Microscopic imaging with electrogenerated chemiluminescence.

The use of electrogenerated chemiluminescence (ECL) at microelectrodes as a light source for scanning optical microscopy is demonstrated. Cone-shaped microelectrodes were constructed by flame etching carbon fibers to a fine point. ECL generated in solution at such electrodes was forced to the apex of the conducting surface by using high-frequency (20-kHz) potential pulses and high concentrations of ECL reagents in the solution. ECL arose from the reaction of 9,10-diphenylanthracene radical cation with the radical anion of benzonitrile, the solvent. The electrode/light source was raster-scanned a finite distance above the sample surface, and images were generated with standard scanning probe software by collecting the transmitted light with a microscope objective. These images compared favorably to optical images of the same sample. A resolution of approximately 600 nm was achieved with this arrangement even though a feedback loop was not employed to control the tip distance from the sample. The source was sufficiently bright (1.82 pW) that well-defined transmittance spectra could be obtained at individual locations on the sample.

Alveolar JE/MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, sequential neutrophil and monocyte influx, and vascular leakage in mice.

The C-C chemokine monocyte chemotactic protein 1 (JE/MCP-1) is a key cytokine for lung monocyte recruitment, and may be detected in high levels in the alveolar space in lung injury. We hypothesized that alveolar JE/MCP-1 might synergize with endotoxin in this compartment to elicit lung inflammatory events. Intratracheal instillation of JE/MCP-1 into BALB/c mice did not provoke increased bronchoalveolar lavage tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and macrophage inflammatory protein 2 (MIP-2) levels, but elicited monocyte recruitment into this compartment. Intratracheal Escherichia coli endotoxin provoked elevated lavage TNF-alpha, IL-6, and MIP-2 levels, peaking after 6 h in parallel with increased alveolar neutrophil numbers, in the absence of vascular leakage. Mice receiving both endotoxin and JE/ MCP-1 showed drastically increased lavage TNF-alpha, IL-6, and MIP-2 levels, 22-fold higher lavage neutrophil numbers, and lung vascular leakage. Moreover, an 8-fold increased alveolar accumulation of monocytes, peaking at 48 h together with expansion of the resident alveolar macrophage pool, was noted. Intraperitoneal instead of alveolar deposition of MCP-1 or endotoxin failed to reproduce the synergistic response, and the same was true for employment of RANTES instead of MCP-1. Blockade of neutrophil recruitment by anti-CD18 did not affect the intra-alveolar cytokine response to MCP-1 plus endotoxin. Together, JE/MCP-1 and endotoxin, when coappearing in the alveolar compartment at low dosage, elicit an early phase of lung inflammatory injury with increased cytokine synthesis and neutrophil recruitment, and a late phase of enhanced monocyte traffic and expansion of the alveolar macrophage pool.

Kokomo family care: automating the clinical practice.

A 14-physician family practice automated its prescription process, telephone messaging and laboratory test reporting to streamline its operations and contain costs.

Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14.

The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-alpha message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are "primed" for enhanced responsiveness to endotoxin with increased cytokine expression.

Antisense oligomers for selective suppression of MCP-1 synthesis in human pulmonary endothelial cells.

Endothelial synthesis of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) has been implicated in the regulation of monocyte recruitment for extravascular pools under both physiologic and inflammatory conditions. We designed and characterized five antisense phosphorothioate oligodeoxynucleotides (PS-ODN) targeting MCP-1 secretion by human pulmonary artery endothelial cells (HPAEC) and pulmonary microvascular endothelial cells (HMVEC-L). The most effective PS-ODN (MCP-1 AS 2) dose-dependently suppressed the secretion of MCP-1 but not the secretion of the C-X-C chemokine interleukin-8 (IL-8) in both HPAEC and HMVEC-L in the nanomolar concentration range. Mismatch controls bearing 2 or 4 bp substitutions showed markedly reduced inhibitory capacity. MCP-1 mRNA levels were not affected even at the highest PS-ODN doses employed (ribonuclease protection assay), suggesting a translational arrest of MCP-1 production. Accordingly, PS-ODN exhibited no nonspecific side effects on immediate-early gene regulation of the transcription factor nuclear factor-kappaB (NF-kappaB), as analyzed by gel shift assays. Antisense pretreatment of HPAEC reduced the monocyte chemotactic bioactivity liberated from tumor necrosis factor-alpha (TNF-alpha)-activated endothelial cells (EC) and reduced the TNF-alpha-induced transendothelial monocyte migration. We conclude that nanomolar concentrations of specific antisense oligodeoxynucleotides effectively inhibit human endothelial MCP-1 synthesis and may thus provide a rational approach to modulate monocyte recruitment under inflammatory conditions.

Cationic lipids employed for antisense oligodeoxynucleotide transport may inhibit vascular cell adhesion molecule-1 expression in human endothelial cells: a word of caution.

Antisense oligodeoxynucleotides (ODN) have become a powerful tool to achieve specific gene inhibition in various cell types, including endothelial cells. The low spontaneous cellular uptake of ODN, however, usually requires the employment of transmembrane carriers, such as the positively charged liposome formulation dioleyloxypropyltrimethyl ammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE). In the present study, we observed that DOTMA/DOPE per se interferes with the inducible expression of vascular cell adhesion molecule-1 (VCAM-1) in human pulmonary artery endothelial cells (HPAEC). By RT-PCR analysis, a dose-dependent suppression of VCAM-1 but not intracellular adhesion molecule-1 (ICAM-1) mRNA levels in tumor necrosis factor-alpha (TNF-alpha)-challenged HPAEC pretreated with DOTMA/DOPE (5-20 microg/ml) was demonstrated. Correspondingly, a strong reduction of TNF-alpha-induced VCAM-1 but not ICAM-1 cell surface expression on HPAEC was observed. These DOTMA/DOPE-induced changes were not due to alterations in VCAM-1 mRNA stability, nor did DOTMA/DOPE inhibit TNF-alpha-induced NF-kappaB-like binding activity in nuclear extracts of HPAEC, as analyzed by electrophoretic mobility shift assay. In contrast, DOTMA/DOPE effected a dose-dependent increase in AP-1-like binding activity in nuclear extracts of HPAEC, as analyzed by Western blotting and EMSA. We conclude that positively charged liposome preparations may per se inhibit TNF-alpha-induced endothelial VCAM-1 expression, and this may be related to changes in AP-1 but not NF-kappaB-dependent transcriptional control. Notably, when used at concentrations below 5 microg/ml, DOTMA/DOPE may be employed for specific antisense-mediated downregulation of VCAM-1 in the absence of vehicle-related side effects on adhesion molecule transcription.

Imaging of nonuniform current density at microelectrodes by electrogenerated chemiluminescence.

The chemiluminescence arising from reaction of electrogenerated radical cations of 9,10-diphenylanthracene (DPA) and benzonitrile (solvent) radical anions has been used to image microelectrodes with dimensions in the micrometer range. Experimental conditions including supporting electrolyte, DPA concentration, and excitation frequency were optimized to affect high luminescent intensity. In solutions of high resistance, the light was found to be temporally delayed with respect to the applied potential due to the increased time required to charge the double layer. Spatially nonuniform light at disk- and band-shaped microelectrodes was observed under certain conditions, with the highest intensity occurring at the region of the electrode with highest curvature. The optimum condition for observation of the nonuniform light was with very high electrode currents. Under this condition, the current density approaches that of the primary current distribution, a circumstance where spatially nonuniform potentials occur. This phenomenon was also examined at a conical electrode as a method of reducing the emission area. A submicrometer-size light source was obtained at high frequencies with an electrode that had a significantly larger uninsulated area.

Experimental examination of factors that affect dust generation by using Heubach and MRI testers.

Four factors that affect dust generation were investigated--type of test material, particle size distribution of the test material, moisture content of the test material, and apparatus used to generate dust. Dust generated from silicon carbide and aluminum oxide was measured by using MRI and Heubach dustiness testers modified to allow the measurement of dust particle size distribution with an Andersen impactor. The two materials investigated generated similar dusts. The size distribution of the test material slightly influenced the amount but strongly influenced the size distribution of the dust generated. Increased moisture content decreased the amount of dust generated; moisture content had little influence on dust size distribution. The two testers generated different amounts of dust; however, the dust particle size distributions generated were similar. These results help explain factors that affect dust generation and the relative importance of alternative methods for dust control.

Intake, digestibility, ruminal characteristics and rate of passage of orchardgrass diets fed to sheep.

Four orchardgrass diets ranging in cell wall content from 60 to 78% were fed to sheep, and relationships among intake, digestion, passage and ruminal measurements were determined. As cell wall concentration increased, dry matter intake, digestible energy intake, dry matter digestibility and excretion rate decreased, while cell wall intake rumen volume, rumen cell wall and retention time increased. Indigestible cell wall intake was similar with each diet. It appeared that as digestible energy intake decreased, the sheep attempted to adapt by increasing ruminal ingesta volume, increasing ruminal ingesta cell wall and decreasing rate of passage; as cell wall concentration increased, indigestible cell wall limitation was manifested in decreased levels of feed and energy intake. Rate and extent of digestion appeared to be related to indigestible cell wall and appeared to be key factors in the control of cell wall turnover and feed intake.

Relationship of dietary selenium to selenium in plasma and milk from dairy cows.

In a 13-wk trial Holstein cows in mid-lactation were fed four diets containing natural selenium alone or supplemented with .1, .2, or .5 mg of inorganic selenium per kilogram of diet. Dietary selenium concentration averaged .334, .385, .456, and .772 mg/kg. Selenium plasma in wk 7 averaged .112 mu/ml with no differences among treatments; milk selenium ranged from .040 to .046 mu/ml and was higher in the two higher selenium diets. In wk 13 selenium in plasma and milk averaged .119 and .054 with no treatment differences. The 7 and 13 wk concentrations were higher than pretrial .084 and .033 mu/ml for plasma and milk during which time dietary selenium concentration was .254 mg/kg. Selenium concentration increased linearly from about .08 to .120 mu/ml of plasma and about .030 to .055 mu/ml of milk as intake of selenium increased from about 2 to 6 mg/day. Increase in selenium intake from 6 to 12 mg/day resulted in little change in plasma and milk selenium. Moderate concentrations of dietary selenium (.3 to .7 mg/kg) do not result in toxic amounts of selenium in milk.