The physicochemical effects of a series of alkanols, alkanediols and glycerol on erythrocyte shape and hemolysis at 4 and 20 degrees C were examined. We calculated the dielectric constant of the incubation medium, Ds, and the dielectric constant of the erythrocyte membrane Dm in the presence of organic solutes. The ratio Ds/Dm = -38.48 at 20 degrees C defines the normal biconcave shape in a medium without hemolytic agents. A decrease in Ds/Dm favors externalization or internalization with consequent hemolysis. Alkanols and alkanediols convert biconcave erythrocytes into echinocytes, which is accompanied by an increase in the projected surface area. Glycerol converts biconcave erythrocytes into stomatocytes, which was accompanied by a marginal decrease in the projected surface area. Progressive externalization in alkanols and alkanediols or internalization in glycerol resulted in a decrease in the projected surface area and the formation of smooth spheres. The degree of shape change induced was related to the degree of hemolysis and the ratio Ds/Dm. A decrease in temperature reduced both the degree of shape change and hemolysis. Our results suggest that physicochemical toxicity may be a result of a temperature dependent hydrophobic interaction between the organic solutes and the membrane and is best interpreted by the ability of the solutes to change Ds and Dm. These results are discussed with respect to the physicochemical constants of the organic solutes.

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We describe a procedure for lipopolysaccharide (LPS) biotinylation using N-biotinyl-L-lysine and application of the biotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (HRP) or streptavidin-gold method. Platelet surfaces were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPS was localized in small vesicles and vacuoles of platelets and in the phagocytic vacuoles of monocytes 60 min post incubation. Bi-LPS provides a reliable, stable, and sensitive tool for determination of LPS binding sites and subcellular distribution.

An important new method for phagocytosis research, confocal scanning fluorescence light microscopy (CSFM), is demonstrated using fluorescent microspheres ingested by murine macrophages. CSFM, in combination with Nomarski differential interference contrast microscopy (DIC), can resolve microspheres inside cells from microspheres attached to the surface of cells. Further, combined CSFM and DIC images can quantitate phagocytosis by individual cells aggregated together. No other method offers these capabilities. A comparison of CSFM and conventional epifluorescence light microscopy (EFM) images shows that CSFM produces significantly higher-resolution images of microspheres than EFM, primarily because CSFM excludes the out-of-focus light artifacts of EFM.

Foreign bodies, in particular sand and soil particles, can cause considerable reaction in wounds and remain in tissues indefinitely. The introduction of foreign material as a contaminant in wounds and intracavitary injuries to military personnel deployed in the Persian Gulf region can be a complicating factor in treatment. Samples of desert sand from the military operational areas of the Persian Gulf were analyzed and the acute local and systemic responses of intraperitoneal contamination determined in an experimental rat model.