Cytotaxins after the sedimentation behavior of human granulocytes.

Human granulocytes from the peripheral blood of healthy donors were subjected to transient gravity sedimentation analysis in Ficoll density gradient columns (37 degrees C) containing different concentrations of Escherichia coli endotoxin-activated serum and medium 199. A dramatic serum concentration-dependent dispersion of the cells based on changes in sedimentation velocity was observed as a function of time, using a new optical scanning instrument. The phenomenon was virtually abolished in the presence of cytochalasin B, a known inhibitor of cellular chemotaxis. The width (second statistical moment) of the sedimenting cell distribution increased in a sigmoid fashion as a function of time regardless of cytotaxin concentration. This indicates that a slow and nonlinear response of the granulocytes to the cytotaxins occurs. This new kinetic method should be useful in examining an alternate manifestation of the chemoresponsiveness of phagocytic cells and of cell interactions in general.

Structural features of rat cardiac ferritins.

Ferritin extracted from rat heart containes two species separable by gel electrophoresis. These were purified and examined for structural characteristics. As in gel electrophoresis, cardiac ferritin preparations yielded only two bands on isoelectric focusing in gels, with pI values of 4.6 and 4.8. After separation by preparative electrophoresis, the two species were found to have a different amino acid composition from each another and from liver ferritin. Similarly, peptide maps showed several components not found in liver ferritin. On dissociation and electrophoresis with sodium dodecyl sulfate, heart ferritins were found to contain subunits of the same sizes as in other rat ferritins but also some larger components. Since cardiac ferritins have apparent molecular weights greater than those of other ferritins, it is concluded they probably contain more subunits, and possibly some of larger size not present in ferritins of other tissues.

Size and charge heterogeneity of rat tissue ferritins.

Ferritins purified from horse spleen and from rat liver, kidney, heart and hepatoma were analyzed by quantitative polyacrylamide gel electrophoresis. From the migration characteristics of these ferritins at several gel concentrations, Ferguson plots were constructed and the molecular sizes and charges (apparent valences) together with their statistical variability were obtained by applying Rodbard computer programs to the data. Finally, ellipses were drawn describing the 95% confidence limits of these data for size and charge and were used to identify those ferritins that differed in size and/or charge. By these criteria, many of the tissue ferritins were differentiated from one another in terms of their molecular size and/or charge. Among the various tissue ferritin monomers, the molecular sizes were essentially similar (420 000-490 000) except for the two heart ferritins which were larger (530 000 and 626 000, respectively). However, the estimated charges on rat liver, kidney and hepatoma monomers (30-38 net protons per molecule) differed from that of spleen monomer (51 net protons per molecule) while the larger rat heart ferritin also had a greater charge (83 net protons) than the smaller (40 net protons). Apoferritins prepared chemically by removal of iron from the holoferritins had migration properties indistinguishable from the parent holoferritins. The migration properties of minor (dimeric) ferritin bands on the gels were compared with those of the monomer bands. The molecular sizes of the minor bands were larger than those of the major bands, and were not inconsistent with a doubling in size. However, charge differences varied, being either similar for major and minor forms (spleen ferritin), approximately twice for the minor form (rat hepatoma ferritin) or five times greater for the minor form (rat liver ferritin). These differences in behavior were confirmed by using minimally sieving gels, on which the major bands of horse spleen ferritin failed to separate whereas those of rat liver ferritin were readily separable. It is concluded that dimers of ferritins from different tissues may associate in different ways.

51Cr and DF32P labeling of human blood cells in leukocyte-rich plasma.

Cell-rich plasma from human peripheral blood was labeled with disodium chromate (51Cr) and diisopropylfluorophosphate (DF32P), and the uptake and distribution of the radionuclides by granulocytes, lymphocytes, monocytes, and erythrocytes were studied. Velocity sedimentation through a 1-2% albumin gradient and electrophoresis in a Ficoll-sucrose gradient demonstrated that granulocytes migrated the fastest, followed by monocytes, and then by the mixture of lymphocyte-red cell populations. Monocytes accumulated four to five times as much 51Cr as the other leukocytes or the red blood cells. Erythrocytes showed a greater uptake of 51Cr than did granulocytes or lymphocytes. Granulocytes were preferentially labeled in vitro by DF32P, and the uptake of DF32P by lymphocytes was about one-third that by granulocytes. No measurable DF32P labeling of monocytes and only slight labeling of erythrocytes was observed by liquid scintillation counting of the labeled cells. These data have implications in the interpretation of in vivo survival studies of radiolabeled leukocytes.

Separation of rat T and B lymphocytes by density gradient electrophoresis.

Density gradient electrophoresis has been employed for the preparative separation of T and B lymphocytes from rat spleen and peripheral blood. The high mobility cells were found to be predominantly T lymphocytes, as determined by their response to phytohemagglutinin and the relative absence of immunoglobulin-positive cells. The low mobility cells were predominantly B cells, as determined by the high percentage of immunoglobulin-positive cells and the total lack of response to PHA, an exclusive T cell mitogen. A better separation of peripheral blood T and B lymphocytes was achieved than with spleen T and B cells.

Density gradient electrophoresis of mouse spleen lymphocytes: age-related differences. A critical thymus-dependent event during development in the young adult mouse.

T and B BALB/c mouse spleen lymphocytes have been separated by preparative density gradient electrophoresis from animals of different ages. Significant age-related differences in the frequency of occurrence of cells exhibiting different mobility were observed in the young adult mouse. In the 6.5-week-old animals, the frequency of occurrence of the high (T lymphocytes) and low (B lymphocytes) mobility cells was changed, so that these lymphocytes exhibit an electrophoretic distribution profile different (essentially unimodal) from younger (3.5--5.5 weeks) or older (7.5--17 weeks) animals. In the latter two, bimodal electrophoretic distributions were observed. However, differences were also found in the frequency of occurrence of high and low mobility cells. The mobility distributions, representing individual cell types, were reproducible. Furthermore, the age-related changes were independent of the method of cell preparation and appeared in all mouse strains examined. Lymphocytes from animals thymectomized at the 5th week of age did not exhibit these changes (i.e. unimodal distribution) by the 6th week of age. Their electrophoretic distributions at the 6th week and thereafter were similar to those obtained from younger (3.5--5.5 weeks old) animals. It is concluded that the observed changes in the electrophoretic distributions of mouse spleen lymphocytes during development are thymus dependent and may be related to thymus involution.

Density gradient electrophoresis of mouse spleen lymphocytes: separation of T and B cell fractions.

Preparative electrophoresis in an isotonic Ficoll--sucrose density gradient has been employed for the separation of mouse (C57Bl/6J) spleen lymphocyte subpopulations. The separated cells were pooled into six fractions according to their relative position (Rp) within the total cell distribution. In general, the high mobility cells were identified as T lymphocytes. These cells exhibited immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated mouse anti-theta globulin and responded in vitro to phytohemagglutinin stimulation. The low mobility cells were activated in vitro by E. coli lipopolysaccharide and showed immunofluorescence upon reaction with fluorescein isothiocyanate-conjugated anti-mouse Ig which is typical of mouse B lymphocytes. Both T and B cells were completely isolated from each other in certain fractions of very high and very low mobility, respectively. Overlapping of the two distributions was observed in the intermediate mobility fractions. The method which utilizes an inexpensive commercially available apparatus should be useful for the preparation of other lymphocyte subpopulations differing in surface charge.

Measurement of the distribution of indium-111 on human plasma proteins using immunoprecipitation.

The distribution of radioactivity on plasma proteins labeled by addition of [111In]oxine to citrated plasma was investigated. Analyses of plasma proteins separated on Sephadex G-200 columns showed that 23-36% of the 111In was associated with proteins with molecular weight greater than 200,000 daltons and the remaining 111In was associated with proteins with molecular weight less than 100,000 daltons, presumably transferrin. Affinity chromatography experiments showed that less than 2% of the radioactivity was associated with albumin. Further identification of the labeled proteins and quantitation of associated radioactivity was performed by precipitating specific proteins with antibodies. These studies showed that the 111In was distributed on transferrin (54-76%), fibrinogen (11-24%), IgM (8-20%), C3 (10-21%), and haptoglobin (3-8%). 111In associated with fibrinogen, IgM, and haptoglobin was over-estimated in some experiments due to binding of 111In-labeled C3 to the antigen-antibody precipitates.

Omental angiogenic lipid fraction and bone repair. An experimental study in the rat.

A lipid material extracted from the omentum has previously been shown to contain a potent angiogenetic activator (20), capable of creating intense vasoproliferation in traumatized tissues (19). This study was undertaken to analyze the efficacy of local administration of this omental lipid fraction on osseous vascularization and bone repair. An osteoperiosteal segmental femoral defect in the rat was replaced by a demineralized allogenic bone graft exposed to continuous local delivery of omental lipid via an implanted miniosmotic pump. Saline solution delivered in the same way served as a control. Neovascularization and bone formation in the transplant were quantitatively evaluated by means of dynamic radioisotopic bone imaging, radiographic photodensitometry, microangiography, and biomechanical testing. Compared with the control group, the omental lipid angiogenic fraction-treated specimens showed an 80% overall increase (p less than 0.001) in bone density as well as a twofold increase (p less than 0.001) in regional blood perfusion, maximal at 2 weeks following surgery. At 12 weeks, biomechanical testing demonstrated significantly higher union rate (p less than 0.05) and strength (p less than 0.01) in the treated specimens as compared with the controls. These data demonstrate that the omental lipid fraction factor has potent angiogenic properties that enhance bone blood perfusion and bone regeneration.

Gravity sedimentation analysis of human blood leukocytes.

A new method of sedimentation analysis of human blood leukocytes is described. Platelets, lymphocytes, monocytes, and polymorphonuclear cells isolated from normal human peripheral blood have been analyzed alone and in mixture by gravity sedimentation, employing a computerized scanning instrument. All four classes could be clearly resolved from each other exhibiting sedimentation velocities of 0.06 +/- 0.00, 1.04 +/- 0.11, 1.27 +/- 0.15 and 1.89 +/- 0.21 x 10(-4) cm/s, respectively, at 37 degrees C in a 2.5--6.25% Ficoll gradient in Medium 199. Less than 10(6) cells can be used for analysis. Possible applications of the method are discussed.

Characterization of feline omentum lipids.

Feline omental lipid extracts, previously reported to be angiogenic in the cornea of rabbits, were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16:0, 18:0, 18:1 and 18:2 fatty acids. Trace quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC)-mass spectrometry, was found to consist of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by high performance thin layer chromatography and HPLC of their perbenzoylated derivatives, were found to consist of glucosyl- and galactosylceramides, galabiosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. The complex glycolipid fraction, obtained from Folch upper phase solvent partition, was found to consist primarily of Forssman glycolipid and gangliosides GM3 and GD3. Smaller amounts of GM1 and other unidentified gangliosides were also present.