Project description:Yamoa⢠is marketed and sold as a dietary supplement with anecdotal positive effects in asthma and hay fever. We determined that Yamoa⢠(ground bark of Funtumia elastica tree) stimulated innate immunity in part by affecting gamma delta T cells. Yamoa⢠had distinct priming effects, very similar to, but more robust than, that of lipopolysaccharide (LPS), on bovine, mouse and human gamma delta T cells. However, the optimal effect was dependent on the presence of accessory cells. Gene expression patterns in bovine gamma delta T cells and monocytes induced by Yamoa⢠were very similar to those induced by ultrapure LPS, but the agonists in Yamoa⢠did not signal entirely through TLR4. Yamoa⢠stimulated human cells to produce cytokines involved innate protection. The bioactive component of Yamoa⢠was delineated to a complex polysaccharide fraction (Yam-I). Intraperitoneal injection of Yamoa⢠and very low doses of Yam-I in mice induced rapid increases peritoneal neutrophils directed by changes chemokine expression. Yamoa⢠and Yam-I were effective as therapeutic treatments in mice with Salmonella enterica serotype Typhimurium (ST) induced enterocolitis that resulted in decreased bacterial counts in feces. This initial characterization of the immune stimulatory properties of polysaccharides derived from Yamoa⢠suggests potential mechanisms for positive effects in asthma and that they have potential for application in infectious disease settings. . Experiment Overall Design: To begin to understand the effects of Yamoa in innate immunity, we investigated the global gene expression profiles of stimulated bovine gamma delta T cells. Peripheral blood from 3 neonatal bovine calves was collected. gamma delta T cells were sorted to >97% purity using a FACS Vantage. Cells were placed in culture and stimulated with either an aqueous extract of Yamoa (32.6ug/ml), ultrapure LPS [uLPS (10ug/ml)] or PBS for 4 hours after which RNA was extracted and processed for microarray analysis.
Project description:The process of early development of mammals is subtly and accurately controlled by the regulation networks of embryo cells. Time course expression data measured at different stages during early embryo development process can give us valuable information by revealing the dynamic expression patterns of genes in genome wide scale. In this study, bovine embryo expression data were generated at oocyte, one cell stage, two cell stage, four cell stage, eight cell stage, sixteen cell stage, morula, and blastocyst; Human embryo expression data were generated at one cell stage, two cell stage, four cell stage, eight cell stage, morula, and blastocyst; Mouse embryo expression data were generated at one cell stage, two cell stage, four cell stage, eight cell stage, morula, and blastocyst. Experiment Overall Design: Bovine, Human, and Mouse embryos were harvested at successive stage from oocyte to blastocyste. Total RNAs were extracted, amplified and hybridized onto Affymetrix microarrays.
Project description:Coordinated interactions between ovarian granulosa and theca cells are required for female endocrine function and fertility. To elucidate these interactions the regulation of the granulosa and theca cell transcriptomes during bovine antral follicle development were investigated. Granulosa cells and theca cells were isolated from small (<5 mm), medium (5-10 mm), and large (>10 mm) antral bovine follicles. A microarray analysis of 24,000 bovine genes revealed that granulosa cells and theca cells each had gene sets specific to small, medium and large follicle cells. Transcripts regulated (i.e., minimally changed 1.5-fold) during antral follicle development for the granulosa cells involved 446 genes and for theca cells 248 genes. Only 28 regulated genes were common to both granulosa and theca cells. Regulated genes were functionally categorized with a focus on growth factors and cytokines expressed and regulated by the two cell types. Candidate regulatory growth factor proteins mediating both paracrine and autocrine cell-cell interactions include macrophage inflammatory protein (MIP1 beta), teratocarcinoma-derived growth factor 1 (TDGF1), stromal derived growth factor 1 (SDF1; i.e., CXCL12), growth differentiation factor 8 (GDF8), glia maturation factor gamma (GMFG), osteopontin (SPP1), angiopoietin 4 (ANGPT4), and chemokine ligands (CCL 2, 3, 5, and 8). The current study examined granulosa cell and theca cell regulated genes associated with bovine antral follicle development and identified candidate growth factors potentially involved in the regulation of cell-cell interactions required for ovarian function. Experiment Overall Design: Granulosacell RNA samples from three groups of follicles different in size - small, medium, and large (pooled untreated ovaries) are compared between each other. Each group has 2 separate biological replicas; each replica contained pooled RNA from 20-40 ovaries from 6-10 different animals.
Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.
Project description:γδ T cells are lymphocytes that function in both innate and adaptive immune responses, and our laboratory has previously reported that both bovine and human γδ T cells are primed for secondary responses by tannin components of the unripe apple peel (APP). In this report we began to investigate the mechanism by which priming occurs. We performed a microarray analysis on sorted bovine γδ T cells treated with APP and observed significant increases in transcripts encoding select inflammatory cytokines, such as GM-CSF, IL-8, and IL-17, but not markers of TCR stimulation such as IFNγ. When injected into the gut of mice, APP induced a robust influx of neutrophils into both the blood and peritoneum as measured by FACS. Importantly, both GM-CSF and IL-8 proteins were detected in these animals. Further studies were performed using the human γδ T cell-line MOLT-14, as large numbers of cells were required for subsequent experiments. Using these cells we found that both the GM-CSF and IL-8 mRNAs were significantly upregulated and stabilized in cells treated with APP. Finally, we show that the ERK1/2 MAPK pathway was activated in APP-treated MOLT-14 cells, and that this pathway plays a role in the mRNA stabilization we observed. Together, our data describe a unique inflammatory response in bovine, murine, and human γδ T cells in response to APP, and suggest that mRNA stability mechanisms could account for the priming phenotype we previously observed.APP has a distinctive gamma delta T cells specific priming activity. Keywords: comparison of 2 treatment types, tannin, gamma delta T cells To begin to understand the effects of APP in innate immunity, we investigated the global gene expression profiles of stimulated bovine gamma delta T cells. Peripheral blood from 2 neonatal bovine calves was collected. gamma delta T cells were sorted to >97% purity using a FACS Vantage. Cells were placed in culture and stimulated with either an aqueous extract of APP (42.2ug/ml) or PBS for 4 hours after which RNA was extracted and processed for microarray analysis following standard protocols from Affymetrix.