RESUMO
Resin cement integrated with zein-incorporated magnesium oxide nanoparticles has previously been found to inhibit oral microbes and decrease bacterial biofilm. However, the bond strength and surface features of this biomaterial have yet to be investigated. The objective of this study was to evaluate the shear bond strength, mode of fracture, and surface roughness of resin cement modified with zein-incorporated magnesium oxide nanoparticles. Characterization of the cement was performed by X-ray diffraction, field emission scanning electron microscopy, and Fourier transform infrared spectroscopy. 126 human teeth were divided into 3 groups and cemented to lithium disilicate ceramic using resin cement with zein-incorporated magnesium oxide nanoparticles at concentrations of 0%, 1%, and 2% (n = 42). 21 samples of each group were subjected to the shear bond strength test, while the other 21 underwent thermocycling for 10,000 cycles before the test, after which all samples were evaluated for the mode of fracture. To assess surface roughness, resin cement disks were analyzed by a profilometer before and after undergoing thermocycling for 10,000 cycles. The shear bond strength of the cement with 1% and 2% nanoparticles was significantly higher than the control before thermocycling. The mode of fracture was found to be mainly adhesive with all groups, with the unmodified cement presenting the highest cohesive failure. There was no significant difference in surface roughness between the groups before or after thermocycling. The addition of zein-incorporated magnesium oxide nanoparticles to resin cement improved or maintained the shear bond strength and surface roughness of the resin cement.
RESUMO
Cathelicidin LL-37âmediated activation of mast cells (MCs) has been implicated in the pathogenesis of rosacea, but the receptor involved and the mechanism of its activation and regulation remain unknown. We found that skin biopsies from patients with rosacea display higher frequencies of MCs expressing MRGPRX2 (mouse counterpart MrgprB2) than normal skin. Intradermal injection of LL-37 in wild-type mice resulted in MC recruitment, expression of inflammatory mediators, and development of rosacea-like inflammation. These responses were substantially reduced in MrgprB2â/â mice and abolished in MC deficient Wsh/Wsh mice. ß-arrestin 2 is an adaptor protein that regulates G protein-coupled receptor function by receptor desensitization and also by activation of downstream signaling. We found that LL-37âinduced rosacea-like inflammation was significantly reduced in mice with MC-specific deletion of ß-arrestin 2 compared with that in control mice. Interestingly, the absence of ß-arrestin 2 resulted in enhanced cofilin phosphorylation and substantial inhibition of LL-37âinduced chemotaxis of mouse peritoneal MCs. Furthermore, LL-37âinduced extracellular signalâregulated kinase 1/2 phosphorylation, NF-κB activation, and proinflammatory cytokine/chemokine production were reduced in ß-arrestin 2â/â peritoneal MCs compared with those in wild-type cells. These findings suggest that MRGPRX2/B2 participates in rosacea and that ß-arrestin 2 contributes to its pathogenesis by promoting cofilin dephosphorylation, extracellular signalâregulated kinase 1/2 and NF-κB phosphorylation, MC chemotaxis, and chemokine/cytokine generation.
Assuntos
NF-kappa B , Rosácea , Camundongos , Animais , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Rosácea/metabolismo , Mastócitos/metabolismo , Inflamação/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Quimiocinas/metabolismo , Mediadores da Inflamação/metabolismo , Fatores de Despolimerização de Actina/metabolismoRESUMO
Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Sinalização do Cálcio , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Knockout , Família MultigênicaRESUMO
The neuropeptide substance P (SP) contributes to neurogenic inflammation through the activation of human mast cells via Mas-related G protein-coupled receptor-X2 (MRGPRX2). Using pertussis toxins and YM-254890, we demonstrated that SP induces Ca2+ mobilization and degranulation via both the Gαi and Gαq family of G proteins in rat basophilic leukemia (RBL-2H3) cells stably expressing MRGPRX2. To determine the roles of MRGPRX2's transmembrane (TM) and intracellular domains on SP-induced responses, we utilized information obtained from both structural modeling and naturally occurring MRGPRX2 missense variants. We found that highly conserved residues in TM6 (I225) and TM7 (Y279) of MRGPRX2 are essential for SP-induced Ca2+ mobilization and degranulation in transiently transfected RBL-2H3 cells. Cells expressing missense variants in the receptor's conserved residues (V123F and V282M) as well as intracellular loops (R138C and R141C) failed to respond to SP. By contrast, replacement of all five Ser/Thr residues with Ala and missense variants (S325L and L329Q) in MRGPRX2's carboxyl-terminus resulted in enhanced mast cell activation by SP when compared to the wild-type receptor. These findings suggest that MRGPRX2 utilizes conserved residues in its TM domains and intracellular loops for coupling to G proteins and likely undergoes desensitization via phosphorylation at Ser/Thr residues in its carboxyl-terminus. Furthermore, identification of gain and loss of function MRGPRX2 variants has important clinical implications for SP-mediated neurogenic inflammation and other chronic inflammatory diseases.
Assuntos
Mutação com Ganho de Função , Mutação com Perda de Função , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/química , Receptores Acoplados a Proteínas G/química , Receptores de Neuropeptídeos/química , Substância P/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismoRESUMO
Host-defense peptides (HDPs) have an important therapeutic potential against microbial infections but their metabolic instability and cellular cytotoxicity have limited their utility. To overcome these limitations, we utilized five small-molecule, nonpeptide HDP mimetics (smHDPMs) and tested their effects on cytotoxicity, antimicrobial activity, and mast cell (MC) degranulation. None of the smHDPMs displayed cytotoxicity against mouse 3T3 fibroblasts or human transformed liver HepG2 cells. However, one compound had both antifungal and antibacterial activity. Surprisingly, all five compounds induced degranulation in a human MC line, LAD2, and this response was substantially reduced in Mas-related G protein-coupled receptor (GPCR)-X2 (MRGPRX2)-silenced cells. Furthermore, all five compounds induced degranulation in RBL-2H3 cells expressing MRGPRX2 but this response was abolished in cells expressing naturally occurring loss-of-function missense variants G165E (rs141744602) and D184H (rs372988289). Mrgprb2 is the likely mouse ortholog of human MRGPRX2, which is expressed in connective tissue MCs (CTMCs) such as cutaneous and peritoneal MCs (PMCs). All five smHDPMs induced degranulation in wild-type PMCs but not in cells derived from Mrgprb2â»/â» mice. These findings suggest that smHDPMs could serve as novel targets for the treatment of drug-resistant fungal and bacterial infections because of their ability to harness CTMCs' host defense functions.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células 3T3 , Animais , Antibacterianos/química , Antifúngicos/química , Degranulação Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Células Hep G2 , Humanos , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto/genética , RatosRESUMO
Human mast cells (MCs) express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). Activation of this receptor by a diverse group of cationic ligands such as neuropeptides, host defense peptides, and Food and Drug Administration-approved drugs contributes to chronic inflammatory diseases and pseudoallergic drug reactions. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P and hemokinin-1), a host defense peptide (human ß-defensin-3) and a Food and Drug Administration-approved cationic drug (bradykinin B2 receptor antagonist, icatibant). We have identified eight missense variants within MRGPRX2's ECL/TM domains from publicly available exome-sequencing databases. We investigated the ability of MRGPRX2 ligands to induce degranulation in rat basophilic leukemia-2H3 cells individually expressing these naturally occurring MRGPRX2 missense variants. Using stable and transient transfections, we found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Thus, diverse MRGPRX2 ligands use common sites on the receptor to induce MC degranulation. These findings have important clinical implications for MRGPRX2 and MC-mediated pseudoallergy and chronic inflammatory diseases.