Biomedical subjects

David A Stenger

Publications and source records attributed to David A Stenger.

11 recordsLinked to original sources

PubMed2004

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Antigens, Bacterial↗

PubMed2004

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses.

The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B(1), B(2), C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.

Acute Disease↗

PubMed2004

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

Animals↗

PubMed2003

Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus.

Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.

Animals↗

PubMed2003

Acute neuropharmacologic action of chloroquine on cortical neurons in vitro.

Chloroquine, a common quinolone derivative used in the treatment of malaria, has been associated with neurologic side-effects including depression, psychosis and delirium. The neuropharmacologic effects of chloroquine were examined on cultured cortical neurons using microelectrode array (MEA) recording and the whole-cell patch clamp technique. Whole-cell patch clamp records under current-clamp mode also showed a chloroquine-induced depression of the firing rate of spontaneous action potentials by approximately 40%, consistent with the observations with the MEA recording, although no changes in either the baseline membrane potential or input resistance were observed. Voltage clamp recordings of spontaneous post-synaptic currents, recorded in the presence of tetrodotoxin, revealed no obvious changes in either the amplitude or rate of occurrence of inward currents with application of chloroquine at 10 microM, suggesting that the fundamental molecular mechanisms underlying spontaneous synaptic transmission may not be affected by acute application of the drug. In contrast, a concentration-dependent inhibition of whole-cell calcium current was observed in the presence of chloroquine. These acute neuropharmacologic changes were not accompanied by cytotoxic actions of the compound, even after exposure of up to 500 microM chloroquine for 7 h. These data suggest that chloroquine can depress in vitro neuronal activity, perhaps through inhibition of membrane calcium channels.

Action Potentials↗

PubMed2003

Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.

Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR, but has not been assessed at the transcriptome level on Affymetrix microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at -20 degrees C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.

Blood Specimen Collection↗

PubMed2003

Effects of collagen matrix on Sindbis virus infection of BHK cells.

The effects of extracellular matrix (ECM) components on the outcomes of alphavirus interaction with cells are not known. Studies that address such interactions have to address several methodological difficulties, including: the survival of the cells within the matrix; the passage of the virus through the matrix to infect embedded cells; and the dissociation of cells and matrix into single-cell suspension, before and after virus infection, for quantitative analysis. Herein, these issues were addressed in the context of a model system of collagen as the ECM component, baby hamster kidney (BHK) cells, and Sindbis virus. The outcomes of Sindbis virus infection of BHK cells, grown in three-dimensional (3D) collagen gel versus on plastic, and on two-dimensional (2D) collagen versus bovine serum albumin (BSA)-coated surfaces were compared. Cell morphology was more slender in 3D and on 2D collagen than on plastic or BSA-coated surfaces. The cells were able to survive in the 3D environment. Using Sindbis virus carrying the green fluorescent protein gene, the virions were found to be capable of penetrating the 3D collagen matrix and infecting the cells. There was more infectious virus in cultures of cells in 3D and on 2D collagen than on plastic or BSA-coated surfaces, respectively. Higher virus titers from cells on 2D collagen compared to BSA-coated surfaces was not associated with uninfected cell number or viability but with increased cell survival after infection. Infected cells on BSA surfaces became detached, while those on 2D collagen remained attached. These experiments establish procedures for analysis of interaction of collagen, BHK cells, and Sindbis virus and suggest that collagen increases infectious Sindbis virus titers from BHK cells by enhancing post-infection cell survival.

Animals↗

PubMed2003

Biological threat detection via host gene expression profiling.

With the increased threat posed by biological weapons, detection techniques for biothreat pathogens are critically needed to monitor and assess the severity of the illness once exposure has occurred. Current approaches for detecting biological threats are either time-consuming or highly specific but provide little information regarding pathogenicity. Genotyping of pathogens by PCR provides a fast and definitive means for identifying pathogens, but reliance on pathogen genotypic endpoints has several limitations. Current progress in DNA microarrays technology provides an alternative way to address the issues faced by traditional detection systems through host gene expression profiles of peripheral blood cells. We discuss the advantages and critical issues facing the use of host gene expression profiling for biological threat detection.

Bioterrorism↗

PubMed2002

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels.

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.

Adult↗

PubMed2002

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells.

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.

Animals↗

PubMed2002

Potential applications of DNA microarrays in biodefense-related diagnostics.

Recent years have witnessed a logarithmic growth in the number of applications involving DNA microarrays. Extrapolation of their use for infectious diagnostics and biodefense-related diagnostics seems obvious. Nevertheless, the application of DNA microarrays to biodefense-related diagnostics will depend on solving a set of substantial, yet approachable, technical and logistical problems that encompass diverse topics from amplification efficiency to bioinformatics.

Bioterrorism↗