RESUMEN
Designing a nanofluidic membrane with high selectivity and fast ion transport property is the key towards high-performance osmotic energy conversion. However, most of reported membranes can produce power density less than commercial benchmark (5â W/m2), due to the imbalance between ion selectivity and permeability. Here, we report a novel nanoarchitectured design of a heterogeneous membrane with an ultrathin and dense zirconium-based UiO-66-NH2 metal-organic framework (MOF) layer and a highly aligned and interconnected branched alumina nanochannel membrane. The design leads to a continuous trilayered pore structure of large geometry gradient in the sequence from angstrom-scale to nano-scale to sub-microscale, which enables the enhanced directional ion transport, and the angstrom-sized (~6.6-7â Å) UiO-66-NH2 windows render the membrane with high ion selectivity. Consequently, the novel heterogeneous membrane can achieve a high-performance power of ~8â W/m2 by mixing synthetic seawater and river water. The power density can be largely upgraded to an ultrahigh ~17.1â W/m2 along with ~48.5 % conversion efficiency at a 50-fold KCl gradient. This work not only presents a new membrane design approach but also showcases the great potential of employing the zirconium-based MOF channels as ion-channel-mimetic membranes for highly efficient blue energy harvesting.
RESUMEN
Elizabethkingia anophelis, a nonfermenting Gram-negative bacterium, causes life-threatening health care-associated infections. E. anophelis harbors multidrug resistance (MDR) genes and is intrinsically resistant to various classes of antibiotics. Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. OMVs specialize and tailor their functions by carrying different components to challenging environments and allowing communication with other microorganisms or hosts. In this study, we sought to understand the characteristics of E. anophelis OMVs under different antibiotic stress conditions. An extensively drug-resistant clinical isolate, E. anophelis C08, was exposed to multiple antibiotics in vitro, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Protein functionality analysis showed that the OMVs were predominantly involved in metabolism, survival, defense, and antibiotic resistance processes, such as the Rag/Sus family, the chaperonin GroEL, prenyltransferase, and an HmuY family protein. Additionally, a protein-protein interaction network demonstrated that OMVs from imipenem-treated E. anophelis showed significant enrichments in the outer membrane, adenyl nucleotide binding, serine-type peptidase activity, the glycosyl compound metabolic process, and cation binding proteins. Collectively, the OMV proteome expression profile indicates that the role of OMVs is immunologically relevant and related to bacterial survival in antibiotic stress environments rather than representing a resistance point. IMPORTANCE Elizabethkingia anophelis is a bacterium often associated with nosocomial infection. This study demonstrated that imipenem-induced E. anophelis outer membrane vesicles (OMVs) are immunologically relevant and crucial for bacterial survival under antibiotic stress conditions rather than being a source of antibiotic resistance. Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by E. anophelis, especially under antibiotic stress. Our findings provide important insights into clinical antibiotic stewardship.
Asunto(s)
Flavobacteriaceae , Proteómica , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , ImipenemRESUMEN
T cells secrete several inflammatory cytokines that play a critical role in the progression of atherosclerosis. Although green tea epigallocatechin-3-gallate (EGCG) exerts anti-inflammatory and anti-atherosclerotic effects in animals, few studies have identified the mechanism underlying these effects in human primary T cells. This study investigated the pathway involved in EGCG modulation of cytokine secretion in activated human primary T cells. We pre-treated human primary T cells with EGCG (0.1, 1, 5, 10, and 20 µM) for 4 h and incubated them with or without phorbol 12-myristate 13-acetate and ionomycin (P/I) for 20 h. The cytokine production, activator protein (AP)-1 binding activity, and level of mitogen-activated protein kinase (MAPK) were assessed using enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, and Western blotting, respectively. At 10 and 20 µM, EGCG decreased interleukin (IL)-2 levels by 26.0% and 38.8%, IL-4 levels by 41.5% and 55.9%, INF-γ levels by 31.3% and 34.7%, and tumor-necrosis factor (TNF)-α levels by 23.0% and 37.6%, respectively. In addition, the level of phosphorylated c-Jun N-terminal (p-JNK) and extracellular signal-regulated kinase (p-ERK) was decreased, but not the level of p-p38 MAPK. EGCG did not alter any of the total protein amounts, suggesting a selective effect on specific types of MAPKs in stimulated human T cells. EGCG tended to inactivate AP-1 DNA-binding activity. The P/I-induced production of IL-2, IL-4, INF-γ, and TNF-α by human T cells was suppressed by AP-1 inhibitor in a concentration-dependent manner. In conclusion, EGCG suppressed cytokine secretion in activated human primary T cells, and this effect was likely mediated by AP-1 inactivation through the ERK and JNK, but not p38 MAPK, pathways. These results may be related to the mechanisms through which EGCG inhibits immune- or inflammation-related atherogenesis.
Asunto(s)
Catequina/análogos & derivados , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Antiinflamatorios/inmunología , Antiinflamatorios/farmacología , Catequina/inmunología , Catequina/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Activación de Linfocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Antimicrobial-resistant (AMR) bacterial infections, including those caused by Acinetobacter baumannii, have emerged as a clinical crisis worldwide. Immunization with AMR determinants has been suggested as a novel approach to combat AMR bacteria, but has not been validated. The present study targeted tigecycline (TGC) resistance determinants in A. baumannii to test the feasibility of this approach. Using bioinformatic tools, four candidates, AdeA, AdeI, AdeK, and TolC, belonging to the resistance-nodulation-division (RND) efflux pump were identified as highly conserved and exposed antigens from 15 A. baumannii genomes. Antisera generated from recombinant proteins showed the capability to reserve Hoechst 33342, a substrate of the efflux pump, in bacterial cells. The rTolC antisera had the highest complement-dependent killing and opsonophagocytosis effect compared to the sera from phosphate-buffered saline immunized mice. Among the antisera, anti-rAdeK-specific antisera decreased the minimal inhibitory concentration of TGC in 26.7% of the tested isolates. Immunization with rAdeK significantly potentiated TGC efficacy in treating TGC-resistant A. baumannii pneumonia in the murine model. The bacterial load (7.5 × 105 vs. 3.8 × 107, p < 0.01) and neutrophil infiltration in the peri-bronchial vasculature region of immunized mice was significantly lower compared to the PBS-immunized mice when TGC was administrated concomitantly. Collectively, these results suggest that active immunization against resistance determinants might be a feasible approach to combat multidrug-resistant pathogens in high risk population.
RESUMEN
Carvedilol (Cav), a nonselective ß-blocker with α1 adrenoceptor blocking effect, has been used as a standard therapy for coronary artery disease. This study investigated the effects of Cav on exosome expression and function, ATP-binding cassette transporter A1 (ABCA1) expression, and cholesterol efflux that are relevant to the process of atherosclerosis. Human monocytic (THP-1) cell line and human hepatic (Huh-7) cells were treated with Cav, and cholesterol efflux was measured. Exosomes from cell culture medium or mice serum were isolated using glycan-coated recognition beads. Low-density lipoprotein receptor knockout (ldlr-/-) mice were fed with high-fat diet and treated with Cav. Cav accentuated cholesterol efflux and enhanced the expressions of ABCA1 protein and mRNA in both THP-1 and Huh-7 cells. In addition, Cav increased expression and function of exosomal ABCA1 in THP-1 macrophage exosomes. The mechanisms were associated with inhibition of nuclear factor-κB (NF-κB) and protein kinase B (Akt). In hypercholesterolemic ldlr-/- mice, Cav enhanced serum exosomal ABCA1 expression and suppressed atherosclerosis by inhibiting lipid deposition and macrophage accumulation. Cav halts atherosclerosis by enhancing cholesterol efflux and increasing ABCA1 expression in macrophages and in exosomes, possibly through NF-κB and Akt signaling, which provides mechanistic insights regarding the beneficial effects of Cav on atherosclerotic cardiovascular disease.
Asunto(s)
Antihipertensivos/farmacología , Aterosclerosis/tratamiento farmacológico , Carvedilol/farmacología , Colesterol/metabolismo , Exosomas/metabolismo , Receptores de LDL/fisiología , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Transporte Biológico , Dieta Alta en Grasa/efectos adversos , Exosomas/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células THP-1RESUMEN
Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas del Sistema Complemento/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Edema Pulmonar/inmunología , Edema Pulmonar/microbiología , Síndrome de Dificultad Respiratoria/microbiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/enzimología , Células Epiteliales Alveolares/enzimología , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/microbiología , Animales , Proteínas Bacterianas/genética , Antígenos CD55/inmunología , Antígenos CD59/inmunología , Citocinas/metabolismo , Glicosilfosfatidilinositoles/inmunología , Glicosilfosfatidilinositoles/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos BALB C , Fosfoinositido Fosfolipasa C/genética , Edema Pulmonar/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
BACKGROUND: Streptococcus pneumoniae is a respiratory pathogen causing severe lung infection that may lead to complications such as bacteremia. Current polysaccharide vaccines have limited serotype coverage and therefore cannot provide maximal and long-term protection. Global efforts are being made to develop a conserved protein vaccine candidate. PrtA, a pneumococcal surface protein, was identified by screening a pneumococcal genomic expression library using convalescent patient serum. The prtA gene is prevalent and conserved among S. pneumoniae strains. Its protective efficacy, however, has not been described. Mucosal immunization could sensitize both local and systemic immunity, which would be an ideal scenario for preventing S. pneumoniae infection. METHODS: We immunized BALB/c mice intranasally with a combination of a PrtA fragment (amino acids 144-1041) and Th17 potentiated adjuvant, curdlan. We then measured the T-cell and antibody responses. The protective efficacy conferred to the immunized mice was further evaluated using a murine model of acute pneumococcal pneumonia and pneumococcal bacteremia. RESULTS: There was a profound antigen-specific IL-17A and IFN-γ response in PrtA-immunized mice compared with that of adjuvant control group. Even though PrtA-specific IgG and IgA titer in sera was elevated in immunized mice, only a moderate IgA response was observed in the bronchoalveolar lavage fluid. The PrtA-immunized antisera facilitated the activated murine macrophage, RAW264.7, to opsonophagocytose S. pneumoniae D39 strain; however, PrtA-specific immunoglobulins bound to pneumococcal surfaces with a limited potency. Finally, PrtA-induced immune reactions failed to protect mice against S. pneumoniae-induced acute pneumonia and bacterial propagation through the blood. CONCLUSIONS: Immunization with recombinant PrtA combined with curdlan produced antigen-specific antibodies and elicited IL-17A response. However, it failed to protect the mice against S. pneumoniae-induced infection.
Asunto(s)
Proteínas Bacterianas/administración & dosificación , Inmunización/métodos , Vacunas Neumococicas/administración & dosificación , Neumonía Neumocócica/prevención & control , Streptococcus pneumoniae , Animales , Proteínas Bacterianas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Fagocitosis/efectos de los fármacos , Fagocitosis/fisiología , Vacunas Neumococicas/inmunología , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/metabolismo , Células RAW 264.7 , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/inmunologíaRESUMEN
Atherosclerosis is a process of imbalanced lipid metabolism in the vascular walls. The underlying pathology mainly involves the deposition of oxidized lipids in the endothelium and the accumulation of cholesterol in macrophages. Macrophages export excessive cholesterol (cholesterol efflux) through ATP-binding cassette transporter A1 (ABCA1) to counter the progression of atherosclerosis. We synthesized novel chalcone derivatives and assessed their effects and the underlying mechanisms on ABCA1 expression in macrophages. Human THP-1 macrophages were treated with synthetic chalcone derivatives for 24 h. In Western blot and flow cytometry analyses, a chalcone derivative, (E)-1-(3,4-diisopropoxyphenyl)-3-(4-isopropoxy-3-methoxyphenyl)prop- 2-en-1-one (1m), was observed to significantly enhance ABCA1 protein expression in THP-1 cells (10 µM, 24 h). Levels of mRNA of ABCA1 and liver X receptor alpha (LXRα) were quantified using a real-time quantitative polymerase chain reaction technique and were found to be significantly increased after treatment with the novel chalcone derivative 1m. Several microRNAs, including miR155, miR758, miR10b, miR145, miR33, and miR106b, which functionally inhibit ABCA1 expression were suppressed after treatment with 1m. Collectively, 1m increases ABCA1 expression in human THP-1 macrophages. The mechanisms involve the activation of the LXRα-ABCA1 pathway and suppression of certain microRNAs that regulate ABCA1 expression.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Chalconas/síntesis química , Chalconas/farmacología , Macrófagos/citología , Regulación hacia Arriba , Chalconas/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores X del Hígado/genética , Macrófagos/efectos de los fármacos , MicroARNs/genética , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Células THP-1RESUMEN
BACKGROUND/AIMS: Hyperlipidemia induces dysfunction in the smooth muscle cells (SMCs) of the blood vessels, and the vascular remodeling that ensues is a key proatherogenic factor contributing to cardiovascular events. Chemokines and chemokine receptors play crucial roles in vascular remodeling. Here, we examined whether the hyperlipidemia-derived chemokine CCL5 and its receptor CCR5 influence vascular SMC proliferation, phenotypic switching, and explored the underlying mechanisms. METHODS: Thoracoabdominal aorta were isolated from wild-type, CCL5 and CCR5 double-knockout mice (CCL5-/-CCR5-/-) fed a high-fat diet (HFD) for 12 weeks. Expression of the contractile, synthetic, and proliferation markers were assayed using immunohistochemical and western blotting. The effects of CCL5 and palmitic acid on cultured SMC proliferation and phenotypic modulation were evaluated using flow cytometry, bromodeoxyuridine (BrdU), and western blotting. RESULTS: Wild-type mice fed an HFD showed markedly increased total cholesterol, triglyceride, and CCL5 serum levels, as well as significantly increased CCL5 and CCR5 expression in the thoracoabdominal aorta vs. normal-diet-fed controls. HFD-fed CCL5-/-CCR5-/- mice showed significantly decreased expression of the synthetic phenotype marker osteopontin and the proliferation marker proliferating cell nuclear antigen, and increased expression of the contractile phenotype marker smooth muscle α-actin in the thoracoabdominal aorta vs. wild-type HFD-fed mice. Human aorta-derived SMCs stimulated with palmitic acid showed significantly increased expression of CCL5, CCR5, and synthetic phenotype markers, as well as increased proliferation. CCL5-treated SMCs showed increased cell cycle regulatory protein expression, paralleling increased synthetic and decreased contractile phenotype marker expression. Inhibition of CCR5 activity by the specific antagonist maraviroc or its expression using small interfering RNA significantly inhibited human aortic SMC proliferation and synthetic phenotype formation. Therefore, CCL5 induces SMC proliferation and phenotypic switching from a contractile to synthetic phenotype via CCR5. CCL5-mediated SMC stimulation activated ERK1/2, Akt/p70S6K, p38 MAPK, and NF-κB signaling. NF-κB inhibition significantly reduced CCR5 expression along with CCR5-induced SMC proliferation and synthetic phenotype formation. CONCLUSIONS: Hyperlipidemia-induced CCL5/CCR5 axis activation serves as a pivotal mediator of vascular remodeling, indicating that CCL5 and CCR5 are key chemokine-related factors in atherogenesis. SMC proliferation and synthetic phenotype transformation attenuation by CCR5 pharmacological inhibition may offer a new approach to treatment or prevention of atherosclerotic diseases associated with hyperlipidemia.
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Proliferación Celular , Quimiocina CCL5/genética , Receptores CCR5/genética , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Línea Celular , Quimiocina CCL5/antagonistas & inhibidores , Quimiocina CCL5/metabolismo , Dieta Alta en Grasa , Humanos , Lípidos/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Osteopontina/metabolismo , Fenotipo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores CCR5/metabolismoRESUMEN
Foam cells are formed when macrophages imbibe low-density lipoprotein (LDL) through scavenger receptors. Here we examined how epigallocatechin-3-gallate (EGCG) influences foam cell formation. We found that EGCG dose-dependently reduced oxidized LDL (oxLDL) uptake in THP-1 (10 µM, 20.0 ± 0.50, p < 0.05) and primary macrophages (134.6 ± 15.6, p < 0.05) and reduced intracellular cholesterol content in these cells, respectively (10 µM, 32.6 ± 0.14, p < 0.05; 31.7 ± 1.26, p < 0.05). EGCG treatment decreased scavenger receptor A expression, but not the expression of CD36 or of reverse cholesterol transporters. Moreover, EGCG stimulated translocation of the p50 and p65 subunits of NF-κB and enhanced NF-κB DNA-binding activity, thus suppressing SR-A promoter activity. EGCG's suppression of SR-A expression was blocked by the NF-κB inhibitor Bay. The present findings suggest that EGCG regulates NF-κB activity and thus suppresses SR-A expression, oxLDL uptake, and foam cell formation.
Asunto(s)
Catequina/análogos & derivados , Células Espumosas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptores Depuradores/genética , Catequina/farmacología , Línea Celular , Colesterol/metabolismo , Células Espumosas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Receptores Depuradores/metabolismoRESUMEN
Glycerophosphodiester phosphodiesterase (GlpQ) metabolizes glycerophosphorylcholine from the lung epithelium to produce free choline, which is transformed into phosphorylcholine and presented on the surfaces of many respiratory pathogens. Two orthologs of glpQ genes are found in Streptococcus pneumoniae: glpQ, with a membrane motif, is widespread in pneumococci, whereas glpQ2, which shares high similarity with glpQ in Haemophilus influenzae and Mycoplasma pneumoniae, is present only in S. pneumoniae serotype 3, 6B, 19A, and 19F strains. Recently, serotype 19A has emerged as an epidemiological etiology associated with invasive pneumococcal diseases. Thus, we investigated the pathophysiological role of glpQ2 in a serotype 19A sequence type 320 (19AST320) strain, which was the prevalent sequence type in 19A associated with severe pneumonia and invasive pneumococcal disease in pediatric patients. Mutations in glpQ2 reduced phosphorylcholine expression and the anchorage of choline-binding proteins to the pneumococcal surface during the exponential phase, where the mutants exhibited reduced autolysis and lower natural transformation abilities than the parent strain. The deletion of glpQ2 also decreased the adherence and cytotoxicity to human lung epithelial cell lines, whereas these functions were indistinguishable from those of the wild type in complementation strains. In a murine respiratory tract infection model, glpQ2 was important for nasopharynx and lung colonization. Furthermore, infection with a glpQ2 mutant decreased the severity of pneumonia compared with the parent strain, and glpQ2 gene complementation restored the inflammation level. Therefore, glpQ2 enhances surface phosphorylcholine expression in S. pneumoniae 19AST320 during the exponential phase, which contributes to the severity of pneumonia by promoting adherence and host cell cytotoxicity.
Asunto(s)
Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Hidrolasas Diéster Fosfóricas/genética , Infecciones Neumocócicas/inmunología , Infecciones del Sistema Respiratorio/inmunología , Streptococcus pneumoniae/patogenicidad , Secuencia de Aminoácidos , Animales , Autólisis/genética , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Expresión Génica/genética , Humanos , Pulmón/citología , Ratones , Ratones Endogámicos BALB C , Fosforilcolina/metabolismo , Infecciones Neumocócicas/microbiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/patología , Alineación de Secuencia , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/inmunologíaRESUMEN
BACKGROUND: Klebsiella pneumoniae has emerged worldwide as a cause of pyogenic liver abscess (PLA) often complicated by meningitis and endophthalmitis. Early detection of this infectious disease will improve its clinical outcome. Therefore, we tried to isolate immunodominant proteins secreted by K. pneumoniae strains causing PLA. RESULTS: The secreted proteins of the NTUH-K2044 strain were separated by two-dimensional electrophoresis and then immunoblotted using convalescent sera from patients with K. pneumoniae PLA. A ~30-kDa immunodominant protein was then identified. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed an open reading frame (KP1_p307) located on the pK2044 plasmid and bioinformatic analysis identified this protein as a signal peptide of unknown function. The KP1_p307 gene was more prevalent in PLA strains and capsular type K1/K2 strains, but disruption of this gene in NTUH-K2044 strain did not decrease virulence in mice. Ten of fourteen (71%) sera from patients with K. pneumoniae PLA were immunoreactive with the recombinant KP1_p307 protein. Seroconversion demonstrated by a rise in serum titer in serial serum samples confirmed that antibodies against the KP1_p307 protein were elicited after infection. CONCLUSIONS: The KP1_p307 protein could be used as an antigen for early serodiagnosis of K. pneumoniae PLA, particularly in K1/K2 PLA strains.
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Proteínas Bacterianas/metabolismo , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/patogenicidad , Absceso Piógeno Hepático/microbiología , Factores de Virulencia/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Cromatografía Liquida , Biología Computacional , Modelos Animales de Enfermedad , Diagnóstico Precoz , Electroforesis en Gel Bidimensional , Femenino , Eliminación de Gen , Humanos , Immunoblotting , Infecciones por Klebsiella/diagnóstico , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/patología , Absceso Piógeno Hepático/diagnóstico , Absceso Piógeno Hepático/inmunología , Absceso Piógeno Hepático/patología , Ratones Endogámicos BALB C , Peso Molecular , Sistemas de Lectura Abierta , Pruebas Serológicas/métodos , Espectrometría de Masas en Tándem , Virulencia , Factores de Virulencia/química , Factores de Virulencia/inmunología , Factores de Virulencia/aislamiento & purificaciónRESUMEN
Ptpn22 encodes PEST domain-enriched tyrosine phosphatase (Pep), which negatively regulates TCR proximal signaling and is strongly associated with a variety of autoimmune diseases in humans. The net effect of Pep on the balance of immunity and tolerance is uncertain because of the simultaneous inhibition of TCR-mediated signaling of effector and regulatory T cells (T(regs)). In this study, we generated transgenic NOD mice that overexpressed Pep in T cells. The transgenic mice had a significantly lower incidence of spontaneous autoimmune diabetes, which was accompanied by fewer IFN-γ-producing T cells, and an increased ratio of CD4(+)Foxp3(+) T(regs)to CD4(+)IFN-γ(+) or to CD8(+)IFN-γ(+) T cells, respectively, in pancreatic islets. Transgenic T cells showed markedly decreased TCR-mediated effector cell responses such as proliferation and Th1 differentiation. By contrast, the inhibitory effect of transgenic Pep on TCR signaling did not affect the differentiation of T(regs) or their suppressive activity. Adoptive transfer experiments showed that transgenic splenocytes exhibited attenuated diabetogenic ability. To examine further the pathogenic features of transgenic T cells, we generated Ptpn22/BDC2.5 doubly transgenic mice and found reduced proliferation and Th1 differentiation in CD4(+) T lymphocytes with additional Pep in pancreatic lymph nodes but not in inguinal lymph nodes of NOD/SCID recipients. This finding indicates that transgenic Pep attenuates T cell functions in an islet Ag-driven manner. Taken together, our results demonstrate that Pep overexpression in T cells attenuates autoimmune diabetes in NOD mice by preferentially modulating TCR signaling-mediated functions in diabetogenic T cells but not in T(regs).
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Diabetes Mellitus Tipo 1/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Tipo 1/epidemiología , Femenino , Factores de Transcripción Forkhead/biosíntesis , Genotipo , Incidencia , Interferón gamma/biosíntesis , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genética , Transducción de Señal , Linfocitos T Reguladores/metabolismoRESUMEN
High levels of reactive oxygen species (ROS), systemic T cell activation, and macrophage infiltration in the kidney are implicated in the acceleration and progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis. However, the pathogenic mechanism of IgAN is still little understood, and it remains a challenge to establish a specific therapeutic strategy for this type of glomerular disorder. Recently, we showed that antroquinonol (Antroq), a pure active compound from Antrodia camphorata mycelium, inhibits renal inflammation and reduces oxidative stress in a mouse model of renal fibrosis. But the anti-inflammatory and immune-regulatory effects of Antroq on the acceleration and progression of primary glomerular disorders have not been determined. In this study, we show that Antroq administration substantially impeded the development of severe renal lesions, such as intense glomerular proliferation, crescents, sclerosis, and periglomerular interstitial inflammation, in mice with induced accelerated and progressive IgAN (AcP-IgAN). Further mechanistic analysis in AcP-IgAN mice showed that, early in the developmental stage of the AcP-IgAN model, Antroq promoted the Nrf2 antioxidant pathway and inhibited the activation of T cells and NLRP3 inflammasome. Significantly improved proteinuria/renal function and histopathology in AcP-IgAN mice of an established stage supported potential therapeutic effects of Antroq on the disease. In addition, Antroq was shown to inhibit activation of NLRP3 inflammasome in vitro by an IgA immune complex (IC) partly involving a reduced ROS production in IgA-IC-primed macrophages, and this finding may be helpful in the understanding of the mode of action of Antroq in the treated AcP-IgAN mice.
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Proteínas Portadoras/antagonistas & inhibidores , Glomerulonefritis por IGA/tratamiento farmacológico , Inflamasomas/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/fisiología , Linfocitos T/efectos de los fármacos , Ubiquinona/análogos & derivados , Animales , Citocinas/sangre , Inmunidad Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , FN-kappa B/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR , Estrés Oxidativo/efectos de los fármacos , Proteinuria/prevención & control , Ubiquinona/farmacología , Ubiquinona/uso terapéuticoRESUMEN
Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease with complex immunological and clinical manifestations. Multiple organ failure in SLE can be caused by immune dysfunction and deposition of autoantibodies. Studies of SLE-susceptible loci and the cellular and humoral immune responses reveal variable aberrations associated with this systemic disease. Invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that control peripheral tolerance. Mounting evidence showing reductions in the proportion and activity of iNKT cells in SLE patients suggests the suppressive role of iNKT cells. Studies using murine lupus models demonstrate that iNKT cells participate in SLE progression by sensing apoptotic cells, regulating immunoglobulin production, and altering the cytokine profile upon activation. However, the dichotomy of iNKT cell actions in murine models implies complicated interactions within the body's milieu. Therefore, application of potential therapy for SLE using glycolipids to regulate iNKT cells should be undertaken cautiously.
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Lupus Eritematoso Sistémico/inmunología , Células T Asesinas Naturales/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Lupus Eritematoso Sistémico/metabolismo , Ratones , Células T Asesinas Naturales/metabolismoRESUMEN
Type 1 diabetes is an autoimmune disorder resulting from lymphocyte-mediated destruction of insulin-producing ß cells in pancreas. Natural killer T cells are regulatory immune components controlling autoreactivity and immune homeostasis. Although early studies supported that amelioration of autoimmune diabetes by natural killer T cells was associated with Th1/2 shift, other Th2-independent regulatory mechanisms were also suggested. Since natural killer T cells are critical for the generation of CD8(+) regulatory T cells controlling anterior chamber-associated immune deviation and CD8(+) regulatory T cells also participate in suppression of immune responses like experimental autoimmune encephalomyelitis, we investigate whether the similar suppressive effects are involved in α-galactosylceramide-induced immune tolerance in non-obese diabetic mice. We demonstrate that repeated exposure of α-galactosylceramide reveals a hyporesponsiveness of total or antigen-presenting cells-depleted splenocytes upon anti-CD3/28 antibodies stimulation. The dispensability of dendritic cells in the hyporesponsiveness is consistent with the comparable expression of costimulatory molecules on CD11c(+) subsets between α-galactosylceramide- and vehicle-treated mice. α-Galactosylceramide treatment not only affects the effector T cell subsets and their cytokine production but also increases the secretion of transforming growth factor-ß by splenocytes, implying the suppressive regulation. The adoptive transfer experiments demonstrate the suppressive effect of T cells from α-galactosylceramide-treated non-obese diabetic mice when co-transferred with vehicle-treated littermates. Finally, it reveals that CD8(+) subset among antigen-presenting cells-depleted splenocytes tends to confer the suppression since the protective ability vanishes upon withdrawal of CD8(+) subset. These results suggest that repeated exposure of α-galactosylceramide ameliorates autoimmune diabetes in non-obese diabetic mice mediated by CD8(+) T cell-associated suppression.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Diabetes Mellitus Tipo 1/inmunología , Galactosilceramidas/farmacología , Animales , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/patología , Femenino , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Páncreas/efectos de los fármacos , Páncreas/inmunología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
Triptolide, a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii, exerts an antitumor effect in KB cancer cells through the induction of apoptosis. In this study, we show that triptolide possesses an anticancer effect on drug-sensitive parental KB cells and multidrug-resistant KB-7D and KB-tax cells that overexpress multidrug resistance protein and MDR, respectively. Our data revealed that triptolide decreases the expression of multidrug resistance protein and MDR in both KB-7D and KB-tax cells. It also induces apoptosis in these multidrug-resistant cancer cells by activating caspase-3, and decreasing Mcl-1 and XIAP. Triptolide not only inhibits tumor growth but also induces apoptosis of these drug-resistant cancer cells in xenograft mouse models. Moreover, we also show that triptolide combined with 5-fluorouracil could be an alternative strategy for chemotherapy enhancement. These results indicate the therapeutic value of triptolide on multidrug-resistant cells, and when combined with 5-fluorouracil for the enhancement of cancer therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Diterpenos/farmacología , Fluorouracilo/farmacología , Fenantrenos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Caspasa 3/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Compuestos Epoxi/farmacología , Humanos , Células KB , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been reported to potentiate the anti-tumor effect in various cancer cells. However, the effect of TPL on oral cancers is not yet evaluated. Herein we first demonstrate that TPL induces prominent growth inhibition and apoptosis in two oral cancer cell lines, SCC25 and OEC-M1 and in KB cells. Our results indicate that TPL induces a dose-dependent apoptosis of these cells at nanomolar concentration. Apoptosis signalings are both activated through time upon TPL treatment detected by elevated caspase-3, 8, 9 activities. In xenograft tumor mouse model, TPL injection successfully inhibits the tumor growth via apoptosis induction which was demonstrated by TUNEL assay. These results demonstrate that TPL exerts anti-tumor effect on oral cancer and KB cells and suggest further the potential of TPL combining with other chemotherapeutic agents or radiotherapy for advanced oral cancer.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Diterpenos/farmacología , Células KB/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Fenantrenos/farmacología , Animales , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Epoxi/farmacología , Humanos , Células KB/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patologíaRESUMEN
Type 1 diabetes (T1D) develops via spontaneous autoimmune destruction of pancreatic beta cells. The immunoprogression and effectors of T1D have been determined using non-obese diabetic (NOD) mouse mode. Transgenic mice overexpressing a variety of transgenes driven by insulin promoter demonstrate that both apoptosis and necrosis lead to islet cells death; furthermore, various immune cells and cytokine effectors are involved in the immunoprogression of T1D. Efficiently halting immune attack in the islet milieu by an effector-specific manner apparently provides the preventive and therapeutic strategies in T1D. Islet transplantation has been reported as an appropriate treatment to accomplish insulin independence and long-term homeostasis of glucose in T1D. However, it is difficult to protect the islet grafts from subsequent immune attack and prolong their survival. In this review, we highlight the transgenic mice that express transgenes restricted in islet cells to depict the complicated interactions of immune cells in inflammatory islets, to investigate the protective efficacy of some immunomodulatory genes, and to develop genetically-modified islets tolerant to immune attack that might be used in future clinical application.
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Diabetes Mellitus Tipo 1/genética , Islotes Pancreáticos/fisiopatología , Antígenos CD/inmunología , Antígeno CTLA-4 , Muerte Celular , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/cirugía , Hemo-Oxigenasa 1/fisiología , Humanos , Inflamación/fisiopatología , Interferón gamma/fisiología , Trasplante de Islotes Pancreáticos , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Linfocitos T Citotóxicos/inmunología , Tiorredoxinas/fisiología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
BACKGROUND: Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease. Capsular serotype K1 and the magA gene have been reported to be associated with this disease. METHODS: The prevalence of magA was determined by polymerase chain reaction (PCR). The sequences of the magA flanking region were completed by inverse PCR and direct sequencing. Serotyping was performed by double immunodiffusion. Insertion mutations and trans-complementation were used to define the K1 genetic determination region. RESULTS: Thirty-five of 42 strains from patients with PLA were magA positive, whereas only 1 of 32 non-PLA strains was magA positive. All 36 magA-positive strains were serotype K1, and the 38 magA-negative strains were not (36/36 vs. 0/38; P<.0001). Sequencing of the magA flanking region revealed a putative capsular polysaccharide synthesis (cps) region; this region was 25 kb in length and contained 20 open reading frames (ORFs); of these ORFs, 9 were cotranscribed as part of an operon and differed from both MGH78578 and the Chedid strain. Mutation of 4 genes in this region turned the mutant strains anti-K1 negative. Trans-complementation restored the K1 phenotype. CONCLUSIONS: The operon containing magA is responsible for capsular serotype K1 of K. pneumoniae. Several loci in the operon are unique determinants of K1 strains.