Electron microscopy of phages in serotypes of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica and Pasteurella multocida strains were examined by transmission electron microscopy for the presence of bacteriophages. Phages were detected in serotype a (SUNY 75) and e (UOH 1705) and in the fresh clinical isolates UOH Q1243 and UOH Q1247 of A. actinomycetemcomitans. Phages were not found in serotype b, c and d strains of A. actinomycetemcomitans, in the fresh clinical isolate UOH Q1244 of this species or in old strains (including reference strains) of related species from the Actinobacillus-Haemophilus-Pasteurella group.

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

Effects of endotoxins from Bacteroides intermedius and Escherichia coli on cytotoxic and lysosomal activity in peritoneal macrophages from endotoxin responder and low responder mouse strains.

Peritoneal macrophages from normal mice strains (C3H/Tif and C57BL/6J) and from the endotoxin (lipopolysaccharide, LPS) low responder strain (C3H/Hej were exposed to two structurally different endotoxins from Bacteroides intermedius and Escherichia coli in vitro. Intracellular activity of a lysosomal enzyme (acid phosphatase) and macrophage mediated cytotoxic activity against a tumor cell line (L929) were tested. Both endotoxins caused increased levels of acid phosphatase activity in normal mice macrophages. No change was obtained in the C3H/Hej macrophages exposed to E. coli LPS; however, the B. intermedius LPS was able to strongly elevate intracellular enzyme level in the C3H/Hej low responder macrophages. Cytotoxic activities were investigated in macrophage supernatants and in co-cultures of stimulated macrophages and target cells. Cytotoxic activity evaluated by measuring release of radioactivity from 14C-thymidine labelled tumor cells was increased with both endotoxins in normal mouse macrophages, but not in non-responder macrophages. When macrophage-mediated effects on tumor cells were tested by counting target cells left per culture, a reduction in target cell number was observed in endotoxin-treated low responder macrophage as well as in normal strain macrophage cultures more pronounced, however, in the normal strain. Cell contact between cytotoxic macrophages and target cells was verified by scanning electron microscopy. The results suggest that LPS effects on macrophages are dependent upon the functional parameters studied, and that the chemical composition of a particular LPS is important for its selective effects on macrophage functions.

The use of frozen monocytes in phagocytosis studies.

Human monocytes were isolated from peripheral blood by Lymphoprep density-gradient centrifugation and adherence to fibronectin. The cells were loosened by ethylene-diamino-tetra-acetate (EDTA), frozen by different freezing methods, thawed, washed and compared to unfrozen cells. After freezing, thawing and washing, cell recovery was calculated and found to vary with the freezing procedure. The best result was about 76% cell recovery. No morphological differences were observed between unfrozen and frozen cells. The experiments also showed that the percentage of cells that attached or phagocytized IgG-opsonized erythrocytes (E-IgG) via the Fc-receptor was unaltered after freezing. Neither was there any difference between unfrozen and frozen monocytes with respect to their ability to phagocytize latex particles. There was no significant difference in reactivity between monocytes frozen for one day and those frozen for six weeks.

The use of frozen erythrocytes in macrophage studies. 2. Attachment and phagocytosis mediated by the two immunological receptors of the macrophages.

Sheep erythrocytes opsonized with IgG or C3b were frozen in various cryoprotective agents, thawed, and compared to corresponding unfrozen erythrocytes exposed to the cryoprotectants and to unfrozen erythrocytes not exposed to the cryoprotectants (controls) as test particles in macrophage attachment and phagocytosis assays. Fc-receptor-mediated attachment and phagocytosis were not influenced by the use of any cryoprotective agent tested or by freezing the erythrocytes. This was also the case with C3b-receptor-mediated attachment. Phagocytosis via this receptor was negligible in normal macrophages, but tended to be slightly more effective when the test particles had been treated with cryoprotective agents. In vitro stimulation of the macrophages with Escherichia coli endotoxin, however, triggered the capacity to internalize treated and untreated erythrocytes equally.

The use of frozen erythrocytes in macrophage studies. 1. Attachment to the foreign surface receptor of the macrophages.

The attachment of red cells to mouse peritoneal macrophages in vitro was tested with erythrocytes (from sheep and man) which had been subjected to different cryoprotective agents and freezing procedures. The experiments showed that with dimethylsulfoxide (DMSO) as the cryoprotective agent no difference in macrophage attachment was seen whether the erythrocytes were frozen or not. With the other cryoprotectants tested, macrophages were more efficient in attaching frozen than unfrozen erythrocytes. This was the case with erythrocytes from both sheep and man. Similar results were obtained with fresh (one week) and old (two weeks) erythrocytes.

The use of frozen erythrocytes in the single radial haemolysis test (SRHT) for antibodies to rubella virus.

The cryoprotection of erythrocytes from newborn chickens was investigated. The cryoprotective agents tested were neutralized polyvinylpyrrolidone (PVP), dimethylsulfoxide (DMSO) and glycerol. Best results were obtained with 10 per cent DMSO, whereas 20 per cent DMSO and glycerol were unfit for use. The red cell concentration and the temperature of freezing and thawing were of importance. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 5 per cent, and best results were obtained when the cells were frozen directly in liquid nitrogen (-196 degrees C). Frozen and thawed chicken cells were used in the single radial haemolysis test (SRHT) for the assay of rubella antibody, and compared with frozen and thawed erythrocytes from hen and sheep. Haemagglutinin (HA) absorbed sheep erythrocytes could be frozen in small quantities and used directly in the SRHT plates whereas frozen erythrocytes from newborn chickens and hen had to be washed before use in the SRHT phase.