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A2ALLGRITS_2009_10_27
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Homo sapiens
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Sunil Kurian
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Liver biopsies from donors of transplants. Comparing biopsy at procurement and post resection.
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CD4 Activated and Unactivated Hu133
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Homo sapiens
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Sunil Kurian
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Compare gene expression in CD4 activated and unactivated
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Controls vs CAN Biopsy Study
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Homo sapiens
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Sunil Kurian
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Biopsies from 33 kidney (11 Well-functioning and 22 Chronic Allograft Nephropathy) transplant patients were profiled on Affymetrix HG-U133 PLus 2.0 arrays.
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Discovery and validation of a gene expression profile for human islet integrity and transplant functionality
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Homo sapiens
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Sunil Kurian
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The pathophysiology of type 1 diabetes (T1DM) is the result of the destruction of the insulin-producing beta cells, and a promising treatment paradigm for T1DM is replacement of the missing beta cells with islet cells isolated from donor organs. Islet transplantation has been shown to improve glycemic control and induce insulin independence in patients with severe type 1 diabetes. In the majority of patients, islet therapy provides one or more years of insulin independence, long-term stability of plasma glucose levels, and, most importantly, several years of freedom from hypoglycemic episodes. There has been rapid progress in the field over the last decade, but there remain some serious obstacles to providing consistent results between the various transplant centers. These include allo- and residual auto-immunity directed against the islet graft, and the availability of consistent, healthy, functional cells for transplantation. This latter issue is further confounded by the lack of reliable methods for gauging the suitability of specific preparations of islet cells for clinical protocols. We identified a set of genes that correctly predicted reversal of diabetes with high accuracy . We also evaluated the gene expression profiles in conjunction with other in vitro islet quality criteria including glucose-responsive oxygen consumption rates and percentages of apoptotic beta cells.
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Genome Wide Analysis of Immune Activation in Human T and B Cells Reveals Distinct Classes of Alternatively Spliced Genes
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Homo sapiens
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Sunil Kurian
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Alternative splicing of pre-mRNA is a mechanism that increases the protein diversity of a single gene by differential exon inclusion during post-transcriptional processing. While alternative splicing is established to occur during lymphocyte activation, little is known about the role it plays during the immune response. Our study is among the first reports of a systematic genome-wide analysis using whole exon DNA microarrays integrating alternative splicing and differential gene expression. Purified human CD2+ T or CD19+ B cells were activated using protocols to model the early events in post-transplant allograft immunity and sampled as a function of time during the process of immune activation. Here we show that 3 distinct classes of alternatively spliced and/or differentially expressed genes change in an ordered manner as a function of immune activation. We mapped our results to function-based canonical pathways and demonstrated that some are populated by only one class of genes, like integrin signaling, while other pathways, such as purine metabolism and T cell receptor signaling, are populated by all three classes of genes. Our studies augment the current view of T and B cell activation in immunity that has been based exclusively upon differential gene expression by providing evidence for a large number of molecular networks populated as a function of time and activation by alternatively spliced genes, many of which are constitutively expressed.
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Isolation and angiogenesis by endothelial progenitors in the fetal liver
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Mus musculus
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Sunil Kurian
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While others have reported that fetal liver contains a population of endothelial progenitors based on expression of cell surface markers or culture assays, this is the first proof of a CD31+Sca1+ progenitor by demonstrating highly efficient in vivo angiogenesis and a direct connection to the host vasculature. We have developed a novel isolation method based on collagenase digestion and culture on a fetal liver-derived feeder layer and demonstrate that the feeder cells or their supernatants are required for endothelial progenitor survival and proliferation.
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Laparoscopic Donor Nephrectomy Gene Expression Profiling Compared to Healthy Control Kidneys
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Homo sapiens
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Sunil Kurian
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We compared gene expression profiles from six donor kidneys prior to surgical manipulation to six kidneys removed after laparoscopic donor nephrectomy (LDN) and several hours of CO2 pneumoperitoneum. Biopsies were obtained from renal cortex and hybridized to Affymetrix HG-U133A GeneChips. For control kidneys we identified 1380 genes present on all 6 samples that had a signal intensity greater than 1000. Functional classification of these revealed genes for cellular signaling (201; 15%), regulation of transcription (156; 11%), cellular transport (144; 10%) and cellular metabolism (111; 8%). A class comparison between the controls and LDN kidneys yielded 865 differentially expressed genes. Functional classification of the 502 genes differentially up-regulated in LDN kidneys identified associations with apoptosis, cell adhesion, cell signaling, regulation of cell growth/proliferation, immune/inflammation, ischemia/stress response and proteolysis/peptidolysis. These data demonstrate an altered renal transcriptome induced by several hours of CO2 pneumoperitoneum and laparoscopic surgery characterized by up-regulation of ischemia and injury associated genes.
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Lentiviral gene delivery of vMIP-II
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Homo sapiens
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Sunil Kurian
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We engineered a lentiviral gene vector with vMIP-II, transduced both mature endothelial cells and endothelial cell progenitors, and transplanted these gene-modified cells in Matrigel templates as an in vivo angiogenesis model. The results are the first demonstration in a cell transplantation setting that vMIP-II is proangiogenic in vivo and can deliver this function by vector-mediated gene delivery to both mature endothelial cells and progenitors.
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Peripheral Blood Gene Expression Profiles of Bipolar Disorder
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Homo sapiens
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Sunil Kurian
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There are to date no objective clinical laboratory blood tests for mood disorders. The current
reliance on patient self-report of symptom severity and on the clinicians’ impression is a ratelimiting
step in effective treatment and new drug development. We propose, and provide proof
of principle for, an approach to help identify blood biomarkers for mood state. We measured
whole-genome gene expression differences in blood samples from subjects with bipolar
disorder that had low mood vs those that had high mood at the time of the blood draw, and
separately, changes in gene expression in brain and blood of a mouse pharmacogenomic
model. We then integrated our human blood gene expression data with animal model gene
expression data, human genetic linkage/association data and human postmortem brain data,
an approach called convergent functional genomics, as a Bayesian strategy for crossvalidating
and prioritizing findings. Topping our list of candidate blood biomarker genes we
have five genes involved in myelination (Mbp, Edg2, Mag, Pmp22 and Ugt8), and six genes
involved in growth factor signaling (Fgfr1, Fzd3, Erbb3, Igfbp4, Igfbp6 and Ptprm). All of these
genes have prior evidence of differential expression in human postmortem brains from mood
disorder subjects. A predictive score developed based on a panel of 10 top candidate
biomarkers (five for high mood and five for low mood) shows sensitivity and specificity for
high mood and low mood states, in two independent cohorts. Our studies suggest that blood
biomarkers may offer an unexpectedly informative window into brain functioning and disease
state.
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Peripheral Blood Gene Expression Profiles of Psychotic Disease
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Homo sapiens
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Tony Mondala
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There are to date no objective clinical laboratory blood tests for psychotic disease states. We provide proof of principle for a convergent functional genomics (CFG) approach to help identify and prioritize blood biomarkers for two key psychotic symptoms, one sensory (hallucinations) and one cognitive (delusions). We used gene expression profiling in whole blood samples from patients with schizophrenia and related disorders, with phenotypic information collected at the time of blood draw, then cross-matched the data with other human and animal model lines of evidence. Topping our list of candidate blood biomarkers for hallucinations, we have four genes decreased in expression in high hallucinations states (Fn1, Rhobtb3, Aldh1l1, Mpp3), and three genes increased in high hallucinations states (Arhgef9, Phlda1, S100a6). All of these genes have prior evidence of differential expression in schizophrenia patients. At the top of our list of candidate blood biomarkers for delusions, we have 15 genes decreased in expression in high delusions states (such as Drd2, Apoe, Scamp1, Fn1, Idh1, Aldh1l1), and 16 genes increased in high delusions states (such as Nrg1, Egr1, Pvalb, Dctn1, Nmt1, Tob2). Twenty-five of these genes have prior evidence of differential expression in schizophrenia patients. Predictive scores, based on panels of top candidate biomarkers, show good sensitivity and negative predictive value for detecting high psychosis states in the original cohort as well as in three additional cohorts. These results have implications for the development of objective laboratory tests to measure illness severity and response to treatment in devastating disorders such as schizophrenia
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Peripheral Blood Lymphocytes in patients with Chronic Allograft Nephropathy
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Homo sapiens
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Sunil Kurian
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Peripheral Blood Lymphocytes in patients with Chronic Allograft Nephropathy experiment. HGU133 Plus 2.0 array controls hybridization and normalization.
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UPENN Living Donor vs Deceased Donor Liver Tx Study
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Homo sapiens
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Sunil Kurian
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Unique patterns of early gene expression are seen in LD and DD liver grafts, correlating with protein expression and clinical results, demonstrating distinct inflammatory profiles and significant down-regulation of metabolic pathways in LD grafts.
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VA Metabolic Study RME 2009-08-11
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Homo sapiens
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Tony Mondala
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Compare various metabolic disorders.
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Whole Blood in patients with Chronic Allograft Nephropathy
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Homo sapiens
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Sunil Kurian
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Whole Blood in patients with Chronic Allograft Nephropathy experiment. HGU133 Plus 2.0 array controls hybridization and normalization.
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Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study
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Homo sapiens
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Sunil Kurian
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Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70ml of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. Results: From two sets of fingerstick collections, about 70uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6ng totRNA with an average RIN of 9.3. The post-amplification yields for 50ng of totRNA averaged at 7.0ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r2 values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.
Conclusions: Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.
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