The delivery of certain living microorganisms in food has long been suggested as having positive health effects in humans. This practice has extended into food animal production, with a variety of ...microorganisms being used; lactic acid bacteria, various Bacillus species and the yeast Saccharomyces cerevisiae have been particularly used in the pig industry. The increased interest in probiotics is essentially due to the problem of microbial resistance to antibiotics and following the ban of the use of antibiotics in animal production, probiotics being considered an alternative means to reduce pathogen infection and improve animal health especially around the time of weaning. However, there is still a need to clarify the probiotic effectiveness in pigs, and the underlying mechanisms. When assessing the efficacy of probiotics one must consider the particular strain of organism being used and the production stage of the pigs being treated. The reproducible delivery of probiotics in industrial pig production is problematic as maintenance of viability is key to their beneficial activity, but difficult to achieve with commonly used feed processing technologies. One specific context where probiotics organisms may be reliably delivered is in systems utilising fermented liquid feeds. Liquid feed may be fermented by the activity of wild lactic acid bacteria or may be stimulated using specific isolates as 'starters'; the latter system has advantages in terms of reproducibility and speed of fermentation. The farm context in which the organism is used is likely to be critical; the use of probiotics is more likely to result in measurable economic gains in animals living in sub-optimal conditions rather than in those reared in the highest welfare and husbandry conditions. The establishment of a beneficial lactic acid bacteria population at birth may lead to healthier animals, this may be most effectively achieved by treating sows, which provide an amplification step and flood the neonatal pigs' environment with desirable bacterial strains. In contrast, it may be sufficient to provide a supportive, protective microbiota around the time of weaning as this is a time of major crisis with instability and loss of certain bacterial populations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We investigated whether spray-dried plasma (SDP) improved growth and health of piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Forty-eight pigs weaned at 21 d (BW = 4.88 +/- 0.43 ...kg) received one of four diets containing 6% SDP or fish proteins (as-fed basis) either nonmedicated (SDP-NM and FP-NM diets) or medicated with 0 or 250 mg/kg of colistine + 500 mg/kg of amoxycycline (SDP-M and FP-M diets), for 15 d. On d 4, pigs were orally challenged with ETEC. On d 15, eight pigs per dietary group were killed, blood and saliva were collected for analysis of K88 fimbriae-specific immunoglobulin (Ig)-A, and jejunum was removed for villi preparation, histological analysis, and cytokine expression. The presence or absence of K88 receptors (K88(+) and K88(-) pigs respectively) was determined by villous adhesion assay. Effects of protein source on ADG (P = 0.04) and ADFI (P < 0.01), as well of medication on ADFI (P < 0.02), of all pigs were observed. In sacrified pigs, there was an effect of protein source on ADG (P = 0.03) and ADFI (P < 0.001), as well an interaction between medication and presence of K88 receptor (P = 0.02) for feed:gain ratio. Plasma K88 specific IgA were low in all K88(-) pigs and higher in K88(+) pigs fed FP-NM compared with all the other groups (P < 0.05), except SDP-M. An interaction was found among protein source, medication, and presence of K88 receptors (P = 0.04). Saliva IgA concentrations were high in all pigs fed FP-NM and low in all other pigs. Jejunum of pigs fed FP-NM showed some ulcerations, edema, and mild inflammatory cell infiltration (ICI). In pigs fed FP-M, edema was reduced. Conversely, only a mild ICI was observed in pigs fed SDP-NM and SDP-M. Crypt depth was increased in K88(+) pigs fed SDP-NM and an interaction between protein source and presence of K88 receptors was observed (P < 0.05). Expressions of tumor necrosis factor-alpha and interleukin (IL)-8 were lower in pigs fed SDP-NM and SDP-M than in those fed FP-NM and FP-M, either K88(-) or K88(+) (P < 0.01). In pigs fed FP diets, expression of IL-8 tended to increase (P = 0.08) in K88(+) compared with K88(-) subjects. Expression of interferon-gamma increased in K88(-) and K88(+) pigs fed FP-M as compared with other pigs (P < 0.01). These results indicate that feeding with SDP improved growth performance and protected against E. coli-induced inflammatory status, and suggest that use of SDP-NM can be considered a valid antibiotic alternative.
Pig weaning period is frequently associated with infectious disease, mainly caused by enterotoxigenic
Escherichia coli (ETEC) K88. Plant extracts exert different beneficial effects and may represent ...antibiotic alternatives to reduce piglet infection. In this study, plant extracts and other natural substances (PENS) have been evaluated on the pig intestinal IPEC-1 cells, for potential protection against ETEC K88 induced membrane damage. Several PENS have been considered: yeast extract, yeast nucleotides, unsaturated oligo-mannuronic acid, ulvan, bromelain and three fractions of bovine colostrums, as anti-inflammatory and immunomodulatory compounds; daidzein and
Chlorella vulgaris extract, as anti-oxidant compounds; allicin, cinnamaldehyde and carvacrol, as anti-bacterial compounds. First, possible toxic effect of PENS on cell membrane permeability was verified by assessing the transepithelial electrical resistance (TEER) and paracellular flux of the extracellular marker phenol red. The highest non-toxic PENS concentration was added to ETEC infected cells to test the protection against membrane damage. The results showed that yeast extract, daidzein, bovine colostrum, bromelain and allicin protected the cells against the increased membrane permeability caused by ETEC, whereas the other PENS did not show this ability. Allicin protection was not due to its anti-bacterial activity, since ETEC growth was unaffected by the presence of allicin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
There is some evidence that zinc oxide (ZnO) protects against intestinal diseases. However, despite the suggestions that ZnO may have an antibacterial effect, the mechanisms of this protective effect ...have not yet been elucidated. We investigated the potential benefits of ZnO in protecting intestinal cells from damage induced by enterotoxigenic Escherichia coli (ETEC, strain K88) and the related mechanisms, using human Caco-2 enterocytes. Cell permeability, measured as transepithelial electrical resistance (TEER), was unaffected by 0.01 and 1 mmol/L ZnO treatments and moderately increased by 5 mmol/L ZnO, compared with untreated cells. Transfer of 14C-inulin was slightly increased by 5 mmol/L ZnO compared with untreated cells; transfer was unaffected by lower concentrations. The TEER and 14C-inulin transfer were lower in ETEC-infected cells than in uninfected cells. Treatment of ETEC exposure with 0.2 mmol/L ZnO prevented disruption of membrane integrity. The ETEC was able to adhere to enterocytes and, to some extent, invade the cells. The ZnO treatment reduced bacterial adhesion and blocked bacterial invasion. The ETEC infection upregulated the expression of the inflammatory cytokines interleukin-8, growth-related oncogene-α and tumor necrosis factor-α, and reduced that of the anti-inflammatory cytokine transforming growth factor-β, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 ...epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698T, against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531T treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly ...explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1β, IL-4, IL-6, IL-10, IL-12, TGF-β, IFN-γ and TNF-α was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7%) were CD3/CD4+ and only a small percentage (14.3%) were CD3/CD8+, all carried the TCR αβ, and had a memory phenotype. The cytokine profile showed high levels of IFN-γ and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.
Evaluation of some immune markers in Italian elderly population in relation to zinc status, gender and antioxidant defence.
Observational study.
Italian population.
Apparently healthy, free-living ...subjects, 56 men and 52 women, aged 70-85 y, enrolled in Italy.
Lymphocytes were unstimulated or stimulated with the mitogen phytohemoagglutinin (PHA). The proliferative capacity was measured as incorporation of 3H-thymidine and reported as stimulation index (SI). Cytokine secretion by lymphocytes was determined by ELISA. The antioxidant enzyme activities were measured using commercial kits.
Dietary zinc intake, as well as zinc in serum, red blood cells and urine were on the normal range of values and did not show any difference between men and women. The proliferative response showed a high variability without significant differences between men and women. The amount of secreted pro- and anti-inflammatory cytokines was similar in men and women. No differences were found in the activity of antioxidant enzymes in lymphocytes, namely superoxide dismutase, glutathione peroxidase and catalase, between men and women. An association between SI and serum zinc level in men was found. SI resulted negatively correlated with interleukin (IL)-1beta (R2 = 0.036 and P = 0.012) and IL-10 (R2 = 0.34 and P = 0.040) only in men. IL-10 of PHA-stimulated lymphocytes was negatively correlated with red blood zinc in men (R2 = 0.41 and P = 0.008), while IL-10 of unstimulated and PHA-stimulated lymphocytes were negatively correlated with serum zinc in women (R2 = 0.38 and P = 0.020; R2 = 0.31 and P = 0.040, respectively). No correlation was observed between immune markers and antioxidant enzyme activities.
Only weak differences on immune response between men and women were observed. However, zinc status appears to have more influence on the ability of lymphocytes to proliferate in men than in women.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to ...affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.
Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the ...retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two
N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP–TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast
Saccharomyces
cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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