RESUMEN
Identifying biomarkers to evaluate the therapeutic effect of immune checkpoint inhibitors (ICIs) is crucial. Regulatory Associated Protein of MTOR Complex 1 (RPTOR), one of the genes in the mTOR pathway, plays a role in regulating tumor progression. However, the connection between RPTOR mutation and the efficacy of ICIs in melanoma remains unclear. The data of ICIs-treated melanoma patients in discovery (n = 384) and validation (n = 320) cohorts were obtained from cBioPortal databases. The genomic data in the two cohorts was used to investigate the connection between RPTOR mutation and immunotherapy efficacy. The underlying mechanisms were explored based on data from the The Cancer Genome Atlas (TCGA)-skin cutaneous melanoma (SKCM) cohort. Compared to melanoma patients with RPTOR wildtype (RPTOR-WT), RPTOR-mutation (RPTOR-Mut) patients achieved prolonged overall survival (OS) in both discovery cohort (median OS of 49.3 months vs. 21.7 months; HR = 0.41, 95% CI: 0.18-0.92; P = 0.026) and validation cohorts (not reached vs. 42.0 months; HR = 0.34, 95% CI: 0.11-1.06; P = 0.049). RPTOR-Mut melanoma patients exhibited a higher objective response rate (ORR) than RPTOR-WT patients in the discovery cohort (55.0% vs. 29.0%, P = 0.022). RPTOR-Mut patients exhibited higher TMB than RPTOR-WT patients in both discovery and validation cohorts (P < 0.001). RPTOR-Mut melanoma patients had an increased number of DNA damage response (DDR) mutations in TCGA-SKCM cohort. Immune cell infiltration analysis suggested that activated CD4 memory T cells were more enriched in RPTOR-Mut tumors. RPTOR-Mut melanoma patients had higher expression levels of immune-related genes than the RPTOR-WT patients. Our results suggest that RPTOR mutation could serve as a predictor of effective immunotherapy for melanoma.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteína Reguladora Asociada a mTOR , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Inmunoterapia , Mutación , Biomarcadores de Tumor/genéticaRESUMEN
Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.
Asunto(s)
Colina/metabolismo , Cocaína/farmacología , Neuronas/fisiología , Núcleo Accumbens/fisiología , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Receptores sigma/metabolismo , Animales , Animales Recién Nacidos , Regulación de la Expresión Génica , Neuronas/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Receptores de Orexina/genética , Orexinas/genética , Ratas , Ratas Sprague-Dawley , Receptores sigma/genética , Vasoconstrictores/farmacología , Receptor Sigma-1RESUMEN
Background: Circulating B cells are crucial for the pathogenesis of IgA nephropathy (IgAN). This study aimed at investigating the relationship between frequency of different subsets of circulating B cells and clinical measures in IgAN patients.Methods: The percentages of different subsets of circulating B cells in 23 IgAN patients and 14 healthy controls (HC) were determined by flow cytometry. Their serum IL-6 levels were measured by Cytometric Bead Array (CBA). Clinical parameters in five patients were measured before and after treatment for 8-12 weeks. The potential relationship between variants was analyzed.Results: In comparison with the HC, the frequency of CD3-CD19+ CD27+ IgD+IgM+ non-switched memory B cells (P = .0038) and CD3-CD19+ CD27hiCD38hi plasmablasts (P = .0467) and serum IL-6 (P = .0392) levels significantly increased in IgAN patients. The percentages of non-switched memory B cells were positively correlated with plasmablasts (R = 0.5781, P = .0039) and serum IL-6 levels (R = 0.6663, P = .0005) in the patients. The percentages of non-switched memory B cells (R = 0.8399, P < .0001), plasmablasts (R = 0.4486, P = .0318) and the levels of serum IL-6 (R = 0.5461, P = .0070) were positively correlated with the values of 24-h urine proteins in IgAN patients. The serum levels of IL-6 were negatively correlated with the eGFR values. Following standard treatment, the frequency of non-switched memory B cells and plasmablasts and the levels of 24-h urine proteins (P = .0317, P = .0079, P < .05) significantly decreased in IgAN patients.Conclusions: Abnormally higher frequency of non-switched memory B cells and plasmablasts may contribute to the pathogenesis of IgAN and be potential biomarkers for IgAN.
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Subgrupos de Linfocitos B/inmunología , Glomerulonefritis por IGA/inmunología , Interleucina-6/sangre , Células Plasmáticas/inmunología , Adolescente , Adulto , Anciano , Femenino , Glomerulonefritis por IGA/sangre , Glomerulonefritis por IGA/patología , Humanos , Memoria Inmunológica/inmunología , Interleucina-6/inmunología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.
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Lisofosfolípidos/química , Piperazinas/química , Pirrolidinas/química , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusión/química , Elemento de Respuesta al Suero , Secuencias de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Cinética , Ligandos , Lisofosfolípidos/farmacología , Simulación del Acoplamiento Molecular , Mutación , Piperazinas/farmacología , Unión Proteica , Pirrolidinas/farmacología , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides delta/química , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Respuesta Sérica/química , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transducción de Señal , Glycine max , Homología Estructural de Proteína , TermodinámicaRESUMEN
Interest in lipoamino acids as endogenous modulators of G-protein coupled receptors has escalated due to their involvement in a variety of physiologic processes. In particular, a role for these amino acid conjugates has emerged in the endocannabinoid system. The study presented herein investigated the effects of N-arachidonoyl glycine (NAGly) on a candidate endocannabinoid receptor, GPR55. Our novel findings reveal that NAGly induces concentration dependent increases in calcium mobilization and mitogen-activated protein kinase activities in HAGPR55/CHO cells. These increases were attenuated by the selective GPR55 antagonist ML193 (N-[4-[[(3,4-Dimethyl-5-isoxazolyl)amino]sulfonyl]phenyl]-6,8-dimethyl-2-(2-pyridinyl)-4-quinolinecarboxamide), supporting receptor mediated signaling. To our knowledge this is the first report identifying GPR55 as a target of the endogenous lipoamino acid, NAGly.
Asunto(s)
Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Glicina/análogos & derivados , Receptores Acoplados a Proteínas G/genética , Animales , Células CHO , Cricetulus , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Glicina/farmacología , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Quinolinas/farmacología , Receptores de Cannabinoides , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The first structure-activity relationships for a benzothiazole scaffold acting as an antagonist at GPR35 is presented. Analogues were designed based on a lead compound that was previously determined to have selective activity as a GPR35 antagonist. The synthetic route was modular in nature to independently explore the role of the middle and both ends of the scaffold. The activities of the analogues illustrate the importance of all three segments of the compound.
Asunto(s)
Benzotiazoles/química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Benzotiazoles/síntesis química , Benzotiazoles/metabolismo , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Relación Estructura-ActividadRESUMEN
In this work, we explored the molecular framework of the known CB1R allosteric modulator PSNCBAM-1 with the aim to generate new bioactive analogs and to deepen the structure-activity relationships of this type of compounds. In particular, the introduction of a NH group between the pyridine ring and the phenyl nucleus generated the amino-phenyl-urea derivative SN15b that behaved as a positive allosteric modulator (PAM), increasing the CB1R binding affinity of the orthosteric ligand CP55,940. The functional activity was evaluated using serum response element (SRE) assay, which assesses the CB1R-dependent activation of the MAPK/ERK signaling pathway. SN15b and the biphenyl-urea analog SC4a significantly inhibited the response produced by CP55,940 in the low µM range, thus behaving as negative allosteric modulators (NAMs). The new derivatives presented here provide further insights about the modulation of CB1R binding and functional activity by allosteric ligands.
Asunto(s)
Compuestos de Fenilurea/farmacología , Piridinas/farmacología , Receptor Cannabinoide CB1/metabolismo , Regulación Alostérica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estructura Molecular , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-ActividadRESUMEN
GPR35 is a G protein-coupled receptor expressed in the immune, gastrointestinal, and nervous systems in gastric carcinomas and is implicated in heart failure and pain perception. We investigated residues in GPR35 responsible for ligand activation and the receptor structure in the active state. GPR35 contains numerous positively charged amino acids that face into the binding pocket that cluster in two distinct receptor regions, TMH3-4-5-6 and TMH1-2-7. Computer modeling implicated TMH3-4-5-6 for activation by the GPR35 agonists zaprinast and pamoic acid. Mutation results for the TMH1-2-7 region of GPR35 showed no change in ligand efficacies at the K1.32A, R2.65A, R7.33A, and K7.40A mutants. However, mutation of arginine residues in the TMH3-4-5-6 region (R4.60, R6.58, R3.36, R(164), and R(167) in the EC2 loop) had effects on signaling for one or both agonists tested. R4.60A resulted in a total ablation of agonist-induced activation in both the ß-arrestin trafficking and ERK1/2 activation assays. R6.58A increased the potency of zaprinast 30-fold in the pERK assay. The R(167)A mutant decreased the potency of pamoic acid in the ß-arrestin trafficking assay. The R(164)A and R(164)L mutants decreased potencies of both agonists. Similar trends for R6.58A and R(167)A were observed in calcium responses. Computer modeling showed that the R6.58A mutant has additional interactions with zaprinast. R3.36A did not express on the cell surface but was trapped in the cytoplasm. The lack of surface expression of R3.36A was rescued by a GPR35 antagonist, CID2745687. These results clearly show that R4.60, R(164), R(167), and R6.58 play crucial roles in the agonist initiated activation of GPR35.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Simulación de Dinámica Molecular , Inhibidores de Fosfodiesterasa/farmacología , Purinonas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Línea Celular , Humanos , Ligandos , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación Missense , Inhibidores de Fosfodiesterasa/química , Estructura Secundaria de Proteína , Purinonas/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genéticaRESUMEN
BACKGROUND: A significant barrier to organ transplantation is the cellular rejection that occurs and mediated by antibodies, T cells, and innate immune cells. This study was aimed to determine the number of CD4(+)CD25(+)Foxp3(+) Treg, CD4(+)IFN-γ(-)IL-17(+) Th17, CD4(+)IFN-γ(+)IL-17(-) Th1 and CD4(+)IFN-γ(+)IL-17(+) Th1/17 cells in renal transplant recipients (RTR). METHODS: Renal transplantation was performed for a total of 35 patients with end-stage renal failure. The number of CD4(+)CD25(+)Foxp3(+) Treg, CD4(+)IFN-γ(-)IL-17(+) Th17, CD4(+)IFN-γ(+)IL-17(-) Th1 and CD4(+)IFN-γ(+)IL-17(+) Th1/17 cells, and the serum level of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17 were measured in pre- and post-transplant patients and 10 healthy controls (HC) using flow cytometry and Cytometric Bead Array (CBA). The association between the number of different subsets of CD4(+) T-cells and clinical parameters were analyzed among the pre- and post-transplant patients, and the healthy controls. RESULTS: The number of CD4(+)IFN-γ(-)IL-17(+) Th17, CD4(+)IFN-γ(+)IL-17(-) Th1 and CD4(+)IFN-γ(+)IL-17(+) Th1/17 cells were significantly increased in patients with End-Stage Renal Failure (ESRF) compared to the HC. Stratification analysis indicated that AMR (Acute antibody mediated acute rejection), AR (acute rejection) and CR (chronic rejection) groups displayed greater number of CD4(+)IFN-γ(-)IL-17(+) Th17, CD4(+)IFN-γ(+)IL-17(-) Th1 and CD4(+)IFN-γ(+)IL-17(+) Th1/17 cells as well as high level of serum IL-2, IFN-γ, TNF-α and IL-17. But, the AMR, AR and CR groups have shown lower level of CD4(+)CD25(+)Foxp3(+) T cells and serum IL-10 compared to transplant stable (TS) patients. Moreover, the number of Tregs were negatively correlated with the number of Th17 cells in RTR patients. The number of Tregs and Th17 cells were positively correlated with the eGFR and serum creatinine values, respectively. CONCLUSION: The imbalance between different types of CD4(+) T cells and dysregulated inflammatory cytokines may contribute towards renal transplantation rejection.
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Citocinas/sangre , Mediadores de Inflamación/sangre , Trasplante de Riñón , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Receptores de Trasplantes , Adulto , Biomarcadores , Femenino , Rechazo de Injerto/inmunología , Humanos , Inmunofenotipificación , Pruebas de Función Renal , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
T helper 17 (Th17) and Th22 cells regulate the development of tumors. However, their roles in the development of colorectal cancer (CRC) are still unclear. A total of 49 patients with CRC and 18 healthy controls (HC) were evaluated for the percentages of circulating Th17 and Th22 cells by flow cytometry. The concentrations of serum interleukin-17A (IL-17A), IL-22 and carcinoembryonic antigen (CEA) were examined. The levels of IL-17A and IL-22 in tumors were determined by real-time PCR. We found that the percentages of Th17 and Th22 cells in the CRC patients were significantly lower than that in the HC and were associated negatively with the pathological stages of CRC. The levels of IL-17A and IL-22 mRNA transcripts were lower in the tumor tissues, particularly in the advanced CRC. After the tumor resection, the percentages of circulating Th17 and Th22 cells increased. These data suggest that decreased Th17 and Th22 responses may be associated with the development of CRC.
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Neoplasias Colorrectales/patología , ARN Mensajero/genética , Linfocitos T Colaboradores-Inductores/patología , Células Th17/patología , Adulto , Anciano , Antígeno Carcinoembrionario/sangre , Antígeno Carcinoembrionario/genética , Estudios de Casos y Controles , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/cirugía , Femenino , Expresión Génica , Humanos , Inmunofenotipificación , Interleucina-17/sangre , Interleucina-17/genética , Interleucinas/sangre , Interleucinas/genética , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Periodo Perioperatorio , ARN Mensajero/sangre , Linfocitos T Colaboradores-Inductores/metabolismo , Células Th17/metabolismo , Interleucina-22RESUMEN
BACKGROUND: Aberrant activation of follicular helper T (TFH) and B cells is associated with the development of autoimmune diseases. However, little is known about the potential role of these cells in the development of primary biliary cirrhosis (PBC). AIM: This study aimed at characterizing the numbers of different subsets of circulating Tfh and B cells as well as evaluating their potential association with the levels of immunoglobulins and autoantibodies in newly diagnosed PBC patients. METHODS: The numbers of circulating CD27(+), CD38(+), CD86(+) and CD95(+) B cells as well as inducible T cell costimulator (ICOS)(+) and programmed death-1 (PD-1)(+), IL-21(+) TFH cells were examined in 58 patients with newly diagnosed PBC and 30 matched healthy controls (HCs). RESULTS: The numbers of circulating CD38(+)CD19(+), CD86(+)CD19(+), and CD95(+)CD19(+) B cells; CD3(+)CD4(+)CXCR5(+)ICOS(+) and CD3(+)CD4(+)CXCR5(+)PD-1(+) Tfh cells; and the levels of serum IL-21 in the PBC patients were significantly greater, but the numbers of CD27(+)CD19(+) B cells were significantly less than those in the HCs (p < 0.05). The numbers of CD3(+)CD4(+)CXCR5(+)ICOS(+) Tfh cells were positively correlated with the numbers of CD38(+)CD19(+) and CD86(+)CD38(+)CD19(+) B cells and the levels of serum anti-mitochondrial antibodies against M2 antigen (AMA-M2), AMA and immunolgubin M (IgM) in the PBC patients. The levels of serum IL-21 were positively correlated with the levels of serum AMA-M2, AMA, IgG and IgM, but negatively with the numbers of CD27(+)CD19(+) B cells in the PBC patients. CONCLUSIONS: Increased numbers of circulating ICOS(+) and IL-21(+) Tfh and CD38(+) plasma cells may be exhibited by patients with recent diagnoses of PBC.
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ADP-Ribosil Ciclasa 1/sangre , Proteína Coestimuladora de Linfocitos T Inducibles/sangre , Cirrosis Hepática Biliar/inmunología , Glicoproteínas de Membrana/sangre , Células Plasmáticas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Adulto , Anciano , Autoanticuerpos/sangre , Autoinmunidad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores/sangre , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Células Cultivadas , Femenino , Humanos , Inmunoglobulinas/sangre , Interleucinas/sangre , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/diagnóstico , Masculino , Persona de Mediana Edad , Células Plasmáticas/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Regulación hacia ArribaRESUMEN
AIMS: This study aimed to assess the differential expression of specific B cell subtypes in patients with chronic viral hepatitis. METHODS: The frequencies of differential expression of specific B cell subtypes in patients with chronic viral hepatitis and healthy controls were assessed by flow cytometry using monoclonal antibodies specific for CD38, CD27, CD86, CD95, TLR-9, and IgD. The effect of adefovir treatment on B cell subsets in HBV patients was determined. The values of clinical parameters in the patients were also measured. RESULTS: The frequency of CD86+ B cells was not significantly different in chronic HBV patients but was higher in HCV patients compared with that in healthy controls. CD95 and IgD levels were lower in HBV and HCV patients than in healthy controls. A significant negative correlation occurred between the proportion of CD95+ B cells and HBV DNA viral load. The frequency of TLR-9 on the B cells in HBV and HCV patients was higher compared with that of healthy controls. After treatment with adefovir, the frequency of CD95 and IgD expressed on B cells was increased in HBV patients. CONCLUSIONS: Activated B cells and exhausted B cells homeostasis were commonly disturbed in HBV and HCV patients.
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Antígeno B7-2/sangre , Hepatitis B Crónica/sangre , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis C Crónica/sangre , Hepatitis C Crónica/tratamiento farmacológico , Receptor Toll-Like 9/sangre , Receptor fas/sangre , Adenina/análogos & derivados , Adenina/uso terapéutico , Adulto , Anciano , Anticuerpos Monoclonales/química , Antivirales/uso terapéutico , Linfocitos B/virología , ADN Viral/sangre , Femenino , Citometría de Flujo , Genotipo , Homeostasis , Humanos , Inmunoglobulina D/sangre , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos/química , Organofosfonatos/uso terapéutico , Fenotipo , Prevalencia , Carga Viral , Adulto JovenRESUMEN
The therapeutic and psychoactive properties of cannabinoids have long been recognized. The type 2 receptor for cannabinoids (CB2) has emerged as an important therapeutic target in several pathologies, as it mediates beneficial effects of cannabinoids while having little if any psychotropic activity. Difficulties associated with the development of CB2-based therapeutic agents have been related to its intricate pharmacology, including the species specificity and functional selectivity of the CB2-initiated responses. We postulated that a plasmalemmal or subcellular location of the receptor may contribute to the differential signaling pathways initiated by its activation. To differentiate between these two, we used extracellular and intracellular administration of CB2 ligands and concurrent calcium imaging in CB2-expressing U2OS cells. We found that extracellular administration of anandamide was ineffective, whereas 2-arachidonoyl glycerol (2-AG) and WIN55,212-2 triggered delayed, CB2-dependent Ca(2+) responses that were Gq protein-mediated. When microinjected, all agonists elicited fast, transient, and dose-dependent elevations in intracellular Ca(2+) concentration upon activation of Gq-coupled CB2 receptors. The CB2 dependency was confirmed by the sensitivity to AM630, a selective CB2 antagonist, and by the unresponsiveness of untransfected U2OS cells to 2-AG, anandamide, or WIN55,212-2. Moreover, we provide functional and morphological evidence that CB2 receptors are localized at the endolysosomes, while their activation releases Ca(2+) from inositol 1,4,5-trisphosphate-sensitive- and acidic-like Ca(2+) stores. Our results support the functionality of intracellular CB2 receptors and their ability to couple to Gq and elicit Ca(2+) signaling. These findings add further complexity to CB2 receptor pharmacology and argue for careful consideration of receptor localization in the development of CB2-based therapeutic agents.
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Señalización del Calcio/fisiología , Membranas Intracelulares/química , Receptor Cannabinoide CB2/química , Benzoxazinas/metabolismo , Benzoxazinas/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Humanos , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Morfolinas/metabolismo , Morfolinas/farmacología , Naftalenos/metabolismo , Naftalenos/farmacología , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/metabolismoRESUMEN
BACKGROUND: IL-10+ regulatory B (Bregs), CD4+Foxp3+ regulatory T (Tregs), and CD4+CXCR5+Foxp3+ follicular regulatory T (TFR) cells regulate the progression of infection disease. This study aimed at examining how those cells associated with the development of chronic hepatitis B (CHB) and chronic hepatitis C (CHC) in a Chinese population. METHODS: The numbers of circulating IL-10+ Bregs, Tregs and TFR cells in 31 CHC, 58 CHB patients and 22 healthy controls (HC) were examined by flow cytometry. The potential association of those cells with clinical measures was analyzed. RESULTS: The numbers of CD5+CD19+CD1dhighIL-10+ Bregs, Tregs and TFR cells and the levels of serum IL-10, IFN-γ and IL-2 in the CHB, and IL-10 and IFN-γ in the CHC patients were significantly higher than that in the HC (p<0.05). Furthermore, the numbers of circulating IL-10+ Bregs and the levels of serum IL-10, but not other cytokines tested were positively correlated with the levels of serum HBV DNA and ALT in the HBeAg- CHB patients as well as HCV RNA and ALT in CHC patients. Additionally, the numbers of circulating TFR cells were positively correlated with the levels of serum HBV DNA and ALT in the CHB patients as well as HCV RNA and ALT in the CHC patients. CONCLUSIONS: Increased numbers of circulating IL-10+ Bregs and TFR cells are associated with poor virus eradication and liver injury in CHB and CHC patients. Furthermore, the levels of serum IL-10 is associated with the hepatic flares.
Asunto(s)
Linfocitos B Reguladores/inmunología , Factores de Transcripción Forkhead/metabolismo , Hepatitis B Crónica/inmunología , Hepatitis C Crónica/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Antígenos CD , ADN Viral/sangre , Demografía , Femenino , Citometría de Flujo , Antígenos e de la Hepatitis B/inmunología , Hepatitis B Crónica/sangre , Hepatitis C Crónica/sangre , Humanos , Interleucina-10/sangre , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: This study aimed at examining the numbers of different subsets of circulating Tfh and B cells in patients with new onset autoimmune hepatitis (AIH). METHODS: A total of 17 patients with new onset AIH and 17 age-/gender-matched healthy controls (HC) were examined for the numbers of ICOS(+) , PD-1(+) and IL-21(+) Tfh cells, CD27(+) , CD38(+) , CD95(+) , CD86(+) and IL-10(+) B cells were quantified by flow cytometry. The concentrations of serum IL-21 and IL-10 were examined. RESULTS: In comparison with that in the HC, significantly increased numbers of circulating CD38(+) , CD86(+) or CD95(+) B cells, ICOS(+) and PD-1(+) Tfh cells and increased levels of serum IL-21, but reduced numbers of CD27(+) , IL-10(+) B cells were detected in the patients. The concentrations of serum IL-21 and IL-10 were positively correlated with the numbers of CD4(+) CXCR5(+) TFH and CD19(+) CD5(+) CD1d(+) B cells respectively. The numbers of ICOS(+) or PD-1(+) Tfh cells were correlated positively with CD86(+) or CD95(+) B cells in those patients respectively. The numbers of CD38(+) B cells, ICOS(+) or PD-1(+) Tfh cells were correlated positively with the concentrations of serum IgG or IgM in the patients respectively; the concentrations of serum IL-21 were correlated positively with serum IgG, IgA and IgM and the concentrations of serum IL-10 were correlated negatively with serum IgG and IgM in the patients. CONCLUSION: Circulating activated Tfh and plasma cells may be associated with hypergammaglobulinaemia during the pathogenic process of AIH in humans.
Asunto(s)
Hepatitis Autoinmune/inmunología , Hipergammaglobulinemia/inmunología , Células Plasmáticas/inmunología , Subgrupos de Linfocitos T/inmunología , Adulto , Anciano , Linfocitos B Reguladores/inmunología , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Interleucina-10/sangre , Interleucinas/sangre , Masculino , Persona de Mediana EdadRESUMEN
The aim of the present study was to assess the long-term impact of entecavir (ETV) on T, B and natural killer (NK) cell immunity in patients with suboptimal responses to adefovir (SRA) chronic hepatitis B (CHB). Thirty SRA CHB patients and 20 age- and gender-matched healthy controls (HC) completed at least 6 months of ETV treatment. Hepatitis B virus (HBV) DNA loads, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the frequency of different subsets of T, B and NK cells in individual subjects were measured. There were smaller numbers of CD3(-) CD56(+) and CD244(+) NK cells, CD3(+) CD8(+) T cells and cytokine-secreting CD4(+) T cells, but greater numbers of CD3(+) CD4(+) , CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(+) CD127(low) T cells and CD19(+) CD27(+) B cells, detected in SRA patients. After switching to ETV monotherapy, the levels of HBV DNA and hepatitis B s antigen, as well as hepatitis B e antigen seropositivity, decreased gradually, accompanied by decreases in ALT and AST levels. Furthermore, the number of NK, CD8(+) and cytokine-secreting CD4(+) T cells increased, whereas the number of CD4(+) , CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(+) CD127(low) T cells and CD19(+) CD27(+) B cells decreased, in SRA CHB patients. The frequency of CD4(+) interferon-γ-positive T cells was negatively associated with serum HBV DNA levels. Thus, treatment with ETV inhibits HBV replication, modulates T and NK cell immunity and improves liver function in SRA CHB patients.
Asunto(s)
Adenina/análogos & derivados , Antivirales/uso terapéutico , Guanina/análogos & derivados , Hepatitis B Crónica/sangre , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos/uso terapéutico , Adenina/uso terapéutico , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Estudios de Casos y Controles , ADN Viral/metabolismo , Guanina/uso terapéutico , Virus de la Hepatitis B/metabolismo , Humanos , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Carga ViralRESUMEN
Clavulanic acid (CLAV) is a component of Augmentin® that preserves antibiotic efficacy by inhibiting ß-lactamase activity. It also enhances cellular glutamate uptake and is a potential CNS therapeutic. Because increased glutamate transmission in brain reward circuits facilitates methamphetamine (METH) locomotor activation and sensitization, we tested the hypothesis that CLAV inhibits acute and sensitized locomotor responses to METH in mice and investigated effects of CLAV on METH-induced changes in glutaminase, the major glutamate-producing enzyme in the brain. Acute METH (3 mg/kg) produced hyperlocomotion that was reduced by CLAV (20 mg/kg but not 10 mg/kg). Mice injected with METH (3 mg/kg) every other day for 9 d and then challenged with METH 27 d later displayed locomotor sensitization. CLAV (10 mg/kg), when injected 15 min before each METH injection during the 9-d exposure interval, blocked locomotor sensitization induced by METH challenge. In METH-sensitized mice, mRNA levels of both isoforms of glutaminase (GLS and GLS2) were altered in the nucleus accumbens compared to mice exposed to a single injection of METH (i.e., GLS decreased and GLS2 increased). CLAV normalized the METH-induced GLS deficit but not the increase in GLS2. In summary, CLAV reduced acute and sensitized locomotor responses to METH and normalized the METH-induced reduction of GLS gene expression in the NAC. Given that glutaminases belong to the ß-lactamase superfamily and CLAV is a ß-lactamase inhibitor, our data point toward studying glutaminase as a therapeutic target of CLAV.
Asunto(s)
Estimulantes del Sistema Nervioso Central , Ácido Clavulánico , Glutaminasa , Metanfetamina , Núcleo Accumbens , ARN Mensajero , Animales , Metanfetamina/farmacología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Glutaminasa/metabolismo , Masculino , Ácido Clavulánico/farmacología , ARN Mensajero/metabolismo , ARN Mensajero/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Ratones Endogámicos C57BL , Ratones , Locomoción/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Relación Dosis-Respuesta a DrogaRESUMEN
The aim of the present study was to investigate the natural killer (NK) cell phenotype and function in chronic hepatitis B virus (HBV) patients and to study the effects of entecavir therapy (10 mg/day, p.o.) on these responses. Peripheral blood NK cells were collected from 18 chronic HBV patients and 14 healthy controls. The effect of entecavir therapy on the phenotype and function of NK cells in chronic HBV patients was characterized by flow cytometry analysis. Concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), HBV viral loads in both groups and potential associations between the frequency of peripheral NK cell subsets and clinical measures were determined. There was a significant reduction in the number of CD3(-)CD56(+) NK cells in chronic HBV patients compared with healthy controls. Furthermore, there were significant increases in the percentage of CD3(-)CD56(+)NKG2D(+) and CD3(-)CD56(+)NKP30(+) NK activating receptors in chronic HBV patients compared with healthy individuals, who exhibited downregulated expression following entecavir treatment. Spearman's correlation analysis revealed that there was a significant positive correlation between the percentage of NKG2D(+) and NKP30(+) NK cells and serum ALT levels. Characterization of NK cell degranulation indicated that the frequency of CD107a(+) NK cells in HBV patients (in response to K562 stimulation) was significantly greater than in healthy controls but decreased following entecavir treatment. Entecavir treatment of hepatitis B e antigen-positive chronic HBV-infected patients not only led to a reduction in HBV DNA loads and normalization of ALT and AST levels, but also resulted in the recovery of NK cell-mediated immunity.
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Antivirales/uso terapéutico , Guanina/análogos & derivados , Hepatitis B Crónica/tratamiento farmacológico , Inmunidad Celular/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Receptores de Células Asesinas Naturales/biosíntesis , Adulto , Antivirales/administración & dosificación , Complejo CD3/biosíntesis , Antígeno CD56/biosíntesis , Regulación hacia Abajo , Femenino , Citometría de Flujo , Guanina/administración & dosificación , Guanina/uso terapéutico , Anticuerpos contra la Hepatitis B/sangre , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Pruebas de Función Hepática , Masculino , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Receptor 3 Gatillante de la Citotoxidad Natural/biosíntesis , Resultado del Tratamiento , Carga Viral , Adulto JovenRESUMEN
UNLABELLED: AIMS. Interleukin-37 (IL-37) is an anti-inflammatory cytokine. This study aims to investigate the concentrations of plasma and cerebrospinal fluid (CSF) IL-37 in patients with Guillain-Barré Syndrome (GBS). METHODS: The levels of plasma and CSF IL-37, IL-17A, IFN- γ , and TNF- α in 25 GBS patients and 20 healthy controls (HC) were determined by enzyme-linked immunoabsorbent assay and flow cytometric bead array assay, respectively. The values of clinical parameters in the patients were also measured. RESULTS: The concentrations of plasma IL-37, IL-17A, IFN- γ , and TNF- α and CSF IL-37 and IL-17A in patients at the acute phase of GBS were significantly higher than those in the HC. The levels of plasma IL-37, IL-17A, IFN- γ , and TNF- α were positively correlated in those patients, and the levels of CSF IL-37 and IL-17A as well as the levels of plasma TNF- α were correlated positively with the GBS disability scale scores (GDSs) in those patients. Treatment with intravenous immunoglobulin significantly reduced the levels of plasma IL-37, IL-17A, IFN- γ , and TNF- α in the drug-responding patients. CONCLUSIONS: Our findings indicate higher levels of plasma and CSF IL-37 and IL-17A and other proinflammatory cytokines in patients with GBS.
Asunto(s)
Síndrome de Guillain-Barré/sangre , Síndrome de Guillain-Barré/líquido cefalorraquídeo , Interleucina-1/sangre , Interleucina-1/líquido cefalorraquídeo , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/uso terapéutico , Inmunoglobulinas Intravenosas/uso terapéutico , Inflamación/metabolismo , Interferón gamma/sangre , Interferón gamma/líquido cefalorraquídeo , Interleucina-17/sangre , Interleucina-17/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/líquido cefalorraquídeo , Adulto JovenRESUMEN
Riluzole, approved to manage amyotrophic lateral sclerosis, is mechanistically unique among glutamate-based therapeutics because it reduces glutamate transmission through a dual mechanism (i.e., reduces glutamate release and enhances glutamate reuptake). The profile of riluzole is favorable for normalizing glutamatergic dysregulation that perpetuates methamphetamine (METH) dependence, but pharmacokinetic and metabolic liabilities hinder repurposing. To mitigate these limitations, we synthesized troriluzole (TRLZ), a third-generation prodrug of riluzole, and tested the hypothesis that TRLZ inhibits METH hyperlocomotion and conditioned place preference (CPP) and normalizes METH-induced changes in mesolimbic glutamate biomarkers. TRLZ (8, 16 mg/kg) reduced hyperlocomotion caused by METH (1 mg/kg) without affecting spontaneous activity. TRLZ (1, 4, 8, 16 mg/kg) administered during METH conditioning (0.5 mg/kg x 4 d) inhibited development of METH place preference, and TRLZ (16 mg/kg) administered after METH conditioning reduced expression of CPP. In rats with established METH place preference, TRLZ (16 mg/kg) accelerated extinction of CPP. In cellular studies, chronic METH enhanced mRNA levels of glutamate carboxypeptidase II (GCPII) in the ventral tegmental area (VTA) and prefrontal cortex (PFC). Repeated METH also caused enhancement of GCPII protein levels in the VTA that was prevented by TRLZ (16 mg/kg). TRLZ (16 mg/kg) administered during chronic METH did not affect brain or plasma levels of METH. These results indicate that TRLZ, already in clinical trials for cerebellar ataxia, reduces development, expression and maintenance of METH CPP. Moreover, normalization of METH-induced GCPII levels in mesolimbic substrates by TRLZ points toward studying GCPII as a therapeutic target of TRLZ.