Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies

Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

doi:10.1371/journal.pone.0046348

Cited by 12 publications

(12 citation statements)

References 42 publications

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“…Data were collected on 20,000 cells by forward and side scatter intensity on an FACs calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and were subsequently analyzed using the Flow Jo software (Tree Star Inc., Ashland, OR). The neuroepithelioma cell line SK-N-MC was grown in DMEM supplemented with 10 % fetal-calf serum at 37 °C with 5 % CO 2 in air atmosphere until the cells reach 80 % confluence [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice

Basso

Boué

Mahiddine

et al. 2016

J Neuroinflammation

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BackgroundT cell-derived opioids play a key role in the control of inflammatory pain. However, the nature of opioids produced by T cells is still matter of debate in mice. Whereas β-endorphin has been found in T lymphocytes by using antibody-based methods, messenger RNA (mRNA) quantification shows mainly mRNA encoding for enkephalins. The objective of the study is to elucidate the nature of T cell-derived opioids responsible for analgesia and clarify discrepancy of the results at the protein and genetic levels.MethodsCD4+ T lymphocytes were isolated from wild-type and enkephalin-deficient mice. mRNA encoding for β-endorphin and enkephalin was quantified by RT-qPCR. The binding of commercially available polyclonal anti-endorphin antibodies to lymphocytes from wild-type or enkephalin knockout mice was assessed by cytofluorometry. Opioid-mediated analgesic properties of T lymphocytes from wild-type and enkephalin-deficient mice were compared in a model of inflammation-induced somatic pain by measuring sensitivity to mechanical stimuli using calibrated von Frey filaments.ResultsCD4+ T lymphocytes expressed high level of mRNA encoding for enkephalins but not for β-endorphin in mice. Anti-β-endorphin polyclonal IgG antibodies are specific for β-endorphin but cross-react with enkephalins. Anti-β-endorphin polyclonal antibodies bound to wild-type but not enkephalin-deficient CD4+ T lymphocytes. Endogenous regulation of inflammatory pain by wild-type T lymphocytes was completely abolished when T lymphocytes were deficient in enkephalins. Pain behavior of immune-deficient (i.e., without B and T lymphocytes) mice was superimposable to that of mice transferred with enkephalin-deficient lymphocytes.ConclusionsRabbit polyclonal anti-β-endorphin serum IgG bind to CD4+ T lymphocytes because of their cross-reactivity towards enkephalins. Thus, staining of T lymphocytes by anti-β-endorphin polyclonal IgG reported in most of studies in mice is because of their binding to enkephalins. In mice, CD4+ T lymphocytes completely lose their analgesic opioid-mediated activity when lacking enkephalins.

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Section: Methodsmentioning

confidence: 99%

Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice

Basso

Boué

Mahiddine

et al. 2016

J Neuroinflammation

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“…Plusieurs études ont révélé l'expression des récepteurs S1PR dans le système nerveux central [25], plus précisément à la surface des astrocytes chez l'homme, et dans l'endothélium chez l'homme et les rongeurs. Les récepteurs S1PR 2-4 seraient, quant à eux, largement distribués et l'expression de S1PR 5 serait restreinte au cerveau et aux lymphocytes T. La localisation de ces récepteurs reste toutefois difficile à déterminer, la production d'anticorps spécifiques n'étant pas aisée [26][27][28]. S1PR 2 auquel ne se lie pas le fingolimod, est exprimé en particulier par les macrophages et les cellules progénitrices d'oligodendrocytes [29].…”

Section: Figure 1 Activation Des Récepteurs De La Sphingosine 1-phosunclassified

La sclérose en plaques et les médicaments immuno-modulateurs des récepteurs de la sphingosine 1-phosphate

Talmont

Hatzoglou

2020

Med Sci (Paris)

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La sclérose en plaques (SEP) est une maladie du système nerveux central à composante inflammatoire, très invalidante qui atteint généralement de jeunes adultes (20 à 40 ans). Cette maladie se caractérise par la destruction progressive, par les cellules du système immunitaire, de la gaine de myéline des axones, ce qui aboutit à une dégénérescence neuronale. Les lymphocytes T et B sont les acteurs principaux de cette maladie qui peut être rémittente ou progressive. Parmi les médicaments utilisés dans le cadre de son traitement, le fingolimod, un immunosuppresseur dont les cibles sont les récepteurs de la sphingosine 1-phosphate, administré par voie orale, agit en empêchant les lymphocytes de quitter le thymus et les ganglions lymphatiques, et de rejoindre les foyers inflammatoires cérébraux. Une recherche intense pour développer des traitements et des médicaments curatifs est actuellement en cours et d’autres immunosuppresseurs interagissant avec les récepteurs de sphingosine 1-phosphate sont en cours de développement.

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“…The use of different approaches for data analysis and statistics by different laboratories can contribute to discordant results (Pan, 2002;Motulsky, 2014). The dearth of well validated antibodies to detect native GPCRs, including for use in proteomic studies, is an important problem in GPCR research (Hutchings et al, 2010;Talmont et al, 2012;Eisen et al, 2013;Marchalant et al, 2014). Use of antibodies to study GPCRs is challenging because the receptors are typically expressed in cells at lower levels than many other cellular proteins.…”

Section: What Are Some Of the Problems And Limitations Of Efforts To mentioning

confidence: 99%

G Protein–Coupled Receptor (GPCR) Expression in Native Cells: “Novel” endoGPCRs as Physiologic Regulators and Therapeutic Targets

et al. 2015

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G protein-coupled receptors (GPCRs), the largest family of signaling receptors in the human genome, are also the largest class of targets of approved drugs. Are the optimal GPCRs (in terms of efficacy and safety) currently targeted therapeutically? Especially given the large number (∼120) of orphan GPCRs (which lack known physiologic agonists), it is likely that previously unrecognized GPCRs, especially orphan receptors, regulate cell function and can be therapeutic targets. Knowledge is limited regarding the diversity and identity of GPCRs that are activated by endogenous ligands and that native cells express. Here, we review approaches to define GPCR expression in tissues and cells and results from studies using these approaches. We identify problems with the available data and suggest future ways to identify and validate the physiologic and therapeutic roles of previously unrecognized GPCRs. We propose that a particularly useful approach to identify functionally important GPCRs with therapeutic potential will be to focus on receptors that show selective increases in expression in diseased cells from patients and experimental animals.

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Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies

Cited by 12 publications

References 42 publications

Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice

Endogenous analgesia mediated by CD4+ T lymphocytes is dependent on enkephalins in mice

La sclérose en plaques et les médicaments immuno-modulateurs des récepteurs de la sphingosine 1-phosphate

G Protein–Coupled Receptor (GPCR) Expression in Native Cells: “Novel” endoGPCRs as Physiologic Regulators and Therapeutic Targets

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