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1.
Eur J Immunol ; 51(11): 2590-2606, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34411303

RESUMEN

The polyimmunoglobulin receptor (pIgR) transcytoses J chain-containing antibodies through mucosal epithelia. In mammals, two cis-duplicates of PIGR, FCMR, and FCAMR, flank the PIGR gene. A PIGR duplication is first found in amphibians, previously annotated as PIGR2 (herein xlFCAMR), and is expressed by APCs. We demonstrate that xlFcamR is the equivalent of mammalian FcamR. It has been assumed that pIgR is the oldest member of this family, yet our data could not distinguish whether PIGR or FCAMR emerged first; however, FCMR was the last family member to emerge. Interestingly, bony fish "pIgR" is not an orthologue of tetrapod pIgR, and possibly acquired its function via convergent evolution. PIGR/FCAMR/FCMR are members of a larger superfamily, including TREM, CD300, and NKp44, which we name the "double-disulfide Ig superfamily" (ddIgSF). Domains related to each ddIgSF family were identified in cartilaginous fish (sharks, chimeras) and encoded in a single gene cluster syntenic to the human pIgR locus. Thus, the ddIgSF families date back to the earliest antibody-based adaptive immunity, but apparently not before. Finally, our data strongly suggest that the J chain arose in evolution only for Ig multimerization. This study provides a framework for further studies of pIgR and the ddIgSF in vertebrates.


Asunto(s)
Antígenos CD/genética , Inmunidad Mucosa/inmunología , Receptores Fc/genética , Receptores Opioides mu/genética , Receptores de Inmunoglobulina Polimérica/genética , Transcitosis/inmunología , Animales , Antígenos CD/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Humanos , Inmunoglobulinas/metabolismo , Filogenia , Transporte de Proteínas/fisiología , Receptores Fc/inmunología , Receptores Opioides mu/inmunología , Receptores de Inmunoglobulina Polimérica/inmunología , Transcitosis/genética , Xenopus laevis
2.
Commun Biol ; 4(1): 238, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33619305

RESUMEN

Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.


Asunto(s)
Anticuerpos/farmacología , Especificidad de Anticuerpos , Ensayos Analíticos de Alto Rendimiento , Proteína Quinasa C/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Sinapsis/efectos de los fármacos , Analgésicos Opioides/farmacología , Animales , Anticuerpos/inmunología , Línea Celular Tumoral , Activación Enzimática , Fentanilo/farmacología , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Morfina/farmacología , Fosforilación , Proteína Quinasa C/inmunología , Proteína Quinasa C/metabolismo , Conejos , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo , Transducción de Señal , Sinapsis/inmunología , Sinapsis/metabolismo , Factores de Tiempo
3.
Methods Mol Biol ; 2201: 71-82, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975790

RESUMEN

Immunohistochemical staining is widely used to identify opioid receptors in specific cell types throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of µ-opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditions in mice. Importantly, we consider the ongoing debate on the specificity of antibodies.


Asunto(s)
Inmunohistoquímica/métodos , Nervios Periféricos/metabolismo , Receptores Opioides mu/inmunología , Analgésicos Opioides/metabolismo , Animales , Axones/metabolismo , Ganglios Espinales/citología , Humanos , Ratones , Nervios Periféricos/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/inmunología , Receptores Opioides/metabolismo , Receptores Opioides mu/metabolismo , Nervio Ciático/citología , Neuropatía Ciática , Células Receptoras Sensoriales/metabolismo , Médula Espinal/metabolismo
4.
Methods Mol Biol ; 2201: 83-95, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975791

RESUMEN

Real-time quantitative reverse transcription-PCR (qRT-PCR ) is a highly sensitive molecular biology method based on the amplification of the cDNA of mRNA to detect and quantify the levels of mRNA of interest. In this chapter, we describe real-time qRT-PCR to detect and quantify mRNA of opioid receptors in immune cells. Specifically, we analyze mouse immune cells isolated from the blood and sciatic nerves exposed to a chronic constriction injury, which represents a model of neuropathic pain. We describe in detail the requirements and techniques to induce the chronic constriction injury, to isolate immune cells from the blood and injured nerves, to isolate the total RNA from immune cells, to perform a cDNA reverse transcription from the total RNA, and to perform real-time qRT-PCR for µ-, δ-, and κ-opioid receptor mRNAs.


Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores Opioides mu/genética , Animales , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuralgia , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/genética , Receptores Opioides/inmunología , Receptores Opioides/metabolismo , Receptores Opioides delta/genética , Receptores Opioides kappa/genética , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo , Transcripción Reversa , Nervio Ciático/lesiones
5.
Methods Mol Biol ; 2201: 99-108, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32975792

RESUMEN

The biological process of opioid analgesic tolerance remains nowadays elusive. In particular the mechanism by which opioid receptor desensitization occurs has not been completely elucidated to date. One possible hypothesis involves the internalization of MOR. Here, we describe a simple in vitro protocol to investigate the localization of MOR-1 after repeated morphine administration in the spinal cord of morphine-tolerant mice, using western blotting and immunofluorescence techniques.


Asunto(s)
Western Blotting/métodos , Receptores Opioides mu/inmunología , Médula Espinal/inmunología , Analgésicos Opioides/farmacología , Animales , Tolerancia a Medicamentos/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Morfina/farmacología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides/genética , Receptores Opioides/inmunología , Receptores Opioides/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Médula Espinal/metabolismo
6.
Immunohorizons ; 4(7): 420-429, 2020 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-32675085

RESUMEN

Opioid peptides are released at sites of injury, and their cognate G protein-coupled opioid receptors (OR) are expressed on immune cells. Exposure of human circulating CD8+ T cells to selective OR agonists differentially regulates thousands of genes. Gene set enrichment analysis reveals that µ-OR more strongly regulates cellular processes than δ-OR. In TCR naive T cells, triggering µ-OR exhibits stimulatory and inhibitory patterns, yet when administered prior to TCR cross-linking, a µ-OR agonist inhibits activation. µ-OR, but not δ-OR, signaling is linked to upregulation of lipid, cholesterol, and steroid hormone biosynthesis, suggesting lipid regulation is a mechanism for immune suppression. Lipid rafts are cholesterol-rich, liquid-ordered membrane domains that function as a nexus for the initiation of signal transduction from surface receptors, including TCR and µ-OR. We therefore propose that µ-OR-specific inhibition of TCR responses in human CD8+ T cells may be mediated through alterations in lipid metabolism and membrane structure.


Asunto(s)
Analgésicos Opioides/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Receptores Opioides mu/inmunología , Transcriptoma/genética , Linfocitos T CD8-positivos/inmunología , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Receptores Opioides mu/agonistas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología
7.
Front Immunol ; 10: 2904, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31921165

RESUMEN

Research on the effects of opioids on immune responses was stimulated in the 1980s by the intersection of use of intravenous heroin and HIV infection, to determine if opioids were enhancing HIV progression. The majority of experiments administering opioid alkaloids (morphine and heroin) in vivo, or adding these drugs to cell cultures in vitro, showed that they were immunosuppressive. Immunosuppression was reported as down-regulation: of Natural Killer cell activity; of responses of T and B cells to mitogens; of antibody formation in vivo and in vitro; of depression of phagocytic and microbicidal activity of neutrophils and macrophages; of cytokine and chemokine production by macrophages, microglia, and astrocytes; by sensitization to various infections using animal models; and by enhanced replication of HIV in vitro. The specificity of the receptor involved in the immunosuppression was shown to be the mu opioid receptor (MOR) by using pharmacological antagonists and mice genetically deficient in MOR. Beginning with a paper published in 2005, evidence was presented that morphine is immune-stimulating via binding to MD2, a molecule associated with Toll-like Receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS). This concept was pursued to implicate inflammation as a mechanism for the psychoactive effects of the opioid. This review considers the validity of this hypothesis and concludes that it is hard to sustain. The experiments demonstrating immunosuppression were carried out in vivo in rodent strains with normal levels of TLR4, or involved use of cells taken from animals that were wild-type for expression of TLR4. Since engagement of TLR4 is universally accepted to result in immune activation by up-regulation of NF-κB, if morphine were binding to TLR4, it would be predicted that opioids would have been found to be pro-inflammatory, which they were not. Further, morphine is immunosuppressive in mice with a defective TLR4 receptor. Morphine and morphine withdrawal have been shown to permit leakage of Gram-negative bacteria and LPS from the intestinal lumen. LPS is the major ligand for TLR4. It is proposed that an occult variable in experiments where morphine is being proposed to activate TLR4 is actually underlying sepsis induced by the opioid.


Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Leucocitos/inmunología , Morfina/efectos adversos , Receptores Opioides mu/inmunología , Receptor Toll-Like 4/inmunología , Citocinas/inmunología , Bacterias Anaerobias Gramnegativas/inmunología , Infecciones por VIH/patología , Heroína , Humanos , Leucocitos/patología , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Morfina/administración & dosificación , Sepsis/inmunología
8.
Int Immunol ; 29(11): 519-524, 2017 12 30.
Artículo en Inglés | MEDLINE | ID: mdl-29281010

RESUMEN

Fc receptors play important roles for a wide array of immune responses. In contrast to the well-defined Fcγ and Fcε receptors, the molecular and functional characteristics of Fc receptors for IgA and IgM have remained incompletely understood for years. Recent progress has unveiled the characteristics of Fc receptors for IgA and IgM, including Fcα/µ receptor (Fcα/µR) (CD351), polymeric immunoglobulin receptor (poly-IgR), Fcα receptor (FcαRI) (CD89) and Fcµ receptor (FcµR). In this review, we summarize the molecular and functional characteristics of Fcα/µR in comparison with poly-IgR, FcµR and FcαRI, and focus particularly on the pro-inflammatory function of Fcα/µR expressed on marginal zone B cells in sepsis.


Asunto(s)
Linfocitos B/inmunología , Inflamación/inmunología , Receptores Fc/inmunología , Receptores Opioides mu/inmunología , Sepsis/inmunología , Animales , Humanos
9.
Biomol NMR Assign ; 11(1): 117-121, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28239762

RESUMEN

Nanobodies are single chain antibodies that have become a highly valuable and versatile tool for biomolecular and therapeutic research. One application field is the stabilization of active states of flexible proteins, among which G-protein coupled receptors represent a very important class of membrane proteins. Here we present the backbone and side-chain assignment of the 1H, 13C and 15N resonances of Nb33 and Nb39, two nanobodies that recognize and stabilize the µ-opioid receptor to opioids in its active agonist-bound conformation. In addition, we present a comparison of their secondary structures as derived from NMR chemical shifts.


Asunto(s)
Camelidae , Resonancia Magnética Nuclear Biomolecular , Receptores Opioides mu/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Animales
10.
J Med Chem ; 58(21): 8647-57, 2015 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-26451468

RESUMEN

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.


Asunto(s)
Analgésicos/química , Analgésicos/uso terapéutico , Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/uso terapéutico , Neuralgia/tratamiento farmacológico , Receptores CCR5/inmunología , Receptores Opioides mu/agonistas , Animales , Enfermedad Crónica , Células HEK293 , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Masculino , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Neuralgia/inmunología , Receptores Opioides mu/inmunología
11.
Sci Rep ; 5: 11384, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-26072707

RESUMEN

Opportunistic lung infection and inflammation is a hallmark of chronic recreational/clinical use of morphine. We show that early induction of IL17 from the bronchial epithelium, following pathogenic encounter is a protective response, which contributes to pathogenic clearance and currently attributed to TLR2 activation in immune cells. Concurrent activation of TLR2 and IL17R in bronchial epithelium results in the sequestration of MyD88 (TLR2 adapter) by Act1/CIKS (IL17R adapter), thereby turning off TLR2 signaling to restore homeostasis. Morphine inhibits the early IL17 release and interaction between Act1 and MyD88, leading to decreased pathogenic clearance and sustained inflammation. Hence, we propose that therapeutically targeting either TLR2 or IL17 in bronchial epithelia, in the context of morphine, can restore inflammatory homeostasis.


Asunto(s)
Interleucina-17/inmunología , Pulmón/inmunología , Morfina/efectos adversos , Infecciones Neumocócicas/inmunología , Receptores de Interleucina-17/inmunología , Receptor Toll-Like 2/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Regulación de la Expresión Génica , Homeostasis , Humanos , Inflamación , Interleucina-17/genética , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones Neumocócicas/etiología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/patología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/inmunología , Receptores de Interleucina-17/genética , Receptores Opioides mu/deficiencia , Receptores Opioides mu/genética , Receptores Opioides mu/inmunología , Transducción de Señal , Streptococcus pneumoniae/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología
12.
Zhongguo Zhen Jiu ; 35(9): 917-21, 2015 Sep.
Artículo en Chino | MEDLINE | ID: mdl-26721148

RESUMEN

OBJECTIVE: To observe the analgesic effect of electroacupuncture (EA) on collagen-induced arthritis (CIA) rats and its regulating effect on inflammation reaction and the endogenous opioid system of synovial tissues. Methods A total of 30 healthy male Wistar rats were randomly divided into a control group, a model group and an EA group, 10 rats in each one. The chronic pain model of CIA rats was made by cattle type-II collagen in the model group and EA group. Rats in the EA group were treated with EA at "Zusanli" (ST 36) and "Kunlun" (BL 60) for 30 min from 16th day after model establishment, once a day for consecutive 10 days. Rats in the control group did not receive any treatment. Rats in the model group were treated with fixation as the EA group. Threshold of pain, arthritis index, paw swelling were measured before model establishment and 16 d, 20 d, 23 d and 25 d after model establishment. The levels of beta-endorphin (ß-END), met-enkephalin (met-ENK), dynorphin A (Dyn A) were measured by radioimmunoassay; the mRNA expressions of mu opioid receptor (MOR), kappa opioid receptor (KOR) and delta opioid receptor (DOR) in synovial tissues of CIA rats were detected by I quantitative polymerase chain reaction (qPCR). RESULTS: Compared with the control group, threshold of pain was reduced (all P<0. 01), arthritis index was increased (all P<0. 01) and paw swelling was increased (all P<0. 01) in the model group on the 16th day, 20th day, 23rd day, 25th day after model establishment. Compared with the model group, the threshold of pain was increased in the EA group (all P<0. 01), arthritis index and paw swelling were reduced (all P<0. 01) on the 23rd day and 25th day after model establishment. Compared with the control group, the level of Dyn A in synovial tissues of CIA rats was increased in the model group (P<0. 01); the mRNA expressions of MOR, KOR and DOR were down-regulated lower than 0. 5 fold of normal level. Compared with the model group, the level of ß-END in synovial tissues of the knee joint was increased in the EA group (P<0. 05), and the mRNA expressions of MOR, KOR and DOR in synovial tissues of CIA rats were up-regulated more than 2 folds of normal level. CONCLUSION: The intervention of EA on chronic pain of CIA rats is superior, which is likely to be related with effects of EA on anti-inflammation and up-regulation of synovial tissue ß-END and MOR, KOR, DOR.


Asunto(s)
Analgésicos Opioides/inmunología , Artritis Reumatoide/terapia , Dolor Crónico/terapia , Electroacupuntura , Líquido Sinovial/inmunología , Analgesia por Acupuntura , Puntos de Acupuntura , Animales , Artritis Reumatoide/inmunología , Bovinos , Dolor Crónico/inmunología , Dinorfinas/genética , Dinorfinas/inmunología , Encefalina Metionina/genética , Encefalina Metionina/inmunología , Humanos , Masculino , Ratas , Ratas Wistar , Receptores Opioides mu/genética , Receptores Opioides mu/inmunología , betaendorfina/genética , betaendorfina/inmunología
13.
Methods Mol Biol ; 1230: 141-54, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25293322

RESUMEN

Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-µC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.


Asunto(s)
Anticuerpos/inmunología , Immunoblotting/métodos , Receptores Opioides mu/aislamiento & purificación , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Glicosilación , Humanos , Ratones , Conejos , Ratas , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo
14.
Cancer Lett ; 344(2): 212-22, 2014 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-24291668

RESUMEN

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.


Asunto(s)
Encefalina Metionina/farmacología , Inmunoterapia Adoptiva/métodos , Sarcoma 180/inmunología , Sarcoma 180/terapia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Calcio/metabolismo , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Encefalina Metionina/inmunología , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Receptores Opioides delta/biosíntesis , Receptores Opioides delta/inmunología , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/inmunología , Sarcoma 180/tratamiento farmacológico
15.
Immunobiology ; 218(5): 798-809, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23182711

RESUMEN

The Fc alpha/mu receptor (Fcα/µR, CD351) is a receptor that has dual specificity for IgA and IgM. Its second extracellular domain (EC2) has an Ig variable region-like structure and is predicted to be the ligand binding domain. EC2 is homologous to the first Ig-like domain (D1) of polymeric Ig receptor (pIgR) and has three complementarity-determining region (CDR)-like loops. A peptide that includes the CDR1-like loop region has been found to be responsible for IgA and IgM binding. However, whether the CDR2- and CDR3-like loops of EC2 contribute to ligand binding has remained unclear. In this work, we made three chimaeric receptors composed of the hFcα/µR backbone but having the CDR1-, CDR2- and CDR3-like loops of EC2 replaced by their counterpart loops from human pIgR D2, which itself does not bind IgA or IgM. Flow cytometry and confocal microscopy analysis showed that substitution of either the CDR1- or the CDR2-like loop abrogated IgA and IgM binding, indicating that both the CDR1- and the CDR2-like loops were important for ligand binding. In comparison, substitution of CDR3-like loop resulted in significant loss of IgM binding but has only a small negative effect on IgA binding. In addition, site-directed mutagenesis of the three CDR-like loops showed that residues Val29, Arg31, Asn54, Gln55, Glu98, Asn99 and Asn100 were involved in both IgA and IgM binding, and substitution of Glu98 within the CDR3-like loop increased IgA binding but decreased IgM binding.


Asunto(s)
Antígenos CD/inmunología , Regiones Determinantes de Complementariedad/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina M/inmunología , Receptores Fc/inmunología , Receptores Opioides mu/inmunología , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/inmunología , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión , Línea Celular Tumoral , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Epítopos , Humanos , Inmunoglobulina A/química , Inmunoglobulina A/genética , Inmunoglobulina M/química , Inmunoglobulina M/genética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Receptores Fc/química , Receptores Fc/genética , Receptores Opioides mu/química , Receptores Opioides mu/genética , Receptores de Inmunoglobulina Polimérica/química , Receptores de Inmunoglobulina Polimérica/genética , Receptores de Inmunoglobulina Polimérica/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia
16.
PLoS One ; 7(9): e46348, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23029489

RESUMEN

G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (µ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.


Asunto(s)
Anticuerpos/inmunología , Inmunoglobulina G/biosíntesis , Receptores de Neuropéptido/inmunología , Receptores Opioides kappa/inmunología , Receptores Opioides mu/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/aislamiento & purificación , Humanos , Inmunización , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pichia/genética , Desnaturalización Proteica , Pliegue de Proteína , Receptores de Neuropéptido/administración & dosificación , Receptores de Neuropéptido/genética , Receptores Opioides kappa/administración & dosificación , Receptores Opioides kappa/genética , Receptores Opioides mu/administración & dosificación , Receptores Opioides mu/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
17.
Ann N Y Acad Sci ; 1261: 1-6, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22823387

RESUMEN

Opioids and cannabinoids modulate T lymphocyte functions. Many effects of the drugs are mediated by µ-opioid receptor and cannabinoid receptor type 1 (CB1), respectively. These two receptors are strikingly similar with respect to their expression in T cells and the mechanisms by which they mediate modulation of T cell activity. Thus, µ-opioid receptors and CB1 not expressed in resting primary human and Jurkat T cells. However, in response to the cytokine IL-4, the epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A, and activation of T cells, functional µ-opioid receptors and CB1 are induced. The induced receptors mediate inhibition of T cell signaling and, thereby, IL-2 production, a hallmark of activated T cells. Although coupled to inhibitory G proteins, µ-opioid receptors and CB1 produce a remarkable increase in cAMP levels in T cells stimulated with opioids and cannabinoids, which is a key mechanism for the inhibition of T cell signaling.


Asunto(s)
Interleucina-4/metabolismo , Neuroinmunomodulación , Receptor Cannabinoide CB1/metabolismo , Receptores Opioides mu/metabolismo , Linfocitos T/metabolismo , Analgésicos Opioides/inmunología , Analgésicos Opioides/farmacología , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Cannabinoides/inmunología , Cannabinoides/farmacología , AMP Cíclico/inmunología , AMP Cíclico/metabolismo , Decitabina , Humanos , Ácidos Hidroxámicos/farmacología , Interleucina-2/antagonistas & inhibidores , Interleucina-2/inmunología , Interleucina-4/inmunología , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Receptor Cannabinoide CB1/inmunología , Receptores Opioides mu/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología
18.
Vis Neurosci ; 29(3): 203-9, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22643230

RESUMEN

The presence of opioid receptors has been confirmed by a variety of techniques in vertebrate retinas including those of mammals; however, in most reports, the location of these receptors has been limited to retinal regions rather than specific cell types. Concurrently, our knowledge of the physiological functions of opioid signaling in the retina is based on only a handful of studies. To date, the best-documented opioid effect is the modulation of retinal dopamine release, which has been shown in a variety of vertebrate species. Nonetheless, it is not known if opioids can affect dopaminergic amacrine cells (DACs) directly, via opioid receptors expressed by DACs. This study, using immunohistochemical methods, sought to determine whether (1) µ- and δ-opioid receptors (MORs and DORs, respectively) are present in the mouse retina, and if present, (2) are they expressed by DACs. We found that MOR and DOR immunolabeling were associated with multiple cell types in the inner retina, suggesting that opioids might influence visual information processing at multiple sites within the mammalian retinal circuitry. Specifically, colabeling studies with the DAC molecular marker anti-tyrosine hydroxylase antibody showed that both MOR and DOR immunolabeling localize to DACs. These findings predict that opioids can affect DACs in the mouse retina directly, via MOR and DOR signaling, and might modulate dopamine release as reported in other mammalian and nonmammalian retinas.


Asunto(s)
Células Amacrinas/metabolismo , Neuronas Dopaminérgicas/metabolismo , Receptores Opioides/biosíntesis , Retina/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Interpretación Estadística de Datos , Femenino , Cabras/inmunología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Receptores Opioides delta/inmunología , Receptores Opioides delta/fisiología , Receptores Opioides mu/inmunología , Receptores Opioides mu/fisiología , Tirosina 3-Monooxigenasa/metabolismo
19.
J Biol Chem ; 287(20): 16734-47, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22451667

RESUMEN

In the natural killer (NK) cells, δ-opiate receptor (DOR) and µ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.


Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Neoplasias Mamarias Animales/inmunología , Multimerización de Proteína/efectos de los fármacos , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Células Asesinas Naturales , Ligandos , Masculino , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/terapia , Multimerización de Proteína/inmunología , Ratas , Ratas Endogámicas F344 , Receptores Opioides delta/agonistas , Receptores Opioides delta/inmunología , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo
20.
Brain Behav Immun ; 26(3): 480-8, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22240038

RESUMEN

Opioids have been shown to affect both innate and adaptive immunity. We previously showed that morphine affects the macrophage production of pro-inflammatory cytokines after LPS in a NFkB dependent manner. Toll like receptors (TLRs) play a crucial role in the signaling pathways which lead to NFkB activation. TLR4 is considered the Lipopolysaccaride (LPS) receptor. The data here presented show that, in murine macrophages, morphine impacts on the immune function acting on the early step of pathogen recognition. Morphine, when added to RAW 264.7 cells and when injected into mice (s.c. 20mg/kg) is in fact able to decrease TLR4 both at mRNA and protein level in RAW cells and peritoneal macrophages. In the same cells, the mu opioid receptor (MOR) antagonist Naltrexone increases TLR4 levels, thus suggesting a role of the endogenous opioid system in TLR4 regulation. The effect of the two drugs is moreover lost in case of co-administration. Experiments with MOR KO mice and with DAMGO (MOR specific agonist) confirm that the effect of morphine on TLR4 mRNA in peritoneal macrophages is due to the MOR activation. Moreover the effect on TLR4 is blocked by PTX thus indicating the involvement of a G(i) protein after MOR binding. This work unveils a clear link between MOR activation and TLR4, suggesting a new possible mechanism at the basis of the peripheral immunosuppressive effect of opioids.


Asunto(s)
Analgésicos Opioides/farmacología , Macrófagos Peritoneales/inmunología , Morfina/farmacología , Receptores Opioides mu/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Animales , Células Cultivadas , Expresión Génica/efectos de los fármacos , Inmunosupresores/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Naltrexona/farmacología , Receptores Opioides mu/inmunología , Receptores Opioides mu/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo
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