RESUMEN
To investigate the effects of Hericium erinaceus polysaccharide (HEP) on immunity in Muscovy duck reovirus (MDRV)-infected ducklings and explore its mechanism of action, an MDRV contact-infection model was established. Then, we investigated the influence of HEP on morphology of main immune organs in MDRV-infected ducklings by HE staining, while antioxidant capacity (T-AOC, MDA), serum protein levels (TP, ALB, GLO), complement levels (C3, C4) and antibody levels (IgA, IgM, IgG) were detected. Apoptotic indexes (apoptosisi rate and FAS-L) were also quantified by TUNEL method and immunohistochemical staining. Meanwhile, FADD and CytC (apoptosis-related genes), were tested by quantitative RT-PCR. Results showed that HEP could reduce the injuries of immune organs caused by MDRV. Additionally, HEP markedly diminished MDA (p < 0.01), while significantly increased T-AOC, TP, ALB, GLO, C3, C4, IgA, IgM and IgG (p < 0.01 or p < 0.05). Then, HEP shifted apoptosis time to an early MDRV-infected stage and reduced apoptosis at later MDRV-infected stage. This was associated with changes of FADD and CytC. Collectively, our data suggested that HEP could reduce the immunesuppression by many ways, such as decreasing organs' injuries, improving antioxidant capacity, serum proteins levels, antibody levels and complement levels, while diminish the apoptosis by lowering the FADD and CytC.
Asunto(s)
Patos/virología , Hericium/química , Sistema Inmunológico/efectos de los fármacos , Polisacáridos/uso terapéutico , Enfermedades de las Aves de Corral/tratamiento farmacológico , Infecciones por Reoviridae/veterinaria , Inmunidad Adaptativa/efectos de los fármacos , Animales , Anticuerpos Antivirales/sangre , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/análisis , Citocromos c/análisis , Evaluación Preclínica de Medicamentos , Proteína de Dominio de Muerte Asociada a Fas/análisis , Linfocitos/efectos de los fármacos , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Tejido Linfoide/virología , Oxidación-Reducción , Polisacáridos/aislamiento & purificación , Polisacáridos/farmacología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/virología , Distribución Aleatoria , Infecciones por Reoviridae/tratamiento farmacológico , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virologíaRESUMEN
FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His(6)-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His(6)-FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.