PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-24 (24)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
Document Types
1.  Transcriptional Signatures as a Disease-Specific and Predictive Inflammatory Biomarker for Type 1 Diabetes 
Genes and immunity  2012;13(8):593-604.
The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery, necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding therapeutic decisions, and monitoring interventions. We previously demonstrated that plasma samples from recent-onset Type 1 diabetes (RO T1D) patients induce a proinflammatory transcriptional signature in freshly drawn peripheral blood mononuclear cells (PBMCs) relative to that of unrelated healthy controls (HC). Here, using cryopreserved PBMC, we analyzed larger RO T1D and HC cohorts, examined T1D progression in pre-onset samples, and compared the RO T1D signature to those associated with three disorders characterized by airway infection and inflammation. The RO T1D signature, consisting of interleukin-1 cytokine family members, chemokines involved in immunocyte chemotaxis, immune receptors, and signaling molecules, was detected during early pre-diabetes and found to resolve post-onset. The signatures associated with cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, patients with confirmed bacterial pneumonia, and subjects with H1N1 influenza all reflected immunological activation, yet each were distinct from one another and negatively correlated with that of T1D. This study highlights the remarkable capacity of cells to serve as biosensors capable of sensitively and comprehensively differentiating immunological states.
doi:10.1038/gene.2012.41
PMCID: PMC4265236  PMID: 22972474
Type 1 diabetes; Cystic Fibrosis; Influenza; Gene expression profiling; Biomarker; Inflammation
3.  Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases 
Diabetes  2013;63(1):68-74.
Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4–1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1−/− recipients, even when CD25+ cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4+CD25+ regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.
doi:10.2337/db13-0192
PMCID: PMC3868049  PMID: 23974926
4.  Requirements for growth and IL-10 expression of highly purified human T regulatory cells 
Journal of clinical immunology  2012;32(5):1118-1128.
Human regulatory T cells (TR) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9D4) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting TR cell proliferation, and under these conditions the proliferative capacity of TR cells was comparable to conventional CD4 lymphocytes. Blocking TGF-β activity abrogated IL-10 expression from these cells, while addition of TGF-β resulted in IL-10 production. These data demonstrate that highly purified populations of TR cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of TR cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-β is critical to enable human TR cells to express IL-10.
doi:10.1007/s10875-012-9701-4
PMCID: PMC4271826  PMID: 22562448
5.  Diubiquitin (Ubd) is a susceptibility gene for virus-triggered autoimmune diabetes in rats 
Genes and immunity  2014;15(3):168-175.
Genetic studies of type 1 diabetes (T1D) have been advanced by comparative analysis of multiple susceptible and resistant rat strains with a permissive class II MHC haplotype, RT1u. LEW.1WR1 (but not resistant LEW.1W or WF) rats are susceptible to T1D induced by a TLR3 agonist (poly I:C) followed by infection with parvovirus. We have mapped genetic loci for virus-induced T1D susceptibility, identifying a major susceptibility locus (Iddm37) near the MHC. Iddm37 homologues on mouse and human chromosomes are also diabetes-linked. We report that a major effect gene within Iddm37 is diubiquitin (Ubd). Gene expression profiling of pancreatic lymph nodes in susceptible and resistant rats during disease induction showed differences in Ubd transcript abundance. The LEW.1WR1 Ubd promoter allele leads to higher inducible levels of UBD than that of LEW.1W or WF. Using zinc finger nucleases (ZFN), we deleted a segment of the LEW.1WR1 Ubd gene and eliminated its expression. UBD-deficient rats show substantially reduced diabetes after viral infection. Complementation studies show that there may be another diabetes gene in addition to Ubd in the Iddm37 interval. These data prove that Ubd is a diabetes susceptibility gene, providing insight into the interplay of multiple genes and environmental factors in T1D susceptibility.
doi:10.1038/gene.2013.72
PMCID: PMC4260472  PMID: 24452267
Autoimmune diabetes; susceptibility genes; virus-induced; rat model
6.  Temporal induction of immunoregulatory processes coincides with age-dependent resistance to viral-induced Type 1 Diabetes 
Genes and immunity  2013;14(6):387-400.
The dilute plasma cytokine milieu associated with Type 1 diabetes (T1D), while difficult to measure directly, is sufficient to drive transcription in a bioassay that uses healthy leukocytes as reporters. Previously, we reported disease-associated, partially IL-1 dependent, transcriptional signatures in both T1D patients and the BioBreeding (BB) rat model. Here we examine temporal signatures in congenic BBDR.lyp/lyp rats that develop spontaneous T1D, and BBDR rats where T1D progresses only after immunological perturbation in young animals. After weaning, the BBDR temporal signature showed early coincident induction of transcription related to innate inflammation as well as IL-10- and TGF-β-mediated regulation. BBDR plasma cytokine levels mirrored the signatures showing early inflammation, followed by induction of a regulated state that correlated with failure of virus to induce T1D in older rats. In contrast, the BBDR.lyp/lyp temporal signature exhibited asynchronous dynamics, with delayed induction of inflammatory transcription and later, weaker induction of regulatory transcription, consistent with their deficiency in regulatory T cells. Through longitudinal analyses of plasma induced signatures in BB rats and a human T1D progressor, we have identified changes in immunoregulatory processes that attenuate a preexisting innate inflammatory state in BBDR rats, suggesting a mechanism underlying the decline in T1D susceptibility with age.
doi:10.1038/gene.2013.31
PMCID: PMC4027975  PMID: 23739610
Type 1 Diabetes; Inflammation; Gene Expression; Cytokine; Immune Regulation; Virus-induced diabetes
7.  Biobreeding rat islets exhibit reduced antioxidative defense and N-acetyl cysteine treatment delays type 1 diabetes 
The Journal of endocrinology  2013;216(2):111-123.
Islet-level oxidative stress has been proposed as a trigger for type 1 diabetes (T1D), and release of cytokines by infiltrating immune cells further elevates reactive oxygen species (ROS), exacerbating β cell duress. To identify genes/mechanisms involved with diabeto-genesis at the β cell level, gene expression profiling and targeted follow-up studies were used to investigate islet activity in the biobreeding (BB) rat. Forty-day-old spontaneously diabetic lymphopenic BB DRlyp/lyp rats (before T cell insulitis) as well as nondiabetic BB DR+/+ rats, nondiabetic but lymphopenic F344lyp/lyp rats, and healthy Fischer (F344) rats were examined. Gene expression profiles of BB rat islets were highly distinct from F344 islets and under-expressed numerous genes involved in ROS metabolism, including glutathione S-transferase (GST) family members (Gstm2, Gstm4, Gstm7, Gstt1, Gstp1, and Gstk1), superoxide dismutases (Sod2 and Sod3), peroxidases, and peroxiredoxins. This pattern of under-expression was not observed in brain, liver, or muscle. Compared with F344 rats, BB rat pancreata exhibited lower GST protein levels, while plasma GST activity was found significantly lower in BB rats. Systemic administration of the antioxidant N-acetyl cysteine to DRlyp/lyp rats altered abundances of peripheral eosinophils, reduced severity of insulitis, and significantly delayed but did not prevent diabetes onset. We find evidence of β cell dysfunction in BB rats independent of T1D progression, which includes lower expression of genes related to antioxidative defense mechanisms during the pre-onset period that may contribute to overall T1D susceptibility.
doi:10.1530/JOE-12-0385
PMCID: PMC4077722  PMID: 23111281
8.  Evaluation of Genomic Evidence for Oxidative Stress in Experimental Radiation Nephropathy 
Background
Chronic persistent oxidative stress has been proposed as a mechanism for late radiation injury to normal tissue. Using biochemical, histological, and pharmacological techniques, we have not been able to confirm this hypothesis for late renal radiation injury. Gene expression may be more revealing, especially since the initial effects of radiation are to damage DNA.
Methods
Gene array studies were done using kidney tissue from irradiated rats, with particular attention to genes pertinent to oxidative stress. The time points were from 1 to 49 days after irradiation. Cellular RNA and mitochondrial DNA were isolated, for gene expression analysis and common deletion testing, respectively.
Results
For the gene expression studies, and from over 30,000 transcripts, only nine related to oxidative stress had 1.4 fold or greater changes in expression. Mitochondrial DNA showed no changes in the common deletion.
Conclusion
These studies do not support the hypothesis of chronic oxidative stress as a mechanism for radiation nephropathy.
doi:10.4172/2327-5790.1000101
PMCID: PMC4013680  PMID: 24818171
Radiation injury; Oxidative stress; Gene array testing; Mitochondrial DNA
9.  IL-10 produced by iTreg cells controls colitis and pathogenic ex-iTreg cells during immunotherapy1 
“Natural” regulatory T (nTreg) cells that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” Treg (iTreg) cells are non-redundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg cell-produced IL-10 and iTreg cell fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naïve CD4+ T cells carrying a non-functional Foxp3 allele. At the onset of weight loss, mice were treated with both iTreg and nTreg cells where one marked subset was selectively IL-10-deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTreg cells were present but IL-10 deficient, iTreg cell-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTreg and ex-iTreg cells, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between Treg subsets that maintains mucosal tolerance. The niche supporting stable iTreg cells is limited and readily saturated, which promotes a large population of ex-iTreg cells with pathogenic potential during immunotherapy.
doi:10.4049/jimmunol.1200936
PMCID: PMC3537488  PMID: 23125413
10.  Predicting Disease-Related Subnetworks for Type 1 Diabetes Using a New Network Activity Score 
Abstract
In this study we investigated the advantage of including network information in prioritizing disease genes of type 1 diabetes (T1D). First, a naïve Bayesian network (NBN) model was developed to integrate information from multiple data sources and to define a T1D-involvement probability score (PS) for each individual gene. The algorithm was validated using known functional candidate genes as a benchmark. Genes with higher PS were found to be more likely to appear in T1D-related publications. Next a new network activity metric was proposed to evaluate the T1D relevance of protein-protein interaction (PPI) subnetworks. The metric considered the contribution both from individual genes and from network topological characteristics. The predictions were confirmed by several independent datasets, including a genome wide association study (GWAS), and two large-scale human gene expression studies. We found that novel candidate genes in the T1D subnetworks showed more significant associations with T1D than genes predicted using PS alone. Interestingly, most novel candidates were not encoded within the human leukocyte antigen (HLA) region, and their expression levels showed correlation with disease only in cohorts with low-risk HLA genotypes. The results suggested the importance of mapping disease gene networks in dissecting the genetics of complex diseases, and offered a general approach to network-based disease gene prioritization from multiple data sources.
doi:10.1089/omi.2012.0029
PMCID: PMC3459426  PMID: 22917479
11.  Identification of a Serum-Induced Transcriptional Signature Associated With Type 1 Diabetes in the BioBreeding Rat 
Diabetes  2010;59(10):2375-2385.
OBJECTIVE
Inflammatory mediators associated with type 1 diabetes are dilute and difficult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing preonset risk, and monitoring therapeutic interventions.
RESEARCH DESIGN AND METHODS
We previously utilized a novel bioassay in which human type 1 diabetes sera were used to induce a disease-specific transcriptional signature in unrelated, healthy peripheral blood mononuclear cells (PBMCs). Here, we apply this strategy to investigate the inflammatory state associated with type 1 diabetes in biobreeding (BB) rats.
RESULTS
Consistent with their common susceptibility, sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induced transcription of cytokines, immune receptors, and signaling molecules in PBMCs of healthy donor rats compared with control sera. Like the human type 1 diabetes signature, the DRlyp/lyp signature, which is associated with progression to diabetes, was differentiated from that of the DR+/+ by induction of many interleukin (IL)-1–regulated genes. Supplementing cultures with an IL-1 receptor antagonist (IL-1Ra) modulated the DRlyp/lyp signature (P < 10−6), while administration of IL-1Ra to DRlyp/lyp rats delayed onset (P = 0.007), and sera of treated animals did not induce the characteristic signature. Consistent with the presence of immunoregulatory cells in DR+/+ rats was induction of a signature possessing negative regulators of transcription and inflammation.
CONCLUSIONS
Paralleling our human studies, serum signatures in BB rats reflect processes associated with progression to type 1 diabetes. Furthermore, these studies support the potential utility of this approach to detect changes in the inflammatory state during therapeutic intervention.
doi:10.2337/db10-0372
PMCID: PMC3279523  PMID: 20682698
12.  A Requisite Role for Induced Regulatory T cells in Tolerance Based on Expanding Antigen Receptor Diversity 
Immunity  2011;35(1):109-122.
SUMMARY
Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential non-redundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.
doi:10.1016/j.immuni.2011.03.029
PMCID: PMC3295638  PMID: 21723159
13.  Global analysis of phase locking in gene expression during cell cycle: the potential in network modeling 
BMC Systems Biology  2010;4:167.
Background
In nonlinear dynamic systems, synchrony through oscillation and frequency modulation is a general control strategy to coordinate multiple modules in response to external signals. Conversely, the synchrony information can be utilized to infer interaction. Increasing evidence suggests that frequency modulation is also common in transcription regulation.
Results
In this study, we investigate the potential of phase locking analysis, a technique to study the synchrony patterns, in the transcription network modeling of time course gene expression data. Using the yeast cell cycle data, we show that significant phase locking exists between transcription factors and their targets, between gene pairs with prior evidence of physical or genetic interactions, and among cell cycle genes. When compared with simple correlation we found that the phase locking metric can identify gene pairs that interact with each other more efficiently. In addition, it can automatically address issues of arbitrary time lags or different dynamic time scales in different genes, without the need for alignment. Interestingly, many of the phase locked gene pairs exhibit higher order than 1:1 locking, and significant phase lags with respect to each other. Based on these findings we propose a new phase locking metric for network reconstruction using time course gene expression data. We show that it is efficient at identifying network modules of focused biological themes that are important to cell cycle regulation.
Conclusions
Our result demonstrates the potential of phase locking analysis in transcription network modeling. It also suggests the importance of understanding the dynamics underlying the gene expression patterns.
doi:10.1186/1752-0509-4-167
PMCID: PMC3017040  PMID: 21129191
14.  Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice  
The Journal of Clinical Investigation  2010;120(9):3167-3178.
Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor–1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1– dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-γ production by natural killer–like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.
doi:10.1172/JCI42629
PMCID: PMC2929901  PMID: 20714108
15.  An Autoinflammatory Disease Due to Homozygous Deletion of the IL1RN Locus 
The New England journal of medicine  2009;360(23):2438-2444.
SUMMARY
We describe a patient with an autoinflammatory disease in which the main clinical features are pustular rash, marked osteopenia, lytic bone lesions, respiratory insufficiency, and thrombosis. Genetic studies revealed a 175-kb homozygous deletion at chromosome 2q13, which encompasses several interleukin-1 family members, including the gene encoding the interleukin-1–receptor antagonist (IL1RN). Mononuclear cells, obtained from the patient and cultured, produced large amounts of inflammatory cytokines, with increasing amounts secreted after stimulation with lipopolysaccharide. A similar increase was not observed in peripheral-blood mononuclear cells from a patient with neonatal-onset multisystem inflammatory disorder (NOMID). Treatment with anakinra completely resolved the symptoms and lesions.
doi:10.1056/NEJMoa0809568
PMCID: PMC2803085  PMID: 19494219
16.  Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary: Insights into TCDD-induced endocrine disruption and reproductive toxicity 
TCDD is a reproductive toxicant and endocrine disruptor, yet the mechanisms by which it causes these reproductive alterations are not fully understood. In order to provide additional insight into the molecular mechanisms that underlie TCDD’s reproductive toxicity, we assessed TCDD-induced transcriptional changes in the ovary as they relate to previously described impacts on serum estradiol concentrations and altered follicular development in zebrafish. In-silico computational approaches were used to correlate candidate regulatory motifs with observed changes in gene expression. Our data suggest that TCDD inhibits follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis, and that interference of estrogen-regulated signal transduction may also contribute to TCDD’s impacts on follicular development. TCDD may also alter ovarian function by disrupting various signaling pathways such as glucose and lipid metabolism, and regulation of transcription. Furthermore, events downstream from initial TCDD molecular-targets likely contribute to ovarian toxicity following chronic exposure to TCDD. Data presented here provide further insight into the mechanisms by which TCDD disrupts follicular development and reproduction in fish, and can be used to formulate new hypotheses regarding previously documented ovarian toxicity.
doi:10.1016/j.reprotox.2007.07.013
PMCID: PMC2693207  PMID: 17884332
TCDD; ovary; zebrafish; follicular development; microarray; endocrine disruptors
17.  Quantitative measurement of pathogen specific human memory T cell repertoire diversity using a CDR3β-specific microarray 
BMC Genomics  2007;8:329.
Background
Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method.
Results
The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p < 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p > 0.05).
Conclusion
This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved.
doi:10.1186/1471-2164-8-329
PMCID: PMC2039751  PMID: 17880719
18.  Comprehensive quality control utilizing the prehybridization third-dye image leads to accurate gene expression measurements by cDNA microarrays 
BMC Bioinformatics  2006;7:378.
Background
Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC.
Results
We found that inadequate amount of bound probes available for hybridization led to significant, gene-specific compression in ratio measurements, increased data variability, and printing pin dependent heterogeneities. The impact of such problems can be captured through the definition of quality scores, and efficiently controlled through quality-dependent filtering and normalization. We compared gene expression measurements derived using our data processing pipeline with the known input ratios of spiked in control clones, and with the measurements by quantitative real time RT-PCR. In each case, highly linear relationships (R2>0.94) were observed, with modest compression in the microarray measurements (correction factor<1.17).
Conclusion
Our microarray analytical and technical advancements enabled a better dissection of the sources of data variability and hence a more efficient QC. With that highly accurate gene expression measurements can be achieved using the cDNA microarray technology.
doi:10.1186/1471-2105-7-378
PMCID: PMC1563483  PMID: 16907976
19.  Cross-species global and subset gene expression profiling identifies genes involved in prostate cancer response to selenium 
BMC Genomics  2004;5:58.
Background
Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations.
Results
Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium.
Conclusions
Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer.
doi:10.1186/1471-2164-5-58
PMCID: PMC516028  PMID: 15318950
20.  Immobilized probe and glass surface chemistry as variables in microarray fabrication 
BMC Genomics  2004;5:53.
Background
Global gene expression studies with microarrays can offer biological insights never before possible. However, the technology possesses many sources of technical variability that are an obstacle to obtaining high quality data sets. Since spotted microarrays offer design/content flexibility and potential cost savings over commercial systems, we have developed prehybridization quality control strategies for spotted cDNA and oligonucleotide arrays. These approaches utilize a third fluorescent dye (fluorescein) to monitor key fabrication variables, such as print/spot morphology, DNA retention, and background arising from probe redistributed during blocking. Here, our labeled cDNA array platform is used to study, 1) compression of array data using known input ratios of Arabidopsis in vitro transcripts and arrayed serial dilutions of homologous probes; 2) how curing time of in-house poly-L-lysine coated slides impacts probe retention capacity; and 3) the retention characteristics of 13 commercially available surfaces.
Results
When array element fluorescein intensity drops below 5,000 RFU/pixel, gene expression measurements become increasingly compressed, thereby validating this value as a prehybridization quality control threshold. We observe that the DNA retention capacity of in-house poly-L-lysine slides decreases rapidly over time (~50% reduction between 3 and 12 weeks post-coating; p < 0.0002) and that there are considerable differences in retention characteristics among commercially available poly-L-lysine and amino silane-coated slides.
Conclusions
High DNA retention rates are necessary for accurate gene expression measurements. Therefore, an understanding of the characteristics and optimization of protocols to an array surface are prerequisites to fabrication of high quality arrays.
doi:10.1186/1471-2164-5-53
PMCID: PMC512283  PMID: 15294027
21.  B.E.A.R. GeneInfo: A tool for identifying gene-related biomedical publications through user modifiable queries 
BMC Bioinformatics  2004;5:46.
Background
Once specific genes are identified through high throughput genomics technologies there is a need to sort the final gene list to a manageable size for validation studies. The triaging and sorting of genes often relies on the use of supplemental information related to gene structure, metabolic pathways, and chromosomal location. Yet in disease states where the genes may not have identifiable structural elements, poorly defined metabolic pathways, or limited chromosomal data, flexible systems for obtaining additional data are necessary. In these situations having a tool for searching the biomedical literature using the list of identified genes while simultaneously defining additional search terms would be useful.
Results
We have built a tool, BEAR GeneInfo, that allows flexible searches based on the investigators knowledge of the biological process, thus allowing for data mining that is specific to the scientist's strengths and interests. This tool allows a user to upload a series of GenBank accession numbers, Unigene Ids, Locuslink Ids, or gene names. BEAR GeneInfo takes these IDs and identifies the associated gene names, and uses the lists of gene names to query PubMed. The investigator can add additional modifying search terms to the query. The subsequent output provides a list of publications, along with the associated reference hyperlinks, for reviewing the identified articles for relevance and interest. An example of the use of this tool in the study of human prostate cancer cells treated with Selenium is presented.
Conclusions
This tool can be used to further define a list of genes that have been identified through genomic or genetic studies. Through the use of targeted searches with additional search terms the investigator can limit the list to genes that match their specific research interests or needs. The tool is freely available on the web at [1], and the authors will provide scripts and database components if requested mdatta@mcw.edu
doi:10.1186/1471-2105-5-46
PMCID: PMC419696  PMID: 15117422
22.  Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays 
BMC Genomics  2004;5:12.
Background
Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray.
Results
Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression).
Conclusions
This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.
doi:10.1186/1471-2164-5-12
PMCID: PMC362869  PMID: 15018646
Spotted oligonucleotide arrays; 70-mers; gene expression analysis
23.  Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility 
Nucleic Acids Research  2003;31(11):e60.
Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R2 = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.
PMCID: PMC156737  PMID: 12771224
24.  Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes 
Nucleic Acids Research  2003;31(4):e14.
Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R2 = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.
PMCID: PMC150246  PMID: 12582259

Results 1-24 (24)