RESUMEN
Type 2 helper T cells (T(H)2) are critically involved in allergies and asthma. Here we demonstrate that extracellular matrix protein-1 (ECM1) is highly and selectively expressed in T(H)2 cells. ECM1 deficiency caused impaired T(H)2 responses and reduced allergic airway inflammation in vivo. Functional analysis demonstrated that although the T(H)2 polarization of ECM1-deficient cells was unimpaired, these cells had a defect in migration and were retained in peripheral lymphoid organs. This was associated with reduced expression of KLF2 and S1P(1). We also found that ECM1 could directly bind the interleukin-2 (IL-2) receptor to inhibit IL-2 signaling and activate S1P(1) expression. Our data identify a previously unknown function of ECM1 in regulating T(H)2 cell migration through control of KLF2 and S1P(1) expression.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Hipersensibilidad/inmunología , Proteínas del Tejido Nervioso/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Células Th2/metabolismo , Traslado Adoptivo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Ganglios Linfáticos/patología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Receptores Mensajeros de Linfocitos/genética , Receptores Mensajeros de Linfocitos/inmunología , Transducción de Señal/inmunología , Células Th2/inmunología , Células Th2/patología , Transgenes/genéticaRESUMEN
Toll-like receptor (TLR) signaling is pivotal to innate and adaptive immune responses and must be tightly controlled. The mechanisms of TLR signaling have been the focus of extensive studies. Here we report that the tripartite-motif protein TRIM30alpha, a RING protein, was induced by TLR agonists and interacted with the TAB2-TAB3-TAK1 adaptor-kinase complex involved in the activation of transcription factor NF-kappaB. TRIM30alpha promoted the degradation of TAB2 and TAB3 and inhibited NF-kappaB activation induced by TLR signaling. In vivo studies showed that transfected or transgenic mice overexpressing TRIM30alpha were more resistant to endotoxic shock. Consistent with that, in vivo 'knockdown' of TRIM30alpha mRNA by small interfering RNA impaired lipopolysaccharide-induced tolerance. Finally, expression of TRIM30alpha depended on NF-kappaB activation. Our results collectively indicate that TRIM30alpha negatively regulates TLR-mediated NF-kappaB activation by targeting degradation of TAB2 and TAB3 by a 'feedback' mechanism.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , FN-kappa B/metabolismo , Receptores Toll-Like/fisiología , Animales , Línea Celular , Retroalimentación Fisiológica/inmunología , Femenino , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/antagonistas & inhibidores , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inhibidoresRESUMEN
Viral infection causes host cells to produce type I IFNs, which play a critical role in viral clearance. IFN regulatory factor (IRF) 7 is the master regulator of type I IFN-dependent immune responses. In this article, we report that N-Myc and STATs interactor (Nmi), a Sendai virus-inducible protein, interacted with IRF7 and inhibited virus-triggered type I IFN production. The overexpression of Nmi inhibited the Sendai virus-triggered induction of type I IFNs, whereas the knockdown of Nmi promoted IFN production. Furthermore, the enhanced production of IFNs resulting from Nmi knockdown was sufficient to protect cells from infection by vesicular stomatitis virus. In addition, Nmi was found to promote the K48-linked ubiquitination of IRF7 and the proteasome-dependent degradation of this protein. Finally, an impairment of antiviral responses is also detectable in Nmi-transgenic mice. These findings suggest that Nmi is a negative regulator of the virus-triggered induction of type I IFNs that targets IRF7.
Asunto(s)
Factor 7 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Infecciones por Respirovirus/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Factor 7 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus Sendai/inmunología , TransfecciónRESUMEN
IRF7 is known as the master regulator in virus-triggered induction of type I IFNs (IFN-I). In this study, we identify GBP4 virus-induced protein interacting with IRF7 as a negative regulator for IFN-I response. Overexpression of GBP4 inhibits virus-triggered activation of IRF7-dependent signaling, but has no effect on NF-κB signaling, whereas the knockdown of GBP4 has opposite effects. Furthermore, the supernatant from Sendai virus-infected cells in which GBP4 have been silenced inhibits the replication of vesicular stomatitis virus more efficiently. Competitive coimmunoprecipitation experiments indicate that overexpression of GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in impaired TRAF6-mediated IRF7 ubiquitination. Our results suggest that GBP4 is a negative regulator of virus-triggered IFN-I production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.
Asunto(s)
Regulación hacia Abajo/inmunología , Proteínas de Unión al GTP/fisiología , Marcación de Gen , Factor 7 Regulador del Interferón/antagonistas & inhibidores , Interferón-alfa/antagonistas & inhibidores , Interferón-alfa/biosíntesis , Virus Sendai/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Regulación hacia Abajo/genética , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Factor 7 Regulador del Interferón/metabolismo , Ratones , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Th cell differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNA interference-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Receptores de Interleucina-2/inmunología , Factor de Transcripción STAT6/inmunología , Células Th2/inmunología , Factores de Transcripción/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , Receptores de Interleucina-2/metabolismo , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/inmunología , Células Th2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Regulación hacia Arriba/inmunologíaRESUMEN
DNA immunization is an efficient method for high-affinity monoclonal antibody generation. Here, we describe the generation of several high-quality monoclonal antibodies (mAbs) against retinol-binding protein 4 (RBP4), an important marker for kidney abnormality and dysfunction, with a combination method of DNA priming and protein boost. The mAbs generated could bind to RBP4 with high sensitivity and using these mAbs, an immunocolloidal gold fast test strip was constructed. The strip can give a result in <5 min and is very sensitive with a detection limit of about 1 ng/ml. A small-scale clinical test revealed that the result of this strip was well in accordance with that of an enzyme-labeled immunosorbent assay kit currently available on the market. Consequently, it could be useful for more convenient and faster RBP4 determination in the clinic.
Asunto(s)
Anticuerpos Monoclonales/inmunología , ADN/inmunología , Oro Coloide , Inmunización/métodos , Enfermedades Renales/patología , Proteínas Plasmáticas de Unión al Retinol/inmunología , Secuencia de Bases , ADN/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Enfermedades Renales/genética , Juego de Reactivos para Diagnóstico , Tiras Reactivas , Proteínas Plasmáticas de Unión al Retinol/genética , Sensibilidad y EspecificidadRESUMEN
Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.
Asunto(s)
Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Disulfuros/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Filogenia , Proteómica , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Análisis de Secuencia de Proteína , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas ViroporinasRESUMEN
To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni2+-NTA agarose. Then the anti-EGFR monoclonal antibody (mAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked immunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Animales , Anticuerpos Monoclonales/genética , Línea Celular Tumoral , Proliferación Celular , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: The 2009 swine-origin influenza virus (S-OIV) H1N1 pandemic has caused more than 18,000 deaths worldwide. Vaccines against the 2009 A/H1N1 influenza virus are useful for preventing infection and controlling the pandemic. The kinetics of the immune response following vaccination with the 2009 A/H1N1 influenza vaccine need further investigation. METHODOLOGY/PRINCIPAL FINDINGS: 58 volunteers were vaccinated with a 2009 A/H1N1 pandemic influenza monovalent split-virus vaccine (15 µg, single-dose). The sera were collected before Day 0 (pre-vaccination) and on Days 3, 5, 10, 14, 21, 30, 45 and 60 post vaccination. Specific antibody responses induced by the vaccination were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). After administration of the 2009 A/H1N1 influenza vaccine, specific and protective antibody response with a major subtype of IgG was sufficiently developed as early as Day 10 (seroprotection rate: 93%). This specific antibody response could maintain for at least 60 days without significant reduction. Antibody response induced by the 2009 A/H1N1 influenza vaccine could not render protection against seasonal H1N1 influenza (seroconversion rate: 3% on Day 21). However, volunteers with higher pre-existing seasonal influenza antibody levels (pre-vaccination HI titer ≥1â¶40, Group 1) more easily developed a strong antibody protection effect against the 2009 A/H1N1 influenza vaccine as compared with those showing lower pre-existing seasonal influenza antibody levels (pre-vaccination HI titer <1â¶40, Group 2). The titer of the specific antibody against the 2009 A/H1N1 influenza was much higher in Group 1 (geometric mean titer: 146 on Day 21) than that in Group 2 (geometric mean titer: 70 on Day 21). CONCLUSIONS/SIGNIFICANCE: Recipients could gain sufficient protection as early as 10 days after vaccine administration. The protection could last at least 60 days. Individuals with a stronger pre-existing seasonal influenza antibody response may have a relatively higher potential for developing a stronger humoral immune response after vaccination with the 2009 A/H1N1 pandemic influenza vaccine.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Animales , Anticuerpos Antivirales/química , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Pruebas de Inhibición de Hemaglutinación/métodos , Hemaglutininas/química , Humanos , Sistema Inmunológico , Cinética , Ratones , Ratones Endogámicos BALB C , Pandemias/prevención & control , Estaciones del Año , Factores de TiempoRESUMEN
T helper cell type 1 (Th1) and 2 (Th2) play central roles in immune regulation. To identify the novel genes differentially expressed between Th1 and Th2 cells, CD4+ T cells were isolated from DO11.10 transgenic mice and induced under Th1 or Th2 conditions. Microarray showed differential expression of neutrophilic granule proteins (NGP) between Th1 and Th2 cells. NGP was first identified as a myeloid-specific granule protein with homology to the cystatin superfamily. Here we confirmed greater expression of NGP in Th2 cells by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction analysis. We also showed that the expression of NGP mRNA had a peak expression after 5 d culture under Th2- but not Th1-biasing conditions. Antibody against NGP was prepared, and in concert with the results of mRNA analysis, the level of NGP protein in Th2 cells detected by Western blot analysis was also higher than that in Th1 cells. GFP-NGP fusion proteins overexpressed in HeLa cells were localized to the cytoplasm. These results suggest NGP is a novel marker distinguishing Th2 from Th1 cells and maybe a novel cytokine secreted by Th2 cells.
Asunto(s)
Proteínas Sanguíneas/genética , Cistatinas/genética , Perfilación de la Expresión Génica , Neutrófilos/química , Vesículas Secretoras/química , Células TH1/metabolismo , Células Th2/metabolismo , Animales , Ratones , Ratones Endogámicos BALB CRESUMEN
Severe acute respiratory syndrome-coronavirus (SARS-CoV) causes an infectious disease through respiratory route. Diagnosing the disease effectively and accurately at early stage is essential for preventing the disease transmission and performing antiviral treatment. In this study, we raised monoclonal antibodies (mAbs) against the nucleocapsid (N) protein of SARS-CoV and mapped epitopes by using different truncated N protein fragments. The mapping of those epitopes was valuable for constructing pair-Abs used in serological diagnosis. The results showed that all of the six raised mAbs were divided into two groups recognizing the region of amino acids 249-317 (A group) or 317-395 (B group). This region spanning amino acids 249-395 contains predominant B cell epitopes located at the C-terminus of N protein. One pair-Abs, consisting of N protein-specific rabbit polyclonal antibody and SARS-CoV N protein-specific mAb, was selected to construct a sandwich ELISA-kit. The kit was able to specifically detect SARS-CoV N proteins in serum samples.
Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Proteínas de la Nucleocápside/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de la Nucleocápside de Coronavirus , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Conejos , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/sangreRESUMEN
This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Naproxeno/metabolismo , Animales , Catálisis , Células Cultivadas , Hidrólisis , Cinética , Ratones , Ratones Endogámicos BALB C , Análisis Espectral , EstereoisomerismoRESUMEN
Described herein is the synthesis of a hapten of biocyclo[2,2,2]octene 5 designed to mimic the exo transition-state of an aza Diels-Alder reaction. Immunization of rabbits with this hapten provided polyclonal antibodies, Aza-BSA-3, which were used to synthesize adduct 4b in the first reported antibody-catalyzed exo Diels-Alder reaction.