RESUMEN
3D measurement methods based on fringe projection have attracted extensive research. However, it is a challenge to deal with overshooting on a high-reflection or specular surface. To eliminate the saturated pixels caused by overshooting, we propose a projection intensity adaptive adjustment method. First, we project three uniform gray-level images and estimate the projection intensity of the measured surface through the captured uniform gray-level images. Then we can obtain the optimal projection fringes in the camera coordinate system. Second, a set of horizontal and vertical gray-coded patterns are used to establish a coordinate matching relationship between the projected image and the captured image. To check the decoding result of the gray-coded patterns, a set of horizontal and vertical sinusoidal fringes are used to calculate the high-reflection mapping area (HRMA) in the projector coordinate system. Through the distribution of HRMA, we can check whether the decoding is reliable or not. Finally, we project the optimal intensity fringes and obtain the measurement results. We develop a measurement system to verify the validity of the proposed method. Experimental results show that the proposed method can effectively avoid overshooting and obtain measurement results with a minimum rms error.
RESUMEN
Background: mcr-1-mediated colistin resistance in Enterobacteriaceae is concerning, as colistin is used in treating multidrug-resistant Enterobacteriaceae infections. We identified trends in human fecal mcr-1-positivity rates and colonization with mcr-1-positive, third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae in Guangzhou, China, and investigated the genetic contexts of mcr-1 in mcr-1-positive 3GC-R strains. Methods: Fecal samples were collected from in-/out-patients submitting specimens to 3 hospitals (2011-2016). mcr-1 carriage trends were assessed using iterative sequential regression. A subset of mcr-1-positive isolates was sequenced (whole-genome sequencing [WGS], Illumina), and genetic contexts (flanking regions, plasmids) of mcr-1 were characterized. Results: Of 8022 fecal samples collected, 497 (6.2%) were mcr-1 positive, and 182 (2.3%) harbored mcr-1-positive 3GC-R Enterobacteriaceae. We observed marked increases in mcr-1 (0% [April 2011] to 31% [March 2016]) and more recent (since January 2014; 0% [April 2011] to 15% [March 2016]) increases in human colonization with mcr-1-positive 3GC-R Enterobacteriaceae (P < .001). mcr-1-positive 3GC-R isolates were commonly multidrug resistant. WGS of mcr-1-positive 3GC-R isolates (70 Escherichia coli, 3 Klebsiella pneumoniae) demonstrated bacterial strain diversity; mcr-1 in association with common plasmid backbones (IncI, IncHI2/HI2A, IncX4) and sometimes in multiple plasmids; frequent mcr-1 chromosomal integration; and high mobility of the mcr-1-associated insertion sequence ISApl1. Sequence data were consistent with plasmid spread among animal/human reservoirs. Conclusions: The high prevalence of mcr-1 in multidrug-resistant E. coli colonizing humans is a clinical threat; diverse genetic mechanisms (strains/plasmids/insertion sequences) have contributed to the dissemination of mcr-1, and will facilitate its persistence.
Asunto(s)
Portador Sano/microbiología , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Portador Sano/epidemiología , Cefalosporinas/farmacología , China/epidemiología , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Heces/microbiología , Humanos , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Prevalencia , Secuenciación Completa del GenomaRESUMEN
CTX-M-140, a novel CTX-M-type extended-spectrum ß-lactamase (ESBL), was identified in cephalosporin-resistant clinical isolates of Proteus mirabilis CTX-M-140 contained an alanine-to-threonine substitution at position 109 compared to its putative progenitor, CTX-M-14. When it was expressed in an Escherichia coli isogenic background, CTX-M-140 conferred 4- to 32-fold lower MICs of cephalosporins than those with CTX-M-14, indicating that the phenotype was attributable to this single substitution. For four mutants of CTX-M-14 that were constructed by site-directed mutagenesis (A109E, A109D, A109K, and A109R mutants), MICs of cephalosporins were similar to those for the E. coli host strain, which suggested that the alanine at position 109 was essential for cephalosporin hydrolysis. The kinetic properties of native CTX-M-14 and CTX-M-140 were consistent with the MICs for the E. coli clones. Compared with that of CTX-M-14, a lower hydrolytic activity against cephalosporins was observed for CTX-M-140. blaCTX-M-140 is located on the chromosome as determined by I-CeuI pulsed-field gel electrophoresis (I-CeuI-PFGE) and Southern hybridization. The genetic environment surrounding blaCTX-M-140 is identical to the sequence found in different plasmids with blaCTX-M-9-group genes among the Enterobacteriaceae Genome sequencing and analysis showed that P. mirabilis strains with blaCTX-M-140 have a genome size of â¼4 Mbp, with a GC content of 38.7% and 23 putative antibiotic resistance genes. Our results indicate that alanine at position 109 is critical for the hydrolytic activity of CTX-M-14 against oxyimino-cephalosporins.
Asunto(s)
Sustitución de Aminoácidos , Antibacterianos/metabolismo , Cefalosporinas/metabolismo , Genoma Bacteriano , Proteus mirabilis/enzimología , beta-Lactamasas/genética , Alanina/metabolismo , Antibacterianos/farmacología , Composición de Base , Cefalosporinas/farmacología , Clonación Molecular , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Tamaño del Genoma , Hidrólisis , Isoenzimas/genética , Isoenzimas/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Proteus mirabilis/efectos de los fármacos , Proteus mirabilis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/metabolismo , beta-Lactamasas/metabolismoRESUMEN
The formation of cytoplasmic stress granules and the innate immune response are two distinct cellular stress responses. Our study investigated the involvement of four innate immune proteins - retinoic-acid-inducible gene I (RIG-I, also known as DDX58), melanoma differentiation-associated gene 5 (MDA5, also known as IFIH1), IFN-ß promoter stimulator (IPS-1, also known as MAVS) and protein kinase regulated by dsRNA (PKR, also known as EIF2AK2) in the formation of stress granules. Knockdown of IPS-1 or PKR significantly decreased the formation of stress granules induced by double-stranded (ds)RNA. IPS-1 depletion markedly attenuated the phosphorylation of PKR and eIF2α that was triggered by dsRNA, and IPS-1 facilitated the in vitro autophosphorylation of PKR. In IPS-1-depleted cells, the dsRNA-mediated dimerization of PKR through its dsRNA-binding domains was significantly abrogated, suggesting that IPS-1 might be involved in PKR dimerization. By co-immunoprecipitation and pulldown assays, our data demonstrate that IPS-1 directly binds to PKR through the IPS-1 caspase activation and recruitment domain (CARD), suggesting that the effect of IPS-1 on the formation of stress granules might be exerted through interacting with PKR and mediating its activation. PKR was recruited into stress granules upon activation, whereas the majority of IPS-1 protein formed clusters on mitochondrial membranes. Our work provides the first evidence that the innate signaling molecule IPS-1 plays an essential role in stress granule formation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Gránulos Citoplasmáticos/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Dimerización , Células HeLa , Calor/efectos adversos , Humanos , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1 , Fosforilación/genética , Unión Proteica , Transporte de Proteínas , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/genética , Receptores Inmunológicos , Esteroles/toxicidad , Estrés Fisiológico/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
We identified New Delhi metallo-ß-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Citrobacter freundii/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/efectos de los fármacos , beta-Lactamasas/genética , Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/genética , Acinetobacter baumannii/aislamiento & purificación , Antibacterianos/uso terapéutico , China , Citrobacter freundii/genética , Citrobacter freundii/aislamiento & purificación , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Heces/microbiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Plásmidos/genética , Plásmidos/aislamiento & purificación , Orina/microbiología , beta-Lactamasas/biosíntesisRESUMEN
Dengue virus (DENV) is a mosquito-borne flavivirus that causes the most prevalent diseases in tropical and subtropical regions. DENV utilizes host factors to facilitate its replication, while host cells intend to restrain virus replication. NS4A of DENV has been implicated to play a crucial role during viral replication. To identify more cellular proteins that are recruited by NS4A, we carried out a tandem affinity purification assay. The mass spectrometry data revealed that human eukaryotic initiation factor 4AI (eIF4AI) was one of potential NS4A-interacting partners. Co-immunoprecipitation data confirmed the interaction between NS4A and eIF4AI, and both the N-terminal ATP-binding domain and C-terminal helicase domain of eIF4AI were involved in their association. Functionally, silencing of eIF4AI by RNAi significantly increased the replication level of DENV1, DENV2 and DENV3. And knockdown of eIF4AI markedly attenuated the phosphorylation of protein kinase regulated by dsRNA-activated (PKR) and eIF2É induced by DENV2 infection. Collectively, these data suggested that a potential role of NS4A in antagonizing host antiviral defense is by recruiting eIF4AI and escaping the translation inhibition mediated by PKR.
Asunto(s)
Virus del Dengue/fisiología , Factor 4A Eucariótico de Iniciación/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virología , Proteínas no Estructurales Virales/metabolismo , Activación Viral/fisiología , Replicación Viral/fisiología , Línea Celular Tumoral , Clonación Molecular , HumanosRESUMEN
Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.
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Autofagia , Regulación de la Expresión Génica , Macrófagos/metabolismo , MicroARNs/metabolismo , Proteínas de Unión al GTP Monoméricas/biosíntesis , Mycobacterium tuberculosis/metabolismo , Neuropéptidos/biosíntesis , Animales , Proteína 7 Relacionada con la Autofagia , Línea Celular , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Viabilidad Microbiana , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Mycobacterium tuberculosis/genética , Neuropéptidos/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/patologíaRESUMEN
Our study presents a first investigation of the effect of the adjuvant PIKA on dengue virus (DENV) replication. PIKA pretreatment decreased the levels of DENV serotype 2 (DENV2) mRNA, protein and viral particles in the hepatoma cell line HepG2. Treatment with PIKA simultaneously with DENV2 infection, but not after infection, resulted in a protective effect. Significant induction of type I and type III interferons (IFNs), as well as interferon-stimulated genes was detected in PIKA-pretreated cells. Neutralization of IFN-ß partially restored the replication levels of DENV2 in PIKA-pretreated cells, suggesting that IFN-ß is one of the mediators involved in the antiviral action of PIKA. Additionally, blockade of TBK-1 signaling largely restored the IFN induction and viral suppression effects mediated by PIKA, further illustrating that PIKA plays its anti-DENV role by promoting innate immunity. These findings suggest that PIKA is an attractive agent to be used in the prevention of DENV diseases.
Asunto(s)
Adyuvantes Inmunológicos , Virus del Dengue/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Replicación Viral/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Carcinoma Hepatocelular , Línea Celular , Culicidae/citología , Virus del Dengue/clasificación , Virus del Dengue/fisiología , Relación Dosis-Respuesta Inmunológica , Regulación de la Expresión Génica/inmunología , Humanos , Inmunidad Innata , Interferones/inmunología , Interferones/metabolismo , Neoplasias Hepáticas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
As a novel family of cell surface receptors, triggering receptors expressed on myeloid cells (TREMs) play an important role in inflammatory responses. However, the role of TREMs in the ocular immune system remains unknown. In this study, we examined the expression and function of TREM-1 in Pseudomonas aeruginosa keratitis, one of the most common sight-threatening ocular diseases. TREM-1 was significantly increased in human corneas after P. aeruginosa infection. Consistent with TREM-1 expression at the human ocular surface, TREM-1 levels (mRNA and protein) were also elevated in the infected corneas of C57BL/6 (B6) mice at 1, 3, and 5 days postinfection. To determine whether TREM-1 dictates the outcome of P. aeruginosa keratitis in susceptible mice, TREM-1 signaling in B6 mice was blocked with a soluble mTREM-1/Fc fusion protein. The results indicated that blockade of TREM-1 reduced the severity of corneal disease, polymorphonuclear neutrophil infiltration, Th1/proinflammatory cytokine expression and Toll-like receptor (TLR) activation but enhanced the production of Th2 cytokines, murine ß-defensin 2 (mBD2), single Ig interleukin-1R-related molecule (SIGIRR), and ST2. Furthermore, we also used agonistic anti-mTREM-1 antibody to activate TREM-1 signaling in B6 mice and found that TREM-1 activation resulted in worsened disease and earlier corneal perforation in infected B6 mouse corneas and elevated production of proinflammatory cytokines and TLR signaling molecules but reduced expression of mBD2, SIGIRR, and ST2. To the best of our knowledge, this study provides the first evidence that TREM-1 functions as an inflammatory amplifier in P. aeruginosa keratitis by modulating TLR signaling and Th1/Th2 responses.
Asunto(s)
Córnea/inmunología , Infecciones Bacterianas del Ojo/inmunología , Glicoproteínas de Membrana/metabolismo , Infecciones por Pseudomonas/inmunología , Receptores Inmunológicos/metabolismo , Receptores Toll-Like/metabolismo , Adulto , Anciano , Animales , Anticuerpos , Córnea/metabolismo , Citocinas/biosíntesis , Citocinas/metabolismo , Infecciones Bacterianas del Ojo/genética , Infecciones Bacterianas del Ojo/metabolismo , Femenino , Humanos , Proteína 1 Similar al Receptor de Interleucina-1 , Queratitis/inmunología , Queratitis/metabolismo , Queratitis/microbiología , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Infiltración Neutrófila , Neutrófilos/inmunología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Receptores Inmunológicos/inmunología , Receptores de Interleucina/biosíntesis , Receptores de Interleucina-1/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células TH1/inmunología , Células Th2/inmunología , Receptores Toll-Like/inmunología , Receptor Activador Expresado en Células Mieloides 1 , beta-Defensinas/biosíntesisRESUMEN
This study was designed to investigate the changes of prostaglandin I2 (PGI2) and nitric oxide (NO) secreted by endothelialized polyurethane small diameter artificial blood vessel. The peripheral blood mononuclear cells of healthy adult were separated and induced into endothelial progenitor cells (EPCs), which were identified by the methods of discrepancy microphage and fluorescent immunology labeling. After the induced cells being seeded on the polyurethane small-diameter artificial vessels, the endothelialized polyurethane small diameter artificial blood vessels were divided into four different experimental groups, including stationary group, low-flow shear stress group (5 dynes/cm2), medium-flow shearstress group (15 dynes/cm2), and high-flow shear stress group (25 dynes/cm2). Then, the levels of 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and NO of different time were measured by enzyme-linked immunosorbent assay and nitrate reductase methods. The peripheral blood mononuclear cells differentiated into EPCs. They presented typical "spindie-shaped" appearance, and they were positively labeled by fluorescent acetylated-LDL, lectin, FLK-1 and vWF. Shear stress enhanced the production of NO and 6-keto-PGF1alpha by EPCs in a dose-dependent manner. Therefore, shear stress increases the secretion of NO and PGI2 by EPC, which suggests that shear stress can improve the antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel.
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Órganos Bioartificiales , Prótesis Vascular , Células Endoteliales/citología , Fibrinolíticos/metabolismo , Poliuretanos/química , Materiales Biocompatibles/química , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Epoprostenol/metabolismo , Humanos , Técnicas In Vitro , Leucocitos Mononucleares/citología , Óxido Nítrico/metabolismo , Estrés MecánicoRESUMEN
Beta-defensins 2 and 3 (BD2 and BD3) are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA). Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1) and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.
Asunto(s)
Autofagia/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Macrófagos/inmunología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , beta-Defensinas/inmunología , Animales , Núcleo Celular/metabolismo , Regulación hacia Abajo/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Macrófagos/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/inmunología , Células RAW 264.7 , ARN Interferente Pequeño/metabolismoRESUMEN
In this study, we evaluated antitumor effects of allotumour RNA-transfected dendritic cells (DCs) cocultured with autologous cytokine-induced killer cells (CIKs) on hormone-refractory prostate cancer. The cocultured cells enhanced prostate cancer cytolysis from 26% (CIKs-induced cytolysis) to 80.8%. They also increased the productions of CD4(+) Th1 (IFN-γ(+)IL-4(-), 55.52%) and CD8(+) T (IFN-γ(+), 69.59%) cells determined by intracellular cytokines IFN-γ /IL-4 staining and reduced the rate of CD4(+) CD25(+) cells from 18.72% (in CIKs) to 9.72%. The cocultured cells significantly inhibited tumor growth in SCID mouse and induced cancer cells necrosis and apoptosis. Our study indicates that tumor RNA-pulsed DCs cocultured with autologous CIKs significantly enhance antitumor immunity, which can be induced by increased CD4(+) Th1 and CD8(+) T cells and decreased CD4(+)CD25(+) regulatory T (T(reg)) cells. This provides a potential immunotherapy strategy for HRPC.
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Antagonistas de Andrógenos/farmacología , Células Asesinas Inducidas por Citocinas/inmunología , Células Dendríticas/inmunología , Resistencia a Antineoplásicos , Neoplasias de la Próstata/terapia , ARN/inmunología , Animales , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Humanos , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , TransfecciónRESUMEN
An adjuvant is usually used to enhance the immune response induced by vaccines. The choice of adjuvant or immune enhancer determines the effectiveness of the immune response. Currently, aluminium (Alum, a generic term for salts of aluminium) is the only FDA-approved adjuvant. Alum predominantly induces the differentiation of Th2 cells and thus mediates an antibody immune response. Therefore, there is an urgent need for new adjuvants that enhance not only humoral but also cellular immune responses. In the present study, we demonstrates that PIKA (a stabilized dsRNA) as an adjuvant directly induces the activation and the proliferation of both B and NK cells in vitro. Injection of PIKA into mice results in the production of cytokines in vivo. In addition, the study demonstrates that PIKA promotes the maturation of bone marrow-derived dendritic cells (BMDCs) including up-regulation of the co-stimulatory molecules CD80, CD86 and CD40, and the induction of cytokines such as IL-12p70, IL-12p40 and IL-6. Importantly, after immunization of mice with HBsAg plus PIKA, the presence of PIKA enhances the titers of HBsAg-specific IgG and HBsAg-specific IFN-gamma production. These results demonstrate that PIKA as an adjuvant can promote both humoral and cellular immune responses. These might have an implication in applying PIKA as an adjuvant to be used in the design and development of both therapeutic and preventive vaccines, and used in the clinical study.
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Adyuvantes Inmunológicos , Formación de Anticuerpos , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Inmunidad Celular , ARN Bicatenario/inmunología , Vacunación , Animales , Antígenos CD/metabolismo , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Anticuerpos contra la Hepatitis B/sangre , Células Asesinas Naturales/inmunología , Cinética , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
In this study, the peripheral blood mononuclear cells of healthy adult were acquired and inducted by vascular endothelial growth factor, et cetera. The differentiated endothelial cells were observed and identified as EPCs by the double positive staining of fluorescent labeled acetylated-LDL and lectin, seeded on the polyurethane small-diameter artificial vessels, treated by shear stress of 15 dyn/cm2, and observed by scanning electronic microscopy. As a result, the peripheral blood mononuclear cells differentiated into EPCs. They were positively stained by labeled acetylated-LDL and lectin. Under observation of scanning electronic microscope, the unseeded polyurethane small-diameter artificial vessel being suited for the growth and spreading of the cells; the cell lineage on surface of artificial vessels of stationary group being arrayed in chaos, and that of shear stress group being arrayed in direction. Therefore, the peripheral cells can differentiate into EPCs, and EPCs can be used as novel source cells for the accelerated endothelialization of small diameter artificial vessel. Shear stress contributes to the mechanic moulding of cell lineage on the surface of artificial vessel.
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Órganos Bioartificiales , Prótesis Vascular , Células Endoteliales/citología , Poliuretanos/química , Células Madre/citología , Materiales Biocompatibles , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Humanos , Leucocitos Mononucleares/citología , Diseño de Prótesis , Resistencia al Corte , Estrés MecánicoRESUMEN
Macrophages play a critical role in the host immune response against mycobacterial infection. Our previous study has demonstrated that microRNA-155 (miR-155), one of the most important small non-coding RNAs in the immune system, promotes oxygen-independent mycobacterial killing in macrophages. However, little is known regarding the role of miR-155 in modulating oxygen-dependent mycobactericidal response in macrophages, including the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In the present study, we demonstrated that miR-155 was increased in macrophages after Mycobacterium bovis bacille Calmette-Guérin (BCG) infection. Moreover, the BCG-induced upregulation of miR-155 in macrophages was dependent on TLR2, NF-κB and JNK signaling pathways. More importantly, our study explored that miR-155 significantly elevated ROS production in macrophages, although miR-155 had no influence on the inducible nitric oxide synthase (iNOS) expression or nitric oxide (NO) production. In addition, our study demonstrated that miR-155 repressed the expression of src homology 2 (SH2) containing inositol 5-phosphatase1 (SHIP1), and knockdown of SHIP1 greatly increased ROS production in BCG-infected macrophages. Collectively, these data indicate that miR-155 modulates ROS but not RNS production by targeting SHIP1, which may provide a better understanding of the host anti-mycobacterial response.
Asunto(s)
MicroARNs/genética , Mycobacterium bovis/inmunología , Monoéster Fosfórico Hidrolasas/genética , Especies Reactivas de Oxígeno/metabolismo , Tuberculosis/inmunología , Animales , Células Cultivadas , Inositol Polifosfato 5-Fosfatasas , Sistema de Señalización de MAP Quinasas , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , FN-kappa B/fisiología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Receptor Toll-Like 2/fisiología , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVES: To investigate the role of miR-146a in dengue virus (DENV) replication. METHODS: Expression levels of miR-146a were measured by real-time PCR and Northern blot. Role of miR-146a was tested by overexpression and inhibition assays. Real-time PCR and 50% tissue culture infective dose (TCID50) assays were used to detect RNA levels and extracellular yields of DENV respectively. Interferon (IFN) levels induced by DENV infection were measured by real-time PCR and ELISA respectively. IFN-ß neutralization and RNAi were used to test the involvement of IFN-ß in the effects of miR-146a. TNFR-associated factor 6 (TRAF6) level was measured by Western-blot. RESULTS: miR-146a expression was significantly increased in primary human monocytes and THP-1 cells upon DENV infection. Overexpression of miR-146a increased DENV2 replication, while inhibition of miR-146a decreased the viral replication. miR-146a impaired the IFN production and the DENV2 replication suppressed by miR-146a inhibition was partially restored by neutralization of IFN-ß or depletion of interferon receptor (IFNAR) 1 or 2. Furthermore, miR-146a targets TRAF6 and overexpression of TRAF6 reversed the effects of miR-146a on IFN-ß induction and viral replication. CONCLUSIONS: DENV infection significantly induced the expression of miR-146a, which facilitated viral replication by targeting TRAF6 and dampening IFN-ß production.
Asunto(s)
Virus del Dengue/inmunología , Virus del Dengue/fisiología , Interferón beta/inmunología , MicroARNs/metabolismo , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Replicación Viral , Northern Blotting , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Humanos , Interferón beta/biosíntesis , MicroARNs/genética , Monocitos/inmunología , Monocitos/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
PURPOSE: We investigated whether the foldable capsular vitreous body (FCVB) could release levofloxacin sustainably in vitro and inhibit endophthalmitis in rabbit models. METHODS: Approximately 1.0 mL levofloxacin (625 µg/mL) was injected into the capsule of nine FCVBS. The levofloxacin release value was determined in the modified franz diffusion cells over time. In the in vivo study, all right eyes of 45 rabbits were infected with Staphylococcus epidermidis AND were divided randomly into three groups at 24 hours after infection: FCVB plus levofloxacin (n = 15), silicone oil plus subconjunctival levofloxacin (n = 15), and an untreated group (n = 15) during a 30-day observation time. Levofloxacin concentrations in the aqueous humor were detected, and therapeutic efficacy was evaluated with clinical evaluation, bacterial counts, cytokine profiles, and histopathology. RESULTS: The FCVB released levofloxacin ranging from 9 to 13.5 ng/mL in vitro and from 42 to 1.6 ng/mL in the aqueous humor during 30 days. In the FCVB and silicone-treated groups, clinical inflammation almost was abolished; no bacteria were detected in the aqueous humor; TNF-α, IL-1ß, and IFN-γ expression decreased; and relatively normal corneal and retinal architecture were kept after the 30-day treatment. CONCLUSIONS: The FCVB could provide us with dual functions, combining a levofloxacin drug delivery system and a vitreous substitute, to treat endophthalmitis in rabbit eyes.
Asunto(s)
Antibacterianos/administración & dosificación , Sistemas de Liberación de Medicamentos , Endoftalmitis/tratamiento farmacológico , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Levofloxacino , Ofloxacino/administración & dosificación , Infecciones Estafilocócicas/tratamiento farmacológico , Cuerpo Vítreo , Animales , Cuerpo Ciliar/metabolismo , Recuento de Colonia Microbiana , Citocinas/genética , Implantes de Medicamentos , Endoftalmitis/genética , Endoftalmitis/microbiología , Infecciones Bacterianas del Ojo/genética , Infecciones Bacterianas del Ojo/microbiología , Pruebas de Sensibilidad Microbiana , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/aislamiento & purificación , Staphylococcus epidermidis/patogenicidadRESUMEN
OBJECTIVE: To investigate the differential expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4) and their potential role in the pathogenesis of chronic suppurative otitis media and cholesteatoma. METHODS: Normal canal skin of 30 patients with tympanosclerosis were enrolled as control, 30 cases with chronic suppurative otitis media and 30 patients with cholesteatoma were studied. Real-time PCR, Western blot and Immunohistochemistry were preformed to detect the expression of TLR2/TLR4 in normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma, and the differential expression were analyzed. RESULTS: (1) the mRNA and protein expression of TLR2 and TLR4 were detected in all normal canal skin, mucosa and granulation tissue of chronic suppurative otitis media, mucosa, granulation tissue, cholesteatoma epithelium of cholesteatoma. (2) Both mRNA and protein level of TLR2/TLR4 in mucosa of chronic suppurative otitis media and cholesteatoma were higher than those in normal canal skin, but lower in cholesteatoma epithelium, there was no significant difference in mucosa of the two otitis media groups. (3) The mRNA and protein expression of TLR2/TLR4 in granulation tissue of chronic suppurative otitis media and cholesteatoma were significant increased when compared with normal canal skin, and TLR2 expression level was higher in granulation tissue of cholesteatoma than in chronic suppurative otitis media. (4) TLR2/TLR4 positive cells mainly infiltrated in granulations, significantly more than in normal skin, while fewer in the epithelium of cholesteatoma. CONCLUSIONS: Differential expression of TLR2 and TLR4 in mucosa suggests middle ear is a TLR2/TLR4 participated functional modulation of the innate immune system and also suggests that they may play a different role in the pathophysiology of chronic otitis media and cholesteatoma.
Asunto(s)
Colesteatoma del Oído Medio/metabolismo , Otitis Media Supurativa/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Estudios de Casos y Controles , Enfermedad Crónica , HumanosRESUMEN
Toll-like receptors (TLRs) play an important role in innate immunity against invading pathogens. Although TLR signaling has been indicated to protect cells from infection of several viruses, the role of TLRs in Dengue virus (DENV) replication is still unclear. In the present study, we examined the replication of DENV serotype 2 (DENV2) by challenging hepatoma cells HepG2 with different TLR ligands. Activation of TLR3 showed an antiviral effect, while pretreatment of other TLR ligands (including TLR1/2, TLR2/6, TLR4, TLR5 or TLR7/8) did not show a significant effect. TLR3 ligand poly(I:C) treatment prior to viral infection or simultaneously, but not post-treatment, significantly down-regulated virus replication. Pretreatment with poly(I:C) reduced viral mRNA expression and viral staining positive cells, accompanying an induction of the type I interferon (IFN-ß) and type III IFN (IL-28A/B). Intriguingly, neutralization of IFN-ß alone successfully restored the poly(I:C)-inhibited replication of DENV2. The poly(I:C)-mediated effects, including IFN induction and DENV2 suppression, were significantly reversed by IKK inhibitor, further suggesting that IFN-ß is the dominant factor involved in the poly(I:C) mediated antiviral effect. Our study presented the first evidence to show that activation of TLR3 is effective in blocking DENV2 replication via IFN-ß, providing an experimental clue that poly(I:C) may be a promising immunomodulatory agent against DENV infection and might be applicable for clinical prevention.
Asunto(s)
Carcinoma Hepatocelular/patología , Virus del Dengue/fisiología , Interferón beta/biosíntesis , Neoplasias Hepáticas/patología , Receptor Toll-Like 3/fisiología , Replicación Viral/fisiología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Virus del Dengue/genética , Humanos , Ligandos , Microscopía Fluorescente , Poli I-C/farmacología , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 3/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of garlicin on the levels of interferon gamma (INF-gamma) and interleukin 4 (IL-4) in blood of allergic rhinitis rat model. METHODS: Thirty healthy female SD rats were randomly divided into 3 groups: control group, negative control group and experimental group, 10 rats for each group. Ten rats (experimental group) were sensitized and intranasally challenged by ovalbumin, aluminium hydroxide hydrate gel and Bordetella pertussis inactive microorganism suspension adjuvants, as allergic rhinitis models, and then injection of garlicin(0.4 ml) intraperitoneally per day for 10 days. Control group rats were immunized as experimental group, and then injection of physiological saline as equal volume as garlicin. Negative control group rats were investigated using physiological saline. Blood of intrajugular vein of rat was extracted for separated plasma Enzyme liked immunosorbent assay (ELISA) was utilized to detect the serum levels of IL-4 and IFN-gamma. RESULTS: The serum levels (x +/- s) of IL4 were (22.81 +/- 8.79) pg/L, (41.43 +/- 4.93) pg/L, (9.93 +/- 2.07) pg/L, and those of IFN-gamma were (22.32 +/- 11.20) pg/L, (11.35 +/- 2.45) pg/L and (21.69 +/- 5.93) pg/L, respectively, among experimental group, control group and negative control group. The serum level of IL-4 in experimental group rats was lower than value of control group rats (t = 3.22, P < 0.05), while higher than negative control group (t = 4.17, P < 0.05). The serum level of IFN-gamma was increased significantly in experimental group rats with significant difference when compared with value of control group rats (t = 3.84, P < 0.05), while no difference was shown between experimental group and negative control group (t = 1.47, P > 0.05). CONCLUSIONS: Garlicin could increase serum level of INF-gamma and decrease serum level of IL4 significantly in allergic rhinitis rat model. It played an important role on regulating serum levels of cytokines of Thl and Th2.