RESUMEN
An olfactory receptor mimetic peptide-modified graphene field-effect transistor (gFET) is a promising solution to overcome the principal challenge of low specificity graphene-based sensors for volatile organic compound (VOC) sensing. Herein, peptides mimicking a fruit fly olfactory receptor, OR19a, were designed by a high-throughput analysis method that combines a peptide array and gas chromatography for the sensitive and selective gFET detection of the signature citrus VOC, limonene. The peptide probe was bifunctionalized via linkage of a graphene-binding peptide to facilitate one-step self-assembly on the sensor surface. The limonene-specific peptide probe successfully achieved highly sensitive and selective detection of limonene by gFET, with a detection range of 8-1000 pM, while achieving facile sensor functionalization. Taken together, our target-specific peptide selection and functionalization strategy of a gFET sensor demonstrates advancement of a precise VOC detection system.
Asunto(s)
Técnicas Biosensibles , Grafito , Receptores Odorantes , Compuestos Orgánicos Volátiles , Técnicas Biosensibles/métodos , Grafito/química , Limoneno , Péptidos , Transistores Electrónicos , Compuestos Orgánicos Volátiles/análisis , Drosophila , AnimalesRESUMEN
High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides. Microbead-assisted capillary electrophoresis (MACE)-SELEX against MTX successfully identified MSmt7 with a similar level of specificity to anti-MTX antibodies within three rounds. The 3'-end of MSmt7 was coupled to a peroxidase-like hemin-DNAzyme to construct a bifunctional oligonucleotide for MTX sensing, where MTX in 50% human serum was detected with a limit of detection (LoD) of 118 nM. Furthermore, amplifying the DNAzyme region with rolling circle amplification significantly improved the sensitivity with an LoD of 290 pM. Presented oligonucleotide-based MTX detection systems will pave the way for antibody-independent MTX detection with reliability and less cost in the laboratory and the clinic.
Asunto(s)
Aptámeros de Nucleótidos , ADN Catalítico , Humanos , Metotrexato , Reproducibilidad de los Resultados , HeminaRESUMEN
Researchers widely apply enzyme inhibition to chemicals such as pesticides, nerve gases, and anti-Alzheimer's drugs. However, application of enzyme inhibition to odorant sensors is less common because the corresponding reaction mechanisms have not yet been clarified in detail. In this study, we propose a new strategy for highly selective detection of odorant molecules by using an inhibitor-specific enzyme. As an example, we analyzed the selective interactions between acetylcholinesterase (AChE) and limoneneâthe major odorant of citrus and an AChE inhibitorâusing molecular dynamics simulations. In these simulations, limonene was found to be captured at specific binding sites of AChE by modifying the binding site of acetylcholine (ACh), which induced inhibition of the catalytic activity of AChE toward ACh hydrolysis. We confirmed the simulation results by experiments using an ion-sensitive field-effect transistor, and the degree of inhibition of ACh hydrolysis depended on the limonene concentration. Accordingly, we quantitatively detected limonene at a detection limit of 5.7 µM. We furthermore distinguished the response signals to limonene from those to other odorants, such as pinene and perillic acid. Researchers will use our proposed odorant detection method for other odorant-enzyme combinations and applications of miniaturized odorant-sensing systems based on rapid testing.
Asunto(s)
Acetilcolinesterasa , Plaguicidas , Acetilcolina/química , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Hidrólisis , LimonenoRESUMEN
TLR7 agonists modulate Th2 immune responses through mechanisms that have not been fully elucidated. Suppression of IL-5 production from Ag- or phytohemagglutinin-stimulated human PBMCs by the TLR7 antedrug AZ12441970 was mediated via type I IFN-dependent and type I IFN-independent mechanisms through TLR7 activation of plasmacytoid dendritic cells, B cells, and monocytes. The type I IFN-dependent inhibition of T cell-derived IL-5 was mediated by IFN-α acting directly on activated T cells. IL-10 was shown not to be involved in the type I IFN-independent inhibition of IL-5 and the mechanism of inhibition required cell-cell interaction. Notch signaling was implicated in the inhibition of IL-5, because addition of a γ-secretase inhibitor blocked the type I IFN-independent suppression of IL-5. Accordingly, AZ12441970 induced high levels of the notch ligands Dll1 and Dll4 mRNA, whereas immobilized DLL4 resulted in the suppression of IL-5 production. Therefore, we have elucidated two mechanisms whereby TLR7 agonists can modulate IL-5 production in human T cells. The suppression of Th2 cytokines, including IL-5, would be of benefit in diseases such as atopic asthma, so we assessed TLR7 function in PBMC from asthmatics and showed equivalent activity compared with healthy volunteers. Demonstrating this function is intact in asthmatics and knowing it links to suppression of Th2 cytokines support the case for developing such compounds for the treatment of allergic disease.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Interferón Tipo I/fisiología , Interleucina-5/antagonistas & inhibidores , Leucocitos Mononucleares/inmunología , Receptores Notch/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 7/fisiología , Células Cultivadas , Humanos , Interferón Tipo I/sangre , Interleucina-5/biosíntesis , Interleucina-5/sangre , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Receptores Notch/sangre , Receptor Toll-Like 7/sangreRESUMEN
Triggering innate immune responses through TLRs is expected to be a novel therapeutic strategy for the treatment of allergic diseases. TLR agonists are able to modulate Th2 immune responses through undefined mechanisms. We investigated the mechanism of action of the suppression of Th2 immune responses with a novel antedrug TLR7 agonist. The antedrug is rapidly metabolized by plasma esterases to an acid with reduced activity to limit systemic responses. Topical administration of this compound inhibited features of the allergic airway inflammatory response in rat and murine allergic airways model. Type I IFN played a role in the suppression of Th2 cytokines produced from murine splenocytes. Inhibition of Th2 immune responses with the antedrug TLR7 agonist was shown to be via a type I IFN-dependent mechanism following short-term exposure to the compound, although there might be type I IFN-independent mechanisms following long-term exposure. We have demonstrated that local type I IFN signaling and plasmacytoid dendritic cells, but not Th1 immune responses, are required for in vivo efficacy against murine airway Th2-driven eosinophilia. Furthermore, migration of dendritic cell subsets into the lung was related to efficacy and is dependent on type I IFN signaling. Thus, the mechanism of action at the cytokine and cellular level involved in the suppression of Th2 allergic responses has been characterized, providing a potential new approach to the treatment of allergic disease.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Profármacos/administración & dosificación , Sistema Respiratorio/efectos de los fármacos , Receptor Toll-Like 7/agonistas , Administración Tópica , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Eosinofilia/complicaciones , Eosinofilia/tratamiento farmacológico , Eosinofilia/inmunología , Eosinofilia/metabolismo , Genes Reporteros , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Ratones , Profármacos/síntesis química , Profármacos/metabolismo , Ratas , Hipersensibilidad Respiratoria/complicaciones , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Sistema Respiratorio/inmunología , Sistema Respiratorio/metabolismo , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/inmunología , Células Th2/citología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Receptor Toll-Like 7/inmunologíaRESUMEN
Replicating the sense of smell presents an ongoing challenge in the development of biomimetic devices. Olfactory receptors exhibit remarkable discriminatory abilities, including the enantioselective detection of individual odorant molecules. Graphene has emerged as a promising material for biomimetic electronic devices due to its unique electrical properties and exceptional sensitivity. However, the efficient detection of nonpolar odor molecules using transistor-based graphene sensors in a gas phase in environmental conditions remains challenging due to high sensitivity to water vapor. This limitation has impeded the practical development of gas-phase graphene odor sensors capable of selective detection, particularly in humid environments. In this study, we address this challenge by introducing peptide-functionalized graphene sensors that effectively mitigate undesired responses to changes in humidity. Additionally, we demonstrate the significant role of humidity in facilitating the selective detection of odorant molecules by the peptides. These peptides, designed to mimic a fruit fly olfactory receptor, spontaneously assemble into a monomolecular layer on graphene, enabling precise and specific odorant detection. The developed sensors exhibit notable enantioselectivity, achieving a remarkable 35-fold signal contrast between d- and l-limonene. Furthermore, these sensors display distinct responses to various other biogenic volatile organic compounds, demonstrating their versatility as robust tools for odor detection. By acting as both a bioprobe and an electrical signal amplifier, the peptide layer represents a novel and effective strategy to achieve selective odorant detection under normal atmospheric conditions using graphene sensors. This study offers valuable insights into the development of practical odor-sensing technologies with potential applications in diverse fields.
Asunto(s)
Técnicas Biosensibles , Grafito , Receptores Odorantes , Odorantes , Grafito/química , Gases , Estereoisomerismo , Receptores Odorantes/química , PéptidosRESUMEN
Topical TLR7 agonists such as imiquimod are highly effective for the treatment of dermatological malignancies; however, their efficacy in the treatment of nondermatological tumors has been less successful. We report that oral administration of the novel TLR7-selective small molecule agonist; SM-276001, leads to the induction of an inflammatory cytokine and chemokine milieu and to the activation of a diverse population of immune effector cells including T and B lymphocytes, NK and NKT cells. Oral administration of SM-276001 leads to the induction of IFNα, TNFα and IL-12p40 and a reduction in tumor burden in the Balb/c syngeneic Renca and CT26 models. Using the OV2944-HM-1 model of ovarian cancer which spontaneously metastasizes to the lungs following subcutaneous implantation, we evaluated the efficacy of intratracheal and oral administration of SM-276001 in an adjuvant setting following surgical resection of the primary tumor. We show that both oral and intratracheal TLR7 therapy can reduce the frequency of pulmonary metastasis, and metastasis to the axillary lymph nodes. These results demonstrate that SM-276001 is a potent selective TLR7 agonist that can induce antitumor immune responses when dosed either intratracheally or orally.
Asunto(s)
Antineoplásicos/administración & dosificación , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/agonistas , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Receptor Toll-Like 7/agonistas , Administración Oral , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antineoplásicos/uso terapéutico , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Evaluación Preclínica de Medicamentos , Femenino , Interferón-alfa/biosíntesis , Subunidad p40 de la Interleucina-12/biosíntesis , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Lectinas Tipo C/biosíntesis , Neoplasias Pulmonares/secundario , Metástasis Linfática/prevención & control , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Linfocitos T/efectos de los fármacos , Receptor Toll-Like 7/metabolismo , Tráquea , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Gas sensing based on graphene field-effect transistors (GFETs) has gained broad interest due to their high sensitivity. Further progress in gas sensing with GFETs requires to detection of various odor molecules for applications in the environmental monitoring, healthcare, food, and cosmetic industries. To develop the ubiquitous odor-sensing system, establishing an artificial sense of smell with electronic devices by mimicking olfactory receptors will be key. Although the application of olfactory receptors to GFETs is straightforward for odor sensing, synthetic molecules with a similar function to olfactory receptors would be desirable to realize the robust performance of sensing. In this work, we designed three new peptides consisting of two domains: a bio-probe to the target molecules and a molecular scaffold. These peptides were rationally designed based on a motif sequence in olfactory receptors and self-assembled into a molecular thin film on GFETs. Limonene, methyl salicylate, and menthol were employed as representative odor molecules of plant flavors to demonstrate the biosensing of odor molecules. The conductivity change of GFETs against the binding to odor molecules with various concentrations and the dynamic response revealed a distinct signature of three different peptides against individual species of the target molecules. The kinetic response of each peptide exhibited characteristic time constants in the adsorption and desorption process, also supported by the principal component analysis. Our demonstration of the graphene odor sensors with the designed peptides opens a way to establish future peptide-array sensors with multi-sequence of peptide, realizing an odor sensing system with higher selectivity.
Asunto(s)
Técnicas Biosensibles , Grafito , Receptores Odorantes , Odorantes , Grafito/química , Transistores Electrónicos , PéptidosRESUMEN
Dendritic cells (DCs) play a crucial role in the immune responses against infections by sensing microbial invasion through toll-like receptors (TLRs). In humans, two distinct DC subsets, CD11c(-) plasmacytoid DCs (PDCs) and CD11c(+) myeloid DCs (MDCs), have been identified and can respond to different TLR ligands, depending on the differential expression of cognate TLRs. In this study, we have examined the effect of TLR-7 ligands on human DC subsets. Both subsets expressed TLR-7 and could respond to TLR-7 ligands, which enhanced the survival of the subsets and upregulated the surface expression of costimulatory molecules such as CD40, CD80, and CD86. However, the cytokine induction pattern was distinct in that PDCs and MDCs produced interferon (IFN)-alpha and interleukin (IL)-12, respectively. In response to TLR-7 ligands, the Th1 cell supporting ability of both DC subsets was enhanced, depending on the cytokines the respective subsets produced. This study demonstrates that TLR-7 exerts its biological effect in a DC subset-specific manner.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/metabolismo , Proteínas de Drosophila , Interferón-alfa/metabolismo , Interleucina-12/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Humanos , Interferón-alfa/sangre , Interleucina-12/sangre , Ligandos , Glicoproteínas de Membrana/genética , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/citología , Receptor Toll-Like 7 , Receptores Toll-LikeRESUMEN
Toll-like receptors (TLRs) play important roles in the innate immune system. In fact, recognition of endogenous immune complexes containing self-nucleic acids as pathogen- or damage-associated molecular patterns contributes to certain autoimmune diseases, and inhibition of these recognition signals is expected to have therapeutic value. We identified dihydropyrrolo[2,3-d]pyrimidines as novel selective TLR9 antagonists with high aqueous solubility. A structure-activity relationship study of a known TLR9 antagonist led to the promising compound 18, which showed potent TLR9 antagonistic activity, sufficient aqueous solubility for parenteral formulation, and druggable properties. Compound 18 suppressed the production of the proinflammatory cytokine IL-6 in CpG-induced mouse model. It is therefore believed that compound 18 has great potential in the treatment of TLR9-mediated systemic uncontrollable inflammatory response like sepsis.
RESUMEN
We investigated the chemical modifications of the nitroquinazoline derivative (1) through the replacement of the NH group at the C(4)-position with several N-alkyl groups to increase the lipophilicity at the C(4)-position. Among them, we found that the N-methyl analogue (5a) showed a 2-fold loss in the inhibitory activity toward tumor necrosis factor-alpha (TNF-alpha) production in vitro as compared with the NH analogue (1); however, 5a exhibited an oral inhibitory activity on TNF-alpha production with an ED50 value of 26 mg/kg, whereas 1 did not. Moreover, the oral bioavailability of 5a was higher than that of 1 (1, F=1%; 5a, F=21%), and the calculated ClogP value for 5a was higher than that for 1. These results suggest that the improved lipophilicity of 5a compared with that of 1 reflects its greater inhibitory activity on TNF-alpha production in vivo as well as oral bioavailability.
Asunto(s)
Quinazolinas/síntesis química , Quinazolinas/farmacología , Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Administración Oral , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Depresión Química , Humanos , Indicadores y Reactivos , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Ratas , Relación Estructura-ActividadRESUMEN
In this study, we have investigated the roles of substituents on the terminal phenyl ring at the C(4)-position of the quinazoline core to complete the structure-activity relationships (SARs) of our NF-kappa B activation inhibitors. Among them, compound 12j afforded highly potent inhibitory activity toward NF-kappa B transcriptional activation with IC(50) value of 2 nM, along with an excellent in vivo efficacy by reducing the edema formation seen in carrageenin-induced inflammation of the rat hind paw.
Asunto(s)
Aminoquinolinas/química , FN-kappa B/antagonistas & inhibidores , Quinazolinas/química , Aminoquinolinas/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Carragenina , Edema/inducido químicamente , Edema/tratamiento farmacológico , Inflamación/inducido químicamente , Concentración 50 Inhibidora , FN-kappa B/metabolismo , Quinazolinas/farmacología , Ratas , Bazo/citología , Bazo/efectos de los fármacos , Bazo/crecimiento & desarrollo , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Exposure of macrophages to lipopolysaccharide (LPS) induces a hypo-responsive state to a second challenge with LPS that is termed LPS tolerance. LPS tolerance is also induced by pre-exposure to lipopeptides and lipoteichoic acid, which trigger Toll-like receptor (TLR) 2-mediated signaling. LPS signaling involves at least two pathways: a MyD88-dependent cascade that is essential for production of inflammatory cytokines and a MyD88-independent cascade that mediates the expression of IFN-inducible genes. We analyzed the induction of LPS tolerance by several microbial components in mouse peritoneal macrophages. Pre-exposure to LPS led to impaired activation of both the pathways. In contrast, mycoplasmal lipopeptides did not affect the MyD88-independent pathway, but impaired the MyD88-dependent signaling by inhibiting LPS-mediated activation of IL-1 receptor-associated kinase (IRAK) 1. The induction of LPS tolerance by recently identified TLR ligands was analyzed. Pretreatment with double-stranded RNA, which triggers the activation of TLR3, led to defective activation of the MyD88-independent, but not the MyD88-dependent, pathway. Imidazoquinoline compounds, which are recognized by TLR7, had no effect on the MyD88-independent pathway, but inhibited LPS-induced activation of MyD88-dependent signaling through down-regulation of IRAK1 expression. Thus, each microbial component induced LPS tolerance in macrophages.
Asunto(s)
Antígenos de Diferenciación/inmunología , Proteínas Bacterianas/inmunología , Tolerancia Inmunológica , Lipopolisacáridos/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Mycoplasma fermentans/inmunología , Oligopéptidos/inmunología , Proteínas Quinasas , Receptores Inmunológicos/inmunología , Salmonella/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/genética , Proteínas Bacterianas/farmacología , Células Cultivadas , Imidazoles/farmacología , Quinasas Asociadas a Receptores de Interleucina-1 , Lipopéptidos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos BALB C , Mycoplasma fermentans/química , Factor 88 de Diferenciación Mieloide , Oligopéptidos/farmacología , Inhibidores de Proteínas Quinasas , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Receptores de Interleucina-1/fisiología , Salmonella/química , Transducción de Señal/efectos de los fármacosRESUMEN
We disclose here a new structural class of low-molecular-weight inhibitors of NF-kappa B activation that were designed and synthesized by starting from quinazoline derivative 6a. Structure-activity relationship (SAR) studies based on 6a elucidated the structural requirements essential for the inhibitory activity toward NF-kappa B transcriptional activation, and led to the identification of the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework. In this series of compounds, 11q, containing the 4-phenoxyphenethyl moiety at the C(4)-position, showed strong inhibitory effects on both NF-kappa B transcriptional activation and TNF-alpha production. Furthermore, 11q exhibited an anti-inflammatory effect on carrageenin-induced paw edema in rats.
Asunto(s)
FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Quinazolinas/química , Quinazolinas/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Carragenina/toxicidad , Edema/inducido químicamente , Edema/tratamiento farmacológico , Miembro Posterior , Humanos , Concentración 50 Inhibidora , Células Jurkat , Masculino , Ratones , Ratones Endogámicos BALB C , Ratas , Bazo/citología , Bazo/efectos de los fármacos , Bazo/crecimiento & desarrollo , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We synthesized various 6-fluoro-7-(1-piperazino)quinazolines based on the structure of 1 and evaluated their inhibitory activities toward both TNF-alpha production and T cell proliferation responses. Among these compounds, 7a, having the 3,4-(methylenedioxy)phenyl moiety at the C(4)-position of the quinazoline ring, showed both inhibitory activities. Furthermore, the oral treatment with 7a exhibited an anti-inflammatory effect in rats with adjuvant arthritis as well as an inhibitory activity toward LPS-induced TNF-alpha production.
Asunto(s)
Quinazolinas/síntesis química , Quinazolinas/farmacología , Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/uso terapéutico , Área Bajo la Curva , Artritis Experimental/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , División Celular/efectos de los fármacos , Concanavalina A/farmacología , Depresión Química , Diseño de Fármacos , Edema/inducido químicamente , Edema/prevención & control , Humanos , Técnicas In Vitro , Lipopolisacáridos , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We synthesized various 6-nitroquinazolines by modifying the structure of compound 1 and evaluated their inhibitory activities toward both TNF-alpha production and T cell proliferation responses. The presence of the unsubstituted piperazine ring at the C(7)-position was required for both inhibitory activities. In this series of compounds, 5d and 5f, containing the 4-fluorophenyl and 3,4-difluorophenyl moiety, respectively, at the C(4)-position, showed the suppressing effects toward both responses with low cell growth inhibition. Furthermore, the oral administration of these compounds mentioned above at doses of 30 and 100 mg/kg also resulted in significant inhibition of TNF-alpha production induced by LPS in vivo.
Asunto(s)
Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/farmacología , Nitroquinolinas/química , Nitroquinolinas/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Linfocitos T/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Evaluación Preclínica de Medicamentos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We investigated the expression of a panel of Toll-like receptors (TLRs) and their functions in human eosinophils. Eosinophils constitutively expressed TLR1, TLR4, TLR7, TLR9, and TLR10 mRNAs (TLR4 greater than TLR1, TLR7, TLR9, and TLR10 greater than TLR6). In contrast, neutrophils expressed a larger variety of TLR mRNAs (TLR1, TLR2, TLR4, TLR6, TLR8 greater than TLR5, TLR9, and TLR10 greater than TLR7). Although the expression levels in eosinophils were generally less prominent compared with those in neutrophils, eosinophils expressed a higher level of TLR7. Furthermore, among various TLR ligands (S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-Cys-Ser-(Lys)(4), poly(I:C), LPS, R-848, and CpG DNA), only R-848, a ligand of TLR7 and TLR8, regulated adhesion molecule (CD11b and L-selectin) expression, prolonged survival, and induced superoxide generation in eosinophils. Stimulation of eosinophils by R-848 led to p38 mitogen-activated protein kinase activation, and SB203580, a p38 mitogen-activated protein kinase inhibitor, almost completely attenuated R-848-induced superoxide generation. Although TLR8 mRNA expression was hardly detectable in freshly isolated eosinophils, mRNA expression of TLR8 as well as TLR7 was exclusively up-regulated by IFN-gamma but not by either IL-4 or IL-5. The up-regulation of the TLRs by IFN-gamma had potentially functional significance: the extent of R-848-induced modulation of adhesion molecule expression was significantly greater in cells treated with IFN-gamma compared with untreated cells. Although the natural ligands for TLR7 and TLR8 have not yet been identified, our results suggest that eosinophil TLR7/8 systems represent a potentially important mechanism of a host-defensive role against viral infection and mechanism linking exacerbation of allergic inflammation and viral infection.
Asunto(s)
Eosinófilos/inmunología , Eosinófilos/metabolismo , Imidazoles/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Moléculas de Adhesión Celular/biosíntesis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/enzimología , Humanos , Imidazoles/farmacología , Interferón gamma/farmacología , Ligandos , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Lipoproteínas/metabolismo , Lipoproteínas/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Poli I-C/metabolismo , Poli I-C/farmacología , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Superóxidos/metabolismo , Receptor Toll-Like 1 , Receptor Toll-Like 10 , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptor Toll-Like 5 , Receptor Toll-Like 7 , Receptor Toll-Like 8 , Receptor Toll-Like 9 , Receptores Toll-Like , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The imidazoquinoline compounds imiquimod and R-848 are low-molecular-weight immune response modifiers that can induce the synthesis of interferon-alpha and other cytokines in a variety of cell types. These compounds have potent anti-viral and anti-tumor properties; however, the mechanisms by which they exert their anti-viral activities remain unclear. Here we show that the imidazoquinolines activate immune cells via the Toll-like receptor 7 (TLR7)-MyD88-dependent signaling pathway. In response to the imidazoquinolines, neither MyD88- nor TLR7-deficient mice showed any inflammatory cytokine production by macrophages, proliferation of splenocytes or maturation of dendritic cells. Imidazoquinoline-induced signaling events were also abolished in both MyD88- and TLR7-deficient mice.
Asunto(s)
Aminoquinolinas/inmunología , Antígenos de Diferenciación/metabolismo , Antivirales/inmunología , Proteínas de Drosophila , Imidazoles/inmunología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adyuvantes Inmunológicos , Animales , Células de la Médula Ósea/inmunología , Células Dendríticas , Imiquimod , Inductores de Interferón/inmunología , Macrófagos Peritoneales/inmunología , Ratones , Ratones Mutantes , Factor 88 de Diferenciación Mieloide , Bazo/citología , Bazo/inmunología , Receptor Toll-Like 7 , Receptores Toll-LikeRESUMEN
Toll-like receptors (TLR) recognize microbial and viral patterns and activate dendritic cells (DC). TLR distribution among human DC subsets is heterogeneous: plasmacytoid DC (PDC) express TLR1, 7 and 9, while other DC types do not express TLR9 but express other TLR. Here, we report that mRNA for most TLR is expressed at similar levels by murine splenic DC sub-types, including PDC, but that TLR3 is preferentially expressed by CD8 alpha(+) DC while TLR5 and TLR7 are selectively absent from the same subset. Consistent with the latter, TLR7 ligand activates CD8 alpha(-) DC and PDC, but not CD8 alpha(+) DC as measured by survival ex vivo, up-regulation of surface markers and production of IL-12p40. These data suggest that the dichotomy in TLR expression between plasmacytoid and non-plasmacytoid DC is not conserved between species. However, lack of TLR7 expression could restrict the involvement of CD8 alpha(+) DC in recognition of certain mouse pathogens.