Effects of Dietary Grape Seed Meal Bioactive Compounds on the Colonic Microbiota of Weaned Piglets With Dextran Sodium Sulfate-Induced Colitis Used as an Inflammatory Model.

Microbiota affects host health and plays an important role in dysbiosis. The study examined the effect of diet including grape seed meal (GSM) with its mixture of bioactive compounds on the large intestine microbiota and short-chain fatty acid synthesis in weaned piglets treated with dextran sodium sulfate (DSS) as a model for inflammatory bowel diseases. Twenty-two piglets were included in four experimental groups based on their diet: control, DSS (1 g/kg/b.w.+control diet), GSM (8% grape seed meal inclusion in control diet), and DSS+GSM (1 g/kg/b.w., 8% grape seed meal in control diet). After 30 days, the colon content was isolated and used for microbiota sequencing on an Illumina MiSeq platform. QIIME 1.9.1 pipeline was used to process the raw sequences. Both GSM and DSS alone and in combination affected the diversity indices and Firmicutes:Bacteroidetes ratio, with significantly higher values in the DSS-afflicted piglets for Proteobacteria phylum, Roseburia, Megasphera and CF231 genus, and lower values for Lactobacillus. GSM with high-fiber, polyphenol and polyunsaturated fatty acid (PUFA) content increased the production of butyrate and isobutyrate, stimulated the growth of beneficial genera like Prevotella and Megasphaera, while countering the relative abundance of Roseburia, reducing it to half of the DSS value and contributing to the management of the DSS effects.

MicroRNA profiling in kidney in pigs fed ochratoxin A contaminated diet.

OTA is a toxic metabolite produced by fungus belonging to Aspergillus and Penicillium genera. Kidney is the main target of this toxin; OTA is considered as one of the etiological factors at the origin of the human Balkan endemic nephropathy. microRNA are short non-coding transcrips (18-22 nucleotides in length) regulating key cellular processes. Various miRNAs have been established to play important roles in development of renal carcinoma and urothelial cancer. The objective of this study is to analyse the miRNA profiling in the kidney of piglets experimentally intoxicated with feed contaminated with OTA. Fifteen piglets (five pigs/group) were randomly distributed into 3 groups, fed normal diet (Group 1: control), or diets contaminated with OTA in two concentrations: 50 µg OTA/kg feed (Group 2: 50 µg OTA/kg feed) or 200 µg OTA/kg feed (Group 3: 200 µg OTA/kg feed) for 28 days. At the end of the experiment blood samples were taken for serological analyses. Animals from control group and 200 µg OTA/kg feed were sacrificed and kidney samples were taken for histological and molecular analyses. As resulted from molecular profiling study there are 8 miRNA differentially expressed in OTA kidney vs control kidney, in which five miRNA were overexpressed in the kidney of OTA intoxicated animals: miR-497 (FC = 6.34), miR-133a-3p (FC = 5.75), miR-423-3p (FC = 5.48), miR-34a (FC = 1.68), miR-542-3p (1.65) while three miRNA were downregulated: miR-421-3p (FC = -3.96); miR-490 (FC = -3.87); miR-9840-3p (FC = -2.13). The altered miRNAs as effect of OTA are strongly connected to the engine of cancer, disturbing nodal points in different pathways, as TP53 signalling. This proof-of-concept study proves the actual utility of miRNAs as biomarkers of mycotoxin exposure, including OTA.

The Impact of Dietary Grape Seed Meal on Healthy and Aflatoxin B1 Afflicted Microbiota of Pigs after Weaning.

The study investigated the effect of grape seed (GS) meal, aflatoxin (AFB1), or their combination on the large intestine microbiota of weanling piglets. Twenty-four piglets were allocated into four groups based on diet composition: (1) Control group; (2) AFB1 (320 g/kg feed) group; (3) GS group (8% inclusion in the diet); (4) AFB1 + GS group. After 30 days of experiment, the colon content was used for microbiota analyses; after isolation of total bacterial genomic DNA, V3/V4 regions of the 16S rRNA amplicons were sequenced using the Illumina MiSeq platform. The raw sequences were analyzed using the v.1.9.1 QIIME pipeline software. 157 numbers of OTUs were identified among all four dietary groups with 26 of them being prevalent above 0.05% in the total relative abundance. GS and AFB1 increase the relative abundance of phylum Bacteroidetes and Proteobacteria, while decreasing the Firmicutes abundance in a synergic manner as compared with the individual treatments. An additive or synergistic action of the two treatments was identified for Lactobacillus, Prevotella and Campylobacter, while rather an antagonistic effect was observed on Lachnospira. The action mechanisms of aflatoxin B1 and grape seed meal that drive the large intestine microbiota to these changes are not known and need further investigations.

Comparative effect of ochratoxin A on inflammation and oxidative stress parameters in gut and kidney of piglets.

Ochratoxin A (OTA) is a secondary metabolite produced by fungi of Aspergillus and Penicillium genra. OTA is mainly nephrotoxic but can also cause hepatotoxicity, mutagenicity, teratogenicity, neurotoxicity and immunotoxicity. As recent studies have highlighted the close relationship between gastrointestinal tract and kidney, as principal organs involved in absorption and respective excretion of xenobiotics, the aim of the present study was to analyze the effect of a subchronic exposure (30 days) to 0.05 mg/kg OTA on immune response and oxidative stress parameters at the level of intestine and kidney of young swine. The experiment was realised on twelve crossbred weaned piglets randomly allotted to both control group or toxin group fed 0.050 mg OTA/kg feed. Our results have shown that a subchronic intoxication with a low dose of OTA for 30 days affected the immune response and the anti-oxidant self-defense at gut and kidney level. The gene expression of both markers of signaling pathways involved in inflammation and inflammatory cytokines were affected in a much higher extent in the gut than in the kidney Of OTA intoxicated piglets.

Dysregulation of anergy-related factors involved in regulatory T cells defects in Systemic Lupus Erythematosus patients: Rapamycin and Vitamin D efficacy in restoring regulatory T cells.

AIM: Systemic Lupus Erythematosus (SLE) patients display dysfunctions in T cell activation and anergy. Therefore the aims of our study were to explore the expression of anergy-related factors in CD4+ T cells in relationship with regulatory T cells (Tregs) frequency in SLE patients and to identify strategies to redress these defects. METHOD: Casitas B-cell lymphoma b (Cbl-b) and 'gene related to anergy in lymphocytes' (GRAIL) proteins were analyzed in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (HD) by immunoblotting. cbl-b, grail, growth response factors (egr)2 and egr3 messenger RNAs (mRNAs) were evaluated by real-time polymerase chain reaction in SLE and HD PBMCs and CD4+ T cells. Phenotypic and functional characterization of CD4+ T cells was performed by flow cytometry. Tregs expansion protocol consisted in culturing CD4+ T cells for 14 or 21 days of experimental activation with anti-CD3 and anti-CD28 monoclonal antibodies, human recombinant interleukin (hrIL)-2, in the absence or presence of rapamycin (Rapa) or 1,25-(OH)2D3 (vitamin D: VitD). RESULTS: SLE PBMCs expressed low levels of Cbl-b and GRAIL proteins. Both SLE PBMCs and CD4+ T cells expressed low levels of egr2/3 mRNAs. SLE patients had a reduced number of Tregs with impaired suppressive activity. An association between egr2 mRNA level in CD4+ T cells and Tregs percentage was identified. Experimental activation of CD4+ T cells in the presence of hrIL-2 and Rapa or VitD induced the expansion of SLE Tregs. However, on long-term, only Rapa exposure of SLE CD4+ T cells yielded high numbers of Tregs with sustained suppressive activity. CONCLUSION: Our results suggest a new strategy to correct defects in CD4+ T cell tolerance mechanisms that may prove beneficial in SLE.

Effects of zearalenone on oxidative stress and inflammation in weanling piglets.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of Fusarium genera. Piglets were fed for 18 days with a control or a ZEN (316 ppb) contaminated diet. At the end of the experiment tissue samples were taken for assessment of: lymphocyte proliferation, monocytes and granulocytes respiratory burst, inflammatory cytokine synthesis in blood and liver, expression of genes involved in oxidative stress or in inflammation, plasma biochemical parameters, total antioxidant status and nitric oxide synthesis. In blood, ZEN increases the respiratory burst of monocytes and the inflammatory cytokine (TNF alpha, IL-1 beta, IFN gamma) synthesis, while in liver, ZEN decreases the synthesis of all inflammatory cytokines investigated. In liver and spleen, different effect on the expression of genes involved in oxidative stress and inflammation was observed. While in liver, ZEN decrease the expression of cyclooxigenase gene, but increase the expression of glutathione peroxydase and catalase genes; in spleen, ZEN induces a decrease of the superoxide dismutase gene expression together with an increase of the cyclooxigenase. In conclusion, our results showed that liver, spleen and blood may also be target tissues in weanling piglets fed ZEN contaminated diet, with different effects on oxidative stress and inflammation.