Biomedical subjects

D M Walters

Publications and source records attributed to D M Walters.

8 recordsLinked to original sources

PubMed2001

Ambient urban Baltimore particulate-induced airway hyperresponsiveness and inflammation in mice.

Airborne particulate matter (PM) is hypothesized to play a role in increases in asthma prevalence, although a causal relationship has yet to be established. To investigate the effects of real-world PM exposure on airway reactivity (AHR) and bronchoalveolar lavage (BAL) cellularity, we exposed naive mice to a single dose (0.5 mg/ mouse) of ambient PM, coal fly ash, or diesel PM. We found that ambient PM exposure induced increases in AHR and BAL cellularity, whereas diesel PM induced significant increases in BAL cellularity, but not AHR. On the other hand, coal fly ash exposure did not elicit significant changes in either of these parameters. We further examined ambient PM-induced temporal changes in AHR, BAL cells, and lung cytokine levels over a 2-wk period. Ambient PM-induced AHR was sustained over 7 d. The increase in AHR was preceded by dramatic increases in BAL eosinophils, whereas a decline in AHR was associated with increases in macrophages. A Th2 cytokine pattern (IL-5, IL-13, eotaxin) was observed early on with a shift toward a Th1 pattern (IFN-gamma). In additional studies, we found that the active component(s) of ambient PM are not water-soluble and that ambient PM-induced AHR and inflammation are dose- dependent. We conclude that ambient PM can induce asthma-like parameters in naive mice, suggesting that PM exposure may be an important factor in increases in asthma prevalence.

Air Pollution↗

PubMed2001

High-density sampling of a bacterial operon using mRNA differential display.

We have implemented a simplified high throughput approach to differential display in order to identify transcriptionally regulated genes in bacteria. In contrast with the few previous applications of differential display to prokaryotes, we use a large number of primers which allows for a high-density sampling of the mRNA population and the identification of many differentially amplified DNA fragments. From the overlap of these short sequences, long contiguous sequences that encode several genes can be assembled. The multiplicity of sampling provides a strong indication that the genes identified are indeed differentially regulated. As a test case, we looked for the genes involved in the degradation of 2,4-dinitrophenol (2,4-DNP) in a Rhodococcus erythropolis strain, HL PM-1. In this experiment a long polycistronic mRNA was sampled repeatedly. The induction of these genes by 2,4-DNP was confirmed by dot blot analysis and two of them were confirmed to be involved in the degradation of 2,4-DNP. This work shows that mRNA differential display is an important tool for the identification of metabolic genes in prokaryotes.

2,4-Dinitrophenol↗

PubMed2001

Methodology for the measurement of mucociliary function in the mouse by scintigraphy.

The objective of the study was to develop a scintigraphic method for measurement of airway mucociliary clearance in small laboratory rodents such as the mouse. Previous investigations have characterized the secretory cell types present in the mouse airway, but analysis of the mucus transport system has been limited to in vitro examination of tissue explants or invasive in vivo measures of a single airway, the trachea. Three methods were used to deposit insoluble, radioisotopic colloidal particles: oropharyngeal aspiration, intratracheal instillation, and nose-only aerosol inhalation. The initial distribution of particles within the lower respiratory tract was visualized by gamma-camera, and clearance of particles was followed intermittently over 6 h and at the conclusion, 24 h postdelivery. Subsets of mice underwent lavage for evidence of tissue inflammation, and others were restudied for reproducibility of the methods. The aspiration and instillation methods of delivery led to greater distributions of deposited activity within the lungs, i.e., approximately 60--80% of the total respiratory tract radioactivity, whereas the nose-only aerosol technique attained a distribution of 32% to the lungs. However, the aerosol technique maximized the fraction of particles that cleared the airway over a 24-h period, i.e, deposited onto airway epithelial surfaces and cleared by mucociliary function such that lung retention at 24 h averaged 57% for delivery by aerosol inhalation and > or =80% for the aspiration or intratracheal instillation techniques. Particle delivery methods did not cause lung inflammation/injury with use of inflammatory cells and chemoattractant cytokines as criteria. Scintigraphy can discern particle deposition and clearance from the lower respiratory tract in the mouse, is noninvasive and reproducible, and includes the capability for restudy and lung lavage when time course or chronic treatments are being considered.

Animals↗

PubMed2000

Assessment of cellular profile and lung function with repeated bronchoalveolar lavage in individual mice.

In this study, we sought to develop procedures that would enable repeated bronchoalveolar lavage (BAL) in individual mice on multiple occasions. To achieve this objective, we first developed the procedures that would allow individual mice to survive a whole lung lavage, and then tested whether, on subsequent days, there was an effect of this initial BAL on the cell profile, lung permeability, and baseline respiratory function. Our results demonstrate that the repeated lavage procedure can be readily carried out in individual mice of different strains on multiple occasions. The lavage procedure itself results in immediate increases in respiratory system resistance and concomitant decreases in compliance, but these parameters return to prelavage values by the 2nd or 3rd day postlavage. Lavage also induces variable increases in inflammatory cells depending on the strain used. However, in all three strains examined here (A/J, BALB/c, and C3H/HeJ), inflammatory cell numbers returned to baseline values within 3 days after an initial lavage procedure. The ability to perform repeated BAL in individual mice should prove to be an extremely useful tool in a variety of functional genomic studies in the lung.

Airway Resistance↗

PubMed1999

Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens.

Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.

Bacterial Proteins↗

PubMed1999

A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.

The ability to successfully intubate the trachea of mice and control their ventilation is important for longitudinal studies requiring recovery from anesthesia and repeated pulmonary function measurements or other evaluations, such as the use of radiological imaging (e.g., computed tomography or magnetic resonance imaging). We describe a method for rapid and repeated intubation of mice, with subsequent pulmonary function measurements at baseline and after an agonist challenge. We describe a simply constructed metal blade used as a laryngoscope to facilitate oropharyngeal exposure, transillumination of the neck to facilitate visualization of the trachea through the oropharynx, readily available polyethylene tubing to intubate the trachea, and a simple solenoid ventilator to maintain physiological ventilation and assess respiratory resistance and compliance. Brief infusions of acetylcholine through a needle into the jugular vein are used to assess the responsiveness of the airway smooth muscle.

Animals↗

PubMed1998

Sonic and mechanical toothbrushes. An in vitro study showing altered microbial surface structures but lack of effect on viability.

The purpose of the present study was to compare the in vitro effects of a mechanical and a sonic toothbrush on the viability of Actinomyces viscosus, the rationale being that induction of irreparable microbial damage resulting from aggressive mechanical action or sonic energy, may inhibit or disrupt the process of successional colonization. Cultures of A. viscosus were grown to a standardized optical density and subdivided into 3 treatment groups of 20 specimens each. Treatment groups consisted of an untreated control and exposure to a mechanical or sonic toothbrush for 15, 30, 45, and 60 s. Subsequent to the prescribed treatment, samples were taken from each specimen dish, subcultured, and the number of CFUs determined. Additional samples were obtained for negative staining and examination by electron microscopy. The mean number of CFUs for each treatment group at each treatment interval were statistically analyzed by ANOVA and multiple pairwise comparisons. Results showed a significant main effect for toothbrushes (p<0.0001) and exposure time (p<0.01), but only marginal significance for the interaction of toothbrush with exposure time (p<0.055). Post-hoc tests showed a significantly greater number of CFUs for the sonic toothbrush compared to both the untreated control and mechanical toothbrush groups. Electron microscopic examination revealed a decrease in aggregation tendency and loss of fimbriae in the sonic toothbrush group. Based on the lack of morphologic evidence that would indicate cell damage and the increase in CFUs over that of the control group, it appeared that neither the mechanical or sonic toothbrushes affected cell viability.

Actinomyces viscosus↗

PubMed1997

Bioavailability of TNT residues in composts of TNT-contaminated soil.

Composting is being explored as a means to remediate 2,4,6-trinitrotoluene (TNT) contaminated soils. This process appears to modify TNT and to bind it to organic matter. The health hazards associated with dusts generated from such materials cannot be predicted without knowing if the association between TNT residues and compost particulate is stable in biological systems. To address this question, single doses of [14C]-TNT, soil spiked with [14C]-TNT, or compost generated with [14C]-TNT-spiked soils were administered to rats by intratracheal instillation. The appearance of 14C in urine and tissues was taken as an indication of the bioavailability of TNT residues from compost particles. In rats instilled with neat [14C]-TNT, about 35% of the 14C dose appeared in urine within 3 d. The 14C excreted in urine by these rats decreased rapidly thereafter, and was undetectable by 4 wk after treatment. Similar results were obtained with soil-treated rats. In contrast, after treatment with [14C]-TNT-labeled compost, only 2.3% of the total 14C dose appeared in urine during the first 3 d. Low levels of 14C continued to be excreted in urine from compost-treated rats for more than 6 mo, and the total amount of 14C in urine was comparable to that in TNT-treated animals. Determination of the radiolabel in tissues showed that 14C accumulated in the kidneys of rats treated with labeled compost but not in rats treated with [14C]-TNT or [14C]-TNT-spiked soil. These results indicate that the association between TNT and particulate matter in compost is not stable when introduced into the lungs. Accumulation of 14C in kidneys suggests the presence of a unique TNT residue in compost-treated rats. The rate of excretion and tissue disposition of 14C in rats treated with TNT-spiked soil indicate that TNT in soil is freely available in the lungs.

Animals↗