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Trastuzumab therapy in HER2+ breast cancer patients has mixed success owing to acquired resistance to therapy. A detailed understanding of downstream molecular cascades resulting from trastuzumab resistance is yet to emerge. In this study, we investigate the cellular mechanisms underlying acquired resistance using trastuzumab-sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG across time. We probe early receptor organization through microscopy and signaling events through multiomics measurements and assess the bioenergetic state through mitochondrial measurements. Integrative analyses of our measurements reveal significant alterations in EGF-treated BT474 HER2 membrane dynamics and robust downstream activation of PI3K/AKT/mTORC1 signaling. EGF-treated BT474R shows a sustained interferon-independent activation of the IRF1/STAT1 cascade, potentially contributing to trastuzumab resistance. Both cell lines exhibit temporally divergent metabolic demands and HIF1A-mediated stress responses. BT474R demonstrates inherently increased mitochondrial activity. HRG treatment in BT474R leads to a pronounced reduction in AR expression, affecting downstream lipid metabolism with implications for treatment response. Our results provide novel insights into mechanistic changes underlying ligand treatment in BT474 and BT474R and emphasize the pivotal role of endogenous ligands. These results can serve as a framework for furthering the understanding of trastuzumab resistance, with therapeutic implications for women with acquired resistance.
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The two subtypes of cannabinoid receptors (CBR), namely CB1R and CB2R, belong to the G protein-coupled receptor (GPCR) superfamily and are confirmed as potential therapeutic targets for a variety of diseases such as inflammation, neuropathic pain, and immune-related disorders. Since CB1R is mainly distributed in the central nervous system (CNS), it could produce severe psychiatric adverse reactions and addiction. In contrast, CB2R are predominantly distributed in the peripheral immune system with minimal CNS-related side effects. Therefore, more attention has been devoted to the discovery of CB2R ligands. In view of the favorable profile of CB2R, many high-binding affinity and selectivity CB2R ligands have been developed recently. This paper reviews recent research progress on CB2R ligands, including endogenous CB2R ligands, natural compounds, and novel small molecules, in order to provide a reference for subsequent CB2R ligand development.
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Canabinoides , Inflamação , Humanos , Receptores de CanabinoidesRESUMO
Tumor necrosis is a recurrent characteristic of head and neck squamous cell carcinomas (HNSCCs). There is a need for more investigations on the influence of biomolecules released by these necrotic foci in the HNSCC tumor microenvironment. It is suspected that a fraction of the biomolecules released by necrotic cells are damage-associated molecular patterns (DAMPs), which are known to be natural endogenous ligands of Toll-like receptors (TLRs), including, among others, proteins and nucleic acids. However, there has been no direct demonstration that biomolecules released by HNSCC necrotic cells can activate TLRs. Our aim was to investigate whether some of these molecules could behave as agonists of the TLR3, either in vitro or in vivo. We chose a functional approach based on reporter cell exhibiting artificial TLR3 expression and downstream release of secreted alkaline phosphatase. The production of biomolecules activating TLR3 was first investigated in vitro using three HNSCC cell lines subjected to various pronecrotic stimuli (external irradiation, serum starvation, hypoxia and oxidative stress). TLR3 agonists were also investigated in necrotic tumor fluids from five oral cancer patients and three mouse tumor grafts. The release of biomolecules activating TLR3 was demonstrated for all three HNSCC cell lines. External irradiation was the most consistently efficient stimulus, and corresponding TLR3 agonists were conveyed in extracellular vesicles. TLR3-stimulating activity was detected in the fluids from all five patients and three mouse tumor grafts. In most cases, this activity was greatly reduced by RNAse pretreatment or TLR3 blocking antibodies. Our data indicate that TLR3 agonists are consistently present in necrotic fluids from HNSCC cells and mainly made of dsRNA fragments. These endogenous agonists may induce TLR3, which might lead to a protumorigenic effect. Regarding methodological aspects, our study demonstrates that direct investigations-including functional testing-can be performed on necrotic fluids from patient tumors.
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Neoplasias de Cabeça e Pescoço , Receptor 3 Toll-Like , Animais , Humanos , Camundongos , Necrose/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Receptor 3 Toll-Like/metabolismo , Receptor Toll-Like 9 , Receptores Toll-Like , Microambiente TumoralRESUMO
The aryl hydrocarbon receptor (AhR) is a decisive regulatory ligand-dependent transcription factor. It binds highly diverse ligands, which can be categorized as either endogenous or exogenous. Ligand binding activates AhR, which can adjust inflammatory responses by modulating immune cells such as dendritic cells (DCs). However, how different AhR ligand classes impact the phenotype and function of human monocyte-derived DCs (hMoDCs) has not been extensively studied in a comparative manner. We, therefore, tested the effect of the representative compounds Benzo(a)pyrene (BP), 6-formylindolo[3,2-b]carbazole (FICZ), and Indoxyl 3-sulfate (I3S) on DC biology. Thereby, we reveal that BP significantly induces a tolerogenic response in lipopolysaccharide-matured DCs, which is not apparent to the same extent when using FICZ or I3S. While all three ligand classes activate AhR-dependent pathways, BP especially induces the expression of negative immune regulators, and subsequently strongly subverts the T cell stimulatory capacity of DCs. Using the CRISPR/Cas9 strategy we also prove that the regulatory effect of BP is strictly AhR-dependent. These findings imply that AhR ligands contribute differently to DC responses and incite further studies to uncover the mechanisms and molecules which are involved in the induction of different phenotypes and functions in DCs upon AhR activation.
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Regulação da Expressão Gênica , Receptores de Hidrocarboneto Arílico , Humanos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Ligantes , Carbazóis/farmacologia , Carbazóis/metabolismo , Indicã/metabolismo , Células Dendríticas , BiologiaRESUMO
The neurobiology drug discovery landscape has transformed over the past decade or so by the discovery of allosteric modulators of receptor superfamilies. A wide range of physiological reactions can occur in response to a limited number of neurotransmitters. This review provides an update on physiological features of the receptors and the signaling pathways that are generated in response to neuroreceptor activation that allow the explanation of this vast array of neurotransmitter responses. Primarily based upon structure, receptors in the nervous system can be classified into four groups: Gprotein coupled receptors, ligand-gated receptors, enzyme-linked receptors, and nuclear receptors. With a particular emphasis on the central nervous system, i.e., brain, spinal cord, and optic nerves, we identify the neuroreceptors, their endogenous agonists, antagonists, sites of expression within the nervous system, current neuropharmacological clinical use, and potential for new drug discovery. New molecular approaches and advances in our knowledge of neuronal communication in processes involved in development, functioning and disorders of the nervous system combined with opportunities to re-purpose existing drugs for new indications continue to highlight the exciting opportunities to improve human health.
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Doenças do Sistema Nervoso Central , Neurofarmacologia , Humanos , Transdução de Sinais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Descoberta de DrogasRESUMO
Receptor for advanced glycation end products (RAGE) is a 45 kDa transmembrane receptor of immunoglobulin family that can bind to various endogenous and exogenous ligands and initiate the inflammatory downstream signaling pathways. RAGE is involved in various disorders including cardiovascular and neurodegenerative diseases, cancer, and diabetes. This review summarizes the structural features of RAGE and its various isoforms along with their pathological effects. Mainly, the article emphasized on the translational significance of antagonizing the interactions of RAGE with its ligands using small molecules reported in the last 5 years and discusses future approaches that could be employed to block the interactions in the treatment of chronic inflammatory ailments. The RAGE inhibitors described in this article could prove as a powerful approach in the management of immune-inflammatory diseases. A critical review of the literature suggests that there is a dire need to dive deeper into the molecular mechanism of action to resolve critical issues that must be addressed to understand RAGE-targeting therapy and long-term blockade of RAGE in human diseases.
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Diabetes Mellitus , Produtos Finais de Glicação Avançada , Diabetes Mellitus/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Ligantes , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Transdução de SinaisRESUMO
Retinoid X receptors (RXRs) present a subgroup of the nuclear receptor superfamily with particularly high evolutionary conservation of ligand binding domain. The receptor exists in α, ß, and γ isotypes that form homo-/heterodimeric complexes with other permissive and non-permissive receptors. While research has identified the biochemical roles of several nuclear receptor family members, the roles of RXRs in various neurological disorders remain relatively under-investigated. RXR acts as ligand-regulated transcription factor, modulating the expression of genes that plays a critical role in mediating several developmental, metabolic, and biochemical processes. Cumulative evidence indicates that abnormal RXR signalling affects neuronal stress and neuroinflammatory networks in several neuropathological conditions. Protective effects of targeting RXRs through pharmacological ligands have been established in various cell and animal models of neuronal injury including Alzheimer disease, Parkinson disease, glaucoma, multiple sclerosis, and stroke. This review summarises the existing knowledge about the roles of RXR, its interacting partners, and ligands in CNS disorders. Future research will determine the importance of structural and functional heterogeneity amongst various RXR isotypes as well as elucidate functional links between RXR homo- or heterodimers and specific physiological conditions to increase drug targeting efficiency in pathological conditions.
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Doenças do Sistema Nervoso , Receptores Citoplasmáticos e Nucleares , Animais , Regulação da Expressão Gênica , Ligantes , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores X de Retinoides/metabolismoRESUMO
Experiments with cell cultures and animal models have provided solid support for the assumption that Nerve Growth Factor (NGF) plays a key role in the regulation of neuronal cell survival and death. Recently, endogenous ligands have been proposed as physiological modulators of NGF biological activity as part of this regulatory cascade. However, the structural and mechanistic determinants for NGF bioactivity remain to be elucidated. We recently unveiled, by an integrated structural biology approach, the ATP binding sites of NGF and investigated the effects on TrkA and p75NTR receptors binding. These results pinpoint ATP as a genuine endogenous modulator of NGF signaling, paving the way to the characterization of not-yet-identified chemical diverse endogenous biological active small molecules as novel modulators of NGF. The present review aims at providing an overview of the currently available 3D structures of NGF in complex with different small endogenous ligands, featuring the molecular footprints of the small molecules binding. This knowledge is essential for further understanding the functional role of small endogenous ligands in the modulation of neurotrophins signaling in physiological and pathological conditions and for better exploiting the therapeutic potentialities of NGF.
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Fator de Crescimento Neural/ultraestrutura , Fatores de Crescimento Neural/ultraestrutura , Receptor de Fator de Crescimento Neural/genética , Receptor trkA/genética , Animais , Sítios de Ligação/genética , Humanos , Ligantes , Fator de Crescimento Neural/química , Fator de Crescimento Neural/genética , Fatores de Crescimento Neural/genética , Neurônios/metabolismo , Neurônios/ultraestrutura , Células PC12 , Ligação Proteica/genética , Ratos , Transdução de Sinais/genéticaRESUMO
The Nerve Growth Factor (NGF) neurotrophin acts in the maintenance and growth of neuronal populations. Despite the detailed knowledge of NGF's role in neuron physiology, the structural and mechanistic determinants of NGF bioactivity modulated by essential endogenous ligands are still lacking. We present the results of an integrated structural and advanced computational approach to characterize the extracellular ATP-NGF interaction. We mapped by NMR the interacting surface and ATP orientation on NGF and revealed the functional role of this interaction in the binding to TrkA and p75NTR receptors by SPR. The role of divalent ions was explored in conjunction with ATP. Our results pinpoint ATP as a likely transient molecular modulator of NGF signaling, in health and disease states.
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The potential use of cannabinoids for therapeutic purposes is at the forefront of cannabinoid research which aims to develop innovative strategies to prevent, manage and treat a broad spectrum of human diseases. This chapter briefly reviews the pivotal role of the endocannabinoid system in modulating the central nervous system and its roles on neurodegenerative diseases and brain disorders. Ligand-induced modulation of cannabinoid 1 and 2 receptors to modulate immune response, decrease neurodegeneration and pain are aspects that are also discussed.
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Canabinoides , Doenças do Sistema Nervoso Central , Canabinoides/uso terapêutico , Doenças do Sistema Nervoso Central/tratamento farmacológico , Endocanabinoides , Humanos , Ligantes , Receptores de CanabinoidesRESUMO
Aryl hydrocarbon receptor (AHR) was initially discovered as a cellular protein involved in mediating the detoxification of xenobiotic compounds. Extensive research in the past two decades has identified several families of physiological ligands and uncovered important functions of AHR in normal development and homeostasis. Deficiency in AHR expression disrupts major signaling systems and transcriptional programs, which appear to be responsible for the development of numerous developmental abnormalities including cardiac hypertrophy and epidermal hyperplasia. This mini review primarily summarizes recent advances in our understanding of AHR functions in normal physiology with an emphasis on the cardiovascular, gastrointestinal, integumentary, nervous, and immunomodulatory systems.
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Trato Gastrointestinal/fisiologia , Sistema Imunitário/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Humanos , Ligantes , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia , Xenobióticos/metabolismoRESUMO
G protein-coupled receptors (GPCRs) represent the largest class of "druggable" proteins in the human genome. For more than a decade, crystal structures and, more recently, cryoEM structures of GPCR complexes have provided unprecedented insight into GPCR drug binding and cell signaling. Nevertheless, structure determination of receptors in complexes with weakly binding molecules or complex polypeptides remains especially challenging, including for hormones, many of which have so far eluded researchers. Nuclear magnetic resonance (NMR) spectroscopy has emerged as a promising approach to determine structures of ligands bound to their receptors and to provide insights into the dynamics of GPCR-bound drugs. The capability to investigate compounds with weak binding affinities has also been leveraged in NMR applications to identify novel lead compounds in drug screening campaigns. We review recent structural biology studies of GPCR ligands by NMR, highlighting new methodologies enabling studies of GPCRs with native sequences and in native-like membrane environments that provide insights into important drugs and endogenous ligands.
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Espectroscopia de Ressonância Magnética/métodos , Neuropeptídeos/química , Receptores Acoplados a Proteínas G/química , Humanos , Ligantes , Bicamadas Lipídicas/química , Modelos Moleculares , Preparações Farmacêuticas/química , Ligação ProteicaRESUMO
Natural killer T (NKT) cells have been placed at the interface between innate and adaptive immunity by a long series of experiments that convincingly showed that beyond cytokine secretion and NK cell recruitment, NKT cells were coordinating dendritic cell and B cell maturation through direct membrane contacts and initiate productive responses. As such, NKT cells are the cellular adjuvant of many immune reactions and have functions that go much beyond what their name encapsulates. In addition, the initial discovery of the ligands of NKT cells is deeply linked to cancer biology and therapy. However, for a host of reasons, animal models in which agonists of NKT cells were used did not translate well to human cancers. A systematic reassessment of NKT cells role in tumorigenesis, especially spontaneous one, is now accessible using single cell analysis technologies both in mouse and man, and should be taken advantage of. Similarly, the migration, localization, phenotype of NKT cells following induced expansion after injection of an agonist can be examined at the single cell level. This technological revolution will help evaluate where and how NKT cells can be used in cancer.
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Monocytes of patients with ankylosing spondylitis (AS) have toll-like receptor 4 (TLR4) overexpression on their monocytes. Myeloid-related protein (MRP) 8/14 protein complexes are calcium-binding proteins, which act as endogenous ligands to TLR4. Thus, we studied the levels of MRP8/14 in adult AS patients. MRP8/14 levels were assessed in 99 adult AS patients satisfying Assessments in Ankylosing Spondylitis International Society 2010 criteria and 71 healthy controls by ELISA. Patient disease parameters like patient and physician global assessment, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), swollen and tender joint count, entheseal count by Maastricht enthesitis index, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were also recorded. Levels were reassessed in 23 patients after 2-5 months of treatment with NSAIDs. All values are in median (IQR). The serum MRP8/14 levels in patients [34.1 (17.94-264.58) µg/ml] were significantly higher than in healthy controls [4.94 (IQR 3.01-8.32) µg/ml (p < 0.0001)]. Patients with peripheral arthritis (n = 50) had higher levels than those with pure axial disease (n = 49) [40.63 (IQR 28.41-73.15) µg/ml vs. 23.72 (11.04-61.55) µg/ml; p = 0.012]. Levels of MRP8/14 correlated with AS Disease Activity Score (DAS)-CRP (r = 0.23, 95%CI = 0.038-0.422, p = 0.02) and CRP (r = 0.28, 95%CI = 0.081-0.45, p = 0.01), and the correlation was better in early disease [≤5 years disease duration; r = 0.40, p = 0.007 and r = 0.57, p = <0.0001, respectively]. Baseline levels were higher in treatment responders than in non-responders [51.17 vs. 32.22 µg/ml; p = 0.02]. Change in MRP8/14 levels correlated with change in BASDAI and ASDAS-CRP (r = 0.489, p = 0.018 and r = 0·498, p = 0.016, respectively). MRP8/14 levels may be used as a biomarker for activity, peripheral arthritis, and response to therapy.
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Artrite/sangue , Calgranulina A/sangue , Calgranulina B/sangue , Espondilite Anquilosante/sangue , Adolescente , Adulto , Artrite/complicações , Biomarcadores/sangue , Sedimentação Sanguínea , Proteína C-Reativa/química , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Imunidade Inata , Inflamação , Masculino , Pessoa de Meia-Idade , Espondiloartropatias/sangue , Espondilite Anquilosante/complicações , Adulto JovemRESUMO
Increased expression of endogenous Toll-like receptor 4 (TLR4) ligands (e.g., Tenascin-C, S100A8/A9, citrullinated fibrinogen (cFb) immune complexes) has been observed in patients with rheumatoid arthritis (RA). However, their roles in RA pathogenesis are not well understood. Here, we investigated the expression kinetics and role of endogenous TLR4 ligands in the murine model of collagen-induced arthritis (CIA). Tenascin-C was upregulated in blood early in CIA, and correlated positively with the clinical score at day 56. Levels of S100A8/A9 increased starting from day 28, peaking at day 42, and correlated positively with joint inflammation. Levels of anti-cFb antibodies increased during the late phase of CIA and correlated positively with both joint inflammation and cartilage damage. Blockade of TLR4 activation at the time of the first TLR4 ligand upregulation prevented clinical and histological signs of arthritis. A TLR4-dependent role was also observed for Tenascin-C and cFb immune complexes in osteoclast differentiation in vitro. Taken together, our data suggests that the pathogenic contribution of TLR4 in promoting joint inflammation and bone erosion during CIA occurs via various TLR4 ligands arising at different stages of disease. The data also suggests that Blockade of TLR4 with monoclonal antibodies is a promising strategy in RA treatment.
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Artrite Reumatoide/imunologia , Osso e Ossos/patologia , Calgranulina A/sangue , Tenascina/sangue , Receptor 4 Toll-Like/metabolismo , Animais , Complexo Antígeno-Anticorpo/sangue , Artrite Experimental/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Calgranulina A/genética , Diferenciação Celular , Colágeno , Modelos Animais de Doenças , Fibrinogênio/imunologia , Articulações/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Osteoclastos/metabolismo , Análise Espaço-Temporal , Tenascina/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
The tumor-associated inflammatory microenvironment may play a pivotal role in epithelial ovarian cancer (EOC) carcinogenesis and outcomes, but a detailed profile in patient-derived tumors is needed. Here, we investigated the expression of TLR4- and MyD88-associated markers in tumors from over 500 EOC patients using immunohistochemical staining. We demonstrate that high expression of TLR4 and MyD88 predicts poorer overall survival in patients with EOC; most likely, this is due to their association with serous histology and features of high tumor burden and aggressiveness, including stage, grade, and ascites at surgery. Combined TLR4 and MyD88 expression appears to serve as an independent risk factor for shortened survival time, even after covariate adjustment (both moderate HR 1.1 [95 % CI 0.7-1.8], both strong HR 2.1 [95 % CI 1.1-3.8], both weak as referent; p = 0.027). We reveal that in EOC tissues with elevated expression of both TLR4 and MyD88 and activated NF-κB signaling pathway, expression of hsp60, hsp70, beta 2 defensin, and HMGB1 are also enriched. In total, these results suggest that activation of TLR4/MyD88/NF-κB signaling by endogenous ligands may contribute to an inflammatory microenvironment that drives a more aggressive phenotype with poorer clinical outcome in EOC patients.
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Inflamação/complicações , Inflamação/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias Epiteliais e Glandulares/etiologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/metabolismo , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Carcinoma Epitelial do Ovário , Transformação Celular Neoplásica , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Inflamação/patologia , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , Transdução de Sinais , Receptor 4 Toll-Like/genética , Microambiente Tumoral/genética , Adulto JovemRESUMO
9-cis-Retinoic acid was identified and claimed to be the endogenous ligand of the retinoidâ X receptors (RXRs) in 1992. Since then, the endogenous presence of this compound has never been rigorously confirmed. Instead, concerns have been raised by other groups that have reported that 9-cis-retinoic acid is undetectable or that its presence occurs at very low levels. Furthermore, these low levels could not satisfactorily explain the physiological activation of RXR. Alternative ligands, among them various lipids, have also been identified, but also did not fulfill criteria for rigorous endogenous relevance, and their consideration as bonaâ fide endogenous mammalian RXR ligand has likewise been questioned. Recently, novel studies claim that the saturated analogue 9-cis-13,14-dihydroretinoic acid functions as an endogenous physiologically relevant mammalian RXR ligand.
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Receptores X de Retinoides/metabolismo , Tretinoína/metabolismo , Alitretinoína , Sítios de Ligação , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Humanos , Isomerismo , Ligantes , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Espectrometria de Massas em Tandem , Ativação Transcricional , Tretinoína/sangue , Tretinoína/químicaRESUMO
High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune complexes (ICs) also leads to activation of dendritic cells (DCs) through TLR9 and TLR7. The initial studies from our lab involved ICs formed by a mixture of autoantibodies and cell debris released from dying cells in culture. To better understand the nature of the mammalian ligands that can effectively activate TLR7 and TLR9, we have developed a methodology for preparing ICs containing defined DNA fragments that recapitulate the immunostimulatory activity of the previous "black box" ICs. As the endosomal TLR7 and TLR9 function optimally from intracellular acidic compartments, we developed a facile methodology to monitor the trafficking of defined DNA ICs by flow cytometry and confocal microscopy. These reagents reveal an important role for nucleic acid sequence, even when the ligand is mammalian DNA and will help illuminate the role of IC trafficking in the response.
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Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ativação Linfocitária/imunologia , Receptores Toll-Like/metabolismo , Animais , Complexo Antígeno-Anticorpo/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Concentração de Íons de Hidrogênio , Camundongos , Microscopia Confocal , Transporte ProteicoRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation leading to tissue destruction and progressive loss of joint function. Here we describe two methods that can be used to assess the contribution of toll-like receptors (TLRs), and their potential ligands, to RA pathogenesis. We focus on the antigen-induced model of murine arthritis and human synovial tissue explant models. Both enable detection of TLR, and TLR ligand, expression, as well as investigation of the effect of inhibition of these molecules. Each offers a unique insight into disease; with murine models allowing kinetic analysis in live animals and explant models allowing examination of inflamed human tissue, which together can help us to dissect the role of TLRs in the onset and progression of RA.
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Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Receptores Toll-Like/metabolismo , Animais , Antígenos/imunologia , Técnicas de Cultura de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Camundongos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Receptores Toll-Like/genéticaRESUMO
As well as being the primary signaling receptor for bacterial endotoxin or lipopolysaccharide Toll-like receptor-4 function is modulated by numerous factors not only in the context of microbial pathogenesis but also autoimmune and allergic diseases. TLR4 is subject to multiple levels of endogenous control and regulation from biosynthesis and trafficking to signal transduction and degradation. On the other hand regulation of TLR4 activity breaks down during Gram -ve sepsis leading to systemic damage, multi organ failure, and death. In this article, we review how TLR4 traffics from the early secretory pathway, the cis/trans Golgi to the cell surface and endolysosomal compartments. We will present evidence about how these processes influence signaling and can potentially lead to increased sensitivity to ligand-dependent activation as well as ligand-independent constitutive activation that may contribute to pathogenesis in sepsis. We will also discuss how sustained signaling may be coupled to endocytosis and consider the potential molecular mechanisms of immuno-modulators that modify TLR4 signaling function including the cat allergen FelD1 and endogenous protein ligands such as the extracellular matrix protein tenascin C and calprotectin (MRP8/14).