RESUMO
Mast cell (MC) exocytosis is organized by prenylated protein, including Rab families. Among Rab proteins, Rab3a, Rab27a, and Rab11 are responsible for exocytosis arrangement. Rab3a and Rab27a are contributed to exocytosis by interacting with other exocytosis proteins. Zoledronate administration disrupted the Rab prenylation process that affected its interaction with other proteins, and finally, its function. The present study has investigated the effect of zoledronate on the histamine release (HR) from RBL-2H3 cells. The main focus is to answer the question of whether zoledronate affects Rab27a/Doc2a interaction. Histamine release on RBL-2H3 cells after zoledronate or clodronate administration was measured using HPLC-fluorometry. Dinitrophenylated bovine serum albumin (DNP-BSA) (20 ng/mL) or ionomycin (1 µM) are used as secretagogues. Calcium (Ca2+) influx observation was performed using Fura-2A/M. In situ proximity ligation assay (PLA) is used to investigate Rab27a/Doc2a interaction after bisphosphonates (BPs) treatment. Histamine concentration measurement with HPLC-fluorometry showed that zoledronate (30, 100 µM) inhibited HR from antigen-activated RBL-2H3 cells. Zoledronate showed less inhibition in cells activated with ionomycin. Intracellular Ca2+ concentration and Ca2+ flux rate from the extracellular compartment was not changed by zoledronate administration. No changes in Rab27a/Doc2a interaction after zoledronate treatment. Histamine release inhibition by zoledronate in DNP-BSA-activated RBL-2H3 cells is not related to the disruption of Rab27a/Doc2a interaction and is not involve the change in Ca2+ influx.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Liberação de Histamina/efeitos dos fármacos , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ácido Zoledrônico/farmacologia , Proteínas rab27 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Ionóforos de Cálcio/farmacologia , Linhagem Celular Tumoral , Exocitose , Histamina , Ionomicina/farmacologia , ProteínasRESUMO
In atopic diseases, the epithelium releases cytokines and chemokines that initiate skin inflammation. Atopic dermatitis (AD) is characterized by a disrupted epidermal barrier and is triggered or exacerbated by environmental stimuli such as house dust mite (HDM) allergens. The proinflammatory cytokine interleukin 33 (IL-33) plays an important role in the pathogenesis of AD, but how IL-33 production in keratinocytes is elicited by HDM is unknown. To that end, here we stimulated monolayer-cultured human keratinocytes and human living skin equivalents with Dermatophagoides pteronyssinus HDM extract to investigate its effects on IL-33 production from keratinocytes. The HDM extract induced intracellular expression of IL-33 and modulated its processing and maturation, triggering rapid IL-33 release from keratinocytes. Group 1 HDM allergen but not group 2 HDM allergen elicited IL-33 production. An ATP assay of keratinocyte culture supernatants revealed an acute and transient accumulation of extracellular ATP immediately after the HDM extract stimulation. Using the broad-spectrum P2 antagonist suramin, the specific purinergic receptor P2Y2 (P2RY2) antagonist AR-C118925XX, and P2RY2-specific siRNA, we discovered that the HDM extract-induced IL-33 expression was mainly dependent on extracellular ATP/P2Y2 signaling mediated by transactivation of epidermal growth factor receptor, followed by activation of the ERK kinase signaling pathway. Moreover, HDM extract-induced release of 25-kDa IL-33 from the keratinocytes depended on an extracellular ATP/P2 signaling-mediated intracellular Ca2+ increase. Our study demonstrates the new mechanism controlling the induction and maturation of keratinocyte-produced IL-33 by HDM allergens, an innate immune process that might play a role in AD development or severity.
Assuntos
Trifosfato de Adenosina/metabolismo , Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Dermatite Atópica/imunologia , Interleucina-33/metabolismo , Queratinócitos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Fibroblastos , Furanos/farmacologia , Humanos , Imunidade Inata , Queratinócitos/imunologia , Piperidinas/farmacologia , Cultura Primária de Células , Antagonistas do Receptor Purinérgico P2Y/farmacologia , RNA Interferente Pequeno/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Tetrazóis/farmacologiaRESUMO
OBJECTIVE: Mice and rats are important animal models for mast cell (MC) study. However, rat Mas-related-GPCR-B3 receptor (MRGPRB3) has been less studied than its mouse counterpart. Therefore, we aimed to characterize rat MRGPRB3. METHODS: Mrgprb3 mRNA expression was assessed in peritoneal cells (RPCs) and peritoneal MCs (RPMCs) of wild-type rats, RPCs of MC-deficient rats, and RBL-2H3 cells by reverse-transcriptase polymerase chain reaction (RT-PCR). RPMCs, MRGPRX2-transfected and non-transfected RBL-2H3 cells were activated by 15-30 min incubation with DNP-BSA, substance-P (SP), or compound-48/80. L732138 or CP96344 was used as a tachykinin/neurokinin-1-receptor antagonist. Histamine release from MCs was measured by HPLC fluorometry. RESULTS: Mrgprb3 mRNA expression was found in all cells, with the highest level in wild-type RPCs. All cells responded to DNP-BSA, but only MRGPRX2-transfected-RBL-2H3 cells and RPMCs responded to all activators. L732138 (0.1-10 µM) and CP96344 (1-100 µM) suppressed SP (10 µM)-induced RPMC activation. L732138 inhibition was dose independent, whereas CP96344 inhibition occurred in a dose-dependent manner. Additionally, only CP96344 suppressed SP (100 µM)- and compound-48/80 (10 µg/mL)-induced RPMC activation. CONCLUSIONS: RPMCs expressing functional MRGPRB3 response upon MRGPRX2 ligands to regulated MC-mediated activities. It`s provide novel insights for future pseudo-allergic studies in rodents.
Assuntos
Mastócitos/efeitos dos fármacos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Substância P/farmacologia , p-Metoxi-N-metilfenetilamina/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Dinitrofenóis , Liberação de Histamina/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Masculino , Mastócitos/imunologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica , Ratos , Receptores de Neuropeptídeos/metabolismo , Soroalbumina Bovina , Taquicininas/químicaRESUMO
The resources of released histamine from activated mast cells, as initial effectors of allergic disease, include not only endogenous prepackaged histamine and newly synthesized histamine, but also histamine that is obtained through the de novo reuptake pathway. To investigate the de novo histamine production pathway, a mast cell line, RBL-2H3 Sc98 in which endogenous histamine production is lacking and only the de novo histamine release pathway via transporters is maintained, was used to dissect histamine reuptake in the present study. Histamine content measurements indicated that RBL-2H3 Sc98 cells took up extracellular histamine for storage in granules and subsequent release after stimulation by an antigen. Profiling and inhibition analysis of possible transporters suggested that the plasma membrane monoamine transporter and organic cation transporter 1 may be candidate transporters for histamine uptake from extracellular spaces, and that vesicular monoamine transporter 2 was responsible for intracellular vesicle uptake. These results may provide the foundation to understand the contribution of exogenous histamine to outward histamine release that is mediated by mechanisms other than conventional exocytosis.
Assuntos
Membrana Celular/metabolismo , Histamina/metabolismo , Mastócitos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Desipramina/farmacologia , Exocitose/efeitos dos fármacos , Lopinavir/farmacologia , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , RatosRESUMO
Chèdiak-Higashi syndrome is a rare autosomal recessive disease that causes hypopigmentation, recurrent infections, mild coagulation defects and neurological problems. Beige mice carry a mutation in the lysosome trafficking regulator (LYST) gene and display some of the key characteristics of human Chèdiak-Higashi syndrome, in particular, a high susceptibility to infection due to aberrant natural killer (NK) cell and polymorphonuclear leucocyte function. Morphological analysis of beige mice reveals the presence of enlarged lysosomes in a variety of cell types, including leucocytes, hepatocytes, fibroblasts and renal tubule cells. To examine the process of granule maturation and degranulation in beige mice mast cells, morphological studies have been conducted using a combination of electrophysiological techniques; however, few functional studies have been conducted with mast cells, such as mediator release. The aim of the present study was to determine the morphological and functional characteristics of skin and peritoneal mast cells and bone marrow-derived mast cells of homozygous (bg/bg) and heterozygous (bg/+) beige mice and wild-type (+/+) mice. The histamine concentration was lower in the peritoneal and bone marrow-derived mast cells of bg/bg mice compared with those of bg/+ and +/+ mice, but the histamine release response was potentiated. In vivo studies of passive cutaneous anaphylaxis showed no differences between bg/bg mice and either bg/+ or +/+ mice. Although bg/bg mast cells with enlarged granules display specific exocytotic processes in vitro, the consequences of mast cell activation in beige mice were similar to those of wild-type mice in vivo.
Assuntos
Síndrome de Chediak-Higashi/imunologia , Grânulos Citoplasmáticos/patologia , Células Matadoras Naturais/imunologia , Lisossomos/patologia , Mastócitos/fisiologia , Neutrófilos/imunologia , Animais , Degranulação Celular , Células Cultivadas , Síndrome de Chediak-Higashi/genética , Modelos Animais de Doenças , Histamina/metabolismo , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação/genética , Proteínas/genética , Proteínas de Transporte VesicularRESUMO
OBJECTIVE: Nigella sativa (N. sativa) has several pharmacological actions which include antioxidant, antidiabetic, anticancer, antitussive, immunomodulator, analgesic, antimicrobial, anti-inflammatory, spasmolytic, and bronchodilator. The purpose of this study is to measure the effectivity of N. sativa ethanol extract as anti-inflammation on peritoneal Wistar rat mast cells. The laboratory experiment was used to investigate the effectivity of N. sativa as an anti-inflammatory on mast cells. Six groups of mast cells were stimulated by C 48/80 to release histamine. Group 1 were without N. sativa, while group 2, 3, 4, 5, and 6 were given N. sativa with concentrations of 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml and 0.5 mg/ml, respectively. Histamine concentration was measured by high-performance liquid chromatography-fluorometry. RESULT: The study showed that N. sativa ethanol extract effectively inhibit histamine release from peritoneal Wistar rat mast cells proportionally to its concentration. N. sativa is effective as an anti-inflammation on mast cells by inhibition of histamine release and has no toxic effect on mast cell. N. sativa could be considered as a potential therapy for asthma therapy and prevention.
Assuntos
Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Histamina/metabolismo , Mastócitos/efeitos dos fármacos , Nigella sativa , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/administração & dosagem , Asma/prevenção & controle , Etanol , Cavidade Peritoneal , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , SementesRESUMO
Mast cells, in addition to endocrine cells and neurons, are typical secretory cells. Their function in allergic inflammation is to secrete inflammatory mediators from secretory vesicles. Intracellular synthesized inflammatory mediators are transported by vesicular monoamine transporters (VMATs) to vesicles where they are stored. After stimulation, the contents of the secretory vesicles are released via exocytosis. This study established a high throughput imaging screening system to monitor the functions of secretory vesicles in mast cells, including molecular uptake via VMAT2 and the exocytotic process, by using a novel fluorescent probe, FFN206, which was developed as a VMAT2 substrate. After loading with FFN206, the rapid uptake of FFN206 was observed and secretory vesicles in mouse bone marrow derived mast cells and a cultured mast cell line were clearly visualized. FFN206 uptake by secretory vesicles was time-dependent and was blocked by reserpine. Furthermore, exocytotic trafficking was monitored dynamically by real-time high-throughput fluorescence quantitation. In the present study, we verified the application of FFN206 for the monitoring of functional vesicles. This high-throughput screening system may benefit instinctive drug evaluation.
Assuntos
Células da Medula Óssea/metabolismo , Exocitose , Ensaios de Triagem em Larga Escala/métodos , Mastócitos/metabolismo , Vesículas Secretórias/metabolismo , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Ratos , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Nitrogen-containing bisphosphonates (NBPs) have been widely used as bone anti-resorptive drugs for the treatment of osteoclast-dependent bone disorders. Zoledronate is currently the most potent NBP, and has potential as an inhibitor of farnesyl pyrophosphate synthase. The present study was undertaken to elucidate the possible effects of zoledronate on FcεRI-dependent mast cell activity in vitro, which is essential for in maintaining homeostasis of the gastrointestinal mucosa. Treatment with zoledronate significantly diminished exocytosis of mast cells, which was reflected by a decrease of FcεRI-dependent histamine release compared to that in vehicle-treated mast cells. Our single-vesicle monitoring and biochemical results suggested that zoledronate modulates intracellular formation of the myosinVa/Rab3a complex and syntaxin4/VAMP7 complex, which are critical in vesicle motility, and therefore disturbs exocytosis via suppression of the velocity of intracellular vesicles and inhibition of membrane fusion. Our findings imply that oral administration of zoledronate could modulate mucosal immune function by blocking mast cell function, and this risk should be of concern in the clinical usage of NBPs.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Mastócitos/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Ácido Zoledrônico/farmacologia , Proteína rab3A de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Exocitose/efeitos dos fármacos , Liberação de Histamina/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Ligação Proteica , Ratos , Vesículas Transportadoras/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) are membrane proteins distributed on the cell surface, and they may be potential drug targets. However, synthesizing GPCRs in vitro can be challenging. Recently, some cell-free protein synthesis systems have been shown to produce a large amount of membrane protein combined with chemical chaperones that include liposomes and glycerol. Liposomes containing high concentrations of glycerol are known as glycerosomes, which are used in new drug delivery systems. Glycerosomes have greater morphological stability than liposomes. Proteoglycerosomes are defined as glycerosomes that contain membrane proteins. Human histamine H1 receptor (HRH1) is one of the most studied GPCRs. In this study, we synthesized wild-type HRH1 (WT-HRH1) proteoglycerosomes and D107A-HRH1, (in which Asp107 was replaced by Ala) in a wheat germ cell-free protein synthesis system combined with asolectin glycerosomes. The mutant HRH1 has been reported to have low affinity for the H1 antagonist. In this study, the amount of synthesized WT-HRH1 in one synthesis reaction was 434 ± 66.6 µg (7.75 ± 1.19 × 103pmol). The specific binding of [3H]pyrilamine to the WT-HRH1 proteoglycerosomes became saturated as the concentration of the radioligand increased. The dissociation constant (Kd) and maximum density (Bmax) of the synthesized WT-HRH1 were 9.76 ± 1.25 nM and 21.4 ± 0.936 pmol/mg protein, respectively. However, specific binding to D107A-HRH1 was reduced compared with WT-HRH1 and the binding did not become saturated. The findings of this study highlight that HRH1 synthesized using a wheat germ cell-free protein synthesis system combined with glycerosomes has the ability to bind to H1 antagonists.
RESUMO
The aim of this study was to investigate the efficacy and safety of YM-58483, a small molecular antagonist of Ca2+ release-activated Ca2+ (CRAC) channels, for the treatment of rheumatoid arthritis (RA), in vivo and ex vivo. YM-58483 was continuously injected subcutaneously in a collagen-induced arthritis (CIA) mouS.E.M.odel using an implanted osmotic pump. The severity of CIA was evaluated using the following parameters: body weight, hind paw volume, clinical score, histological analysis, cytokine levels, Ca2+ influx, and specific IgG production. The efficacy of long-term application of YM-58483 was also verified ex vivo in RA patient-derived peripheral blood monocytes. Assessment of the clinical severity of CIA, cytokine profile in serum and joint protein extracts, and specific IgG production showed that continuous application of YM-58483 suppressed synovial inflammation by inhibiting immune cell activity. Chemical screening and hepatography indicated that long-term subcutaneous delivery of YM-58483 was safer than oral administration for systemic application. Moreover, constant preincubation with YM-58483 at an IC50 of 0.1-1â¯nM altered proinflammatory cytokine production ex vivo in peripheral T cells derived from RA patients. Our findings suggest that continuous long-term application of appropriate CRAC inhibitors such as YM-58483 is a potential therapeutic strategy for global immunosuppression in RA.
Assuntos
Anilidas/administração & dosagem , Anilidas/farmacologia , Anti-Inflamatórios/farmacologia , Artrite Reumatoide/tratamento farmacológico , Tiadiazóis/administração & dosagem , Tiadiazóis/farmacologia , Anilidas/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Autoanticorpos/biossíntese , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Injeções Subcutâneas , Masculino , Camundongos , Segurança , Baço/imunologia , Líquido Sinovial/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tiadiazóis/uso terapêuticoRESUMO
OBJECTIVE: To investigate the effects of friedelin (terpenoid) and 8-hydroxyisocapnolactone-2-3-diol (coumarin) with concentration 10 µM, 30 µM, and 100 µM on inhibiting mast cells (MCs) degranulation. METHODS: The investigation was performed in vitro by administering each compound into rat peritoneal MCs and rat basophilic leukemia-2H3 cells followed by activation with 50 µg/mL of compound 48/80 or 1 µM of ionomycin. The concentration of histamine released from each group was measured by a high-performance liquid chromatography-fluorometry system with post-column derivatization using o-phthalaldehyde. RESULTS: 8-Hydroxyisocapnolactone-2-3-diol inhibited degranulation of compound 48/80 activated-rat peritoneal MCs with the histamine release percentages of 74.57%, 72.21% and 51.79% when the 10 µM, 30 µM and 100 µM concentrations were used, respectively. Where as about 81% histamine was released by the control group. Degranulation inhibition ability was also observed in ionomycin-activated rat basophilic leukemia-2H3 cells. In contrast, friedelin failed to inhibit degranulation in either cell type. The inhibition of 8-hydroxyisocapnolactone-2-3-diol was not related to the depletion of histamine synthesis as implied by the total histamine measurement. CONCLUSIONS: These results exhibit the promising of 8-hydroxyisocapnolactone-2-3-diol is a potential parent structure for developing a MCs stabilizer.
RESUMO
Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which block the mevalonate pathway. The activity of statins not only decreases cholesterol levels but also ameliorates inflammation and modulates the immune system. In this study, we investigated the effects of simvastatin on histamine release using rat basophilic leukaemia (RBL-2H3) cells, and examined its interaction with proteins involved in the exocytosis process. Treatment with simvastatin for 24h inhibited histamine release in RBL-2H3 cells in a concentration-dependent manner after stimulation with dinitrophenylated bovine serum albumin (DNP-BSA, as an antigen), ionomycin (a calcium ion [Ca2+] ionophore), and thapsigargin (an inhibitor of Ca2+-ATPase in the endoplasmic reticulum). Simvastatin-induced inhibition was counteracted by co-administration of mevalonolactone or geranylgeraniol, but not farnesol. Indeed, several exocytotic proteins were post-translationally modified by isoprenylation, which is required for proper localization in the lipid membrane. RBL-2H3 cells express proteins involved in the fusion of granules and the plasma membrane, such as Ras-like protein in the brain 27a (Rab27a), synaptosome-associated protein 23 (SNAP23), and vesicle-associated membrane protein 7 (VAMP7), as well as Ca2+ binding proteins, such as double C2 alpha (Doc2a), synaptotagmin2, and mammalian uncoordinated13-4 (munc13-4). The interaction of Rab27a and Doc2a proteins was detected using proximity ligation assays. Antigen stimulation caused these proteins to interact, and this interaction could be disrupted by co-administration of simvastatin. In conclusion, simvastatin inhibited the mevalonate pathway, which suppressed the geranylgeranylation of Rab27a by depleting geranylgeranyl pyrophosphate and interfering with the Rab27a-Doc2a interaction. This activity resulted in the inhibition of exocytosis in RBL-2H3 cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Ácido Mevalônico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinvastatina/farmacologia , Proteínas rab27 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Exocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Liberação de Histamina/efeitos dos fármacos , Prenilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.
Assuntos
Anilidas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Artrite Reumatoide/terapia , Linfócitos B/efeitos dos fármacos , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Imunoterapia/métodos , Linfócitos T/efeitos dos fármacos , Tiadiazóis/uso terapêutico , Anilidas/efeitos adversos , Animais , Anticorpos Neutralizantes/efeitos adversos , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Células Cultivadas , Modelos Animais de Doenças , Xenoenxertos , Humanos , Hiperglicemia/etiologia , Terapia de Imunossupressão , Camundongos , Camundongos SCID , Linfócitos T/imunologia , Tiadiazóis/efeitos adversosRESUMO
Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA) patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC) channels; also known as ORAI1) may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs) of mice with collagen-induced arthritis (CIA) was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-γ, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients.
Assuntos
Artrite Experimental/terapia , Artrite Reumatoide/terapia , Terapia Genética , Proteína ORAI1/genética , Animais , Artrite Experimental/sangue , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Citocinas/sangue , Citocinas/imunologia , Inativação Gênica , Articulações/imunologia , Articulações/patologia , Lentivirus/genética , Linfonodos , Masculino , Camundongos Endogâmicos DBA , Ligante RANK/sangue , Ligante RANK/imunologia , RNA Interferente Pequeno/genética , Baço/citologia , Membrana Sinovial/citologiaRESUMO
With the aim of controlling disease relapse and bone deformation of individual joints, the application of gene therapy in rheumatoid arthritis (RA) has slowly progressed on a trial-and-error basis. Several new therapeutic targets have been identified in preclinical studies in animal models, although a limited number of gene-based clinical trials have been conducted. In this article, we summarize the status of gene therapy for RA by addressing issues related to innovating drug development. More disease- and target-specific preclinical tests are required to overcome the insufficient information regarding pharmacokinetics and toxicokinetics, which are related to safety issues in the field of RA gene therapy.
Assuntos
Artrite Reumatoide/terapia , Terapia Genética , Animais , HumanosRESUMO
Ca(2+) release-activated calcium channel 3 (CRACM3) is a unique member of the CRAC family of Ca(2+)-selective channels. In a non-excitable exocytosis model, we found that the extracellular L3 domain and the cytoplasmic C-terminus of CRACM3 interacted in an activity-dependent manner with the N-peptide of syntaxin4, a soluble N-ethylmaleimide-sensitive factor attachment receptor protein. Our biochemical, electrophysiological and single-vesicle studies showed that knockdown of CRACM3 suppressed functional exocytosis by decreasing the open time of the vesicle fusion pore without affecting Ca(2+) influx, the activity-dependent membrane capacitance (Cm) change, and the total number of fusion events. Conversely, overexpressing CRACM3 significantly impaired cell exocytosis independent of Ca(2+), led to an impaired Cm change, decreased the number of fusion events, and prolonged the dwell time of the fusion pore. CRACM3 changes the stability of the vesicle fusion pore in a manner consistent with the altered molecular expression. Our findings imply that CRACM3 plays a greater role in exocytosis than simply acting as a compensatory subunit of a Ca(2+) channel.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Exocitose/fisiologia , Proteínas Qa-SNARE/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Linhagem Celular Tumoral , Exocitose/efeitos dos fármacos , Fusão de Membrana/fisiologia , Proteínas Qa-SNARE/genética , Ratos , Vesículas Secretórias/metabolismo , Análise de Célula Única , Tapsigargina/farmacologiaRESUMO
PURPOSE: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. MATERIALS AND METHODS: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. RESULTS: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. CONCLUSIONS: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.
Assuntos
Mitocôndrias/enzimologia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Proteínas Repressoras/metabolismo , Células A549 , Trifosfato de Adenosina/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Estresse Oxidativo , ProibitinasRESUMO
PURPOSE: Epidermal growth factor receptor (EGFR) gene mutations are the most established genomic biomarkers for the efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). The most frequent deletion in exon 19 is delE746_750, followed by del747_753insS and del747_750insP. Since investigations of delE746 have not been reported previously, it is unclear if delE746 conveys sensitivity to TKI effect of TKI on EGFR delE746. The objective was to characterize delE746 of the EGFR gene and to explore the effects of TKIs on the delE746. METHODS: We assessed the ability of gefitinib to inhibit phosphorylation of clonal L929 cell lines expressing EGFR with delE746. 3-D structures of the EGFR proteins were also used to investigate the interaction with gefitinib. RESULTS: The delE746 mutant EGFR-expressing cells exhibited gefitinib-sensitive autophosphorylation, which altered the structure of the EGFR and increased the instances of docking during docking simulations of gefitinib with the EGFR-TK. This mutant revealed that it exhibited molecular conformation alterations, and more frequent binding with gefitinib compared to wild-type EGFR. We administered EGFR-TKI, gefitinib to a Japanese woman with lung cancer that contained delE746. The patient achieved partial response after a 5 month of treatment with gefitinib. CONCLUSION: Our study revealed biological, structural, and probably clinical features of the delE746 form of EGFR.
Assuntos
Receptores ErbB , Deleção de Genes , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Animais , Linhagem Celular Tumoral , Códon , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Éxons , Gefitinibe , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Camundongos , Simulação de Acoplamento Molecular , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Quinazolinas/administração & dosagem , Quinazolinas/uso terapêutico , Resultado do TratamentoRESUMO
Antihistamines inhibit histamine signaling by blocking histamine H1 receptor (H1R) or suppressing H1R signaling as inverse agonists. The H1R gene is upregulated in patients with pollinosis, and its expression level is correlated with the severity of nasal symptoms. Here, we show that antihistamine suppressed upregulation of histidine decarboxylase (HDC) mRNA expression in patients with pollinosis, and its expression level was correlated with that of H1R mRNA. Certain antihistamines, including mepyramine and diphenhydramine, suppress toluene-2,4-diisocyanate (TDI)-induced upregulation of HDC gene expression and increase HDC activity in TDI-sensitized rats. However, d-chlorpheniramine did not demonstrate any effect. The potencies of antihistamine suppressive effects on HDC mRNA elevation were different from their H1R receptor binding affinities. In TDI-sensitized rats, the potencies of antihistamine inhibitory effects on sneezing in the early phase were related to H1R binding. In contrast, the potencies of their inhibitory effects on sneezing in the late phase were correlated with those of suppressive effects on HDC mRNA elevation. Data suggest that in addition to the antihistaminic and inverse agonistic activities, certain antihistamines possess additional properties unrelated to receptor binding and alleviate nasal symptoms in the late phase by inhibiting synthesis and release of histamine by suppressing HDC gene transcription.
Assuntos
Expressão Gênica/efeitos dos fármacos , Antagonistas dos Receptores Histamínicos/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologia , Histidina Descarboxilase/genética , Histidina Descarboxilase/metabolismo , Receptores Histamínicos H1/metabolismo , Tolueno 2,4-Di-Isocianato/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Depressão Química , Relação Dose-Resposta a Droga , Histamina/fisiologia , Agonistas dos Receptores Histamínicos , Antagonistas dos Receptores Histamínicos H1 , Humanos , Hipersensibilidade/tratamento farmacológico , Hipersensibilidade/genética , Masculino , Terapia de Alvo Molecular , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The effect of reserpine on histamine (HA) and tele-methylhistamine (N(τ)-MHA) in hypothalamus and cortex of rats was analyzed and compared to catecholamines. IP injection of reserpine (5 mg/kg) confirmed the effectiveness of reserpine treatment on noradrenaline and dopamine levels. Our in-vitro experiment with synaptosomal/crude mitochondrial fraction from hypothalamus and cortex confirmed that while mono amine oxidase (MAO) is an efficient metabolic enzyme for catecholamines, HA is not significantly affected by its enzymatic action. HMT activity after reserpine, pargyline and L-histidine treatment showed no differences compared to the control values. However HDC was significantly increased in both hypothalamus and cortex. In this study, Ws/Ws rats with deficiency of mast cells were used to clarify aspects of HA metabolism in HAergic neurons by eliminating the contribution of mast cells. The irreversible MAO-B inhibitor Pargyline (65 mg/kg) failed to accumulate N(τ)-MHA in the hypothalamus. However, when animals treated with reserpine and pargyline/reserpine were compared, the last group showed higher N(τ)-MHA values (p < 0.01). Moreover, the precursor of HA, L-histidine (1 g/kg), produced an increase of HA in the hypothalamus to 166% and the cortex to 348%. In conclusion, our results suggest that the effect of reserpine on the HA pools in the brain might be different. The neuronal HA pools are more resistant to reserpine as compared to those of catecholamine. Moreover, the HAergic pool appears to be more resistant to depletion than mast cells' pool, and thus HDC/HMT activity and its localization may play a key role in the understanding of HA metabolism in brain after reserpine treatment.