The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
The role of platelets in the maintenance of endothelial barrier is examined in an in vitro model of the microvasculature. Human platelets (6,000/microliters) perfused through a cell column of endothelial-covered microcarriers decrease paracellular permeability of sodium fluorescein (mol wt 342) to 63% of baseline values. This effect is reversible and a second application and removal of platelets produces a similar response. This effect occurs within 5 min and reverses within 10 min after platelet removal. The reduction in permeability is not due to mechanical obstruction of endothelial junctions, since the number of recirculating platelets is not reduced and releasate from unstimulated 2-h platelet incubations also decreases permeability. Releasate from platelets stimulated with 0.1 U/ml of thrombin for 15 min have the same permeability reducing effect. In this system, the platelet factors serotonin (10(-3) M) and ADP (10(-4) M) have no effect on permeability. However, the platelet factors adenosine (10(-4) M), ATP (10(-5) M), and beta-agonists decrease permeability. None of these appear to account for platelet permeability activity, since activity is not blocked by agents directed against these mediators (adenosine deaminase, apyrase, 8-phenyltheophylline, or propranolol). The active factor(s) is stable at -20 degrees C, heat stable, sensitive to trypsin, and has an apparent molecular weight > 100. We conclude that unstimulated platelets release a factor(s) that enhances endothelial barrier in vitro and may be important in maintenance of the normal in vivo barrier.
Small unilamellar liposomes were used in this study of shear stress effects on the trans-bilayer flux of calcium ions (Ca2+). Liposome suspensions were prepared from 99% egg phosphatidylcholine by a microporous filter extrusion technique. The inner aqueous phase of the unilamellar liposomes contained indo-1(5-), a fluorescent indicator of free Ca2+. The external aqueous phase was composed of Hepes-buffered saline containing normal physiological levels of common ionic species. Calcium ion levels were set at 100 nM and 1 mM in the inner and outer aqueous phases, respectively. Liposome suspensions were exposed to graded levels of uniform shear stress in an optically modified rotational viscometer. Intraliposome Ca2+ concentration was estimated from continuous measurement of indo-1(5-) fluorescence. Electronically measured particle size distribution was used to determine liposome surface area for estimation of trans-bilayer Ca2+ flux. Trans-bilayer Ca2+ flux increased linearly with applied shear rate from 27 s-1 to 2700 s-1. Diffusional resistance of the lipid bilayer, not the convective resistance of the surrounding fluid, was the limiting step in the transport of Ca2+. Liposome permeability to Ca2+ increased by nearly two orders of magnitude over the physiologically relevant shear rate range studied. Solute transport in injectable liposome preparations may be dramatically influenced by cardiovascular fluid stress. Solute delivery rates determined in liposomes exposed to static conditions may not accurately predict in vivo, cardiovascular solute transport.
Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.