RESUMEN
Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1ß and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.
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Inflamasomas , Toxoplasma , Ratones , Animales , Humanos , Femenino , Embarazo , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Piroptosis , Trofoblastos/metabolismo , Catepsina B/metabolismo , Catepsina B/farmacología , Placenta/metabolismo , ARN Interferente Pequeño , Caspasas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas NLR/metabolismoRESUMEN
Herein, three In(III)-based metal-organic frameworks (In-MOFs) with different degrees of interpenetration (DOI), namely In-MOF-1, In-MOF-2, and In-MOF-3, constructed by In3+ and Y-shaped ligands 4,4',4â³-s-triazine-2,4,6-triyltribenzoate (H3TATB), are successfully synthesized through the ionothermal/solvothermal method. Subsequently, three novel In-MOFs, including noninterpenetration polycatenation, 2-fold interpenetrated, and 4-fold interpenetrated structure, are employed as the platform for systematically investigating the separation efficiency of CO2/N2, CO2/CH4, and CO2/CH4/N2 mixture gas system. Among them, In-MOF-2 shows the highest CO2 uptake capacities at 298 K and simultaneously possesses the low adsorption enthalpy of CO2 (26.4 kJ/mol at low coverage), a feature desirable for low-energy-cost adsorbent regeneration. The CO2/N2 (v: v = 15/85) selectivity of In-MOF-2 reaches 37.6 (at 298 K and 1 bar), also revealing outstanding selective separation ability from flue gases and purifying natural gas, affording a unique robust separation material as it has moderate DOI and pore size. In-MOF-2 shows exceptional stability and feasibility to achieve reproducibility. Aperture adjustment makes In-MOF-2 a versatile platform for selectively capturing CO2 from flue gases or purifying natural gas.
RESUMEN
Oily water caused in the process of industry leads to not only the waste of resources, but also environmental pollution. Membrane separation, as a facile and efficient separation technology, has attracted widespread attention in the field of oil/water separation. The development of membrane materials with high separation performance is one of the key elements to improve separation efficiency. In this work, a superhydrophobic membrane composited with a trifluoromethyl-containing covalent organic framework (COF) is prepared, which exhibits excellent performance on separations of oil/water mixtures and water-in-oil emulsions. For different composition of oil/water mixtures, the highest flux of oil is up to 32 000 L m-2 h-1 and oil/water separation efficiency is above 99%. Moreover, the high oil/water separation efficiency remains unchanged after successive cycles. This work provides a feasible scheme for the design of high-efficiency oil/water separation membranes.
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Estructuras Metalorgánicas , Membranas , Contaminación Ambiental , Tecnología , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Adenosine is a purine nucleoside involved in numerous physiological processes; however, the role of adenosine receptors in T. gondii-induced trophoblast cell function has not been investigated until now. The goal of the present study was to evaluate the intracellular signaling pathways regulated by adenosine receptors using a HTR-8/SVneo trophoblast cell model of T. gondii infection. HTR8/SVneo human extravillous trophoblast cells were infected with or without T. gondii and then evaluated for cell morphology, intracellular proliferation of the parasite, adenosine receptor expression, TNF-α production and mitogen-activated protein (MAP) kinase signaling pathways triggered by adenosine A3 receptor (A3AR). HTR8/SVneo cells infected with T. gondii exhibited an altered cytoskeletal changes, an increased infection rate and reduced viability in an infection time-dependent manner. T. gondii significantly promoted increased TNF-α production, A3AR protein levels and p38, ERK1/2 and JNK phosphorylation compared to those observed in uninfected control cells. Moreover, the inhibition of A3AR by A3AR siRNA transfection apparently suppressed the T. gondii infection-mediated upregulation of TNF-α, A3AR production and MAPK activation. In addition, T. gondii-promoted TNF-α secretion was dramatically attenuated by pretreatment with PD098059 or SP600125. These results indicate that A3AR-mediated activation of ERK1/2 and JNK positively regulates TNF-α secretion in T. gondii-infected HTR8/SVneo cells.
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MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptor de Adenosina A3/fisiología , Toxoplasmosis/metabolismo , Trofoblastos/metabolismo , Trofoblastos/parasitología , Factor de Necrosis Tumoral alfa/metabolismo , Células Cultivadas , HumanosRESUMEN
There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.
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Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Vacunas Virales/inmunología , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Modelos Animales de Enfermedad , Fiebre Aftosa/inmunología , Fiebre Aftosa/patología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/patogenicidad , Ratones , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Porcinos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/aislamiento & purificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación , Vacunas Virales/administración & dosificación , Vacunas Virales/aislamiento & purificaciónRESUMEN
Toxoplasma gondii is an apicomplexan zoonotic protozoan parasite that infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicate a need for the development of an effective vaccine. T. gondii GRA8 is a member of the dense granules protein family and is used as a marker of acute infection. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pDsRed2-GRA8, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with the pDsRed2-GRA8 plasmid and then challenged by infection with the highly virulent GFP-RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii of this vaccine were analyzed by measuring cytokine and serum antibody titers, splenocyte proliferation assays, and the survival times of mice after challenge. Our results showed that mice immunized with pDsRed2-GRA8 demonstrated specific humoral and cellular responses, induced higher IgG antibody titers with predominant IgG2a production; increased levels of IL-10, IL-12 (p70), IFN-γ, TNF-α, and splenocyte proliferation; and prolonged survival times compared to those of control mice. The present study showed that DNA immunization with pDsRed2-GRA8 induced humoral and cellular immune responses, and all immunized mice showed greater Th1-type immune responses and longer survival times than those of control mice. These results indicated that T. gondii GRA8 DNA immunization induces a partial protective effect against acute toxoplasmosis.
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Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/prevención & control , Vacunas de ADN/inmunología , Enfermedad Aguda , Animales , Anticuerpos Antiprotozoarios/sangre , Proliferación Celular , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Bazo/citología , Bazo/inmunologíaRESUMEN
Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines, which are important for innate immunity. NLRs, i.e., nucleotide-binding oligomerization domain (NOD)-like receptors, play a crucial role as innate immune sensors and form multiprotein complexes called inflammasomes, which mediate caspase-1-dependent processing of pro-IL-1ß. To elucidate the role of inflammasome components in T. gondii-infected THP-1 macrophages, we examined inflammasome-related gene expression and mechanisms of inflammasome-regulated cytokine IL-1ß secretion. The results revealed a significant upregulation of IL-1ß after T. gondii infection. T. gondii infection also upregulated the expression of inflammasome sensors, including NLRP1, NLRP3, NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP, in a time-dependent manner. The infection also upregulated inflammasome adaptor protein ASC and caspase-1 mRNA levels. From this study, we newly found that T. gondii infection regulates NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and neuronal apoptosis inhibitor protein (NAIP) gene expressions in THP-1 macrophages and that the role of the inflammasome-related genes may be critical for mediating the innate immune responses to T. gondii infection.
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Expresión Génica , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Macrófagos/parasitología , Proteínas NLR/metabolismo , Toxoplasma/inmunología , Línea Celular , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Inflamasomas/genética , Proteínas NLR/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.
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Apoptosis , Macrófagos/parasitología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Toxoplasma/fisiología , Proteína Letal Asociada a bcl/metabolismo , Androstadienos/farmacología , Animales , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Diferenciación Celular , Línea Celular Tumoral/citología , Supervivencia Celular , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Macrófagos/citología , Ratones , Ratones Endogámicos BALB C , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Organismos Libres de Patógenos Específicos , WortmaninaRESUMEN
Aim: To investigate the anticancer mechanisms of silver nanoparticles (AgNPs) in colorectal cancer. Methods: Anticancer effects of AgNPs were determined in colorectal cancer HCT116 cells and xenograft mice using cellular and molecular methods. Results: AgNPs induced mitochondrial reactive oxygen species production, mitochondrial dysfunction and endoplasmic reticulum (ER) stress responses through NOX4 and led to HCT116 cell apoptosis. Pretreatment with DPI or 4-PBA significantly inhibited mitochondrial reactive oxygen species production, apoptosis, ER stress response, NOX4 expression and mitochondrial dysfunction in AgNP-treated HCT116 cells. AgNPs also significantly suppressed HCT116 cell-based xenograft tumor growth in nude mice by inducing apoptosis and ER stress responses. Conclusion: AgNPs exert anticancer effects against colorectal cancer via ROS- and ER stress-related mitochondrial apoptosis pathways.
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Neoplasias Colorrectales , Nanopartículas del Metal , Animales , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Neoplasias Colorrectales/tratamiento farmacológico , Estrés del Retículo Endoplásmico , Humanos , Ratones , Ratones Desnudos , Mitocondrias , NADPH Oxidasa 4 , Especies Reactivas de Oxígeno , PlataRESUMEN
BACKGROUND: Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. METHODS: FHs 74 Int cells infected with T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis. RESULTS: Under normal and T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1ß, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells. CONCLUSIONS: This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.
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Células Epiteliales/parasitología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata , Inflamasomas/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas NLR/genética , Línea Celular , Humanos , Inflamasomas/inmunología , Intestino Delgado/citología , Intestino Delgado/parasitología , Proteínas NLR/clasificación , Proteínas NLR/inmunología , ARN MensajeroRESUMEN
Silver nanoparticles (AgNPs) have cytotoxic effects on various human cell types. The endoplasmic reticulum (ER) is very sensitive to cytotoxic damage. Retina tissue is easily affected by internal and external stimuli. However, the effect of AgNPs on human retinal cells is not known. This study examined the effect of AgNPs on ER stress induction and their mechanism of action in human retinal pigment epithelium (RPE) ARPE-19 cells. We found that AgNPs significantly increased ARPE-19 cell cytotoxicity and stimulated caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage, as well as mitochondrial membrane potential (MMP) depolarization, in ARPE-19 cells in a dose-dependent manner (0.2-5 µg/mL for 18 h). AgNPs (5 µg/mL for 18 h) induced several signature ER stress markers, as indicated by the upregulated expressions of CCAAT/enhancer-binding protein-homologous protein (CHOP), phosphorylated protein kinase RNA-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), and inositol-requiring protein 1 (IRE1), and cleaved activating transcription factor 6 (ATF6). AgNPs also activated ASK1 and JNK in ARPE-19 cells, and induced increases in Bax and Puma expressions, as well as a decrease in Mcl-1 expression. However, inhibition of the ER stress response by pretreatment with 4-PBA included apparently and dose-dependently reduced levels of p-PERK, p-IRE1, CHOP, cleaved ATF6, p-ASK1, p-JNK, cleaved caspase-3, procaspase-12, and MMP depolarization in AgNP-treated ARPE-19 cells; it also led to significantly increased Mcl-1 protein levels in a dose-dependent manner in ARPE-19 cells. Pretreatment with JNK inhibitor SP600125 significantly attenuated caspase-3 cleavage and MMP depolarization and increased Mcl-1 protein levels in AgNPs-treated ARPE-19 cells in a dose-dependent manner. Hence, our study demonstrated that AgNPs induced apoptosis in human RPE ARPE-19 cells by ER stress response and ER stress-dependent mitochondrial apoptosis via the IRE1/ASK1/JNK/Mcl-1 pathways.
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Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Endorribonucleasas/metabolismo , Nanopartículas del Metal/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Plata/toxicidad , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Endorribonucleasas/genética , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Humanos , MAP Quinasa Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.
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Toxoplasma , Proliferación Celular , Humanos , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Toxoplasma/metabolismo , Factor A de Crecimiento Endotelial VascularRESUMEN
PURPOSE: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity. MATERIALS AND METHODS: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4-/ - mice. RESULTS: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed. CONCLUSION: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.
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Apoptosis/efectos de los fármacos , Nanopartículas del Metal/química , NADPH Oxidasa 4/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/parasitología , Plata/farmacología , Toxoplasmosis/patología , Animales , Autofagia/efectos de los fármacos , Línea Celular , Forma de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Fase G1/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Fosforilación/efectos de los fármacosRESUMEN
BACKGROUND: Toxoplasma gondii can invade and replicate in all nucleated cells in a wide range of host species, and infection induces IL-1ß production. IL-1ß plays central roles in the stimulation of the innate immune system and inflammation. However, little is known of the innate immune responses in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection. METHODS: FHs 74 Int cells were infected with the T. gondii GFP-RH strain. Then, IL-1ß production and its mechanisms of action were evaluated using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection. RESULTS: Infection of FHs 74 Int cells by T. gondii triggered significant time- and dose-dependent IL-1ß production. Although T. gondii activated NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in FHs 74 Int cells, NLRP3 levels were consistently and significantly time-dependently increased, while the other inflammasomes were not. Transfection with siRNA targeting NLRP3, cleaved caspase-1 (Casp-1) or ASC significantly reduced T. gondii-induced IL-1ß production, whereas T. gondii proliferation was markedly increased. Toxoplasma gondii infection activated P2X7 receptor (P2X7R) levels in FHs 74 Int cells in a time-dependent manner; however, transfection with siRNA targeting P2X7R significantly reduced T. gondii-induced IL-1ß secretion and substantially increased T. gondii proliferation, which is mediated by decreased protein expression levels of NLRP3, cleaved Casp-1 and ASC. Collectively, NLRP3-dependent IL-1ß secretion is mediated by P2X7R in small intestinal epithelial cells in response to T. gondii infection, thereby controlling parasite proliferation. CONCLUSIONS: This study revealed that the P2X7R/NLRP3 pathway plays important roles in IL-1ß secretion and inhibition of T. gondii proliferation in small intestinal epithelial cells. These results not only contribute to our understanding of the mucosal immune mechanisms of T. gondii infection but also offer new insight into the identification of innate resistance in the gut epithelium.
Asunto(s)
Células Epiteliales/parasitología , Interacciones Huésped-Patógeno , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Western Blotting , Línea Celular , Supervivencia Celular , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/fisiología , Técnica del Anticuerpo Fluorescente , Formazáns/análisis , Silenciador del Gen , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Sales de Tetrazolio/análisisRESUMEN
Autophagy and apoptosis are critical for controlling Toxoplasma gondii (T. gondii) infection. T. gondii infection during pregnancy can damage the fetus and cause birth defects; however, the molecular mechanisms of this process are poorly understood. This study aims to determine the activities of autophagy and apoptosis as well as their regulatory mechanisms during T. gondii infection by using human umbilical cord mesenchymal stem cells (hUC-MSCs) as a model of congenital diseases. LC3B, a hallmark protein of autophagy was incrementally upregulated with the infection duration, whereas p62 was downregulated in T. gondii-infected hUC-MSCs. Concurrent to this result, the invasion of T. gondii into hUC-MSCs increased in a time-dependent manner. The expression levels of Bcl-2 family proteins including Bcl-2, Bcl-xL, Bim, Bax, Bid and Bak were not altered; however, Mcl-1 levels in hUC-MSCs were dramatically decreased upon T. gondii infection. In addition, at 24 h post-infection, cleaved PARP and cleaved caspase-3 protein levels were elevated in hUC-MSCs. Importantly, Mcl-1 overexpression reduced the levels of autophagy- and apoptosis-related proteins in T. gondii-infected hUC-MSCs. Mcl-1 proteins were primarily expressed in the fraction containing mitochondria and strongly interacted with Beclin-1 under normal conditions; however, these interactions were remarkably attenuated by T. gondii infection. These results suggest that mitochondrial Mcl-1 is an essential signaling mediator regulating the activation of autophagy and apoptosis during T. gondii infection.
Asunto(s)
Apoptosis , Autofagia , Regulación hacia Abajo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Toxoplasma/fisiología , Cordón Umbilical/citología , Beclina-1 , Supervivencia Celular , Humanos , Mitocondrias/metabolismo , Modelos Biológicos , Unión Proteica , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 µM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.
Asunto(s)
Interleucina-12/biosíntesis , Interleucina-23/biosíntesis , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Toxoplasma , Toxoplasmosis/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Toxoplasmosis/patología , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.
RESUMEN
Foot-and-mouth disease (FMD) is a highly contagious infectious disease, and the use of vaccines is known to be effective for its prevention. In 2010/2011, there was an epidemic of the South East Asia (SEA) topotype in East Asian countries. We adapted the SEA topotype virus isolated in November 2010 in Korea in cells to analyze the characteristics of the virus and evaluate its possibility as a vaccine. After cell culture adaptation, the FMD virus particle 146S was purified to develop an inactivated oil vaccine for SEA or other topotypes. To measure its immunogenicity, pigs were inoculated with the experimental vaccine at different concentrations of the antigen. The results indicated that the groups immunized with at least 7.5 µg antigen were protected from homologous challenge. The immunized pigs were also protected against heterologous virus (ME-SA topotype) challenge. The genetic variations between the two field isolates and the adapted vaccine strains were identified in six amino acids by complete genome sequencing.
Asunto(s)
Fiebre Aftosa/prevención & control , Enfermedades de los Porcinos/prevención & control , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Asia Sudoriental/epidemiología , Fiebre Aftosa/epidemiología , Virus de la Fiebre Aftosa/clasificación , Virus de la Fiebre Aftosa/genética , Genoma Viral , Sus scrofa/inmunología , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/virología , Vacunas de Productos Inactivados/inmunologíaRESUMEN
A recombinant infectious bovine enterovirus (BEV) vector was constructed to express a foot-and-mouth disease virus (FMDV) capsid protein (VP1) epitope. Sequences encoding the VP1 epitope (amino acid residues 141-160) of FMDV (vaccine strain O1/Manisa/Turkey/69) were inserted into pBLUBEV at the VP1/2A junction. The growth characteristics of the parental virus and viruses derived from recombinant plasmids (pBLUBEV, pBLUBEV-Manisa-epi) were determined by plaque assay and one-step growth curve analysis. There were no significant differences in the growth kinetics and plaque morphologies between transfectant viruses and their parental virus. The expressed VP1 epitope was detected successfully by using indirect immunofluorescence assay with a polyclonal antibody against the FMDV VP1 epitope from Madin Darby bovine kidney (MDBK) cells infected with BEV-Manisa-epi transfectant virus. This study demonstrated a novel alternative live viral vector that may be utilized as a candidate vaccine vector for veterinary applications.
Asunto(s)
Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Enterovirus Bovino/genética , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Virus de la Fiebre Aftosa/genética , Animales , Anticuerpos Antivirales/inmunología , Bovinos , Perros , Enterovirus Bovino/crecimiento & desarrollo , Epítopos/genética , Epítopos/inmunología , Fiebre Aftosa/genética , Fiebre Aftosa/inmunología , Fiebre Aftosa/prevención & control , Virus de la Fiebre Aftosa/inmunología , Vectores Genéticos , Células de Riñón Canino Madin Darby , ARN Viral/genética , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
Toxoplasma gondii is distributed worldwide and infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicates the need for the development of a vaccine. To evaluate the protective efficacy of a multiantigenic DNA vaccine expressing GRA7 and ROP1 of T. gondii with or without a plasmid encoding murine interleukin-12 (pIL12), we constructed DNA vaccines using the eukaryotic plasmids pGRA7, pROP1, and pGRA7-ROP1. Mice immunized with pGRA7, pROP1, or pGRA7-ROP1 showed significantly increased serum IgG2a titers; production of gamma interferon (IFN-γ), IL-10, and tumor necrosis factor alpha (TNF-α); in vitro T cell proliferation; and survival, as well as decreased cyst burdens in the brain, compared to mice immunized with either the empty plasmid, pIL12, or vector with pIL12 (vector+pIL12). Moreover, mice immunized with the multiantigenic DNA vaccine pGRA7-ROP1 had higher IgG2a titers, production of IFN-γ and TNF-α, survival time, and cyst reduction rate compared to those of mice vaccinated with either pGRA7 or pROP1 alone. Furthermore, mice immunized with either a pGRA7-ROP1+pIL12 or a single-gene vaccine combined with pIL12 showed greater Th1 immune response and protective efficacy than the single-gene-vaccinated groups. Our data suggest that the multiantigenic DNA antigen pGRA7-ROP1 was more effective in stimulating host protective immune responses than separately injected single antigens, and that IL-12 serves as a good DNA adjuvant.