Project description:gd T cells have an important yet incompletely defined role in inflammation associated with a variety of infectious and autoimmune conditions. To better understand the precise roles of gd T cells relative to ab T cells in a specific infection, we utilized Salmonella Enterica Serovar Typhimurium (S. typhimurium) infection in cattle as it is a leading cause of disease in cattle and closely approximates S. typhimurium-induced enterocolitis in humans. To best represent phenotype and gene expression changes occurring in the gut mucosa early in S. typhimurium infection, gd and ab T cells were collected directly from the mesenteric lymphatic ducts and analyzed by FACS or immediately sorted and processed for microarray analysis. Gene expression profiles were compared at intervals during infection within T cell subsets. The majority of gene expression changes in both subsets occurred 48 hours after infection. In response to S. typhimurium infection there was an increase in expression of several genes in gd T cells which were indicative of activation, proliferation and innate function, whereas in ab T cells gene expression changes suggested a lack of S. typhimurium-specific response. This work represents the first focus on gene expression trends in tissue-derived T lymphocytes in an in vivo model that is highly relevant to human S. typhimurium-induced enterocolitis. Experiment Overall Design: For one mock infection (calf 156) and two experimental S. typhimurium infections (calves 112 and 162), gd and ab lymphatic T cells were stained with GD3.8 directly conjugated to FITC, washed, and sorted on a Vantage SE cell sorter (BD Immunocytometry Systems) as previously described. Sorted gd and ab T cells were collected directly into TRIzol reagent (Invitrogen; calf 112) and lysed or suspended in Buffer RLT (Qiagen; calves 156 and 162) and lysed using Qiashredder columns, then frozen at -80oC. RNA was extracted following the manufacturerâs protocol for Trizol (Invitrogen) extraction, or RNeasy (Qiagen) column purification, assessed on a Bioanalyzer 2100 (Agilent Technologies), and amplified either using Affymetrix Two-cycle (calf 112) target labeling protocol with 100 ng total RNA or the One-cycle protocol (calves 156 and 162) with approximately 1.6 micrograms of total RNA as described in the GeneChipÂ® Expression Analysis Technical Manual (June 2004). Hybridizations to GenechipÂ® Bovine Genome Arrays (Affymetrix) were performed with 15 micrograms biotin labeled cRNA. Washing and staining was performed in the GeneChipÂ® Fluidics Station 450 using the Midi_euk2v3 protocol. Chip scans were performed on the Affymetrix GeneChipÂ® Scanner 3000. GeneChipÂ® Operating Software (GCOS v.1.1, Affymetrix) was used for data collection. Experiment Overall Design: Table I represents annotated genes of potential interest that changed 2 fold or greater in expression between 0 and 48 hours post-Salmonella infection (calves 112 and 162) or mock-infection (calf 156) in T cell subsets.

Project description:Yamoaâ¢ is marketed and sold as a dietary supplement with anecdotal positive effects in asthma and hay fever. We determined that Yamoaâ¢ (ground bark of Funtumia elastica tree) stimulated innate immunity in part by affecting gamma delta T cells. Yamoaâ¢ 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 Yamoaâ¢ were very similar to those induced by ultrapure LPS, but the agonists in Yamoaâ¢ did not signal entirely through TLR4. Yamoaâ¢ stimulated human cells to produce cytokines involved innate protection. The bioactive component of Yamoaâ¢ was delineated to a complex polysaccharide fraction (Yam-I). Intraperitoneal injection of Yamoaâ¢ and very low doses of Yam-I in mice induced rapid increases peritoneal neutrophils directed by changes chemokine expression. Yamoaâ¢ 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 Yamoaâ¢ 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.

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