy could be of value for circumventing tolerance to poorly immunogenic proteins.


Subject(s)
Autoantibodies/immunology , DNA, Complementary/genetics , Electroporation , Immunization/methods , Prions/genetics , Prions/immunology , Vaccines/immunology , Animals , Epitopes, T-Lymphocyte/immunology , Genetic Vectors/genetics , Humans , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Species Specificity , Tetanus Toxin/genetics , Tetanus Toxin/immunology , Th2 Cells/immunology , Transfection
11.
J Neuroimmunol ; 209(1-2): 50-6, 2009 Apr 30.
Article in English | MEDLINE | ID: mdl-19232746

ABSTRACT

Prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans, are a group of devastating neurodegenerative disorders for which no therapy is yet available. However, passive immunotherapy appears to be a promising therapeutic approach, given that antibodies against the cellular prion protein (PrPc) have been shown in vitro to antagonize deposition of the disease-associated prion protein (PrPSc). Nevertheless, in vivo deleterious side effects of injected anti-PrP antibodies have been reported, mainly due to their Fc fragments and divalence. In this context, we examined here the ability of five Fabs (monovalent fragments devoid of the Fc part), prepared from antibodies already characterized in the laboratory, to inhibit prion replication in infected neuronal cells. We show that all Fabs (which all retain the same apparent affinity for PrPc as their whole antibody counterpart, as measured in EIA experiments) recognize quite well membrane bound-PrP in neuronal cells (as shown by flow cytometry analysis) and inhibit PrPSc formation in infected cells in a dose-dependent manner, most of them (four out of five) exhibiting a similar efficiency as whole antibodies. From a fundamental point of view, this report indicates that the in vitro curative effect of antibodies i) is epitope independent and only related to the efficiency of recognizing the native, membrane-inserted form of neuronal PrP and ii) probably occurs by directly or indirectly masking the PrPc epitopes involved in PrPSc interaction, rather than by cross-linking membrane bound PrPc. From a practical point of view, i.e. in the context of a possible immunotherapy of prion diseases, our data promote the use of monovalent antibodies (either Fabs or engineered recombinant fragments) for further in vivo studies.


Subject(s)
Antibodies/pharmacology , Peptide Fragments/pharmacology , Prion Diseases/drug therapy , Prion Diseases/immunology , Prions/antagonists & inhibitors , Prions/immunology , Animals , Antibodies/chemistry , Cell Line, Tumor , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/immunology , Dose-Response Relationship, Drug , Epitopes/immunology , Mice , Neurons/drug effects , Neurons/immunology , Peptide Fragments/chemical synthesis , PrPC Proteins/chemistry , PrPC Proteins/drug effects , PrPC Proteins/immunology , PrPSc Proteins/antagonists & inhibitors , PrPSc Proteins/chemistry , PrPSc Proteins/immunology , Prion Diseases/physiopathology , Prions/chemistry , Protein Binding/drug effects , Protein Binding/physiology
12.
Mol Immunol ; 46(6): 1076-83, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19012967

ABSTRACT

Immunization with anti-idiotypic (anti-Id) antibodies, used as surrogate antigens, has led to promising results, notably in active immunotherapy of cancers, essentially because it breaks immunological tolerance against self-tumor-associated antigens. The aim of the present study was to provide a proof-of-principle that this vaccination approach could be envisaged also in the field of prion diseases, caused by the accumulation of an aggregated pathological isoform of the highly tolerogenic self-prion protein (PrP), and for which no therapy is available. We investigated the possibility of raising anti-Id antibodies mimicking the human PrP (hPrP), using as immunogens either a peptide derived from the paratope of an anti-PrP mAb or the entire antibody. To this end, we cloned and sequenced SAF61 mAb, an anti-PrP antibody already produced in the laboratory, directed against a critical epitope of PrP involved in the aggregation process. A synthetic peptide (denoted CDR3L) was designed from the identification of a 17-amino-acid sequence encompassing the CDR3 region of the light chain whose hydropathic profile was opposed to that of PrP epitope. CDR3L peptide was directly demonstrated to bind hPrP, confirming the role of hydropathic complementarity in antigen-antibody interactions. When injected into rabbits, CDR3L generated anti-SAF61 anti-Id polyclonal antibodies that exclusively recognized SAF61 mAb but were unable to compete with hPrP for antibody binding. By contrast, immunizations with the entire SAF61 mAb generated anti-Id antibodies specifically competing with soluble or membrane-bound hPrP (in EIA or flow cytometry experiments, respectively) for binding not only SAF61 mAb but also other anti-PrP mAbs directed against similar epitopes, i.e. behaving as "internal images" of this disease-related PrP epitope. These results could open the way to raising PrP-like mAbs, which might serve as surrogate antigens in a new active immunotherapeutic approach to prion diseases.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Prions/immunology , Amino Acid Sequence , Animals , Cell Line , Epitopes , Humans , Mice , Molecular Mimicry , Molecular Sequence Data , Peptides/immunology , Rabbits
13.
J Immunol Methods ; 341(1-2): 41-9, 2009 Feb 28.
Article in English | MEDLINE | ID: mdl-19027745

ABSTRACT

Generation of therapeutic antibodies against human proteins is hampered by the difficulty of obtaining large quantities of correctly folded immunogens when following classic immunization procedures. Here we compared several genetic immunization protocols for their potential ability to generate high levels of antibodies against proteins expressed in their native form. We chose as a model the prion protein (PrP) because it has been demonstrated that the recognition of the native conformation of PrP is an absolute prerequisite for anti-PrP antibodies to be used as therapeutic tools for prion diseases, a group of lethal neurodegenerative disorders. We designed two human PrP-DNA vectors, containing or not a stimulatory T cell epitope, which were injected into mice following four different protocols: in the naked form with or without electroporation, or protected by cationic polymers or block copolymers. For comparison, other animals received conventional injections of recombinant human PrP with Freund's adjuvant or alum. We found that genetic immunization, carried out especially through DNA electroporation and, to a lesser extent, through injection of block copolymer-protected DNA, was able to generate high amounts of antibodies recognizing native PrP as expressed on the cell surface. Conversely, protein immunizations led to very high levels of antibodies against PrP immobilized on microtiter plates, but unable to recognize the native cell membrane-bound PrP. This clearly demonstrates the usefulness of genetic immunization, when performed under well defined conditions, in raising antibodies to native proteins. These results are of interest not only in view of passive immunotherapy of prion diseases, but also, more generally, in view of generating antibodies to human membrane proteins for immunotherapeutic or immunodiagnostic purposes.


Subject(s)
Antibodies, Monoclonal/immunology , DNA/pharmacology , Genetic Vectors/pharmacology , Immunization/methods , Prion Diseases/immunology , Prions/immunology , Animals , Cell Line , DNA/genetics , DNA/immunology , Electroporation/methods , Female , Genetic Vectors/genetics , Genetic Vectors/immunology , Humans , Mice , Prion Diseases/diagnosis , Prions/analysis , Prions/genetics , Protein Folding
14.
Mol Immunol ; 44(8): 1888-96, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17140664

ABSTRACT

Since antibodies currently constitute the most rapidly growing class of human therapeutics, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. Using as model a monoclonal antibody directed against the human prion protein that we prepared previously and tested for its therapeutic value, we describe here experimental conditions allowing the production of large quantities (up to 35 mg/l of bacterial culture) of correctly refolded and totally functional single chain fragment variable (scFv). These quantities were sufficient to characterize the binding properties of this small recombinant fragment through in vitro and ex vivo approaches. Interestingly, this scFv retains full binding capacity for its antigen, i.e. the human prion protein, when compared with the corresponding Fab or whole antibody, and recognizes soluble, solid-phase-adsorbed, and membrane-bound prion protein. This strongly suggests that from the mAb cloning step to the refolding of the recombinant fragment, each stage is well controlled, leading to almost 100% functional scFv. These results are of interest not only in view of possible immunotherapy for prion diseases, but also more generally in emphasizing the great promise of these small recombinant molecules in the context of targeted therapies.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Immunoglobulin Variable Region/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Escherichia coli/genetics , Escherichia coli/growth & development , Humans , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/therapeutic use , Prion Diseases/drug therapy , Prion Diseases/immunology , Prions/genetics , Prions/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use
15.
DNA Cell Biol ; 23(6): 395-402, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15231073

ABSTRACT

DNA vaccination appears as a very promising approach to raise protective antibodies against a variety of proteins from pathogens or tumor cells, but is often hindered by the low immunogenicity of the genetic vectors used for the immunizations. To enhance the humoral response through improvement of the antigenic presentation of newly synthesized proteins upon vaccination, we engineered a plasmid coding for a low immunogenic protein (an scFv, i.e. the single-chain Fragment variable of a well-characterized antibody) fused to a small-size universal T-helper cell epitope derived from tetanus toxin, whose efficiency in classical protein-based immunization protocols has already been demonstrated. We found that immunization of C57Bl/6 mice using this vector greatly enhanced the production not only of specific antibodies recognizing essentially conformational epitopes on the undenatured scFv protein but also of antibodies against linear epitopes on the denatured protein. Since this T-epitope is known to be accommodated by several haplotypes of H-2 molecules in mice, as well as by various class II MHC molecules in humans, the results reported here allow us to conclude that this method could be of general interest for future applications of genetic immunization, including DNA-based vaccinations in humans.


Subject(s)
Antibody Formation/immunology , Epitopes/immunology , Gene Expression , Peptide Fragments/immunology , Plasmids/genetics , Vaccines, DNA/immunology , Animals , Antibodies/immunology , Base Sequence , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , DNA, Complementary/genetics , DNA, Complementary/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/metabolism , Female , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Mice , Molecular Sequence Data , Oligonucleotides , Peptide Fragments/genetics , Plasmids/immunology , Tetanus Toxin/genetics , Transfection
16.
J Neurochem ; 86(1): 246-54, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12807444

ABSTRACT

Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood-brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-alpha, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-alpha treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-alpha and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-alpha was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1beta. Concomitantly, TNF-alpha induced IL-1beta mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1beta mRNA synthesis and IL-1beta protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-alpha and IL-1beta between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.


Subject(s)
Astrocytes/metabolism , Capillary Permeability/drug effects , Endothelin-1/pharmacology , Endothelium, Vascular/drug effects , Interleukin-1/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Astrocytes/cytology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/physiology , Brain/blood supply , Capillary Permeability/physiology , Cells, Cultured , Coculture Techniques , Endothelin-1/biosynthesis , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Interleukin-1/genetics , Interleukin-1/metabolism , Microcirculation/cytology , RNA, Messenger/biosynthesis
17.
Eur J Pharmacol ; 468(3): 175-82, 2003 May 16.
Article in English | MEDLINE | ID: mdl-12754055

ABSTRACT

Two peptides were derived from the structural analysis of a previously described monoclonal antibody [Mol. Immunol. 37 (2000) 423] against the tachykinin NK(1) receptor for the neuropeptide substance P. Here we show that these two peptides were able to inhibit the inositol phosphate transduction pathway triggered both by substance P and neurokinin A, another high-affinity endogenous ligand for the tachykinin NK(1) receptor. They also reduced the cAMP production induced by substance P. By contrast, only one antagonist peptide was able to prevent substance P and neurokinin A from binding the receptor, as revealed both by biochemical and autoradiographic studies. First, these results illustrate the generality of the antibody-based strategy for developing new bioactive peptides. Second, they indicate that antagonists, even exhibiting very close amino acid composition, can interact with the tachykinin NK(1) receptor at different contact sites, some of them clearly distinct from the contact domains for endogenous agonists.


Subject(s)
Antibody Formation/immunology , Complementarity Determining Regions/biosynthesis , Complementarity Determining Regions/pharmacology , Peptide Biosynthesis , Peptide Fragments/pharmacology , Receptors, Neurokinin-1/immunology , Substance P/immunology , Animals , Autoradiography , Cattle , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/biosynthesis , Inositol Phosphates/biosynthesis , Inositol Phosphates/pharmacokinetics , Neurokinin A/antagonists & inhibitors , Neurokinin A/drug effects , Neurokinin A/metabolism , Peptide Fragments/biosynthesis , Radioligand Assay , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/metabolism , Signal Transduction , Substance P/antagonists & inhibitors , Substance P/metabolism
18.
Bioconjug Chem ; 14(3): 629-41, 2003.
Article in English | MEDLINE | ID: mdl-12757389

ABSTRACT

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone-6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC(50) = 100 nM +/- 20). The IC(50) value was not altered in the case of 2-chloro compound 1 (IC(50) = 100 nM +/- 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC(50) = 500 nM +/- 80). A drastic reduction in binding (IC(50) > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.


Subject(s)
Halogens/metabolism , Naphthyridines/chemical synthesis , Naphthyridines/metabolism , Receptors, Neurokinin-1/metabolism , Tomography, Emission-Computed/methods , Animals , Biotransformation , CHO Cells , Cricetinae , Drug Evaluation, Preclinical/methods , Halogens/chemical synthesis , Hydrolysis , Ligands , Radioligand Assay/methods , Tomography, Emission-Computed, Single-Photon/methods
19.
J Neuroimmunol ; 128(1-2): 23-30, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12098507

ABSTRACT

Antibodies against the native form of the human NK1 receptor (hNK1R) for the neuropeptide substance P (SP), an important immunoregulator, are difficult to produce using classical immunization techniques. We show here that mice immunized with a plasmid harboring hNK1R cDNA developed antibodies recognizing extracellular epitopes of native hNK1R expressed on CHO cell membranes, as shown by FACS and immunofluorescence analysis, some antibodies being specifically directed against the second extracellular loop (E2) of the receptor. This original strategy, DNA immunization, thus efficiently generated new immunological tools to further analyse the role of SP in the regulation of immune cell functions.


Subject(s)
Antibodies/genetics , Antibodies/immunology , DNA, Complementary/genetics , DNA, Complementary/immunology , Immunization/methods , Receptors, Neurokinin-1/genetics , Receptors, Neurokinin-1/immunology , Animals , Blotting, Western , CHO Cells , COS Cells , Cell Membrane/genetics , Cell Membrane/immunology , Cricetinae , Epitopes/genetics , Epitopes/immunology , Female , Flow Cytometry , Genetic Vectors , Humans , Mice , Neuroimmunomodulation/genetics , Neuroimmunomodulation/immunology , Plasmids/genetics , Plasmids/immunology , Protein Denaturation/immunology , Protein Structure, Tertiary/genetics , Substance P/immunology , Substance P/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
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