Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α-tocopherol, performance, haematology and blood chemistry in male Holstein calves.

Vitamin E (vit E), an essential antioxidant for maintaining the stability of biological membranes and the function of the immune system, is considered to support adaptive immune responses and performance in cattle. The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. Active and passive immunization of calves with Shiga toxoids (rStxMUT ) was recently shown to reduce the STEC shedding. Here, we examined the influence of vit E on calves' serum α-tocopherol, performance, haematology, blood chemistry and its interaction with rStxMUT immunization. Data from calves having received passive (colostrum from immunized cows) and active (intramuscularly at 5th and 8th weeks of life) vaccination with rStxMUT (n = 24) were compared to unvaccinated controls (n = 24; fed with low anti-Stx colostrum, placebo injected). For each vaccination group, data were analysed according to the level of vit E supplementation offered by milk replacer (188 IU all-rac-α-tocopheryl acetate daily [VitEM ] vs. 354 IU [VitEH ]). An increase by 79% in daily vit E supplementation led to slightly higher serum α-tocopherol level and earlier concentrate intake at the beginning of the experiment without significant differences in live weight gain, haematology, blood chemistry parameters and peripheral CD4+ and CD8+ T-cell subpopulations. rStxMUT vaccination modulated the CD4+ /CD8+ ratio irrespective of vit E supplementation but decreased concentrate intake in VitEH in a time-dependent manner. Results of our study indicate that an increase in daily vit E supplementation vastly fails to exert effects on laboratory parameters and growth performance. However, observed interactive effects of vit E supply and vaccination on the regulation of feed intake deserves further attention.

Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids.

The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. We examined whether immunization with genetically inactivated recombinant Shiga toxoids (rStx1MUT/rStx2MUT) influences STEC shedding in a calf cohort. A group of 24 calves was passively (colostrum from immunized cows) and actively (intra-muscularly at 5th and 8th week) vaccinated. Twenty-four calves served as unvaccinated controls (fed with low anti-Stx colostrum, placebo injected). Each group was divided according to the vitamin E concentration they received by milk replacer (moderate and high supplemented). The effective transfer of Stx-neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assay. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16th week of life. Using the expression of activation marker CD25 on CD4+CD45RO+ cells and CD8αhiCD45RO+ cells as flow cytometry based read-out, cells from vaccinated animals responded more pronounced than those of control calves to lysates of STEC and E. coli strains isolated from the farm as well as to rStx2MUT in the 16th week. Summarized for the entire observation period, less fecal samples from vaccinated calves were stx1 and/or stx2 positive than samples from control animals when calves were fed a moderate amount of vitamin E. This study provides first evidence, that transfer to and induction in young calves of Stx-neutralizing antibodies by Shiga toxoid vaccination offers the opportunity to reduce the incidence of stx-positive fecal samples in a calf cohort.

Metabolite Profile and Antiproliferative Effects in HaCaT Cells of a Salix reticulata Extract.

Phenolic constituents of Salix reticulata (Salicaceae) and antiproliferative activity of an extract and individual compounds were investigated in immortalized human non-tumorigenic keratinocytes (HaCaT). A MeOH extract from aerial parts afforded several flavonoids, including luteolin and apigenin glycosides (2-5 and 9) and catechin (1), two procyanidin fractions, and the phenolic glucosides picein (6), triandrin (7), and salicortin (8). In an adenosine triphosphate assay, the MeOH extract reduced cell viability by approximately 60 % at a concentration of 100 µg/mL. Cell proliferation was assessed with a BrdU incorporation ELISA assay. The extract inhibited proliferation of HaCaT cells in a concentration-dependent manner, with approximately 50 % inhibition at 100 µg/mL. In time-lapse assays, the extract showed distinct inhibitory effects on cell migration at concentrations of 12.5, 25, and 50 µg/mL. The activity of selected constituents was also determined. Luteolin-7-O-ß-glucuronide (3) significantly inhibited cell proliferation at concentrations of 10 and 50 µM. In contrast, luteolin-7-O-ß-glucopyranoside (2) and a procyanidin fraction (P1) had only weak effects, while picein (6) and salicortin (8) did not affect cell proliferation. Luteolin-7-O-ß-glucuronide (10 µM) and, to a lesser extent, the procyanidin fraction (10 µg/mL) also inhibited cell migration.

The Antiatherogenic Effect of Fish Oil in Male Mice Is Associated with a Diminished Release of Endothelial ADAM17 and ADAM10 Substrates.

BACKGROUND: Growing evidence suggests that disintegrin and metalloprotease (ADAM) 17 (ADAM17) and ADAM10 contribute to the pathogenesis of vascular diseases. ADAM17 promotes inflammatory processes by liberating tumor necrosis factor α, interleukin 6 receptor (IL-6R), and tumor necrosis factor receptor 1 (TNFR1). ADAM17 and ADAM10 modulate vascular permeability by cleaving endothelial adhesion molecules such as junctional adhesion molecule A (JAM-A) and vascular endothelial cadherin (VE-cadherin), respectively. OBJECTIVE: This study was designed to investigate whether a link might exist between the protective effects of fish oil (FO) supplementation against atherosclerosis and ADAM function. METHODS: Male LDL receptor knockout (LDLR(-/-)) mice and male wild-type (WT) mice were fed a Western diet (200 g/kg fat, 1.5 g/kg cholesterol) containing either 20% lard (LDLR(-/-)-lard and WT-lard groups) or 10% lard combined with 10% FO (LDLR(-/-)-FO and WT-FO groups) for 12 wk. Atherosclerotic lesion development and fatty acid composition of liver microsomes were evaluated. ADAM10 and ADAM17 expression was determined by quantitative real-time polymerase chain reaction and immunoblot analyses. Concentrations of soluble ADAM substrates in plasma and liver extracts were measured by ELISA. RESULTS: Diets supplemented with FO markedly reduced development of early atherosclerotic lesions in LDLR(-/-) mice (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 29.6 ± 6.1% vs. 22.5 ± 4.2%, P < 0.05). This was not accompanied by changes in expression of ADAM17 or ADAM10 in the aorta or liver. No dietary effects on circulating TNFR1 (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.22 ± 0.23 vs. 1.39 ± 0.28, P > 0.2) or IL-6R (1.06 ± 0.12 vs. 0.98 ± 0.09 fold of WT-lard group, P > 0.1), classical substrates of ADAM17 on macrophages, and neutrophil granulocytes were observed. However, a reduction in atherosclerotic lesions in the LDLR(-/-)-FO group was accompanied by a significant reduction in the circulating endothelial cell adhesion molecules JAM-A (LDLR(-/-)-lard group vs. LDLR(-/-)-FO group mean ± SD: 1.42 ± 0.20 vs. 0.95 ± 0.56 fold of WT-lard group, P < 0.05), intercellular adhesion molecule 1 (1.15 ± 0.14 vs. 0.88 ± 0.17 fold of WT-lard group, P < 0.05), and VE-cadherin (0.88 ± 0.12 vs. 0.72 ± 0.15 fold of WT-lard group, P < 0.05), reflecting reduced ADAM activity in endothelial cells. CONCLUSION: FO exerted an antiatherogenic effect on male LDLR(-/-) mice that was accompanied by a reduced release of ADAM17 and ADAM10 substrates from endothelial cells. It is suggested that FO-decreased ADAM activity contributes to improved endothelial barrier function and thus counteracts intimal lipoprotein insudation and macrophage accumulation.

Vitamin D receptor deficiency and low vitamin D diet stimulate aortic calcification and osteogenic key factor expression in mice.

Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR(-/-)) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR(-/-)) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D(3) over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas.