Biomedical subjects

Bernard H Brownstein

Publications and source records attributed to Bernard H Brownstein.

11 recordsLinked to original sources

PubMed2006

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Gene Expression Profiling↗

PubMed2006

Microfluidic isolation of leukocytes from whole blood for phenotype and gene expression analysis.

Technologies that enable the isolation of cell subtypes from small samples of complex populations will greatly facilitate the implementation of proteomics and genomics to human diseases. Transcriptome analysis of blood requires the depletion of contaminating erythrocytes. We report an automated microfluidic device to rapidly deplete erythrocytes from whole blood via deionized water lysis and to collect enriched leukocytes for phenotype and genomic analyses. Starting with blood from healthy subjects, we demonstrate the utility of this microfluidic cassette and lysis protocol to prepare unstimulated leukocytes, and leukocytes stimulated ex vivo with Staphylococcal enterotoxin B, which mimics some of the cellular effects seen in patients with severe bacterial infections. Microarrays are used to assess the global gene expression response to enterotoxin B. The results demonstrate that this system can isolate unactivated leukocytes from small blood samples without any significant loss, which permits more information to be obtained from subsequent analysis, and will be readily applicable to clinical settings.

Cell Separation↗

PubMed2006

The number of genes changing expression after chronic exposure to code division multiple access or frequency DMA radiofrequency radiation does not exceed the false-positive rate.

Experiments with cultured C3H 10T 1/2 cells were performed to determine if exposure to cell phone radiofrequency (RF) radiations induce changes in gene expression. Following a 24 h exposure of 5 W/kg specific adsorption rate, RNA was extracted from the exposed and sham control cells for microarray analysis on Affymetrix U74Av2 Genechips. Cells exposed to 0.68 Gy of X-rays with a 4-h recovery were used as positive controls. The number of gene expression changes induced by RF radiation was not greater than the number of false positives expected based on a sham versus sham comparison. In contrast, the X-irradiated samples showed higher numbers of probe sets changing expression level than in the sham versus sham comparison.

Animals↗

PubMed2005

Commonality and differences in leukocyte gene expression patterns among three models of inflammation and injury.

The aim of this study was to compare gene expression profiles of leukocytes from blood (white blood cells; WBCs) and spleen harvested at an early time point after injury or sham injury in mice subjected to trauma/hemorrhage, burn injury, or lipopolysaccharide (LPS) infusion at three experimental sites. Groups of injured or LPS-infused animals and sham controls were killed at 2 h after injury and resuscitation, blood and spleen were harvested, and leukocyte populations were recovered after erythrocyte lysis. RNA was extracted from postlysis leukocyte populations. Complementary RNA was synthesized from each RNA sample and hybridized to microarrays. A large number (500-1,400) of genes were differentially expressed at the 2-h time point in injured or LPS-infused vs. sham animals. Thirteen of the differentially expressed genes in blood, and 46 in the spleen, were upregulated or downregulated in common among all three animal models and may represent a common, early transcriptional response to systemic inflammation from a variety of causes. The majority of these genes could be assigned to pathways involved in the immune response and cell death. The up- or downregulation of a cohort of 23 of these genes was validated by RT-PCR. This large-scale microarray analysis shows that, at the 2-h time point, there is marked alteration in leukocyte gene expression in three animal models of injury and inflammation. Although there is some commonality among the models, the majority of the differentially expressed genes appear to be uniquely associated with the type of injury and/or the inflammatory stimulus.

Animals↗

PubMed2005

A network-based analysis of systemic inflammation in humans.

Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.

Acute Disease↗

PubMed2005

Application of genome-wide expression analysis to human health and disease.

The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

Cluster Analysis↗

PubMed2005

Expression of the proto-oncogene Fos after exposure to radiofrequency radiation relevant to wireless communications.

In this study the expression levels of the proto-oncogene Fos were measured after exposure to radiofrequency (RF) radiation at two relatively high specific absorption rates (SARs) of 5 and 10 W/kg for three types of modulated signals: 847.74 MHz code division multiple access (CDMA), 835.62 MHz frequency division multiple access (FDMA), and 836.55 MHz time division multiple access (TDMA). This work was undertaken to confirm a previous report by Goswami et al. (Radiat. Res. 151, 300-309, 1999) that CDMA and FDMA radiation caused small but statistically significant increases in Fos levels as cells entered plateau phase during exposure. No effects on Myc or Jun levels were observed in that study. Therefore, in the present study, analyses were restricted to Fos expression during the transition from exponential growth to plateau phase. Fos expression was measured using the real-time polymerase chain reaction (RT-PCR) technique. Serum-stimulated C3H 10T(1/2) cells were used as a positive control for Fos expression. Possible influences of final cell number or pH variability on Fos expression were evaluated. Expression of Fos mRNA in C3H 10T(1/2) cells was not significantly different from that found after sham exposure at either SAR level for any signal modulation. Therefore, the results of Goswami et al. could not be confirmed.

Animals↗

PubMed2004

Whole blood and leukocyte RNA isolation for gene expression analyses.

The analysis of gene expression data in clinical medicine has been plagued by the lack of a critical evaluation of accepted methodologies for the collection, processing, and labeling of RNA. In the present report, the reliability of two commonly used techniques to isolate RNA from whole blood or its leukocyte compartment was compared by examining their reproducibility, variance, and signal-to-noise ratios. Whole blood was obtained from healthy subjects and was either untreated or stimulated ex vivo with Staphylococcus enterotoxin B (SEB). Blood samples were also obtained from trauma patients but were not stimulated with SEB ex vivo. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system or from isolated leukocytes. Biotin-labeled cRNA was hybridized to Affymetrix GeneChips. The Pearson correlation coefficient for gene expression measurements in replicates from healthy subjects with both techniques was excellent, exceeding 0.985. Unsupervised analyses, including hierarchical cluster analysis, however, revealed that the RNA isolation method resulted in greater differences in gene expression than stimulation with SEB or among different trauma patients. The intraclass correlation, a measure of signal-to-noise ratio, of the difference between SEB-stimulated and unstimulated blood from healthy subjects was significantly higher in leukocyte-derived samples than in whole blood: 0.75 vs. 0.46 (P = 0.002). At the P < 0.001 level of significance, twice as many probe sets discriminated between SEB-stimulated and unstimulated blood with leukocyte isolation than with PAXgene. The findings suggest that the method of RNA isolation from whole blood is a critical variable in the design of clinical studies using microarray analyses.

Adolescent↗

PubMed2004

Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major.

To complete its life cycle, protozoan parasites of the genus Leishmania undergo at least three major developmental transitions. However, previous efforts to identify genes showing stage regulated changes in transcript abundance have yielded relatively few. Here we used expression profiling to assess changes in transcript abundance in three stages: replicating promastigotes and infective non-replicating metacyclics, which occur in the sand fly vector, and in the amastigote stage residing with macrophage phagolysosomes in mammals. Microarrays were developed containing 11,484 PCR products that included a number of known genes and 10,464 random 1 kb genomic DNA fragments. Arrays were hybridized in triplicate and genes showing two-fold or greater changes in 2/3 experiments were scored as differentially expressed. Remarkably, only about one percent of the DNAs expression varied by this criteria, in either stage comparison. Northern blot analysis confirmed the predicted change in mRNA abundance for most of these (68%). This set of genes included most of those previously identified in the literature as differentially regulated as well as a number of novel genes. Notably, Leishmania maxicircle transcripts showed strong up-regulation in metacyclic and amastigote parasites, probably associated with changes in parasite energy metabolism. However, current data suggest that expression profiling using shotgun DNA libraries significantly underestimates the extent of regulated transcripts.

Animals↗

PubMed2004

Typing single-nucleotide polymorphisms in Toxoplasma gondii by allele-specific primer extension and microarray detection.

Genotyping is an important tool for epidemiological and population genetic studies in protozoan parasites. The most commonly used method for genotyping is polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis of single nucleotide polymorphisms (SNPs). However, PCR-RFLP analysis is labor intensive, and only a proportion of the SNPs are recognized by currently available restriction enzymes. Here, we have developed a more efficient microarray-based method to genotype SNPs in the protozoan parasite Toxoplasma gondii. This method is sensitive, accurate, and capable of analyzing multiple SNPs simultaneously in a high-throughput format.

Alleles↗

PubMed2002

Putting the Leishmania genome to work: functional genomics by transposon trapping and expression profiling.

Leishmania are important protozoan pathogens of humans in temperate and tropical regions. The study of gene expression during the infectious cycle, in mutants or after environmental or chemical stimuli, is a powerful approach towards understanding parasite virulence and the development of control measures. Like other trypanosomatids, Leishmania gene expression is mediated by a polycistronic transcriptional process that places increased emphasis on post-transcriptional regulatory mechanisms including RNA processing and protein translation. With the impending completion of the Leishmania genome, global approaches surveying mRNA and protein expression are now feasible. Our laboratory has developed the Drosophila transposon mariner as a tool for trapping Leishmania genes and studying their regulation in the form of protein fusions; a classic approach in other microbes that can be termed 'proteogenomics'. Similarly, we have developed reagents and approaches for the creation of DNA microarrays, which permit the measurement of RNA abundance across the parasite genome. Progress in these areas promises to greatly increase our understanding of global mechanisms of gene regulation at both mRNA and protein levels, and to lead to the identification of many candidate genes involved in virulence.

Animals↗