RESUMEN
Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates µ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express µ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of µ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between µ-opioid and TLR4 receptors.
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Analgésicos Opioides , Trampas Extracelulares , Humanos , Animales , Ratones , Analgésicos Opioides/farmacología , Receptor Toll-Like 4/metabolismo , Metadona/farmacología , Mastocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Médula Ósea/metabolismo , Calcio/metabolismo , Trampas Extracelulares/metabolismo , Receptor Toll-Like 2/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent ß-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1ß mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment.
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Receptor Cannabinoide CB2 , Receptores Acoplados a Proteínas G , Ratones , Humanos , Animales , Receptores Acoplados a Proteínas G/genética , Receptor Cannabinoide CB2/genética , Quimiotaxis , Mastocitos , Citocinas , Actinas , Receptores de Cannabinoides/genética , Lisofosfolípidos/farmacología , Lisofosfolípidos/fisiologíaRESUMEN
Alcohol consumption has been linked to numerous pathologic conditions, including infectious diseases and several types of cancer. Alcohol exerts its modulatory effects on the immune system (IS) in a dose- and time-dependent manner. Numerous studies indicate that these alterations affect responses such as peripheral inflammation or decreased antibody production and promote chronic inflammation, leading to cell death. The molecular mechanisms underlying these effects involve generating an oxidative tissue environment, producing cell damage-associated molecular patterns (DAMPs), and activating pattern recognition receptors. In particular, toll-like receptors and their signaling system emerge as central elements whose activity is altered by alcohol intake. There is also some epidemiological evidence demonstrating the causal role of alcohol in the development of various types of cancer, such as head-and-neck cancer, esophageal cancer, colorectal cancer, liver cancer, and breast cancer. Most recent evidence suggests that factors related to alcohol consumption and cancer include increased levels of acetaldehyde, production of reactive oxygen species, alteration in DNA methylation, and modifications in retinoid metabolism. In addition, changes associated with alcohol use on the IS and intestinal microbiota may favor the growth of some types of tumors.
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Neoplasias de la Mama , Etanol , Humanos , Femenino , Etanol/metabolismo , Acetaldehído/metabolismo , Consumo de Bebidas Alcohólicas/efectos adversos , InflamaciónRESUMEN
Huntington´s disease (HD) is a pathological condition that can be studied in mice by the administration of quinolinic acid (QUIN), an agonist of the N-methyl-d-aspartate receptor (NMDAR) that induces NMDAR-mediated cytotoxicity and neuroinflammation. Mast cells (MCs) participate in numerous inflammatory processes through the release of important amounts of histamine (HA). In this study, we aimed to characterize the participation of MCs and HA in the establishment of neural and oxidative damage in the QUIN-induced model of HD. C57BL6/J mice (WT), MC-deficient c-KitW-sh/W-sh (Wsh) mice and Wsh mice reconstituted by intracerebroventricular (i.c.v.) injection of 5 × 105 bone marrow-derived mast cells (BMMCs), or i.c.v. administered with HA (5 µg) were used. All groups of animals were intrastriatally injected with 1 µL QUIN (30 nmol/µL) and 3 days later, apomorphine-induced circling behavior, striatal GABA levels and the number of Fluoro-Jade positive cells, as indicators of neuronal damage, were determined. Also, lipid peroxidation (LP) and reactive oxygen species production (ROS), as markers of oxidative damage, were analyzed. Wsh mice showed less QUIN-induced neuronal and oxidative damage than WT and Wsh-MC reconstituted animals. Histamine administration restored the QUIN-induced neuronal and oxidative damage in the non-reconstituted Wsh mice to levels equivalent or superior to those observed in WT mice. Our results demonstrate that MCs and HA participate in the neuronal and oxidative damages observed in mice subjected to the QUIN -induced model of Huntington's disease.
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Histamina/inmunología , Enfermedad de Huntington/inmunología , Enfermedad de Huntington/patología , Mastocitos/inmunología , Neuronas/patología , Animales , Modelos Animales de Enfermedad , Femenino , Histamina/metabolismo , Enfermedad de Huntington/inducido químicamente , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácido Quinolínico/toxicidadRESUMEN
Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (-)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes' activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.
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Fentanilo , Morfina , Analgésicos Opioides/farmacología , Animales , Núcleo Dorsal del Rafe/metabolismo , Fentanilo/farmacología , Masculino , Morfina/farmacología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas NLR , Piroptosis , Ratas , Ratas Wistar , Receptores Opioides/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Hypoxia is a condition that together with low pH, high amounts of reactive oxygen species (ROS), and increased adenosine levels characterize tumor microenvironment. Mast cells (MCs) are part of tumor microenvironment, but the effect of hypoxia on the production of MC-derived cytokines has not been fully described. Using the hypoxia marker pimonidazole in vivo, we found that MCs were largely located in the low-oxygen areas within B16-F1 mice melanoma tumors. In vitro, hypoxia promoted ROS production, a ROS-dependent increase of intracellular calcium, and the production of MCP 1 (CCL-2) in murine bone marrow-derived MCs. Hypoxia-induced CCL-2 production was sensitive to the antioxidant trolox and to nifedipine, a blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). Simultaneously with CCL-2 production, hypoxia caused the ROS-dependent glutathionylation and membrane translocation of the α1c subunit of Cav1.2 LVDCCs. Relationship between ROS production, calcium rise, and CCL-2 synthesis was also observed when cells were treated with H2O2 In vivo, high CCL-2 production was detected on hypoxic zones of melanoma tumors (where tryptase-positive MCs were also found). Pimonidazole and CCL-2 positive staining diminished when B16-F1 cell-inoculated animals were treated with trolox, nifedipine, or the adenosine receptor 2A antagonist KW6002. Our results show that MCs are located preferentially in hypoxic zones of melanoma tumors, hypoxia-induced CCL-2 production in MCs requires calcium rise mediated by glutathionylation and membrane translocation of LVDCCs, and this mechanism of CCL-2 synthesis seems to operate in other cells inside melanoma tumors, with the participation of the adenosine receptor 2A.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Quimiocina CCL2/metabolismo , Mastocitos/inmunología , Melanoma Experimental/inmunología , Microambiente Tumoral/inmunología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Antioxidantes/farmacología , Biopsia , Bloqueadores de los Canales de Calcio/farmacología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/inmunología , Línea Celular Tumoral/trasplante , Quimiocina CCL2/inmunología , Peróxido de Hidrógeno/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Melanoma Experimental/patología , Ratones , Especies Reactivas de Oxígeno/metabolismo , Receptor de Adenosina A2A/metabolismo , Microambiente Tumoral/efectos de los fármacosRESUMEN
Mast cells (MCs) contribute to the control of local inflammatory reactions and become hyporesponsive after prolonged TLR4 activation by bacterial LPS. The molecular mechanisms involved in endotoxin tolerance (ET) induction in MCs are not fully understood. In this study, we demonstrate that the endocannabinoid 2-arachidonoylglycerol (2-AG) and its receptor, cannabinoid receptor 2 (CB2), play a role in the establishment of ET in bone marrow-derived MCs from C57BL/6J mice. We found that CB2 antagonism prevented the development of ET and that bone marrow-derived MCs produce 2-AG in a TLR4-dependent fashion. Exogenous 2-AG induced ET similarly to LPS, blocking the phosphorylation of IKK and the p65 subunit of NF-κB and inducing the synthesis of molecular markers of ET. LPS caused CB2 receptor trafficking in Rab11-, Rab7-, and Lamp2-positive vesicles, indicating recycling and degradation of the receptor. 2-AG also prevented LPS-induced TNF secretion in vivo, in a MC-dependent model of endotoxemia, demonstrating that TLR4 engagement leads to 2-AG secretion, which contributes to the negative control of MCs activation. Our study uncovers a functional role for the endocannabinoid system in the inhibition of MC-dependent innate immune responses in vivo.
Asunto(s)
Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Glicéridos/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Lipopolisacáridos/toxicidad , Mastocitos/inmunología , Receptor Cannabinoide CB2/inmunología , Receptor Toll-Like 4/inmunología , Animales , Tolerancia Inmunológica/inmunología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Proteína 2 de la Membrana Asociada a los Lisosomas/inmunología , Ratones , Ratones Noqueados , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptor Cannabinoide CB2/genética , Receptor Toll-Like 4/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/inmunología , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/inmunología , Proteínas de Unión a GTP rab7RESUMEN
BACKGROUND: Huntington's disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases. METHODS: Bone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of ß-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed. RESULTS: R6/1 BMMCs showed normal ß-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-ß). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice. CONCLUSIONS: Our data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.
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Citocinas/metabolismo , Proteína Huntingtina/genética , Mastocitos/metabolismo , Transporte de Proteínas/genética , Receptor Toll-Like 4/metabolismo , Animales , Endotoxemia/metabolismo , Inflamación/metabolismo , Lipopolisacáridos , Ratones , Ratones Transgénicos , Receptores de IgE/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Transforming growth factor-ß (TGF-ß) is a potent mast cell (MC) chemoattractant able to modulate local inflammatory reactions. The molecular mechanism leading to TGF-ß-directed MC migration is not fully described. Here we analyzed the role of the Src family protein kinase Fyn on the main TGF-ß-induced cytoskeletal changes leading to MC migration. Utilizing bone marrow-derived mast cells (BMMCs) from WT and Fyn-deficient mice we found that BMMC migration to TGF-ß was impaired in the absence of the kinase. TGF-ß caused depolymerization of the cortical actin ring and changes on the phosphorylation of cofilin, LIMK and CAMKII only in WT cells. Defective cofilin activation and phosphorylation of regulatory proteins was detected in Fyn-deficient BMMCs and this finding correlated with a lower activity of the catalytic subunit of the phosphatase PP2A. Diminished TGF-ß-induced chemotaxis of Fyn-deficient cells was also observed in an in vivo model of MC migration (bleomycin-induced scleroderma). Our results show that Fyn kinase is an important positive effector of TGF-ß-induced chemotaxis through the control of PP2A activity and this is relevant to pathological processes that are related to TGF-ß-dependent mast cell migration.
Asunto(s)
Actinas/metabolismo , Quimiotaxis , Mastocitos/fisiología , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Factores Despolimerizantes de la Actina/metabolismo , Animales , Mastocitos/inmunología , Ratones , Ácido Ocadaico/farmacología , Fosforilación , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Transducción de Señal , Proteína Smad2/metabolismoRESUMEN
Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/ß, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/ß activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/ß necessary for the secretion of TNF by VAMP3(+) carriers.
Asunto(s)
Regulación de la Expresión Génica , Mastocitos/inmunología , Proteína Quinasa C-alfa/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Lipopolisacáridos/inmunología , Mastocitos/efectos de los fármacos , Ratones , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-fyn/deficiencia , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
The Wnt/ß-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/ß-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/ß-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and ß-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with ß-catenin to promote cell proliferation.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Papillomavirus Humano 18/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Factor de Transcripción 4/metabolismo , Factores de Transcripción/genética , Neoplasias del Cuello Uterino/virología , Empalme Alternativo , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/genética , Humanos , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación hacia Arriba , Neoplasias del Cuello Uterino/metabolismo , Vía de Señalización WntRESUMEN
Cacalolides are a kind of sesquiterpenoids natural compounds synthesized by Psacalium decompositum (A. Gray) H. Rob. & Brettell or Psacalium peltatum (Kunth) Cass. Antioxidant and hypoglycemic effects have been found for cacalolides such as cacalol, cacalone or maturine, however, their effects on inflammatory processes are still largely unclear. The main aim of this study was to investigate the biological activities of secondary metabolites from P. decompositum and P. peltatum through two approaches: (1) chemoinformatic and toxicoinformatic analysis based on ethnopharmacologic background; and (2) the evaluation of their potential anti-inflammatory/anti-allergic effects in bone marrow-derived mast cells by IgE/antigen complexes. The bioinformatics properties of the compounds: cacalol; cacalone; cacalol acetate and maturin acetate were evaluated through Osiris DataWarrior software and Molinspiration and PROTOX server. In vitro studies were performed to test the ability of these four compounds to inhibit antigen-dependent degranulation and intracellular calcium mobilization, as well as the production of reactive oxygen species in bone marrow-derived mast cells. Our findings showed that cacalol displayed better bioinformatics properties, also exhibited a potent inhibitory activity on IgE/antigen-dependent degranulation and significantly reduced the intracellular calcium mobilization on mast cells. These data suggested that cacalol could reduce the negative effects of the mast cell-dependent inflammatory process.
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Mastocitos/metabolismo , Psacalium/química , Receptores de IgE/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Inflamación/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologíaRESUMEN
Fyn kinase is widely expressed in neuronal and glial cells of the brain, where it exerts multiple functional roles that affect fundamental physiological processes. The aim of our study was to investigate the, so far unknown, functional role of Fyn in the retina. We report that Fyn is expressed, in vivo, in a subpopulation of Müller glia. We used a mouse model of Fyn genetic ablation and Müller-enriched primary cultures to demonstrate that Fyn deficiency induces morphological alterations in the mature retina, a reduction in the thickness of the outer and inner nuclear layers and alterations in postnatal Müller cell physiology. These include shortening of Müller cell processes, a decrease in cell proliferation, inactivation of the Akt signal transduction pathway, a reduced number of focal adhesions points and decreased adhesion of these cells to the ECM. As abnormalities in Müller cell physiology have been previously associated to a compromised retinal function we evaluated behavioral responses to visual stimulation. Our results associate Fyn deficiency with impaired visual optokinetic responses under scotopic and photopic light conditions. Our study reveals novel roles for Fyn kinase in retinal morphology and Müller cell physiology and suggests that Fyn is required for optimal visual processing.
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Células Ependimogliales/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Animales , Adhesión Celular , Proliferación Celular , Células Cultivadas , Células Ependimogliales/citología , Células Ependimogliales/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Transducción de Señal , Visión OcularRESUMEN
We evaluated the antihypertensive properties of 4-tert-buthyl-2,6-bis(thiomorpholine-4-ilmethyl)phenol (TBTIF). Spontaneously hypertensive rats were treated with TBTIF or captopril (both at 1 mg·kg⻹·d⻹ intramuscularly for 4 days), and their blood pressure (BP) was assessed. In some experiments, concentration response curves to angiotensin I or angiotensin II were generated in rat aortic rings and in the absence or presence of Ang-(1-7), N(G)-monomethyl L-arginine, or both; additionally, the angiotensin-converting enzyme (ACE) and ACE2 mRNA levels were quantified in the aortic rings using reverse transcription-polymerase chain reaction. TBTIF diminished BP and reduced angiotensin I- or angiotensin II-induced vasoconstriction. The presence of Ang-(1-7) induced a greater reduction in vasoconstriction, and this effect was reversed by L-N(G)-monomethyl arginine. Moreover, TBTIF decreased the mRNA of ACE and increased the mRNA of ACE2. In conclusion, TBTIF diminished rat BP through nitric oxide-dependent and nitric oxide-independent mechanisms. In contrast to captopril, TBTIF exhibits better antihypertensive properties through mechanisms that involve ACE2.
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Antihipertensivos/farmacología , Aorta Torácica/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Morfolinas/farmacología , Fenoles/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Aorta Torácica/fisiopatología , Captopril/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Endogámicas SHR , Regulación hacia ArribaRESUMEN
We have previously shown that morphine pretreatment inhibits mast cell-dependent TNF production after LPS injection in the murine peritoneal cavity. In this study, we used bone marrow-derived mast cells (BMMCs) to investigate the molecular mechanisms of that inhibition. We found that morphine prevented LPS-induced TNF secretion in these cells. The observed inhibition was not due to morphine-induced TLR4 internalization and it was related to the blockage of preformed TNF secretion. LPS-induced TNF exocytosis in BMMCs was dependent on tetanus toxin-insensitive vesicle-associated membrane proteins and calcium mobilization, as well as PI3K, MAPK, and IκB kinase (IKK) activation. TNF secretion was also associated to the phosphorylation of synaptosomal-associated protein 23 (SNAP-23), which was found forming a complex with IKK in LPS-activated BMMCs. Morphine pretreatment prevented TLR4-dependent ERK and IKK phosphorylation. Analyzing the signaling events upstream of IKK activation, we found diminished TGF-ß-activated kinase 1 (TAK1) phosphorylation and TNFR-associated factor (TRAF) 6 ubiquitination in BMMCs pretreated with morphine and stimulated with LPS. Morphine pretreatment provoked a marked increase in the formation of a molecular complex composed of TRAF6 and ß-arrestin-2. Naloxone and a combination of µ and δ opioid receptor antagonists prevented morphine inhibitory actions. In conclusion, our results show that activation of µ and δ opioid receptors with morphine suppresses TLR4-induced TNF release in mast cells, preventing the IKK-dependent phosphorylation of SNAP-23, which is necessary for TNF exocytosis, and this inhibition correlates with the formation of a ß-arrestin-2/TRAF6 complex. To our knowledge, these findings constitute the first evidence of molecular crosstalk between opioid receptors and the TLR4 signal transduction system in mast cells.
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Arrestinas/metabolismo , Mastocitos/efectos de los fármacos , Morfina/farmacología , Narcóticos/farmacología , Proteínas Qb-SNARE/inmunología , Proteínas Qc-SNARE/inmunología , Transducción de Señal/efectos de los fármacos , Factor 6 Asociado a Receptor de TNF/metabolismo , Animales , Activación Enzimática , Citometría de Flujo , Quinasa I-kappa B/metabolismo , Immunoblotting , Inmunoprecipitación , Lipopolisacáridos/inmunología , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Arrestina beta 2 , beta-ArrestinasRESUMEN
UNLABELLED: Immunosuppressive morphine actions are well characterized, but other opiates are less studied. The objectives of this work were: (a) to compare the acute effects of morphine and fentanyl to inhibit early peritoneal LPS-induced TNFα release; (b) to find if, as in the case of morphine, intraperitoneal mast cells (MCs) are the target of fentanyl's immunosuppressive actions; and (c) to analyze if repeated opiate administration induces tolerance to opiate immunosuppressive effects. Independent groups of mice received a single i.p. injection of morphine (0.1-10mg/kg) or fentanyl (0.001-0.1mg/kg) 10min prior to LPS (1mg/kg). Peritoneal TNFα levels were determined 1h later. The effects of fentanyl were analyzed in MC-deficient mice (W-sh) and in W-sh mice reconstituted with bone marrow-derived MCs. Other animals received 6 or 10 doses of morphine (10mg/kg, 3×/day) or fentanyl (0.1mg/kg 3×/day) and were then challenged with LPS. Fentanyl was equally effective and 1000× more potent than morphine to inhibit i.p. LPS-induced TNFα release and this was dependent on intraperitoneal MCs. Repeated morphine administration induced tolerance to both antinociception and inhibition of response to endotoxin. Repeated fentanyl injection did not induce significant antinociceptive tolerance, but, interestingly, produced sensitization to LPS. IN CONCLUSION: (1) opiates with different analgesic potency also differ in their potency to induce immunosuppression; (2) MCs are the cellular target of the immunosuppressive actions of fentanyl here studied; (3) in contrast with morphine, tolerance to fentanyl's immunosuppressive actions can be dissociated from tolerance to its antinociceptive effects.
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Analgésicos/farmacología , Fentanilo/farmacología , Inmunosupresores/farmacología , Lipopolisacáridos/farmacología , Morfina/farmacología , Umbral del Dolor/efectos de los fármacos , Analgésicos/uso terapéutico , Animales , Fentanilo/uso terapéutico , Inmunosupresores/uso terapéutico , Masculino , Ratones , Morfina/uso terapéutico , Dolor/tratamiento farmacológico , Dolor/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Human papillomavirus (HPV)-positive Head and Neck Squamous Cell Carcinomas (HNSCC) comprise a particular cancer entity traditionally associated with better clinical outcomes. Around 25% of HNSCC are HPV positive, HPV16 being the most prevalent type. Nevertheless, close to 30% of the HPV-positive patients have an unfavorable prognosis, revealing that this type of tumor exhibits great heterogeneity leading to different clinical behaviors. Efforts have been made to identify RNA molecules with prognostic value associated with the clinical outcome of patients with HPV-positive HNSCC, with the aim of identifying patients at high risk of metastasis, disease recurrence, and poor survival, who would require closer clinical follow-up and timely intervention. Moreover, the molecular identification of those HPV-positive HNSCC patients with good prognosis will allow the implementation of de-escalating therapeutic strategies, aiming to reduce side effects, resulting in a better quality of life. This review compiles a series of recent studies addressing different methodological and conceptual approaches aimed at searching for potential gene expression-based biomarkers associated with the prognosis of patients with HPV-positive HNSCC.
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Biomarcadores de Tumor , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/virología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Pronóstico , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/genética , Neoplasias de Cabeza y Cuello/virología , Neoplasias de Cabeza y Cuello/genética , Papillomaviridae/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
This work aimed to show which treatments showed efficacy against coronavirus disease 2019 (COVID-19); therefore, the results of 37 clinical trials started in 2020 and completed in 2021 are reviewed and discussed here. These were selected from databases, excluding vaccines, computational studies, in silico, in vitro, and those with hyperimmune sera from recovered patients. We found 34 drugs, one vitamin, and one herbal remedy with pharmacological activity against symptomatic COVID-19. They reduced mortality, disease progression, or recovery time. For each treatment, the identifier and type of trial, the severity of the disease, the sponsor, the country where the trial was conducted, and the trial results are presented. The drugs were classified according to their mechanism of action. Several drugs that reduced mortality also reduced inflammation in the most severe cases. These include some that are not considered anti-inflammatory, such as Aviptadil, pyridostigmine bromide, anakinra, imatinib, baricitinib, and bevacizumab, as well as the combination of ivermectin, aspirin, dexamethasone, and enoxaparin. Nigella sativa seeds with honey have also been reported to have therapeutic activity. On the other hand, tofacitinib, novaferon with ritonavir, and lopinavir were also effective, as well as in combination with antiviral therapies such as danoprevir with ritonavir. The natural products colchicine and Vitamin D3 were only effective in patients with mild-to-moderate COVID-19, as was hydroxychloroquine. Drug repositioning has been the main tool in the search for effective therapies by expanding the pharmacological options available to patients.
El objetivo del presente trabajo fue conocer qué tratamientos mostraron efectividad contra COVID-19, para lo cual se revisan y discuten los resultados de 37 estudios clínicos iniciados durante 2020 y concluidos en 2021. Estos fueron seleccionados de bases de datos, excluyendo vacunas, estudios computacionales, in silico, in vitro y con sueros hiperinmunes de pacientes recuperados. Se documentaron 34 fármacos, una vitamina y un remedio herbolario, con actividad farmacológica ante COVID-19 sintomático. Estos redujeron la mortalidad, el progreso de la enfermedad, o el tiempo de recuperación. Para cada tratamiento se presenta identificador y tipo de estudio, la gravedad de la enfermedad, patrocinador, país donde se realizó, así como sus resultados. Los fármacos se clasificaron de acuerdo con su mecanismo de acción. Varios fármacos que redujeron la mortalidad también disminuyeron la inflamación en los casos más graves. Esto incluyendo algunos no considerados antiinflamatorios, como el aviptadil, el bromuro de piridostigmina, el anakinra, el imatinib, el baricitinib y el bevacizumab, así como la combinación de ivermectina, aspirina, dexametasona y enoxaparina. También se reportaron con actividad terapéutica las semillas de Nigella sativa con miel. Además, resultaron efectivos el tofacitinib, el novaferón con ritonavir y lopinavir, así como los antivirales en terapias combinadas como el danoprevir con ritonavir. Los productos naturales colchicina y vitamina D3, solo tuvieron actividad en los pacientes en estado leve a moderado de la COVID-19, así como la hidroxicloroquina. El reposicionamiento de fármacos fue la principal herramienta para buscar terapias efectivas ampliando las opciones farmacológicas accesibles a los pacientes.
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Productos Biológicos , COVID-19 , Humanos , Ritonavir/uso terapéutico , Antivirales/uso terapéutico , Antivirales/farmacología , SARS-CoV-2 , PandemiasRESUMEN
Allergic reactions are highly prevalent pathologies initiated by the production of IgE antibodies against harmless antigens (allergens) and the activation of the high-affinity IgE receptor (FcεRI) expressed in the surface of basophils and mast cells (MCs). Research on the mechanisms of negative control of those exacerbated inflammatory reactions has been intense in recent years. Endocannabinoids (eCBs) show important regulatory effects on MC-mediated immune responses, mainly inhibiting the production of pro-inflammatory mediators. However, the description of the molecular mechanisms involved in eCB control of MC activation is far from complete. In this review, we aim to summarize the available information regarding the role of eCBs in the modulation of FcεRI-dependent activation of that cell type, emphasizing the description of the eCB system and the existence of some of its elements in MCs. Unique characteristics of the eCB system and cannabinoid receptors (CBRs) localization and signaling in MCs are mentioned. The described and putative points of cross-talk between CBRs and FcεRI signaling cascades are also presented. Finally, we discuss some important considerations in the study of the effects of eCBs in MCs and the perspectives in the field.
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Hipersensibilidad , Receptores de IgE , Humanos , Receptores de IgE/metabolismo , Inmunoglobulina E/metabolismo , Inmunoglobulina E/farmacología , Endocannabinoides/metabolismo , Endocannabinoides/farmacología , Mastocitos/metabolismo , Hipersensibilidad/metabolismoRESUMEN
Toll-like receptors (TLRs) are central players in innate immunity responses. They are expressed in glial cells and neurons, and their overactivation leads to the production of proinflammatory molecules, neuroinflammation, and neural damage associated with many neurodegenerative pathologies, such as Huntington's disease (HD). HD is an inherited disorder caused by a mutation in the gene coding for the protein Huntingtin (Htt). Expression of mutated Htt (mHtt) causes progressive neuronal degeneration characterized by striatal loss of GABAergic neurons, oxidative damage, neuroinflammatory processes, and impaired motor behavior. The main animal models to study HD are the intrastriatal injection of quinolinic acid (QA) and the transgenic B6CBA-Tg (HDexon1)61Gpb/1 J mice (R6/1). Those models mimic neuronal damage and systemic manifestations of HD. The objective of this work was to study the participation of TLR4 in the manifestations of neuronal damage and HD symptoms in the two mentioned models. For this purpose, C57BL6/J and TLR4-KO mice were administered with QA, and after that motor activity, and neuronal and oxidative damages were measured. R6/1 and TLR4-KO were mated to study the effect of low expression of TLR4 on the phenotype manifestation in R6/1 mice. We found that TLR4 is involved in motor activity, and neurological and oxidative damage induced by intrastriatal injection of QA, and the low expression of TLR4 causes a delay in the onset of phenotypic manifestations by the mHtt expression in R6/1 mice. Our results show that TLR4 is involved in both models of HD and focuses then as a therapeutic target for some deleterious reactions in HD.