We have developed an improved method for measuring the filamentous (F) actin content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleton. Fresh whole blood (100 microliters) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer pre-cooled to -8 degrees C and subsequently fixed at 4 degrees C for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugation, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothiocyanate (FITC)-phalloidin and quantified by means of flow cytometry. In healthy test subjects, PMN stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly increased F-actin assembly, while in patients with multiple organ failure, two subpopulations arose: one with greater F-actin content and a second with lower F-actin content in comparison with the unstimulated blood sample. This simple and fast method may be a useful tool in basic and clinical research.

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Polymorphonuclear leukocyte functions as predictive markers for infections after organ transplantation-Abstract Infectious complications

The aim of the study was to demonstrate an activation of polymorpho-nuclear leukocytes (PMNs) in chronic progressive atherosclerosis (ATH). A group of patients with ATH, and a group of ATH patients under aspirin (ASA) therapy were compared with control persons without atherosclerotic alterations (healthy controls). Each group comprised 15 male age-matched subjects. The following inflammatory parameters related to PMN activities were measured: the polymorphonuclear leukocyte (PMN) blood count; blood PMN migration and reactive oxygen species release in vitro; the blood levels of PMN elastase, malondialdehyde, antibodies to oxidized LDL and soluble ICAM-1. In ATH patients, the PMN blood counts and the share of blood PMNs migrating upon platelet activating factor and leukotriene B4 stimulation were significnatly above the values of the healthy controls, while the other parameters were not significantly altered. ASA treatment attenuated the inflammatory response and reduced the differences between ATH and the healthy controls. It can be concluded that, in patients with chronic progressive atherosclerosis, PMNs are involved in the inflammatory process underlying the disease.