Cell-mediated immune responses of cattle to Pasteurella haemolytica and responses of goats to alternative routes of exposure to Pasteurella haemolytica

Goats were inoculated with Pasteurella haemolytica A1 by three different routes to evaluate a caprine model of reproduction of pneumonic pasteurellosis. Lungs were examined and compared grossly, microscopically, and by bacterial culture. All of three goats inoculated intratracheally, one of three goats inoculated intravenously, and none of three goats inoculated intratonsillarly developed pneumonic lesions. Pneumonic lesions in the intravenously-inoculated goat were similar in gross and microscopic aspects to the lesions present in the lungs of intratracheally-inoculated goats. In a separate study, there was evidence of replication of P. hemolytica organisms in the lungs of cattle after subcutaneous vaccination with a live P. haemolytica vaccine, and evidence that the organisms may have gained access to the lung via the vascular system;Humoral immune responses of cattle to P. haemolytica have been thoroughly studied, and humoral immune responses sometimes do not correlate with protection against experimental disease. Other factors, including cell-mediated immune responses, may be involved in protection against pneumonic pasteurellosis. To evaluate the cell-mediated immune responses to P. haemolytica, calves were vaccinated with a commercial P. haemolytica bacterin, or a commercial live P. haemolytica vaccine, or they were infected intratracheally with P. haemolytica. Lymphocyte cultures from peripheral blood mononuclear cells or lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro lymphocyte proliferative responses and gamma interferon production as measures of cell-mediated immunity. In response to stimulation with an outer membrane protein preparation from P. haemolytica, strong proliferative responses and gamma interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves;All calves were challenge-exposed to P. haemolytica, and cell-mediated immune and humoral immune responses were correlated with the degree of pneumonia after experimental challenge. Neither of the cell-mediated immune responses nor the humoral immune response was strongly correlated with the degree of pneumonia present after the experimental challenge

Detoxification of T-2 toxin and feeding behavior changes associated with T-2 toxin

http://www.worldcat.org/oclc/1809270

From junior to senior Pinocchio: A cross-sectional lifespan investigation of deception

We present the first study to map deception across the entire lifespan. Specifically, we investigated age-related difference in lying proficiency and lying frequency. A large community sample (n = 1005) aged between 6 and 77 were surveyed on their lying frequency, and performed a reaction-time (RT) based deception task to assess their lying proficiency. Consistent with the inverted U-shaped pattern of age-related changes in inhibitory control that we observed in a stop signal task, we found that lying proficiency improved during childhood (in accuracy, not RTs), excelled in young adulthood (in accuracy and RTs), and worsened throughout adulthood (in accuracy and RTs). Likewise, lying frequency increased in childhood, peaked in adolescence, and decreased during adulthood. In sum, we observed important age-related difference in deception that generally fit with the U-shaped pattern of age-related changes observed in inhibitory control. Theoretical and practical implications are discussed from a cognitive view of deception

A Practical Platform for Blood Biomarker Study by Using Global Gene Expression Profiling of Peripheral Whole Blood

Background: Although microarray technology has become the most common method for studying global gene expression, a plethora of technical factors across the experiment contribute to the variable of genome gene expression profiling using peripheral whole blood. A practical platform needs to be established in order to obtain reliable and reproducible data to meet clinical requirements for biomarker study. Methods and Findings: We applied peripheral whole blood samples with globin reduction and performed genome-wide transcriptome analysis using Illumina BeadChips. Real-time PCR was subsequently used to evaluate the quality of array data and elucidate the mode in which hemoglobin interferes in gene expression profiling. We demonstrated that, when applied in the context of standard microarray processing procedures, globin reduction results in a consistent and significant increase in the quality of beadarray data. When compared to their pre-globin reduction counterparts, post-globin reduction samples show improved detection statistics, lowered variance and increased sensitivity. More importantly, gender gene separation is remarkably clearer in post-globin reduction samples than in pre-globin reduction samples. Our study suggests that the poor data obtained from pre-globin reduction samples is the result of the high concentration of hemoglobin derived from red blood cells either interfering with target mRNA binding or giving the pseudo binding background signal. Conclusion: We therefore recommend the combination of performing globin mRNA reduction in peripheral whole blood samples and hybridizing on Illumina BeadChips as the practical approach for biomarker study

Characterization of whole blood gene expression profiles as a sequel to globin mRNA reduction in patients with sickle cell disease

Global transcriptome analysis of whole blood RNA using microarrays has been proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA. This is a particular problem in patients with sickle cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation. In order to accurately measure the steady state blood transcriptome in sickle cell patients we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA for genome-wide transcriptome analyses using microarrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle cell disease patients. This led to an improvement in microarray data quality by reducing data variability, with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. Analysis of differences between the whole blood transcriptome and PBMC transcriptome revealed important erythrocyte genes that participate in sickle cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases

Global Transcriptomic Profiling Using Small Volumes of Whole Blood: A Cost-Effective Method for Translational Genomic Biomarker Identification in Small Animals

Blood is an ideal tissue for the identification of novel genomic biomarkers for toxicity or efficacy. However, using blood for transcriptomic profiling presents significant technical challenges due to the transcriptomic changes induced by ex vivo handling and the interference of highly abundant globin mRNA. Most whole blood RNA stabilization and isolation methods also require significant volumes of blood, limiting their effective use in small animal species, such as rodents. To overcome these challenges, a QIAzol-based RNA stabilization and isolation method (QSI) was developed to isolate sufficient amounts of high quality total RNA from 25 to 500 μL of rat whole blood. The method was compared to the standard PAXgene Blood RNA System using blood collected from rats exposed to saline or lipopolysaccharide (LPS). The QSI method yielded an average of 54 ng total RNA per μL of rat whole blood with an average RNA Integrity Number (RIN) of 9, a performance comparable with the standard PAXgene method. Total RNA samples were further processed using the NuGEN Ovation Whole Blood Solution system and cDNA was hybridized to Affymetrix Rat Genome 230 2.0 Arrays. The microarray QC parameters using RNA isolated with the QSI method were within the acceptable range for microarray analysis. The transcriptomic profiles were highly correlated with those using RNA isolated with the PAXgene method and were consistent with expected LPS-induced inflammatory responses. The present study demonstrated that the QSI method coupled with NuGEN Ovation Whole Blood Solution system is cost-effective and particularly suitable for transcriptomic profiling of minimal volumes of whole blood, typical of those obtained with small animal species

Use of bioanalyzer electropherograms for quality control and target evaluation in microarray expression profiling studies of ocular tissues

Expression profiling with DNA microarrays has been used to examine the transcriptome of a wide spectrum of vertebrate cells and tissues. The sensitivity and accuracy of the data generated is dependent on the quality and composition of the input RNA. In this report, we examine the quality and array performance of over 200 total RNA samples extracted from ocular tissues and cells that have been processed in a microarray core laboratory over a 7-year period. Total RNA integrity and cRNA target size distribution were assessed using the 2100 Bioanalyzer. We present Affymetrix GeneChip array performance metrics for different ocular samples processed according to a standard microarray assay workflow including several quality control checkpoints. Our review of ocular sample performance in the microarray assay demonstrates the value of considering tissue-specific characteristics in evaluating array data. Specifically, we show that Bioanalyzer electropherograms reveal highly abundant mRNAs in lacrimal gland targets that are correlated with variation in array assay performance. Our results provide useful benchmarks for other gene expression studies of ocular systems

Gene expression profiling of human whole blood samples with the Illumina WG-DASL assay

<p>Abstract</p> <p>Background</p> <p>Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole genome DASL assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We assessed the utility of the whole genome DASL assay in an analysis of peripheral whole blood gene expression profiles.</p> <p>Results</p> <p>We find that gene expression detection is significantly increased with the use of whole genome DASL compared to the standard IVT-based direct hybridization. Additionally, globin-probe negative whole genome DASL did not exhibit significant improvements over globin-probe positive whole genome DASL. Globin reduction further increases the detection sensitivity and reliability of both whole genome DASL and IVT-based direct hybridization with little effect on raw intensity correlations. Raw intensity correlations between total RNA and globin reduced RNA were 0.955 for IVT-based direct hybridization and 0.979 for whole genome DASL.</p> <p>Conclusions</p> <p>Overall, the detection sensitivity of the whole genome DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies.</p

Gene expression analyses in breast cancer epidemiology: the Norwegian Women and Cancer postgenome cohort study

Introduction The introduction of high-throughput technologies, also called -omics technologies, into epidemiology has raised the need for high-quality observational studies to reduce several sources of error and bias. Methods The Norwegian Women and Cancer (NOWAC) postgenome cohort study consists of approximately 50,000 women born between 1943 and 1957 who gave blood samples between 2003 and 2006 and filled out a two-page questionnaire. Blood was collected in such a way that RNA is preserved and can be used for gene expression analyses. The women are part of the NOWAC study consisting of 172,471 women 30 to 70 years of age at recruitment from 1991 to 2006 who answered one to three questionnaires on diet, medication use, and lifestyle. In collaboration with the Norwegian Breast Cancer Group, every NOWAC participant born between 1943 and 1957 who is admitted to a collaborating hospital for a diagnostic biopsy or for surgery of breast cancer will be asked to donate a tumor biopsy and two blood samples. In parallel, at least three controls are approached for each breast cancer case in order to obtain blood samples from at least two controls per case. The controls are drawn at random from NOWAC matched by time of follow-up and age. In addition, 400 normal breast tissues as well as blood samples will be collected among healthy women participating at the Norwegian Mammography Screening program at the Breast Imaging Center at the University Hospital of North-Norway, Tromsø. Results The NOWAC postgenome cohort offers a unique opportunity (a) to study blood-derived gene expression profiles as a diagnostic test for breast cancer in a nested case-control design with adjustment for confounding factors related to different exposures, (b) to improve the reliability and accuracy of this approach by adjusting for an individual's genotype (for example, variants in genes coding for hormone and drug-metabolizing and detoxifying enzymes), (c) to study gene expression profiles from peripheral blood as surrogate tissue to biomonitor defined exposure (for example, hormone) and its association with disease risk (that is, breast cancer), and (d) to study gene variants (single nucleotide polymorphisms and copy number variations) and environmental exposure (endogenous and exogenous hormones) and their influence on the incidence of different molecular subtypes of breast cancer. Conclusion The NOWAC postgenome cohort combining a valid epidemiological approach with richness of biological samples should make an important contribution to the study of the etiology and system biology of breast cancer

Comparison of Whole Blood and Peripheral Blood Mononuclear Cell Gene Expression for Evaluation of the Perioperative Inflammatory Response in Patients with Advanced Heart Failure

Background: Heart failure (HF) prevalence is increasing in the United States. Mechanical Circulatory Support (MCS) therapy is an option for Advanced HF (AdHF) patients. Perioperatively, multiorgan dysfunction (MOD) is linked to the effects of device implantation, augmented by preexisting HF. Early recognition of MOD allows for better diagnosis, treatment, and risk prediction. Gene expression profiling (GEP) was used to evaluate clinical phenotypes of peripheral blood mononuclear cells (PBMC) transcriptomes obtained from patients’ blood samples. Whole blood (WB) samples are clinically more feasible, but their performance in comparison to PBMC samples has not been determined. Methods: We collected blood samples from 31 HF patients (57¡15 years old) undergoing cardiothoracic surgery and 7 healthy age-matched controls, between 2010 and 2011, at a single institution. WB and PBMC samples were collected at a single timepoint postoperatively (median day 8 postoperatively) (25–75% IQR 7–14 days) and subjected to Illumina single color Human BeadChip HT12 v4 whole genome expression array analysis. The Sequential Organ Failure Assessment (SOFA) score was used to characterize the severity of MOD into low (# 4 points), intermediate (5–11), and high ($ 12) risk categories correlating with GEP. Results: Results indicate that the direction of change in GEP of individuals with MOD as compared to controls is similar when determined from PBMC versus WB. The main enriched terms by Gene Ontology (GO) analysis included those involved in the inflammatory response, apoptosis, and other stress response related pathways. The data revealed 35 significant GO categories and 26 pathways overlapping between PBMC and WB. Additionally, class prediction using machine learning tools demonstrated that the subset of significant genes shared by PBMC and WB are sufficient to train as a predictor separating the SOFA groups. Conclusion: GEP analysis of WB has the potential to become a clinical tool for immune-monitoring in patients with MO