Kehe Huang

Effects of different selenium sources on tissue selenium concentrations, blood GSH-Px activities and plasma interleukin levels in finishing lambs

Thirty-two wether lambs of Tan sheep were randomly assigned into four dietary treatment groups (eight per group) for an 8-wk study and then fed a basal diet deficient in Se (0.06 mg/kg) or diets supplemented to provide 0.10 mg/kg Se from sodium selenite, selenized yeast, and selenium-enriched probiotics, respectively. Blood samples were collected at d 0, 28, and 56 of the experiment and tissue samples were collected at experiment termination. Tissue and blood Se concentrations, blood glutathione peroxidase (GSH-Px) activities, and plasma interleukin levels were analyzed. The results showed that the concentrations of Se in the kidney, liver, and muscle increased in all of the supplemented groups (p<0.01) compared with the control group. However, the Se concentrations in the kidney, liver, and muscle in the groups supplemented with Se yeast and Se-enriched probiotics were higher than those in the group supplemented with sodium selenite (p<0.01). The activities of GSH-Px and the concentrations of Se in blood also increased in all of the supplemented groups during the period of supplementation (p<0.01) compared with the control group. The activities of GSH-Px and the concentrations of Se in the whole blood of the lambs fed with selenized yeast and Se-enriched probiotics were higher than those of lambs fed with sodium selenite (p<0.01 or p<0.05). The concentrations of interleukin-1 and interleukin-2 in plasma significantly increased in all of the supplemented groups during the entire period of experiment (p<0.01) compared with the control group, but had no significant differences among all of the supplemented groups. In conclusion, a diet supplemented with Se for finishing lambs was able to increase the concentrations of Se in tissue and blood, activities of GSH-Px in blood, and levels of interleukins in plasma. Organic Se sources (selenized yeast and Se-enriched probiotics) were more effective than the inorganic Se source (sodium selenite) in increasing tissue and blood Se concentrations and blood GSH-Px activities of lambs. However, there were no significant differences in plasma interleukin levels of lambs between organic and inorganic Se sources.

Reactive oxygen species regulate the replication of porcine circovirus type 2 via NF-κB pathway

Intracellular redox state has been suggested to have various effects on the replication of different viruses within host cells. The aim of the present study was to investigate the influence of reactive oxygen species (ROS) on replication of porcine circovirus type 2 (PCV2), in PK15 cells. Following PCV2 infection there was a time-dependent increase in ROS. Antioxidant N-acetyl-l-cysteine treatment of cells resulted in lower ROS levels and lower PCV2 replication. In contrast, treatment by buthionine sulfoximine (BSO), a GSH synthesis inhibitor, resulted in elevation of ROS levels and increased PCV2 replication. Furthermore, inhibiting the activity of NF-κB, a redox-responsive transcription factor, suppressed BSO-mediated increase of PCV2 replication, indicating that increased PCV2 replication likely occurs via ROS activation of NF-κB. Taken together, our results indicate that the generation of ROS during PCV2 infection is involved in its replication and this progression is associated with the alteration in NF-κB activity induced by ROS.

Selenium Promotes T-Cell Response to TCR-Stimulation and ConA, but Not PHA in Primary Porcine Splenocytes

There is controversy in the literature over whether the selenium (Se) influences cellular immune responses, and the mechanisms possibly underlying these effects are unclear. In this study, the effects of Se on T-cell proliferation and IL-2 production were studied in primary porcine splenocytes. Splenocytes were treated with different mitogens in the presence of 0.5–4 µmol/L sodium selenite. Se significantly promoted T-cell receptor (TCR) or concanavalin A (ConA)-induced T-cell proliferation and IL-2 production but failed to regulate T-cell response to phytohemagglutinin (PHA). In addition, Se significantly increased the levels of cytosolic glutathione peroxidase (GPx1) and thioredoxin reductase 1 (TR1) mRNA, the activity of GPx1 and the concentration of reduced glutathione (GSH) in the unstimulated, or activated splenocytes. These results indicated that Se improved the redox status in all splenocytes, including unstimulated, TCR, ConA and PHA -stimulated, but only TCR and ConA-induced T-cell activation was affected by the redox status. N-acetylcysteine (NAC), a pharmacological antioxidant, increased T-cell proliferation and IL-2 production by TCR and ConA stimulated splenocytes but had no effect on the response to PHA in primary porcine splenocytes confirming that PHA-induced T-cell activation is insensitive to the redox status. We conclude that Se promotes GPx1 and TR1 expression and increases antioxidative capacity in porcine splenocytes, which enhances TCR or ConA -induced T-cell activation but not PHA-induced T-cell activation. The different susceptibilities to Se between the TCR, ConA and PHA -induced T-cell activation may help to explain the controversy in the literature over whether or not Se boosts immune responses.

Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection.

Due to drawbacks of live attenuated vaccines, much more attention has been focused on screening of Brucella protective antigens as subunit vaccine candidates. Brucella is a facultative intracellular bacterium and cell mediated immunity plays essential roles for protection against Brucella infection. Identification of Brucella antigens that present T-cell epitopes to the host could enable development of such vaccines. In this study, 45 proven or putative pathogenesis-associated factors of Brucella were selected according to currently available data. After expressed and purified, 35 proteins were qualified for analysis of their abilities to stimulate T-cell responses in vitro. Then, an in vitro gamma interferon (IFN-γ) assay was used to identify potential T-cell antigens from B. abortus. In total, 7 individual proteins that stimulated strong IFN-γ responses in splenocytes from mice immunized with B. abortus live vaccine S19 were identified. The protective efficiencies of these 7 recombinant proteins were further evaluated. Mice given BAB1_1316 (CobB) or BAB1_1688 (AsnC) plus adjuvant could provide protection against virulent B. abortus infection, similarly with the known protective antigen Cu-Zn SOD and the license vaccine S19. In addition, CobB and AsnC could induce strong antibodies responses in BALB/c mice. Altogether, the present study showed that CobB or AsnC protein could be useful antigen candidates for the development of subunit vaccines against brucellosis with adequate immunogenicity and protection efficacy.

Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo

The anticryptosporidial effect of sodium selenite (selenium) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system and an immunosuppressed C57BL/6N adult mouse model. Parasite numbers in cell culture were significantly reduced (p<0.01) following treatment with selenium (Se) at concentrations of 6, 9, and 12 µM at 48 h postinoculation (PI) and at 1.5, 3, and 6 µM at 96 h PI. Parasite reduction was greater than 50% at 48 h PI when 9 and 12 µM Se was used, and at 96 h PI when 6 µM Se was used. Such Se-induced reduction of Cryptosporidium parvum infection was significantly (p<0,0001) blocked when using free-radical scavengers such as mannitol (20 mM). A combined solution of mannitol (20 mM) and reduced glutathione (0.5 mM) enhanced the blockage to almost 100%. Adult C57BL/6N mice were immunosuppressed with dexamethasone phosphate administered ad libitum (16 µg/mL) in drinking water and inoculated with 105 oocysts/mouse. Significantly fewer (p<0.001) oocysts were shed in the feces of mice treated with Se administered ad libitum (12 µM) in drinking water than in untreated mice. The survival time of mice was also significantly increased (p<0.001) following Se treatment. Collectively, these results indicate that Se plays an important role in cryptosporidiosis, and oxidative stress caused by Se is probably a major mechanism in inhibition of C. parvum infection.

Protective Effect of Selenomethionine on Aflatoxin B1-Induced Oxidative Stress in MDCK Cells

AFB1 is a mycotoxin which exerts their cytotoxicity through increasing oxidative damage in target organ. Kidney is one of target organs vulnerable to damage caused by AFB1. In this study, Madin-Darby canine kidney (MDCK) cells were used to evaluate the AFB1-induced cell damage by the MTT assay. The results revealed that the toxic effect of AFB1 on MDCK cells is both dose and time dependent. Half maximal toxic concentration (IC50) was noted at 0.25xa0μg/ml of AFB1. Further, protective effect of six different concentrations (0.2, 0.8, 1, 2, 4, and 8xa0μM) of selenomethionine (SeMet) was observed against 0.25xa0μg/ml of AFB1-induced damage. The results showed that 0.25xa0μg/ml of AFB1 caused significant increase in oxidative stress, which was demonstrated by significant increase of malondialdehyde (MDA) level, reduction of intracellular GSH level, as well as GPX1 activity and mRNA level in MDCK cells when compared with control. SeMet protected the cells from AFB1-induced oxidative damage in a dose-dependant manner. Good protection could be achieved between 1 and 4xa0μM of concentration. Amid this range, MDA level significantly decreased while intracellular GSH level and GPX1 activity in addition to mRNA level significantly increased. Moreover, cell viability was significantly improved. It could be concluded that SeMet is a potential antioxidative agent to alleviate AFB1-induced oxidative stress.

Effects of Selenium-Enriched Probiotics on Lipid Metabolism, Antioxidative Status, Histopathological Lesions, and Related Gene Expression in Mice Fed a High-Fat Diet

A total of 80 female albino mice were randomly allotted into five groups (nu2009=u200916) as follows: (A) normal control, (B) high-fat diet (HFD),; (C) HFDu2009+u2009probiotics (P), (D) HFDu2009+u2009sodium selenite (SS), and (E) HFDu2009+u2009selenium-enriched probiotics (SP). The selenium content of diets in groups A, B, C, D, and E was 0.05, 0.05, 0.05, 0.3, and 0.3xa0μg/g, respectively. The amount of probiotics contained in groups C and E was similar (Lactobacillus acidophilus 0.25u2009×u20091011/mL and Saccharomyces cerevisiae 0.25u2009×u2009109/mL colony-forming units (CFU)). The high-fat diet was composed of 15xa0% lard, 1xa0% cholesterol, 0.3xa0% cholic acid, and 83.7xa0% basal diet. At the end of the 4-week experiment, blood and liver samples were collected for the measurements of lipid metabolism, antioxidative status, histopathological lesions, and related gene expressions. The result shows that HFD significantly increased the body weights and liver damages compared to control, while P, SS, or SP supplementation attenuated the body weights and liver damages in mice. P, SS, or SP supplementation also significantly reversed the changes of alanine aminotransferase (AST), aspartate aminotransferase (ALT), total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), total protein (TP), high-density lipoprotein (HDL), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalasa (CAT), and malondialdehyde (MDA) levels induced by HFD. Generally, adding P, SS, or SP up-regulated mRNA expression of carnitine palmitoyltransferase-I (CPT1), carnitine palmitoyltransferase II (CPT2), acetyl-CoA acetyltransferase II (ACAT2), acyl-coenzyme A oxidase (ACOX2), and peroxisome proliferator-activated receptor alpha (PPARα) and down-regulated mRNA expression of fatty acid synthase (FAS), lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol regulatory element-binding protein-1 (SREBP1) involved in lipid metabolism. Among the group, adding SP has a maximum effect in improving lipid metabolism, antioxidative status, histopathological lesions, and related gene expression in mice fed a HFD.

Oxalate-degrading capacities of lactic acid bacteria in canine feces.

In this study, lactic acid bacteria in canine feces were isolated and identified, and their oxalate-degrading capacities were evaluated. The oxalate-degrading capacities were determined for 24 of 47 (51.06%) lactic acid bacteria isolates. Of these, 8 isolates [Leuconostoc mesenteroides (RL75), Lactococcus garvieae (CD2), Lactococcus subsp. lactis (CS21), Enterococcus faecium (CL71 and CL72), and Enterococcus faecalis (CD14, CS62, and CD12)] degraded more than 5% of the oxalate present, while the others degraded less than 5% of the oxalate in vitro. Isolates that degraded more than 5% of the oxalate present were selected for further examination. The oxalate-degrading capacities of individual isolates, a mixture of Enterococcus, a mixture of Lactococcus, and a mixture of the eight isolates were evaluated in media containing different concentrations of glucose (sufficient, insufficient, or no glucose). In comparison with the control medium, all of the individual isolates and mixtures of isolates could degrade oxalate in all three groups (P<0.05). In most cases, the isolates growing in medium with 20 g/L of glucose had higher oxalate-degrading capacities than those growing in medium with 2.5 g/L of glucose or no glucose. The mixture of all isolates showed higher oxalate-degrading capacity than the individual isolates and other mixtures. The oxalate-degrading capacities of the isolates were isolate dependent.

Preparation of Selenium/Zinc-Enriched Probiotics and Their Effect on Blood Selenium and Zinc Concentrations, Antioxidant Capacities, and Intestinal Microflora in Canine

The aim of this study was to prepare Se/Zn-enriched probiotics and investigate their effect on blood Se and Zn concentrations, blood antioxidant capacities, and intestinal microflora in canine. The preparation was performed in a single-factor experiment. The optimal culture conditions were as follows: the initial concentrations of Se4+ and Zn2+ were 5 and 150xa0µg/ml, respectively; the inoculation volume was 5%; and the liquid volume of the medium was 50xa0ml in a 250-ml flask. The medium was then cultured at 32°C for 36xa0h. The biomass of the final product was 26.83xa0g/l, organic Se concentration was 173.35xa0µg/g, organic Zn concentration was 4.38xa0mg/g, Candida utilis biomass was 8.69xa0lg colony-forming units (CFU)/ml, and Lactobacillus biomass was 9.61xa0lg CFU/ml. In vivo, 20 indigenous dogs were randomly assigned to two dietary treatment groups for a 35-day study and fed a basal diet or a diet supplemented with 2.0xa0g of Se/Zn-enriched probiotics. Blood and fecal samples were collected on days 0, 15, and 30 of the experiment. Compared with the control group, the blood Se concentration; the blood Zn concentration; the activities of glutathione peroxidase, superoxide dismutase, and total antioxidant capacity in the blood; and the amount of Lactobacillus and Bifidobacterium in the feces increased in the supplemented group during the period of supplementation (Pu2009<u20090.01), while malondialdehyde level in the blood and the amounts of Escherichia coli, Staphylococcus, and Enterococcus in the feces decreased in the supplemented group (Pu2009<u20090.01).

Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice

Brucella has been considered as a non-motile, facultative intracellular pathogenic bacterium. However, the genome sequences of different Brucella species reveal the presence of the flagellar genes needed for the construction of a functional flagellum. Due to its roles in the interaction between pathogen and host, we hypothesized that some of the flagellar proteins might induce protective immune responses and these proteins will be good subunit vaccine candidates. This study was conducted to screening of protective antigens among these flagellar proteins. Firstly, according to the putative functional roles, a total of 30 flagellar genes of Brucella abortus were selected for in vitro expression. 15 of these flagellar genes were successfully expressed as his-tagged recombinant proteins in Escherichia coli ER2566. Then, these proteins were purified and used to analyze their T cell immunity induction activity by an in vitro gamma interferon (IFN-γ) assay. Five of the flagellar proteins could stimulate significantly higher levels of IFN-γ secretion in splenocytes from S19 immunized mice, indicating their T cell induction activity. Finally, immunogenicity and protection activity of these 5 flagellar proteins were evaluated in BALB/c mice. Results showed that immunization with FlgJ (BAB1_0260) or FliN (BAB2_0122) plus adjuvant could provide protection against B. abortus 544 infection. Furthermore, mice immunized with FlgJ and FliN developed a vigorous immunoglobulin G response, and in vitro stimulation of their splenocytes with immunizing proteins induced the secretion of IFN-γ. Altogether, these data suggest that flagellar proteins FlgJ and FliN are protective antigens that could produce humoral and cell-mediated responses in mice and candidates for use in future studies of vaccination against brucellosis.