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This study explores the synchronization of multimodal physiological data streams, in particular, the integration of electroencephalography (EEG) with a virtual reality (VR) headset featuring eye-tracking capabilities. A potential use case for the synchronized data streams is demonstrated by implementing a hybrid steady-state visually evoked potential (SSVEP) based brain-computer interface (BCI) speller within a fully immersive VR environment. The hardware latency analysis reveals an average offset of 36 ms between EEG and eye-tracking data streams and a mean jitter of 5.76 ms. The study further presents a proof of concept brain-computer interface (BCI) speller in VR, showcasing its potential for real-world applications. The findings highlight the feasibility of combining commercial EEG and VR technologies for neuroscientific research and open new avenues for studying brain activity in ecologically valid VR environments. Future research could focus on refining the synchronization methods and exploring applications in various contexts, such as learning and social interactions.
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Cocaine and amphetamine-regulated transcript (CART) is expressed in pancreatic islet cells and neuronal elements. We have previously established insulinotropic actions of CART in human and rodent islets. The receptor for CART in the pancreatic beta cells is unidentified. We used RNA sequencing of Cartpt knockdown (KD) INS-1 832/13 cells and identified GPR162 as the most Cartpt-regulated receptor. We therefore tested if GPR162 mediates the effects of CART in beta cells. Binding of CART to GPR162 was established using proximity ligation assay, radioactive binding, and co-immunoprecipitation, and KD of Gpr162 mRNA caused reduced binding. Gpr162 KD cells had blunted CARTp-induced exocytosis, and reduced CARTp-induced insulin secretion. Furthermore, we identified a hitherto undescribed GPR162-dependent role of CART as a regulator of cytoskeletal arrangement. Thus, our findings provide mechanistic insight into the effect of CART on insulin secretion and show that GPR162 is the CART receptor in beta cells.
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Inflammatory chemokines are often elevated in disease settings, where the largest group of CC-chemokines are the macrophage inflammatory proteins (MIP), which are promiscuous for the receptors CCR1 and CCR5. MIP chemokines, such as CCL3 and CCL5 are processed at the N terminus, which influences signaling in a highly diverse manner. Here, we investigate the signaling capacity of peptides corresponding to truncated N termini. These 3-10-residue peptides displayed weak potency but, surprisingly, retained their signaling on CCR1. In contrast, none of the peptides generated a signal on CCR5, but a CCL3-derived tetrapeptide was a positive modulator boosting the signal of several chemokine variants on CCR5. In conclusion, chemokine N termini can be mimicked to produce small CCR1-selective agonists, as well as CCR5-selective modulators.
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Quimiocinas , Receptores de Quimiocinas , Quimiocina CCL3 , Quimiocina CCL4 , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiocinas/metabolismo , Proteínas Inflamatórias de Macrófagos/química , Proteínas Inflamatórias de Macrófagos/metabolismoRESUMO
Extensive ligand-receptor promiscuity in the chemokine signaling system balances beneficial redundancy and specificity. However, this feature poses a major challenge to selectively modulate the system pharmacologically. Here, we identified a conserved cluster of three aromatic receptor residues that anchors the second extracellular loop (ECL2) to the top of receptor transmembrane helices (TM) 4 and 5 and enables recognition of both shared and specific characteristics of interacting chemokines. This cluster was essential for the activation of several chemokine receptors. Furthermore, characteristic motifs of the ß1 strand and 30s loop make the two main CC-chemokine subgroups-the macrophage inflammatory proteins (MIPs) and monocyte chemoattractant proteins (MCPs)-differentially dependent on this cluster in the promiscuous receptors CCR1, CCR2, and CCR5. The cluster additionally enabled CCR1 and CCR5 to discriminate between closely related MIPs based on the N terminus of the chemokine. G protein signaling and ß-arrestin2 recruitment assays confirmed the importance of the conserved cluster in receptor discrimination of chemokine ligands. This extracellular site may facilitate the development of chemokine-related therapeutics.
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Quimiocinas , Receptores de Quimiocinas , Quimiocinas/metabolismo , Ligantes , Estrutura Secundária de Proteína , Receptores CCR5/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Manifestations and outcomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not well documented in patients with common variable immunodeficiency disorder (CVID). METHODS: A Danish nationwide retrospective clinician-reported survey. RESULTS: Eleven patients with CVID and SARS-CoV-2 infection were identified. The median age was 50 years (range 22-72). All were on immunoglobulin replacement therapy. Eight patients had other pre-existing co-morbidities. Three patients were asymptomatic during the SARS-CoV-2 infection while seven developed mild coronavirus disease 2019 (COVID-19). One patient had more severe disease with hypoxia and required oxygen therapy. This patient had multiple co-morbidities including well known risk factors for severe COVID-19. All patients recovered. CONCLUSIONS: The results suggest that CVID may not be a risk factor for severe COVID-19. However, further monitoring of this immunodeficient population is needed to confirm our observation.
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COVID-19 , Imunodeficiência de Variável Comum , Adulto , Idoso , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/epidemiologia , Dinamarca/epidemiologia , Humanos , Pessoa de Meia-Idade , Morbidade , Estudos Retrospectivos , SARS-CoV-2 , Adulto JovemRESUMO
Biased ligands that selectively confer activity in one pathway over another are pharmacologically important because biased signaling may reduce on-target side effects and improve drug efficacy. Here, we describe an N-terminal modification in the incretin hormone glucagon-like peptide (GLP-1) that alters the signaling capabilities of the GLP-1 receptor (GLP-1R) by making it G protein biased over internalization but was originally designed to confer DPP-4 resistance and thereby prolong the half-life of GLP-1. Despite similar binding affinity, cAMP production, and calcium mobilization, substitution of a single amino acid (Ala8 to Val8) in the N-terminus of GLP-1(7-36)NH2 (GLP-1 Val8) severely impaired its ability to internalize GLP-1R compared to endogenous GLP-1. In-depth binding kinetics analyses revealed shorter residence time for GLP-1 Val8 as well as a slower observed association rate. Molecular dynamics (MD) displayed weaker and less interactions of GLP-1 Val8 with GLP-1R, as well as distinct conformational changes in the receptor compared to GLP-1. In vitro validation of the MD, by receptor alanine substitutions, confirmed stronger impairments of GLP-1 Val8-mediated signaling compared to GLP-1. In a perfused rat pancreas, acute stimulation with GLP-1 Val8 resulted in a lower insulin and somatostatin secretion compared to GLP-1. Our study illustrates that profound differences in molecular pharmacological properties, which are essential for the therapeutic targeting of the GLP-1 system, can be induced by subtle changes in the N-terminus of GLP-1. This information could facilitate the development of optimized GLP-1R agonists.
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Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of ß-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K1303.26, to be important for G protein signaling, but not ß-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors.
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Quimiocina CCL19/imunologia , Quimiocina CCL21/imunologia , Receptores CCR7/imunologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Linhagem Celular Tumoral , Quimiocina CCL19/genética , Quimiocina CCL19/metabolismo , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Cricetinae , Cricetulus , Humanos , Ligantes , Camundongos , Mutação , Ligação Proteica , Receptores CCR7/genética , Receptores CCR7/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genéticaRESUMO
Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val1, Gly2, Ser3, and Glu4) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys11 modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu4 of XCL1 and Tyr117 and Arg273 of XCR1. Subsequent mutation of Tyr117 and Arg273 led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.
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Quimiocinas C/metabolismo , Quimiocinas/metabolismo , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Células COS , Quimiocinas/química , Quimiocinas/genética , Quimiocinas C/química , Quimiocinas C/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Ligação Proteica , Conformação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in ß-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors.
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The intra-islet theory states that glucagon secretion is suppressed when insulin secretion is stimulated, but glucagon's role in intra-islet paracrine regulation is controversial. This study investigated intra-islet functions of glucagon in mice. We examined glucagon-induced insulin secretion using isolated perfused pancreata from wild-type, GLP-1 receptor (GLP-1R) knockout, diphtheria toxin-induced proglucagon knockdown, ß cell-specific glucagon receptor (Gcgr) knockout, and global Gcgr knockout (Gcgr-/-) mice. We found that glucagon stimulates insulin secretion through both Gcgr and GLP-1R. Moreover, loss of either Gcgr or GLP-1R does not change insulin responses, whereas combined blockage of both receptors significantly reduces insulin secretion. Active GLP-1 is identified in pancreatic perfusate from Gcgr-/- but not wild-type mice, suggesting that ß cell GLP-1R activation results predominantly from glucagon action. Our results suggest that combined activity of glucagon and GLP-1 receptors is essential for ß cell secretory responses, emphasizing a role for paracrine intra-islet glucagon actions to maintain appropriate insulin secretion.
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Glucagon/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Transdução de Sinais , Animais , Células COS , Chlorocebus aethiops , AMP Cíclico/metabolismo , Exenatida/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos Endogâmicos C57BL , PerfusãoRESUMO
CXCL12 is a chemotactic cytokine that attracts many different cell types for homeostasis and during inflammation. Under stress conditions, macrophages and granulocytes produce factors such as peroxynitrite as a consequence of their oxidative response. After short incubations of CXCL12 with peroxynitrite, the gradual nitration of Tyr7, Tyr61, or both Tyr7 and Tyr61 was demonstrated with the use of mass spectrometry, whereas longer incubations caused CXCL12 degradation. Native CXCL12 and the nitrated forms, [3-NT61]CXCL12 and [3-NT7/61]CXCL12, were chemically synthesized to evaluate the effects of Tyr nitration on the biological activity of CXCL12. All CXCL12 forms had a similar binding affinity for heparin, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3. However, nitration significantly enhanced the affinity of CXCL12 for chondroitin sulfate. Internalization of CXCR4 and ß-arrestin 2 recruitment to CXCR4 was significantly reduced for [3-NT7/61]CXCL12 compared to CXCL12, whereas ß-arrestin 2 recruitment to ACKR3 was similar for all CXCL12 variants. [3-NT7/61]CXCL12 was weaker in calcium signaling assays and in in vitro chemotaxis assays with monocytes, lymphocytes and endothelial cells. Surprisingly, nitration of Tyr61, but not Tyr7, partially protected CXCL12 against cleavage by the specific serine protease CD26. In vivo, the effects were more pronounced compared to native CXCL12. Nitration of any Tyr residue drastically lowered lymphocyte extravasation to joints compared to native CXCL12. Finally, the anti-HIV-1 activity of [3-NT7]CXCL12 and [3-NT7/61]CXCL12 was reduced, whereas CXCL12 and [3-NT61]CXCL12 were equally potent. In conclusion, nitration of CXCL12 occurs readily upon contact with peroxynitrite and specifically nitration of Tyr7 fully reduces its in vitro and in vivo biological activities.
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Quimiocina CXCL12 , Quimiotaxia/efeitos dos fármacos , Células Endoteliais/imunologia , Linfócitos/imunologia , Monócitos/imunologia , Ácido Peroxinitroso , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Quimiocina CXCL12/química , Quimiocina CXCL12/imunologia , Quimiotaxia/imunologia , Cricetulus , Linfócitos/citologia , Camundongos , Monócitos/citologia , Ácido Peroxinitroso/química , Ácido Peroxinitroso/farmacologia , Receptores CXCR4/química , Receptores CXCR4/imunologiaRESUMO
Chemokine-chemokine receptor (CKR) interactions are traditionally described by a two-step/two-site mechanism that details the major contact points between chemokine ligands and CKRs leading to ligand recognition and receptor activation. Chemokine recognition site 1 (CRS1) encompasses interactions between the CKR N-terminus and the globular chemokine core. Chemokine recognition site 2 (CRS2) includes interactions between the unstructured chemokine N-terminus and the binding pocket of the receptor. The two-step/two-site paradigm has been an adequate framework to study the intricacies of chemokine:CKR interactions, but emerging studies highlight the limitations of this model. Here, we present studies of CRS2 interactions between the chemokine CCL20 and its cognate receptor CCR6 driven by the hypothesis that CCL20 interacts with CCR6 as described by the two-step/two-site model. CCL20 is a chemokine with an unusually short N-terminus of 5 residues (NH2 -ASNFD), compared to the average length of 10 residues for chemokine ligands. We have investigated how well CCL20 tolerates manipulation of the N-terminus by monitoring binding affinity of variants and their ability to activate the receptor. We show the CCL20 N-terminus tolerates truncation of up to 3 residues, extension by up to 5 additional residues, and point mutations at 4 of 5 positions with minimal loss of binding affinity and minimal impairment in ability to stimulate calcium mobilization, inositol triphosphate accumulation, chemotaxis, and ß-arrestin-2 recruitment. Mutation of the fifth residue, aspartate, to alanine or lysine has a dramatic impact on binding affinity for CCR6 and ligand potency. We postulate CCL20 does not activate CCR6 through the canonical two-step/two-site mechanism of CKR activation.
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Quimiocina CCL20/química , Quimiocina CCL20/metabolismo , Receptores CCR6/metabolismo , Sequência de Aminoácidos , Análise Mutacional de DNA , Humanos , Células Jurkat , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Human cytomegalovirus has hijacked and evolved a human G-protein-coupled receptor into US28, which functions as a promiscuous chemokine 'sink' to facilitate evasion of host immune responses. To probe the molecular basis of US28's unique ligand cross-reactivity, we deep-sequenced CX3CL1 chemokine libraries selected on 'molecular casts' of the US28 active-state and find that US28 can engage thousands of distinct chemokine sequences, many of which elicit diverse signaling outcomes. The structure of a G-protein-biased CX3CL1-variant in complex with US28 revealed an entirely unique chemokine amino terminal peptide conformation and remodeled constellation of receptor-ligand interactions. Receptor signaling, however, is remarkably robust to mutational disruption of these interactions. Thus, US28 accommodates and functionally discriminates amongst highly degenerate chemokine sequences by sensing the steric bulk of the ligands, which distort both receptor extracellular loops and the walls of the ligand binding pocket to varying degrees, rather than requiring sequence-specific bonding chemistries for recognition and signaling.
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Quimiocina CX3CL1/química , Receptores de Quimiocinas/química , Receptores Acoplados a Proteínas G/química , Transdução de Sinais , Proteínas Virais/química , Animais , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/farmacologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/agonistas , Proteínas Virais/metabolismoRESUMO
OBJECTIVE: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. METHODS: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. RESULTS: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or ß-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. CONCLUSION: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.
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Ácidos e Sais Biliares/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Mucosa Intestinal/metabolismo , Pâncreas/metabolismo , Peptídeo YY/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Resolution of peri-implant inflammation and re-osseointegration of peri-implantitis affected dental implants seem to be dependent on bacterial decontamination. The aims of the study were to evaluate the antimicrobial effects of 3 different instrumentations on a micro-textured dental implant surface contaminated with an avirulent or a virulent Porphyromonas gingivalis strain and to determine alterations to the implant surface following instrumentation. Forty-five dental implants (Straumann SLA) were allocated to 3 treatment groups: Er:YAG laser, chitosan brush, and titanium curette (10 implants each) and a positive (10 implants) and a negative (5 implants) control. Each treatment group and the positive control were split into subgroups of 5 implants subsequently contaminated with either the avirulent or virulent P. gingivalis strain. The antimicrobial effect of instrumentation was evaluated using checkerboard DNA-DNA hybridization. Implant surface alterations were determined using a light interferometer. Instrumentation significantly reduced the number of attached P. gingivalis ( P < .001) with no significant differences among groups ( P = .310). A significant overall higher median score was found for virulent compared with avirulent P. gingivalis strains ( P = .007); the Er:YAG laser uniquely effective removing both bacterial strains. The titanium curette significantly altered the implant surface micro-texture. Neither the Er:YAG laser nor the chitosan brush significantly altered the implant surface. The 3 instrumentations appear to have a similar potential to remove P. gingivalis. The titanium curette significantly altered the microstructure of the implant surface.
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Anti-Infecciosos/farmacologia , Implantes Dentários/microbiologia , Porphyromonas gingivalis/efeitos dos fármacos , Instrumentos Odontológicos , Propriedades de SuperfícieRESUMO
Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.
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Varicela/virologia , Herpes Zoster/virologia , Herpesvirus Humano 3/genética , Leucócitos/fisiologia , Proteínas do Envelope Viral/genética , Animais , Linhagem Celular , Movimento Celular , Quimiocinas/metabolismo , Varicela/imunologia , Drosophila melanogaster , Células Epiteliais/virologia , Genes Reporter , Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 3/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Mutação , Tonsila Palatina/virologia , Domínios Proteicos , Linfócitos T/virologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , VírionRESUMO
The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH2-terminal amino acids. CD26-truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3-68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3-68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP3), Akt or extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of ß-arrestin 2 to the cell membrane via CXCR4 by CXCL12(3-68) was abolished, whereas a weakened but significant ß-arrestin recruitment remained via ACKR3. CXCL12-induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra-articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra-articular lymphocyte infiltration was significantly increased in sitagliptin-treated mice and CXCL12(3-68) failed to induce migration under both CD26-inhibiting and non-inhibiting conditions. In conclusion, CD26-cleavage skews CXCL12 towards ß-arrestin dependent recruitment through ACKR3 and destroys the CXCR4-mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26-blockade in tissues may enhance CXCL12-induced inflammation.
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Quimiocina CXCL12/metabolismo , Dipeptidil Peptidase 4/metabolismo , Linfócitos/metabolismo , Receptores CXCR4/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células Endoteliais/metabolismo , Glicosaminoglicanos/metabolismo , CamundongosRESUMO
[This corrects the article on p. 568 in vol. 7, PMID: 28018341.].
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The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and ß-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.
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BACKGROUND: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency. RESULTS: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. CONCLUSIONS: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.