Protective effects of miRNA-589 on cerebral ischemia-reperfusion injury.

To uncover the potential influence of microRNA-589 (miRNA-589) on cerebral ischemia-reperfusion injury (IRI) and the underlying mechanism, BV2 cells were stimulated by lipopolysaccharide (LPS) or conditioned medium (CM) of primary cortical neurons undergoing oxygen-glucose deprivation (OGD). Regulatory effects of miRNA-589 on the release of inflammatory factors in BV2 cells induced with LPS or CM of primary cortical neurons undergoing OGD were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The interaction between miRNA-589 and TRAF6 was finally assessed by dual-luciferase reporter gene assay. MiRNA-589 was downregulated in BV2 cells induced with LPS or CM of primary cortical neurons undergoing OGD. Overexpression of miRNA-589 reduced the release of inflammatory factors in LPS or CM-induced BV2 cells. TRAF6 was verified to be the downstream gene of miRNA-589, and its level was negatively regulated by miRNA-589. MiRNA-589 is downregulated following cerebral IRI and alleviates inflammatory response through negatively regulating TRAF6.

Efficacy study and field application of an inactivated new type gosling viral enteritis virus vaccine for domestic geese.

New type gosling viral enteritis virus (NGVEV) caused a serious disease in naive juvenile goslings. In the described studies the performance of 2 vaccines was analyzed: a vaccine containing adjuvanted inactivated NGVEV and a vaccine containing adjuvanted inactivated NGVEV and recombinant goose IL-2. Breeder geese were subcutaneously vaccinated at the beginning of the egg production period with the vaccines. Breeder geese sham vaccinated with PBS served as control. The cellular and humoral immune responses of the vaccinated breeder geese, as well as the presence of maternally derived antibody to NGVEV, were investigated by ELISA, virus neutralization test, and lymphocyte proliferation assay, respectively. A significantly higher immunogenicity (P < 0.05) was induced by the inactivated NGVEV-recombinant goose IL-2 adjuvant vaccine compared with the inactivated NGVEV vaccine. The offspring of the vaccinated birds were challenged with virulent NGVEV (100 50% lethal dose) and the protective efficacy of the vaccines was determined. Furthermore, in a field trial the efficacy of the inactivated NGVEV vaccine was recorded from years 2003 to 2007. No clinical signs or abnormal health status were observed in the vaccinated breeder geese and the progeny. After a single application, >80% protection was shown in the progeny of geese vaccinated against NGVEV challenge for approximately 5 mo. The extensive field trials further demonstrated that vaccination of breeder geese with the inactivated NGVEV vaccine could be a safe and efficacious means to control NGVE disease. Moreover, the level of maternally derived NGVEV antibody titer in the egg yolk reflected the level of NGVEV antibodies in the breeder geese, suggesting that the egg yolk could be used to monitor the vaccination efficacy in commercial goose breeder flocks.

Development and application of an indirect immunohistochemical method for the detection of duck plague virus vaccine antigens in paraffin sections and localization in the vaccinated duckling tissues.

The objective of the present study was to develop and apply a streptavidin-alkaline phosphatase labeling system of indirect immunohistochemistry (SP-IHC) to detect antigenic distribution and localization regularity of duck plague virus (DPV) vaccine antigens in paraformaldehyde-fixed paraffin-embedded tissues of experimentally vaccinated ducklings. Male New Zealand rabbits were immunized with purified DPV antigens, which were engaged by a combination of differential centrifugation and sucrose-density gradient ultracentrifugation. The rabbit anti-DPV polyclonal antibodies were purified and used as the primary antibodies. Forty-eight 28-d-old DPV-free Pekin ducklings were subcutaneously inoculated with attenuated DPV vaccine in the immunization group and sterile PBS in the control group. The tissues were collected at sequential time points between 4 h and 18 wk postvaccination (PV) and were prepared for SP-IHC observation. The presence of DPV-specific antigens was first observed in the liver and spleen at 12 h PV; in the bursa of Fabricius, thymus, Harderian gland, esophagus, and intestinal tract at 1 d PV; and in the heart, lung, kidney, pancreas, and brain at 3 d PV. The positive staining reaction could be detected in the vaccinated duckling tissues until 18 wk PV, and no positive staining cells could be observed in the controls. The highest levels of positive staining reaction were found in the liver, spleen, bursa of Fabricius, thymus, and intestinal tract, whereas a few DPV vaccine antigens were distributed in the heart, pancreas, and esophagus. The target cells had a ubiquitous distribution, especially in the mucosal epithelial cells, lamina propria cells, macrophages, hepatocytes, and lymphocytes, which served as the principal sites for antigen localization. These findings demonstrated that SP-IHC was a reliable method for detecting antigenic distribution and localization regularity of DPV vaccine antigens in routine paraffin sections. The present study may be useful for describing proliferation and distribution regularity of DPV vaccine in the vaccinated duckling tissues and enhance further studies and clinical application of attenuated DPV vaccine.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification.

AIM: The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. METHODS: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). RESULTS: The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies µl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. CONCLUSIONS: LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.

Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing.

Oxidized low density lipoprotein suppresses the expression of tumor necrosis factor-alpha mRNA in stimulated murine peritoneal macrophages.

In the present report we have examined expression of the gene encoding the inflammatory monokine TNF-alpha in murine peritoneal macrophages treated with different forms of low density lipoprotein (LDL). LDL modified by oxidation in vitro is unable to stimulate inflammatory gene expression in peritoneal macrophages. However, treatment of macrophage cultures with oxidized LDL for 6 h or more resulted in a concentration and time-dependent suppression of TNF-alpha mRNA expression induced in response to stimulation with either LPS or maleylated BSA. This suppression was maximal after 12 h of exposure to oxidized LDL and at a concentration of 100 to 200 micrograms LDL cholesterol/ml of culture medium. The suppressive effect was restricted to oxidatively modified LDL as treatment with native LDL or acetylated LDL did not affect TNF-alpha mRNA expression, despite the fact that both acetylated and oxidized LDL lead to intracellular lipid accumulation. The expression of maleyl albumin-stimulated TNF-alpha mRNA expression could be reproduced by lipid extracts of oxidized LDL provided to macrophages at the same cholesterol concentration as from the intact lipoprotein particle. Extracts from native LDL were ineffective. These results suggest that oxidized lipid accumulation in monocytes infiltrating the arterial wall may lead to the suppression of certain inflammatory functions which, in turn, may influence the development of mature atherosclerotic lesions.