Epithelial cells in the intestinal mucosa maintain gut homeostasis by interacting with different types of microbiota. Proper appropriate immune responses in the intestinal epithelium are essential for the preservation of the intestinal homeostasis. In the present study, we aimed to identify genotypic and phenotypic changes in mice following oral feeding of various substances which has been shown to differentially affect intestinal homeostasis. We orally fed C57BL/6 mice for either one or seven days with one of the four substances: dextran sulfate sodium (DSS); Typhoid VI Polysaccharide vaccine (Vi vaccine); antibiotic cocktails (AB) of ampicillin, vancomycin, neomycin, and metronidazole; or(probiotics)consisting of Lactobacillus Rhamnosus R0011and L. Acidophilus R0052.While DSS and AB feeding resulted in severe gut pathology characterized by infiltration of inflammatory cells, epithelium shedding, and distortion of paneth cells. Vi vaccine and probiotics feeding resulted in phenotypic improvement of the gut health characterized by epithelial cell proliferation and increased formation of tight junctions between epithelial cells. Interestingly, microarray data showed significant increase in the expression levels of genes regulating cell proliferation and intestinal homeostasis in the gut epithelium of probiotics-and Vi vaccine-fed mice compared to DSS-or AB-fed mice. In addition, expression levels of genes regulating cell death and inflammation were significantly increased in the gut epithelium of DSS- and AB-fed mice. These results suggest that intestinal homeostasis play a pivotal role in maintaining gut health and, subsequently, in protecting host against enteric bacteria and external pathogens infection.
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Specimen part
View SamplesOver one million prostate biopsies are performed in the U.S. every year. However, pathology examination is not definitive in a significant percentage of cases due limited diagnostic tumor. We have observed that the microenvironment of prostate tumor cells exhibits numerous differential gene expression changes and have asked whether such information can be used to distinguish tumor from nontumor. We initially compared expression analysis data (Affymetrix U133plus2) from 18 volunteer biopsy specimens to 17 specimens containing largely tumor-adjacent stroma and identified 964 significant (p_adj < 0.01 and B > 0) expression changes. These genes were filtered to eliminate possible aging-related genes and genes expressed in tumor cells > 10% of the stroma cell expression level leading to 23 candidate genes (28 Affymetrix probe sets). A classifier based on the 28 probe sets was tested on 289 independent cases, including 195 tumor-bearing cases, 99 nontumor cases (normal biopsies, normal autopsies, remote stroma as well as pure tumor adjacent stroma) all with accuracies >85%, sensitivities >90% and specificities >85%. These results indicate that the prostate cancer microenvironment exhibits reproducible changes useful for categorization as tumor and nontumor.
In silico estimates of tissue components in surgical samples based on expression profiling data.
Subject
View SamplesProstate cancer gene expression profiles were studied in this project. A total RNA from 148 prostate sample with various amount of different cell types were hybridized to Affymetrix U133A arrays. The percentage of different cell types vary considerably among samples and were determined by pathologist. Cell type specific genes can be determined by linear regression using the methods of Stuart et al, PNAS, 2004.
In silico estimates of tissue components in surgical samples based on expression profiling data.
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View SamplesAdjuvants are critical for the success of vaccines, and agonists for microbial pattern recognition receptors are promising new candidates. A mechanism for the immune enhancing role of adjuvants is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double stranded RNA (poly ICLC), a ligand for TLR3 and MDA-5 cytosolic RNA helicase. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC showed upregulation of genes involved in multiple innate immune pathways in all subjects, including interferon and inflammasome signaling. Blocking of type I interferon receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate pathways were similarly induced in volunteers immunized with the highly efficacious Yellow Fever Vaccine. Therefore a chemically defined microbial agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immunity, here for a live attenuated viral vaccine in humans.
Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans.
Time
View SamplesThe Her-2/Neu-positive mouse breast cancer cell line was serially co-cultured with minced brain, bone marrow, and lung tissue in an intravital microscopy chamber mounted on the dorsal skinfold of nude mice, alternating with growth in vitro. Gene expression analysis was performed on the cells grown in culture after sorting and further growth in vitro. Gene expression under these growth conditions differed in time and according to the co-cultivated organ tissue. This study reveals genes that are expressed by cells as they adapt differentially to various foreign tissue microenvironments, and may represent a paradigm to discover gene expression changes that occur immediately upon extravasation when cancer metastasizes.
Effects of different tissue microenvironments on gene expression in breast cancer cells.
Cell line
View SamplesEffector CD8+ T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF- and TNF-. We investigated the difference between CXCR1+ and CXCR1- subsets of human effector CD27-CD28-CD8+ T cells. Both subsets similarly expressed cytolytic molecules and exerted substantial cytolytic activity, whereas only the CXCR1- subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1+ subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1- subset and that of pro-apoptotic DAPK1 in the CXCR1+ subset. The IL-2 producers were more frequently found in the IL-7R+ subset of the CXCR1- effector CD8+ T cells than in the IL-7R- subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1- subset. The present study has highlighted a novel subset of effector CD8+ T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8+ T cells.
Functional heterogeneity of human effector CD8+ T cells.
Sex, Specimen part
View SamplesPrevious studies have revealed that UV-stimulation of a variety of cells leads to activation of the EGF receptor, induction of Egr1, growth inhibition and apoptosis. On the other hand both Egr1 and EGF receptor activation are implicated in promoting the progression of prostate cancer. We treated M12 tumorigenic prostate epithelial cells which express little Egr1 with UV irradiation which rapidly activated the EGF receptor and elevated Egr1. Treatment with specific EGFR and ERKI/II inhibitors (PD153035 and UO126, respectively) confirmed that the upregulation of Egr1 was downstream of EGFR and ERKI/II Map kinase pathway. ChIP on chip experiments using Egr1 antibody identified 288 significantly bound promoters upon UV stimulation. Of these target genes, 40% had consensus Egr1 site in their promoters, considerably greater than that expected by chance (p < 0.005). The array binding results were validated by PCR analysis of 25 genes using DNA from conventional IP experiment. Affymetrix gene expression analysis of UV treated and control cells confirmed that a significant number of these bound promoters showed gene expression changes. Addition of siRNA to Egr1 confirmed that the gene expression changes were dependent upon Egr1 expression. Addition of EGF led to similar expression changes for nine tested genes. Proliferation and apoptosis assays confirmed that M12 cells undergo growth arrest and apoptosis following UV irradiation. Moreover, addition of EGF also promoted significant growth inhibition. These results indicate the M12 cells undergo a EGF receptor dependent apoptosis response upon UV-stimulation and that Egr1 mediates the regulation of numerous genes downstream of the EGF receptor that are associated with this response.
Egr1 regulates the coordinated expression of numerous EGF receptor target genes as identified by ChIP-on-chip.
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